Bioconjugate Chem. 2008, 19, 2427–2431
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A New PEG-β-Alanine Active Derivative for Releasable Protein Conjugation Gianfranco Pasut,*,† Anna Mero,† Francesca Caboi,‡ Silvia Scaramuzza,‡ Luigi Sollai,‡ and Francesco M. Veronese† Department of Pharmaceutical Sciences, University of Padua, via F. Marzolo 5, 35131 Padua, Italy, and Bio-Ker S.r.l., Piscinamanna 09010 Pula (CA), Italy. Received July 7, 2008; Revised Manuscript Received November 3, 2008
A new PEGylating agent, PEG-βAla-NHCO-OSu, has been studied for protein amino conjugation using human growth hormone (hGH) and granulocyte colony stimulating factor (G-CSF) as model therapeutic proteins. This new activated PEG possesses a convenient property for protein modification when compared to other activated carboxylate PEGs, namely, lower reactivity. When this polymer reacts with a protein, its features lead to fewer PEG-protein conjugate isomers because it preferentially binds the most nucleophilic and exposed amines. Furthermore, the conjugates obtained with PEG-βAla-NHCO-OSu showed an interesting slow release of polymer chains upon incubation under physiological conditions. Further investigations determined that the PEG chains released are those coupled to histidine residues, and this finally yields less PEGylated species as well as free protein. This release allows a partial recovery of protein activity that is often remarkably and permanently reduced after stable PEGylation, and it occurs in water or blood without the involvement of enzymes. On the other hand, the rate of PEG release, tuned by the chemical structure of this new PEGylating agent, is not too high, and therefore, the achievement of a desired prolongation of protein half-life in vivo is still feasible. The pharmacokinetics of hGH-PEG6k-βAla conjugate was compared to that of native hGH in rats and monkeys, and the blood residence times were increased by 10- and 7-fold, respectively. The conjugate potency was evaluated in hypophysectomized rats demonstrating a superior pharmacodynamic profile with respect to native hGH.
INTRODUCTION Biotechnology products are becoming more successful as therapeutic agents because of the important role of monoclonal antibodies (1). Besides important and promising results, several proteins still present relevant limitations for a safe therapeutic use, such as (i) the tendency to promote an immunological response, particularly in the case of heterologous proteins but also for human proteins administered at high doses (2), (ii) the physicochemical instability, both during shelf storage and in vivo after administration, and (iii) the short body residence time, especially for proteins with molecular weights below the kidney clearance threshold. Most of these problems can be overcome by PEGylation (the chemical link of polyethylene glycol), because it increases molecular weight of native drugs, shields critical protein sites (epitopes or amino acid sequences degraded by enzymes), reduces aggregation, and enhances water solubility (3-6). Altogether, these advantages lead to improved therapeutics that can be less frequently administered with respect to parent drugs, thus increasing patient compliance. PEGylation has already reached important results as demonstrated by the eight marketed PEG-protein conjugates (5). Despite its potentials, PEGylation is not devoid of open problems, such as the marked activity reduction of conjugated proteins that can in some cases prevent the exploitation of a conjugate, although this is often counterbalanced by the greater prolongation of conjugate in vivo half-life. A typical example is PEGylated interferon R-2a (PEGASYS), which retains only 7% of the * Corresponding author. Gianfranco Pasut, Department of Pharmaceutical Sciences, University of Padua, via Marzolo 5, 35100 Padova, Italy. Phone: +39(0)49-8275693. Fax: +39(0)49-8275366. e-mail:
[email protected]. † University of Padua. ‡ Bio-Ker S.r.l.
native protein activity, but because of the half-life increase from 2.1 to 15 h, it has become a product with enhanced therapeutic value (7). Several research groups are now addressing this problem with interesting “releasable PEGs”, although none of the conjugates reported so far entered the market. These PEGs take advantage of cleavable spacers that allow and control the release of conjugated protein in its fully active native form. Often, these systems are based on ester bonds which trigger the release, and the rate can be tuned by changing the neighboring chemical groups (8-11). We recently developed a new PEGylating derivative, PEGβAla-NHCO-OSu, which exhibited lower reactivity toward amines and higher stability in alkaline solutions, as compared to other N-hydroxysuccinimide activated PEGs (12). For protein PEGylation, this derivative was found useful because it preferentially reacts with the most nucleophilic and solvent-exposed protein amino groups, yielding a less heterogeneous mixture of conjugate isomers. This behavior was investigated with two therapeutic proteins, namely, human growth hormone (hGH) and granulocyte colony stimulating factor (G-CSF). Interestingly, the conjugates obtained with this PEGylating agent possess the property to slowly release some polymer chains, forming either fewer PEGylated protein isomers or native proteins also. To better understand the release mechanism, PEGβAla-NHCO-OSu was conjugated to simple models revealing that the polymer is released when coupled to histidines. The PEG release rate was slow enough to ensure the conjugate halflife prolongation obtained by PEGylation. The hGH conjugate was also investigated for the pharmacokinetic profile, in rats and monkeys, and the potency in hypophysectomized rats. It is worth noting that a single weekly
10.1021/bc800281s CCC: $40.75 2008 American Chemical Society Published on Web 11/24/2008
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subcutaneous injection of hGH-PEG-βAla was equally potent to a daily subcutaneous injection of native hGH.
MATERIALS AND METHODS Lyophilized rh-GH and rh-G-CSF were supplied by Bio-Ker (Pula, Cagliari, Italy). PEG-NH2 of different molecular weights was purchased from Nektar (Huntsville, AL). N,N-Dicychlohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), 2,4,6trinitrobenzensulfonic acid (TNBS), p-nitrophenyl chloroformate, and organic solvents were purchased from Aldrich Chemie (Steinheim-West-Germany). Salts of analytical grade were from Merck. β-Alanine (βAla) was a Fluka product. Boc-NH-βAlaNHS was purchased from Novabiochem (Germany). Gel filtration chromatography was performed with a Shimadzu (Kyoto, Japan) analytical HPLC system, using a Zorbax GF250 column (250 × 4.6 mm). Elution was carried out at a flow rate of 0.3 mL/min with 0.1 M PBS, 0.3 M NaCl containing 20% of acetonitrile. The effluent was monitored by recording the absorbance at 226 nm or at 280 nm. Conjugate purification was carried out using an ion exchange SP-Sepharose fast flow (GE Healthcare, Uppsala, Sweden) loaded column (300 × 25 mm), with a Shimadzu HPLC (Kyoto, Japan). Elution was carried out at flow rate of 0.5 mL/min with a linear gradient of 0.1 M NaCl in water from 0% to 100% in 60 min. The effluent was monitored by recording the absorbance at 226 nm. Protein concentrations were determined spectrophotometrically on a Perkin-Elmer Lambda-25 spectrophotometer. The concentrations of stock solutions of proteins and PEG-protein conjugates were evaluated from their absorbance at 280 nm. Extinction coefficients at 280 nm for PEG-proteins were considered unchanged from the native proteins. PEG-βAla-NHCO-OSu Synthesis. PEG-βAla-NHCO-OSu was synthesized as previously reported (12). Briefly, PEG-NH2 6 kDa (0.05 mmol), dissolved in CH2Cl2, reacted with BocNH-βAla-NHS (0.15 mmol) in the presence of Et3N (0.1 mmol) to yield PEG-βAla-NH-Boc, which was purified by precipitation in Et2O. The Boc protection group was removed by TFA hydrolysis, forming PEG-βAla-NH2 that was recovered by evaporation of the solvent in a Rotavap, followed by precipitation in Et2O. The dried PEG-βAla-NH2 (0.042 mmol) dissolved in DMSO and reacted with di(N-succinimidyl) carbonate (0.21 mmol), for 12 h at 50 °C, gave the desired PEG-βAla-NHCOOSu that was recovered by precipitation from Et2O (yield 72%). The degree of PEG activation was evaluated as reported elsewhere (13); briefly, an equimolar solution of PEG-βAlaNHCO-OSu and Gly-Gly was reacted and the unreacted amino groups determined by Snyder assay (14). The percentage of PEG-βAla-NHCO-OSu activation was 81%. Percentages of PEG dimer (below 5%), formed during the last step of the synthesis, and unreactive PEG (without the amino group, about 7%), present in the commercial batch of PEG-NH2, were also determined. This may account for the reported percentage of PEG-βAla-NHCO-OSu activation. 1 H NMR (CDCl3, 300 MHz) analysis of PEG-βAla-NHCOOSu (PEG-βAla-NHCO-OSu) was performed by irradiating the sample with a pulse of 1444 Hz to suppress the signal of PEG(OCH2CH2)n hydrogens: σ 2.466 (t, 2H, NH-CO-CH2-CH2), σ 2.797 (s, 4H, -CH2-, NHS), σ 3.364 (s, 3H, CH3O), σ 3.538 (q, 2H, NH-CO-CH2-CH2). Protein Conjugation with PEG-βAla-NHCO-OSu. PEGβAla-NHCO-OSu was conjugated to either hGH or G-CSF as follows: a solution of 5 mg/mL of protein in 10 mM phosphate buffer pH 7 was left to react for 3 h at 4 °C with 3 molar excess of PEG-βAla-NHCO-OSu per each protein’s amino group. The reaction was monitored by gel permeation chromatography (GPC), and after 3 h, the total amount of protein had reacted
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with PEG, as determined by GPC and MALDI-TOF analysis (see Supporting Information). The PEG-protein conjugate mixtures were fractionated by ion exchange chromatography, and the conjugate fractions were pooled and concentrated by ultrafiltration. The conjugate purity was checked by GPC, and the samples were eventually further purified by a second chromatographic step. The final solution was dialyzed against water, and a solution of trehalose was added to reach a ratio of 0.2 mg of trehalose for 0.8 mg of protein and lyophilized. Characterization was carried out by gel filtration chromatography. PEG Release from Conjugates. The purified conjugate mixtures were dissolved in physiological buffer (10 mM Na2HPO4, 0.15 M NaCl; pH 7.4), at a concentration of 1 mg/ mL (expressed as protein content) and incubated for 96 h at room temperature. At predetermined times, samples were withdrawn and analyzed by GPC using a Zorbax GF250 column (250 × 4.6 mm). Elution was carried out at a flow rate of 0.3 mL/min with 0.1 M PBS, 0.3 M NaCl, containing 20% of acetonitrile. The effluent was monitored by recording the absorbance at 226 nm or at 280 nm. The degree of PEG release was evaluated by both the variation in the peak area of the conjugates and the appearance of free protein peak. Solutions of hGH and G-CSF were studied under the same conditions. Peptide Model Conjugation with PEG-βAla-NHCO-OSu. Triptorelin (pGlu-His-Trp-Ser-Tyr-DTrp-Leu-Arg-Pro-GlyNH2), Boc-Tyr, and Gly-Phe-Leu-Gly were used as models to evaluate the reactivity of the secondary amine of histidine, the tyrosine hydroxyl group, and the N-terminal amino group, respectively. Each peptide or protected amino acid (1 equiv) was dissolved in DMSO, and PEG-βAla-NHCO-OSu (0.33 equiv) was added under stirring. After dissolution, 1.5 equiv of Et3N was added, the reaction was allowed to proceed for 12 h, diluted 1:1 with CH2Cl2, and finally washed with acidic water. The organic phase, dried over anhydrous sodium sulfate, was precipitated in diethyl ether. The product was recovered by filtration, dried under vacuum, and incubated in a solution of 10 mM Na2HPO4, 0.15 M NaCl, pH 7.4, at room temperature. The conjugate stability was evaluated by GPC or by RP-HPLC. Animal Studies. All animal experiments were performed in accordance with the procedure described in the Guide for the Care and Use of Laboratory Animals. Pharmacokinetic Study in Rats. Pharmacokinetic study in rats has been performed on male Sprague-Dawley rats, weighing about 300-350 g (Harlan Nossan). hGH content in serum samples was quantified by ELISA, using the Active Human Growth Hormone Elisa Kit supplied by Diagnostic System Laboratories Inc. (DSL), 445 Medical Centre Boulevard, Webster, TX. Four rats (group 1) have been administered once with 2.5 mg/kg b.w. of hGH-PEG-βAla subcutaneously on the back. Blood has been withdrawn at predose and 1, 2, 4, 8, 24, 32, 48, and 72 h after PEGylated h-GH administration. Four rats (group 2) have been administered once with 0.5 mg/kg b.w. of reference product (native hGH). Blood has been withdrawn at predose and 5, 15, 30, 60, and 90 min and 2, 3, 4, and 6 h after hGH administration. hGH content in serum samples was quantified by ELISA. Pharmacokinetic Study in Monkeys. Pharmacokinetic study in monkeys has been done on young adult male rhesus monkeys (Macaca mulatta) 4 years old (4-7 kg). hGH content in the serum was quantified by ELISA as reported above. Three monkeys (group 1) have been administered once with 1.5 mg/ kg b.w. of hGH-PEG-βAla, by subcutaneous injection. Blood (at least 1.0 mL) has been withdrawn at predose and 0.5, 1, 2, 4, 8, 12, 18, 24, 32, and 48 h after administration and every 24 h for 1 week. Three monkeys (group 2) have been administered once with 0.5 mg/kg b.w. of the reference product (native hGH), by subcutaneous injection. Blood (at least 1.0
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Scheme 1. Synthesis of PEG-βAla-NHCO-OSu and Its Coupling with a Protein
mL) has been withdrawn at predose, 5, 15, and 30 min, and 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 h after administration. hGH content in serum samples was quantified by ELISA. Pharmacodynamic Study in Hypophysectomized Rats (Weight Gain Test). Pharmacodynamic study in rats was performed by treating hypophysectomized Sprague-Dawley rats, weighing 80-100 g. Animals were divided in three groups, eight rats in each: the first group was administered once with 240 µg/rat (protein equiv) of hGH-PEG-βAla by subcutaneous route on the back. The second group received daily 60 µg/rat/ day of native hGH for 6 days by subcutaneous route on the back. The third was treated with vehicle by subcutaneous route on the back. Animal weight was monitored every two days until the end of the study.
tion in PBS buffer at pH 7.4; Figure 2 shows the free protein release. After 96 h, the amount of released free hGH or G-CSF was found to be 21% and 29% of the starting conjugated proteins, respectively. In both cases, the GPC analysis revealed a decrease of diPEGylated species peak area during the time of
RESULTS AND DISCUSSION Preparation and Characterization of hGH and G-CSF Conjugates with PEG-βAla-NHCO-OSu. In Scheme 1, the synthesis of PEG-βAla-NHCO-OSu and the subsequent conjugation to protein amino groups are shown. Coupling reactions between PEG-βAla-NHCO-OSu and hGH or G-CSF were monitored by gel permeation chromatography (GPC) as reported in Figure 1 (panels A and B). After 3 h of incubation, nearly all protein amounts disappeared from the reaction mixture, yielding two new peaks that, on the basis of GPC and MALDITOF analysis, corresponded to mono- and di-PEGylated conjugates with traces of tri-PEGylated species (for MALDI-TOF analysis, see Supporting Information). In order to purify the conjugates from the excess of unreacted polymer and the free protein, a cationic exchange chromatography was carried out and purified reaction mixtures were checked by GPC. The lower reactivity of PEG-βAla-NHCO-OSu with respect to the PEG-O-CH2COOSu (PEG-SCM), a well-known PEGylating agent, was confirmed by studying both their chemical stability in alkaline solution and the conjugate isomers obtained after a coupling reaction with hGH (see Supporting Information). In particular, the half-life of NHS hydrolysis was 6.35 and 1.05 min for PEG-βAla-NHCO-OSu and PEG-O-CH2COOSu, respectively. When PEG-SCM was conjugated to hGH under the same conditions of PEG-βAla-NHCO-OSu, diPEGylated and triPEGylated isomers were formed to a considerable degree while PEG-βAla-NHCO-OSu formed mainly monoPEGylated conjugates (see Supporting Information). PEG Release from the Conjugates. The stability of hGH and G-CSF conjugates was assessed by GPC following incuba-
Figure 1. Gel filtration profile of reaction mixture of hGH (panel A) and G-CSF (panel B) with PEG-βAla-NHCO-OSu, after 3 h of reaction. (A) PEG/hGH reaction: peak at TR ) 7.52 min is di-PEGylated hGH conjugates, and peak at TR ) 7.98 min is mono-PEGylated hGH conjugates (as determined by MALDI-TOF analysis). (B) PEG/G-CSF reaction: peak at TR ) 7.32 min is di-PEGylated G-CSF conjugates, and peak at TR ) 7.82 min is mono-PEGylated G-CSF conjugates. The peaks at 10.16 min and 10.25 min are N-hydroxysuccinimide. The shoulders eluting with the first peaks of each chromatogram correspond to the tri-PEGylated conjugates. In both chromatograms, the peaks at about 9 min correspond to the unreacted protein.
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Figure 2. Protein release from hGH-PEG-βAla (() and G-CSF-PEGβAla (0) conjugates during incubation in a solution of 10 mM Na2HPO4, 0.15 M NaCl, pH 7.4 at room temperature. The released hGH or G-CSF were evaluated by GPC analysis.
Figure 3. Triptorelin release after incubation of triptorelin-PEG-βAla conjugate in a solution of 10 mM Na2HPO4, 0.15 M NaCl, pH 7.4 at room temperature. The release was determined by GPC on the basis of formation of free peptide and decrease of conjugate amount.
the experiment with a concomitant small increase of monoPEGylated conjugates and the formation of a peak of free protein. PEG Release from Peptide Models. To gain insight into the release mechanism of PEG-βAla-NHCO-OSu, this polymer was studied with suitable models, namely, Triptorelin (pGluHis-Trp-Ser-Tyr-DTrp-Leu-Arg-Pro-Gly-NH2), Gly-Phe-LeuGly, and Boc-Tyr used to mimic the conjugation at His side chain; N-terminal amino group, and hydroxyl group of Tyr, respectively. The conjugate stability was studied following incubation in PBS. The Gly-Phe-Leu-Gly and Boc-Tyr conjugates showed negligible PEG release after 5 days of incubation, demonstrating the stability of the amide or carbamate bond between PEG-βAla-NHCO-OSu and the amine group of Gly or the hydroxyl group of Tyr, respectively. On the other hand, a release was observed in the case of triptorelin, where PEG is linked to the histidine secondary amine (see Figure 3). We expect that the coupling of PEG-βAla-NHCO-OSu to the Tyr hydroxyl group of a protein will probably occur to a very limited extent during common reaction conditions in water solutions,
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Figure 4. Pharmacokinetic profile of native hGH and hGH-PEG-βAla in rats (dose 2.5 mg/kg). PEGylation raised the hGH half-life from 0.8 to 8.3 h.
Figure 5. Pharmacokinetic profile of native hGH and hGH-PEG-βAla in monkeys (dose 1.5 mg/kg). As shown, after PEGylation the hGH half-life increased from 3.1 to 20.8 h.
where stronger nucleophiles are present, e.g., amino groups of lysines and N-terminal amino acid. Pharmacokinetic Studies of hGH-PEG-βAla Conjugate in Rats and Monkeys. Pharmacokinetics studies were carried out in rats and monkeys for the hGH-PEG-βAla diconjugate fraction separated by cation exchange chromatography. The conjugation led to a great increase in blood residence time in both animal models; in fact, hGH half-life increased from 0.8 to 8.3 h in rats and from 3.1 to 20.8 h in monkeys, as shown in Figures 4 and 5, respectively. These data are comparable to those reported by other authors on hGH-PEG obtained by stable PEGylation (15), although a thorough comparison is difficult because several parameters are different in these two studies, as, for instance, the mass of PEG. In Vivo Activity of hGH-PEG-βAla in Animal Model. The biological activity of hGH-PEG-βAla conjugate mixtures and their pharmacodynamic profiles were tested in hypophysectomized rats by evaluating the weight gain during one week observation of animals. A single subcutaneous injection of hGHPEG-βAla (1 × 240 µg/rat, protein equiv) was found to be similar or slightly more potent than the same total amount of free hGH but given daily over a period of six days (6 × 40 µg/rat) (see Figure 6). Clark and co-workers reported similar results for hGH conjugated to 5 PEG 5 kDa chains (15). The
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ACKNOWLEDGMENT The research was supported by Bio-Ker. Supporting Information Available: Experimental conditions of hGH conjugation with PEG-SCM, MALDI-TOF mass spectra of PEG-βAla-NHCO-OSu, PEG-βAla-NHCO-OSu/hGH reaction mixture and PEG-SCM/hGH reaction mixture. Half-life hydrolysis of PEG-βAla-NHCO-OSu and PEG-SCM in water. This material is available free of charge via the Internet at http:// pubs.acs.org.
LITERATURE CITED
Figure 6. Weight gain in hypophysectomized rats given daily subcutaneous injections of hGH (6 × 40 µg/rat) or one weekly dose of hGHPEG-βAla (1 × 240 µg/rat, protein equiv).
hGH-(PEG)5 conjugate prepared by these authors had a halflife longer (15 h) than the hGH-PEG-βAla reported here (8.3 h), thus extending theoretically the protein activity over a longer period. The activity of these two different hGH-PEG conjugates are comparable, and this seems more in favor of the new hGH-PEG, which is still able to lead to a great pharmacodynamic response although showing a shorter half-life. Probably, this relevant response is partially due to the ability of this new PEG-βAla conjugate to slowly release in part the PEG chains, yielding either fewer PEGylated species or native hGH that are in any case more biologically active than the starting conjugate. Furthermore, the rate of PEG release does not compromise the beneficial half-life increase of the protein obtained after PEGylation.
CONCLUSION Our study demonstrates that the new PEGylating agent, PEGβAla-NHCO-OSu, can enhance the potential of PEG-protein conjugation. In fact, this polymer showed a higher stability in water at alkaline pH values, six times higher than that of PEGSCM, and a lower reactivity toward amines that direct the coupling only toward the strongest and most solvent-exposed nucleophiles of a protein (12). Therefore, it forms fewer PEG-protein isomers than other commonly used PEG reagents. Furthermore, this PEG derivative showed the propensity to be slowly released from the protein conjugate when the polymer is attached to the side chain of histidine. As it is a phenomenon extremely relevant to recovering the protein activity that is often markedly reduced in the conjugated protein, this is one of the major limitations of PEGylation. The possibility to address this problem by using a simple approach, such as the coupling with PEG-βAla-NHCO-OSu, is particularly needed, and it could lead to the preparation of improved protein bioconjugates. In fact, beside the advantage of a prolonged in vivo half-life, these conjugates will possess the ability to recover the protein activity by slowly releasing the PEG chains attached. The pharmacokinetic and pharmacodynamic data presented here support the effectiveness of PEG-βAla-NHCO-OSu as a new PEGylating agent for protein modification.
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