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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 659-665, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4866

Polyethylene Glycol Conjugation at Cys232 Prolongs the Half-Life of {alpha}1 Proteinase Inhibitor

André M. Cantin, Donald E. Woods, Diane Cloutier, Erick K. Dufour and Richard Leduc

Pulmonary Research Unit and Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec; and Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada

Address correspondence to: André M. Cantin, M.D., Pulmonary Research Unit, Faculty of Medicine, University of Sherbrooke, 3001 12ième Avenue Nord, Fleurimont, PQ, J1H 5N4 Canada. E-mail: Andre.Cantin{at}usherbrooke.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{alpha}1 Proteinase inhibitor ({alpha}1PI), a natural inhibitor of the serine proteinase leukocyte elastase, is also an intravenous therapeutic agent used to treat hereditary emphysema and may be useful in other respiratory disorders. However, to achieve sustained suppression of leukocyte elastase, {alpha}1PI must be given frequently and in large amounts, thus limiting its clinical use. We hypothesized that conjugating {alpha}1PI with polyethylene glycol (PEG) at Cys232 could extend the in vivo half-life of {alpha}1PI in blood and lung. We present evidence that site-specific conjugation with either 20 or 40 kD PEG at Cys232 of nonglycosylated recombinant human {alpha}1PI (rh{alpha}1PI) results in an active inhibitor with prolonged in vivo stability. In addition, 72 h after airway instillation PEG-rh{alpha}1PI was found to be significantly better than glycosylated {alpha}1PI in protecting the lung against leukocyte elastase–mediated lung hemorrhage. We conclude that thiol-specific PEGylation markedly improves the in vivo pharmacokinetic profile of rh{alpha}1PI and represents a simple, novel strategy to address the therapeutic goal of human leukocyte elastase inhibition.

Abbreviations: {alpha}1 proteinase inhibitor, {alpha}1PI • bronchoalveolar lavage fluid, BALF • cystic fibrosis, CF • human leukocyte elastase, HLE • human neutrophil elastase, HNE • isopropyl ß-D-thiogalactoside, IPTG • relative molecular mass, Mr • N-ethylmaleimide, NEM • polyethylene glycol, PEG • recombinant human {alpha}1PI, rh{alpha}1PI • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human leukocyte elastase (HLE) is thought to play an important role in the pathophysiology of several respiratory disorders, including emphysema, cystic fibrosis, pulmonary hypertension, and pulmonary fibrosis (1–5). Although many synthetic human leukocyte elastase inhibitors have been developed, none are approved for use in the treatment of any respiratory disorders and the only currently available therapeutic option for HLE inhibition is intravenous {alpha}1 proteinase inhibitor ({alpha}1PI), approved for use in patients with hereditary emphysema (6). The large quantities and the frequent infusions of {alpha}1PI that are needed to restore protective levels of this protease inhibitor are significant factors that limit access to this therapy for patients with {alpha}1PI deficiency. The short supply of {alpha}1PI, which is currently purified from pooled blood products, has also been a limiting factor in the development of clinical trials to explore the potential usefulness of HLE suppressive therapy in other diseases such as cystic fibrosis, where the rationale for testing {alpha}1PI therapy is compelling (7–11).

Potential therapeutic alternatives to blood-derived {alpha}1PI are currently being explored. Because nonglycosylated {alpha}1PI has a short half-life in blood, strategies have focused on production systems that allow the addition of carbohydrates at the glycosylation sites of {alpha}1PI. One such strategy involves the production of human recombinant {alpha}1PI in the milk of transgenic sheep (12). Such an approach may prove to be successful, but little is currently known about the feasibility of this strategy or the effects of ovine post-translational modifications on the protein's pharmacokinetic properties in humans. In this context, we chose to explore the pharmacokinetics and HLE inhibitory activity of an alternate HLE inhibitor in the form of recombinant human {alpha}1PI covalently conjugated with polyethylene glycol.

Polyethylene glycol is a polyalkylene oxide polymer which, when linked to proteins (a process referred to as PEGylation), has been shown to markedly increase the biologic stability and decrease the immunogenicity of therapeutic proteins (13). Recently, a simple effective method of PEGylating proteins has been described in which PEG is activated with a maleimide group capable of specifically forming a covalent bond with free thiols on the surface of proteins (14). The reaction time is short and the procedure avoids the use of toxic chemicals. In addition to markedly increasing biologic half-life, the conjugation of proteins with maleimido-PEG does not adversely affect protein function, thus prompting investigators to propose the use of this technology to develop useful therapeutic molecules such as PEGylated human antibody fragments (14).

To conjugate PEG-maleimide, the desired protein must have a cysteine residue with an exposed thiol group at the protein surface. {alpha}1 Protease inhibitor contains a unique, free thiol at Cys232 (15). Three-dimensional studies have shown that this residue is exposed at the surface of the protein (Figure 1). We therefore hypothesized that PEGylation at Cys232 of {alpha}1PI using maleimido-PEG may result in an HLE inhibitor with markedly improved pharmacokinetic properties. Moreover, because previous studies suggested that modification of the Cys232 thiol alters the capacity of {alpha}1PI to inhibit HLE (16), we also studied the kinetics of HLE inhibition by thiol-specific PEGylated rh{alpha}1PI both in vitro and in vivo in a murine model of HLE-induced lung injury.



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  		Figure 1. The general structure of serpinA1 (human {alpha}1 proteinase inhibitor, {alpha}1-antitrypsin) based on the crystallographic coordinates (36). Representation was performed using InsightII (MSI) from the Protein Data Bank file 1QLP. Those residues that constitute the scissile bond P1-P1' (Met358-Ser359), the N-glycosylation sites (Asn46, Asn83, and Asn247), and the free Cys232 are shown.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
{alpha}1 Proteinase Inhibitor Preparations
Human {alpha}1PI was purified from the blood-derived {alpha}1PI preparation, Prolastin (Bayer Inc., Etobicoke, ON, Canada). Prolastin was diluted to a concentration of 100 mg/ml in 50 mM Tris, 50 mM NaCl at pH 8.0. To remove albumin, a 500-µl aliquot of this preparation was deposited on a 10-ml Cibacron Blue-Sepharose column (Amersham Pharmacia Biotech Inc., Baie d'Urfé, PQ, Canada) (17). The unbound protein fraction was collected and filtered through a CNBr-activated Sepharose-4B gel (Amersham Pharmacia Biotech) to which antihuman serum albumin antibodies (Sigma-Aldrich Canada Ltd, Oakville, ON, Canada) were covalently linked. The unbound protein fraction was then filtered through an anti–whole serum affinity column prepared by attaching anti–whole serum antibodies specifically depleted of {alpha}1PI antibodies to CnBr-activated Sepharose 4B beads as previously described (18).

Recombinant human {alpha}1PI (rh{alpha}1PI) was produced in a procaryotic system. The cDNA encoding human {alpha}1PI was subcloned in pQE31 (Qiagen Inc., Mississauga, ON, Canada). The final construct (pQE31–rh{alpha}1PI) consisted of a His-tag at the N-terminus of the molecule to facilitate purification, followed by the {alpha}1PI sequence excluding the signal peptide. rh{alpha}1PI was expressed in the BL21 Escherichia coli strain as previously described (19). Cultures were grown to an OD600 of 0.7–0.8, upon which induction was performed using 1 mM isopropyl ß-D-thiogalactoside (IPTG), and growth was continued for a further 5 h at 37°C. Cells were then harvested and proteins were prepurified according to published procedures (19) and rh{alpha}1PI was purified on Ni+2-NTA resin followed by affinity purification as previously described (20). The purity of the preparation was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the relative molecular mass (Mr) estimated by comparing the relative mobility to low-range protein standards (Bio-Rad Laboratories, Hercu-les, CA).

Polyethylene Glycol Conjugation of {alpha}1PI
Human {alpha}1PI has a unique free thiol at Cys232. We therefore chose to covalently bind to the thiol side-chain of this residue either 5, 20, or 40 kD polyethylene glycol that had been previously activated to react specifically with thiols through an attached maleimide group (Shearwater Corporation, Huntsville, AL). The maleimido-PEG derivative was incubated with either {alpha}1PI (throughout the text, Prolastin-derived protein is referred to as {alpha}1PI) or rh{alpha}1PI in phosphate-buffered saline (pH 7.4) at a molar ratio of PEG:protein of 5:1 for 2 h at 37°C. The {alpha}1PI preparations were quantitated by radial immunodiffusion assays (Dade Behring Inc., Newark, DE). Because PEG can alter the immunoreactivity of conjugated proteins, rh{alpha}1PI–PEG conjugates were analyzed by electrophoresis on a 7.5% SDS-PAGE and was stained with Coomassie blue to ensure purity. Total protein content was then determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Mississauga, ON, Canada) which is based on the Bradford method (21), using bovine serum albumin as standard. The {alpha}1PI concentrations measured using the Bio-Rad Protein Assay were found to be identical to those obtained with radial immunodiffusion, indicating that bovine serum albumin was an appropriate standard. To determine whether PEGylation was specific to Cys232, {alpha}1PI was incubated with N-ethylmaleimide (NEM), a reagent that specifically and covalently binds thiol residues. SDS-PAGE (data not shown) subsequently revealed that NEM-treated {alpha}1PI could no longer be PEGylated, thus confirming that PEG was specifically attached to the single thiol residue of {alpha}1PI, Cys232.

Neutrophil Elastase Inhibitory Capacity
To ensure an accurate determination of the human leukocyte elastase activity used in all kinetic experiments, trypsin was titrated against {alpha}1PI using the active site titrant 4-methylumbelliferyl 4-guanidinobenzoate (22), and HLE was then titrated against {alpha}1PI. The HLE inhibitory capacity of each preparation was determined by incubating 0–25 nM inhibitor with 20 nM HLE for 1 h at 37°C. Residual HLE activity was determined using the specific chromogenic substrate methoxy-succinyl-alanyl-alanyl-prolyl-valyl p-nitroanilide as previously described (18). To ensure that the inhibitors could form irreversible complexes with HLE, each inhibitor was incubated with HLE at a 1:0.5 molar ratio for 1 h at 23°C and subsequently analyzed on a 7.5% SDS-PAGE. The rates of association (Kassoc) of {alpha}1PI and {alpha}1PI-PEG20 were also determined according to previously published methods (23, 24).

Iodination of {alpha}1PI and Determination of In Vivo Distribution
Protein preparations were iodinated in the presence of chlora-mine-T (Sigma-Aldrich) (25). Sufficient 125I-labeled and unlabeled {alpha}1PI preparations were added to prepare solutions at a concentration of 300 µg/ml with a specific activity of 2.9 x 105 cpm/µg protein. CD1 mice received either {alpha}1PI, rh{alpha}1PI, or PEGylated preparations, intravenously (100 µl) or intranasally (50 µl) as previously described (26). Mice were killed at either 4, 24, or 48 h and both blood and bronchoalveolar lavage fluid (BALF) were collected as previously described (26) to determine the sample radioactivity and to perform autoradiography. To obtain the autoradiograms, blood and BALF were separated on a 7.5% SDS-PAGE followed by autoradiography (27). Because free, unbound PEG was not separated from {alpha}1PI-PEG, we determined whether unbound PEG could affect the {alpha}1PI half-life. To do this, {alpha}1PI, in which the Cys232 was blocked by covalent linkage with NEM, was incubated with PEG-20 and labeled as described above, before intravenous injection into mice. Mice were then killed at 4 and 24 h to determine blood radioactivity levels as an index of {alpha}1PI half-life.

Lung Anti-HLE Protection Assay
To compare the capacity of {alpha}1PI and rh{alpha}1PI versus PEGylated rh{alpha}1PI to protect the lung against HLE up to 72 h after nasal instillation, C57BL/6 mice were anaesthetized and instilled intranasally with 50 µl of either saline, 9 µM {alpha}1PI, 9 µM rh{alpha}1PI, 9 µM 5 or 20 kD PEGrh{alpha}1PI. At 24 and 72 h, the mice were subsequently instilled with 50 µl of 9 µM HLE and killed 1 h later to determine the hemoglobin content of BALF as an index of HLE-mediated lung injury as previously described (26).

Statistical Analysis
The results are expressed as the mean ± SEM. Statistical analysis was performed by ANOVA and applying post hoc analysis with Fisher's protected LSD test using the SuperANOVA software package (Abacus Concepts Inc., Berkeley, CA). A P value less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Conjugation of PEG to {alpha}1PI and rh{alpha}1PI
The reaction of {alpha}1PI with PEG of either 5, 20, or 40 kD resulted in the formation of protein–PEG complexes that could be identified by SDS-PAGE as Coomasie blue–stained bands that shifted from the expected Mr of native {alpha}1PI at 52 kD (Figure 2, left panel) to higher molecular weights. Although most of the {alpha}1PI was found to be linked to PEG in each lane, a small amount of unPEGylated {alpha}1PI persisted, as indicated by the band at 52 kD. The reaction of 20 kD PEG with rh{alpha}1PI resulted in a single band (Figure 2, right panel). The difference in migration pattern between {alpha}1PI (Mr 52 kD) and rh{alpha}1PI (Mr 47 kD) is due to the glycosylation state of the former.



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  		Figure 2. Polyacrylamide gel electrophoresis (Coomassie blue staining) of blood-derived human {alpha}1 proteinase inhibitor ({alpha}1PI, left panel) or recombinant human {alpha}1PI (rh{alpha}1PI, right panel), either alone or covalently conjugated to 5, 20, or 40 kD PEG. The PEG-{alpha}1PI samples were prepared by reacting {alpha}1PI with maleimido-PEG reagent in a 1:5 ratio for 2 h at 37°C.

 
HLE Inhibition In Vitro
To determine whether covalent conjugation of PEG at Cys232 had an adverse effect on the HLE inhibitory capacity of {alpha}1PI, the residual HLE activity was determined after 1 h incubation with either {alpha}1PI, rh{alpha}1PI, or their PEG derivatives. All protein preparations inhibited HLE efficiently. Complete HLE inhibition was observed in all preparations, with and without PEG, at a stoichiometric 1:1 molar ratio (Figure 3A). Similar results (results not shown) were also obtained after 24 h incubation, suggesting that the thiol modification with PEG did not promote dissociation of the HLE/PEG{alpha}1PI complexes. The {alpha}1PI and rh{alpha}1PI with and without conjugated PEG were equally capable of binding HLE and forming complexes as assessed on SDS-PAGE (Figure 3B). Furthermore, we observed that the covalent binding of PEG did not adversely affect the association rate constant of binding between HLE and {alpha}1PI ({alpha}1PI Kassoc = 1.05 ± 0.08 x 107 M-1 s-1; {alpha}1PI-PEG20 Kassoc = 0.95 ± 0.01 x 107 M-1 s-1, n = 3, P > 0.5).



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  		Figure 3. Human leukocyte elastase inhibitory activity of glycosylated and recombinant human {alpha}1 proteinase inhibitor ({alpha}1PI) alone or conjugated to either 5 (PEG-5) or 20 (PEG-20) kD polyethylene glycol residues. (A) Each inhibitor was incubated at the shown concentrations in the presence of 20 nM human leukocyte elastase for 1 h, followed by an elastase activity assay using the chromogenic substrate MeO-suc-AAPVpNA. Each data point represents the mean ± SEM of triplicate determinations. (B) Preparations of {alpha}1PI, rh{alpha}1PI, {alpha}1PI-PEG20, and rh{alpha}1PI-PEG20 were incubated with HLE at a molar ratio of 1:0.5 for 1 h at 23°C, followed by SDS-PAGE. Arrows correspond to inhibitor–HLE complex formation. Lanes 1 and 5, {alpha}1PI and rh{alpha}1PI; lanes 2 and 6, {alpha}1PI + HLE and rh{alpha}1PI + HLE; lanes 3 and 7, {alpha}1PI-PEG20 and rh{alpha}1PI-PEG20; lanes 4 and 8, {alpha}1PI-PEG20 + HLE and rh{alpha}1PI-PEG20 + HLE.

 
Effect of PEG Size on rh{alpha}1PI Half-Life In Vivo
To determine the optimal size of conjugated PEG to increase {alpha}1PI half-life in vivo, blood levels of radiolabeled rh{alpha}1PI with and without 5, 20, and 40 kD PEG were measured at various times. As can be seen in Figure 4, rh{alpha}1PI was rapidly cleared from blood, with levels at 4 h markedly lower than those of glycosylated {alpha}1PI (rh{alpha}1PI = 57,333 ± 6,566 cpm/ml versus {alpha}1PI = 328,000 ± 55,381 cpm/ml, P < 0.005). The conjugation of 5 kD PEG increased protein persistence in blood, but levels remained lower than those of {alpha}1PI at 8 and 24 h (8 h: rh{alpha}1PI-PEG5 = 86,333 ± 5,457 cpm/ml, {alpha}1PI = 190,333 ± 13,094 cpm/ml; 24 h: rh{alpha}1PI-PEG5 = 28,000 ± 4,619 cpm/ml, {alpha}1PI = 113,667 ± 9,025 cpm/ml, P < 0.005 rh{alpha}1PI-PEG5 compared with {alpha}1PI at each time point). In contrast, the conjugation of 20 and 40 kD PEG to rh{alpha}1PI completely restored the clearance kinetic properties by increasing blood levels at 4, 8, and 24 h to values similar to {alpha}1PI (Figure 4: rh{alpha}1PI-PEG20 and rh{alpha}1PI-PEG40: P > 0.05 all times compared with {alpha}1PI; P < 0.005 compared with rh{alpha}1PI, all times). Importantly, the presence of free, unbound PEG was found to have no effect on the half-life of either {alpha}1PI or rh{alpha}1PI (Table 1).



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  		Figure 4. Effect of the molecular weight of the covalently linked PEG conjugate on the persistence of radiolabeled, recombinant human {alpha}l proteinase inhibitor in the blood of CD1 mice. Preparations of glycosylated human {alpha}1 proteinase inhibitor ({alpha}1PI), rh{alpha}1PI produced in E. coli bacteria, and rh{alpha}1PI covalently linked to PEG of either 5, 20, or 40 kD, were radio-iodinated (specific activity = 2.9 x 105 cpm per µg protein, 2.9 x 106 cpm/100 µl). Subsequently, 100 µl of each sample was injected intravenously in the tail vein of each mouse. At 4, 8, and 24 h, animals were killed and blood was obtained for determination of radioactivity. Each column represents the mean ± SEM, n = 3. *P < 0.005 versus {alpha}1PI, {dagger}P < 0.005 versus rh{alpha}1PI.

 

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  		TABLE 1 Effect of the presence of free PEG20 on blood half-life of AT and rhAT after intravenous injection.

 
Effect of PEGylation on rh{alpha}1PI Half-Life in Blood and Airway
To compare the in vivo half-life of rh{alpha}1PI with that of rh{alpha}1PI conjugated to PEG, a preparation containing similar amounts of rh{alpha}1PI and rh{alpha}1PI linked to PEG of 20 kD (PEG20) was radiolabeled and analyzed by autoradiography. In contrast to non-PEGylated [125I]rh{alpha}1PI, which migrated as a single band (Figure 5A, left lane), the preparation combining rh{alpha}1PI and [125I]rh{alpha}1PI-PEG20 migrated as two distinct bands of similar intensity (Figure 5A, right lane). Autoradiograms of blood obtained from mice injected intravenously with this preparation are shown in Figure 5B, whereas autoradiograms of BALF obtained from mice instilled intranasally with samples containing [125I]rh{alpha}1PI-PEG20 and [125I]rh{alpha}1PI are shown in Figure 5C. The first lanes in Figures 5B and 5C represent starting material before injection in the mice (0 h). The rh{alpha}1PI clearly persisted for longer times in BALF than it did in blood. In both BALF and blood the rh{alpha}1PI-PEG20 persisted for longer times than the non-PEGylated form.



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  		Figure 5. In vivo persistence of radiolabeled, recombinant human {alpha}1-proteinase inhibitor conjugated to polyethylene glycol. (A) Autoradiogram of [125I]rh{alpha}1PI without and with conjugated PEG20. (B) Autoradiograms of blood obtained from mice injected intravenously with [125I]rh{alpha}1PI-PEG20 and [125I]rh{alpha}1PI. Time 0 represents the starting material before injection into animals. At each of the 4, 24, and 48 h time points, animals were killed and blood was separated on a 7.5% PAGE followed by autoradiography. (C) Autoradiograms of BALF obtained from mice instilled intranasally with samples containing [125I]rh{alpha}1PI-PEG20 and [125I]rh{alpha}1PI. Animals were killed at the indicated times, and BALF was separated on a 7.5% PAGE before analysis by autoradiography. Each row represents a different animal. The non-PEGylated rh{alpha}1PI was found to persist for longer times in bronchoalveolar lavage fluid than in blood. In both BALF and blood the rh{alpha}1PI-PEG20 persisted for longer times than the non-PEGylated form.

 
In Vivo HLE Inhibitory Activity
Because both rh{alpha}1PI and PEG-rh{alpha}1PI were found to be effective inhibitors of HLE in vitro, we next verified their capacity to inhibit HLE-mediated lung damage in vivo (Figure 6A). The instillation of 9 µM HLE alone at the nares of C57Bl mice resulted in lung hemorrhage which could be clearly detected 1 h later by BAL (saline BAL, results not shown, Hb = 27 ± 7 µg/ml, HLE control BAL Hb = 646.0 ± 32.6 µg/ml, P < 0.0001). Nasal instillation of 9 µM {alpha}1PI 24 h before the addition of HLE provided significant protection against HLE-mediated lung hemorrhage (BAL Hb = 319.6 ± 32.7, P < 0.0001 versus control). Interestingly, nasally instilled nonglycosylated rh{alpha}1PI was also highly effective at protecting the lung 24 h later against an equimolar concentration of HLE, and the conjugation of PEG (5 kD) resulted in a complex equally effective in protecting the lung against HLE (rh{alpha}1PI BAL Hb = 179.7 ± 37.1, PEG-5-rh{alpha}1PI Hb = 109.6 ± 11.9, P < 0.0001 each against control).



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  		Figure 6. Comparative protection by recombinant human {alpha}1-proteinase inhibitor with and without PEG at 24 h (A) and 72 h (B), against human leukocyte elastase–induced pulmonary hemorrhage in C57Bl/6 mice. (A) Mice were instilled intranasally with 50 µl of either saline solution (control), 9 µM blood-derived human {alpha}1 proteinase inhibitor ({alpha}1PI), recombinant human {alpha}1 proteinase inhibitor (rh{alpha}1PI), or 9 µM rh{alpha}1PI covalently linked to 5 kD PEG (rh{alpha}1PI-PEG5). After 24 h, 50 µl of 9 µM human leukocyte elastase was instilled intranasally and the animals were killed 1 h later for bron-choalveolar lavage and determination of hemoglobin concentration in the BALF. Each column represents the mean ± SEM, n = 7. *P < 0.0001 compared with control, {dagger}P < 0.02 compared with {alpha}1PI. (B) C57Bl/6 mice were instilled intranasally with 50 µl of either saline solution (control), 9 µM glycosylated human {alpha}1 proteinase inhibitor ({alpha}1PI), 9 µM recombinant human {alpha}1 proteinase inhibitor (rh{alpha}1PI), or 9 µM rh{alpha}1PI covalently linked to 20 kD PEG (rh{alpha}1PI-PEG20). After 72 h, 50 µl of 9 µM human leukocyte elastase was instilled intranasally and the animals were killed 1 h later for bronchoalveolar lavage and determination of hemoglobin concentration in the BALF. Each column represents the mean ± SEM, n = 15. * P < 0.02 compared with control.

 
To determine whether the conjugation of 20 kD PEG to rh{alpha}1PI extended the duration of protection against HLE-induced lung injury beyond that of rh{alpha}1PI or glycosylated {alpha}1PI, HLE-induced lung hemorrhage was assessed 72 h after nasal instillation of the inhibitors. Untreated animals developed readily detectable lung hemorrhage, and mice pretreated (3 d earlier) with either {alpha}1PI or rh{alpha}1PI showed similar levels of lung hemorrhage, suggesting that neither recombinant nor glycosylated {alpha}1PI was protective 72 h after instillation (Figure 6B). In contrast, mice treated with rh{alpha}1PI conjugated to 20 kD PEG continued to be protected against HLE-induced lung hemorrhage 72 h after instillation (BAL Hb: control = 467 ± 45, {alpha}1PI = 585.5 ± 59, rh{alpha}1PI = 436 ± 81, P > 0.05 each versus control; PEG20{alpha}1PI = 252 ± 43 µg/ml, P < 0.02 versus control).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
{alpha}1PI is a protein of therapeutic interest, but limited amounts of glycosylated {alpha}1PI originating from human blood are available for use in experimental and clinical studies (28). One solution to this problem would be to produce rh{alpha}1PI in high-output expression systems such as bacteria. However, it has been reported that the lack of glycosylation of protein produced in procaryotes markedly decreases the half-life of recombinant {alpha}1PI and limits the use of this system for {alpha}1PI production (29). The presence of a single free thiol residue at the surface of human {alpha}1PI protein provides a unique opportunity to apply a novel, simple, and rapid procedure that markedly increases a protein's biological half-life while preserving its function. The procedure involves the covalent linkage of a maleimido-PEG to proteins of therapeutic interest, through specific modification at a free thiol residue (14). The current study provides evidence that rh{alpha}1PI produced in a procaryotic system can be readily conjugated to PEG using maleimido-PEG. Although previous investigators have reported that thiol-specific modification of {alpha}1PI can decrease its elastase inhibitory activity (16), we found that not only is the anti-HLE activity of thiol-modified {alpha}1PI preserved in vitro, but it is also markedly improved in vivo. We observed that the size of the PEG polymer linked to Cys232 residue of {alpha}1PI influences the half-life of the protein in blood, and that the optimal PEG size is 20 kD.

The conjugation of PEG was found to be specific to the thiol residue of {alpha}1PI as determined by the absence of PEGylation when the Cys232 of {alpha}1PI was blocked through covalent linkage of NEM before the addition of maleimido-PEG (data not shown). In addition, free, unbound PEG had no effect on the half-life of {alpha}1PI, indicating that it is the conjugation of PEG at the thiol residue which is responsible for the improved pharmacokinetic properties of {alpha}1PI. We also observed that the PEGylated proteins tended to migrate at a much higher apparent molecular weight than the one predicted from the sum of the molecular weights of both {alpha}1PI and PEG. This abnormal migration of PEGylated proteins had been previously observed by Kurfurst (30), who reported that PEGylation of proteins decreases their electrophoretic mobility.

The concept of using PEG conjugation to increase the stability and half-life of rh{alpha}1PI has been investigated in the past. Mast and coworkers first reported that PEG activated with 1,1' carbonyldiimidazole could be coupled to recombinant {alpha}1PI and that the resulting complex showed improved plasma retention times over rh{alpha}1PI (31, 32). However, the preparation of the PEG-rh{alpha}1PI conjugates for these studies was based on the methods of Beauchamp and colleagues, and involved the use of toxic chemicals such as dioxane (33). Furthermore, the preparation required prolonged reaction steps of up to 120 h, during which time ~ 33% of the protein was inactivated. In contrast, the site-specific PEGylation of rh{alpha}1PI reported in the current study involved no toxic reagents, required reaction times of only 2 h, and resulted in fully active protein as determined by its capacity (i) to inhibit HLE, (ii) to form complexes with HLE on SDS-PAGE, and (iii) to bind HLE with an association rate constant similar to that of native {alpha}1PI.

One of the potential problems with site-specific modifications at Cys232 of {alpha}1PI is the possibility that thiol-modified {alpha}1PI may show decreased HLE inhibitory activity as reported by Tyagi (16). The decrease in protease inhibitory activity of thiol-modified {alpha}1PI was suggested to be related to a change in the predominantly hydrophobic environment surrounding Cys232, which, after thiol modification, becomes partially accessible to water. Strikingly, in the current study the conjugation of maleimido-PEG to rh{alpha}1PI did not result in any decrease in anti-HLE activity. The mechanism by which maleimido-PEG allows the preservation of anti-HLE activity despite the thiol modification is not clear, and is beyond the scope of the current study. However, we speculate that one possible explanation may be that the PEG residue maintains a sufficiently hydrophobic environment around Cys232 to allow an optimal reaction of PEG-rh{alpha}1PI with HLE and preserve its full inhibitory capacity.

Although the therapeutic use of {alpha}1PI has been proposed or investigated for several diseases, the only currently approved indication for the therapeutic use of {alpha}1PI is for subjects with a hereditary deficiency of {alpha}1PI. Our results clearly show that thiol-specific conjugation of PEG results in a protein complex with a prolonged half-life in blood. Although conjugation of PEG 5 kD to rh{alpha}1PI improved the half-life of rh{alpha}1PI in blood, PEG of 20 kD was needed to restore the pharmacokinetic properties to those equivalent to glycosylated {alpha}1PI. We found no advantage of PEG 40 kD over PEG 20 kD in our studies of blood-infused rh{alpha}1PI.

It has been suggested that the glycosylation of {alpha}1PI plays a much more important role in determining the half-life of the protein in blood than it does at the airway surface (29, 34). This concept was strengthened in the current study by autoradiography studies of labeled rh{alpha}1PI indicating the persistence of rh{alpha}1PI in BALFs for more than 24 h but a marked decrease in blood rh{alpha}1PI levels at 4 h. However, PEG-modified rh{alpha}1PI instilled in the airways persisted at the airway epithelial surface for longer times than rh{alpha}1PI. These pharmacokinetic properties may be of therapeutic importance because patients with diseases such as cystic fibrosis (CF), in which airway delivery of {alpha}1PI is preferable to intravenous administration, may require less frequent aerosolizations and/or lower levels of PEG-modified {alpha}1PI to achieve therapeutic efficacy. In addition, PEG conjugation has clearly been shown to protect proteins against nonspecific proteolysis (35). Because human neutrophil elastase (HNE)-mediated inactivation of {alpha}1PI is a major concern in CF, the PEG-modified {alpha}1PI may prove to be particularly advantageous in the highly proteolytic environment of CF airway secretions. Finally, the in vivo protection afforded by PEG-rh{alpha}1PI instilled in the airways 72 h before an HNE challenge was superior not only to rh{alpha}1PI but also to blood-derived, human {alpha}1PI, thus suggesting that PEGylation rendered the in vivo stability of the airway-delivered protein at least equivalent to that of glycosylated {alpha}1PI.

In summary, the current study provides evidence that it is possible, through a simple procedure involving thiol-specific maleimido-PEG conjugation, to markedly improve the in vivo half-life of rh{alpha}1PI delivered either directly to the lung surface or to the blood compartment. The anti-HLE activity of such thiol-modified {alpha}1PI is fully preserved in vitro. The in vivo protection of rh{alpha}1PI-PEG20 against HNE-mediated lung hemorrhage persisted for at least 72 h, thus rendering this conjugate more protective than either rh{alpha}1PI or {alpha}1PI. We suggest that rh{alpha}1PI conjugated at Cys232 to 20 kD PEG may provide both a potent and useful tool for experimental investigations of HNE inhibitory therapy and a rational therapeutic alternative in defined diseases.


   Acknowledgments
 
The authors thank Marc Martel and Ginette Bilodeau for expert technical assistance. This study was funded by grants from the Bayer Canadian Blood Services/Héma Québec Partnership Fund, the Canadian Cystic Fibrosis Foundation, and the Canadian Institutes for Health Research. E.K.D. is a recipient of a studentship from the Fonds de la Recherche en Santé du Québec (FRSQ). A.M.C. and R.L. are scholars of the FRSQ.

Received in original form March 26, 2002

Received in final form June 17, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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{alpha}1-Antitrypsin: Not Just an Antiprotease: Extending the Half-Life of a Natural Anti-Inflammatory Molecule by Conjugation with Polyethylene Glycol
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