Novel Poly(ethylene glycol) Derivatives for Preparation of Ribosome

Bioconjugate Chem. 2002, 13, 757−765

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Novel Poly(ethylene glycol) Derivatives for Preparation of Ribosome-Inactivating Protein Conjugates Silvia Arpicco,† Franco Dosio,† Andrea Bolognesi,‡ Chiara Lubelli,‡ Paola Brusa,† Barbara Stella,† Maurizio Ceruti,§ and Luigi Cattel*,† Dipartimento di Scienza e Tecnologia del Farmaco, Via Pietro Giuria 9, 10125 Torino, Italy, Dipartimento di Patologia Sperimentale, Via S. Giacomo 14, 40126 Bologna, Italy, and Dipartimento Farmacochimico, Tossicologico e Biologico, Via Archirafi 32, 90123 Palermo, Italy. Received November 12, 2001; Revised Manuscript Received May 9, 2002

This study describes the synthesis, characterization, and reactivity of new methoxypoly(ethylene glycol) (mPEG) derivatives containing a thioimidoester reactive group. These activated polymers are able to react with the lysyl -amino groups of suitable proteins, generating an amidinated linkage and thereby preserving the protein’s positive charge. mPEG derivatives of molecular weight 2000 and 5000 Da were used, and two spacer arms were prepared, introducing chains of different lengths between the hydroxyl group of the polymer and the thioimidate group. These mPEG derivatives were used to modify gelonin, a cytotoxic single-chain glycoprotein widely used in preparation of antitumoral conjugates, whose biological activity is strongly influenced by charge modification. The reactivity of mPEG thioimidates toward lysil -amino groups of gelonin was evaluated, and the results showed an increased degree of derivatization in proportion to the molar excesses of the polymer used and to the length of the alkyl spacer. Further studies showed that the thioimidate reactive is able to maintain gelonin’s significant biological activity and immunogenicity. On the contrary, modification of the protein with N-hydroxysuccinimide derivative of mPEG strongly reduces the protein’s cytotoxic activity. Evaluation of the pharmacokinetic behavior of native and PEG-grafted gelonin showed a marked increase in plasma half-life after protein PEGylation; in particular, the circulating life of the conjugates increased with increased molecular weight of the polymer used. The biodistribution test showed lower organ uptake after PEGylation, in particular by the liver and spleen.

INTRODUCTION 1

Ribosome-inactivating proteins (RIPs) are a group of naturally occurring plant and fungal proteins that have been widely studied for their potential applications, especially as anticancer agents (1). All RIPs exhibit RNA N-glycosidase activity, depurinating rRNA at a specific universally conserved position (i.e., cleavage of N-glycosidic bond of a specific adenine of 28S rRNA) (2). More recently, it has appeared that RIPs are also capable of inactivating many nonribosomal nucleic acid substrates (3) and hence can be considered polynucleotide:adenosine glycosidases. These insights have renewed interest in RIPs because understanding the enzymatic activity enhances exploitation of the properties and activities of RIPs for diverse applications. Indeed, such biomolecules are being exploited for designing immunotoxins/hormonotoxins obtained by chemical * Corresponding author. Phone: ++39.011.6707697. Fax: ++39.011.6707695. E-mail: [email protected]. † Dipartimento di Scienza e Tecnologia del Farmaco. ‡ Dipartimento di Patologia Sperimentale. § Dipartimento Farmacochimico. 1 Abbreviations: RIPs, ribosome-inactivating proteins; mPEG, methoxypoly(ethylene glycol); PEG, poly(ethylene glycol); SPDP, N-succinimidyl-3-(2-pyridyldithio)-propionate; SS-5PEG, methoxypoly(ethylene glycol) succinimidyl succinate with Mr of 5 kDa; SS-20PEG, methoxypoly(ethylene glycol) succinimidyl succinate with Mr of 20 kDa; TNBS, 2,4,6-trinitrobenzenesulfonic acid; THF, tetrahydrofuran; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ELISA, enzymelinked immunosorbent assay; FCS, fetal calf serum.

conjugation or by production of chimeric proteins using gene splicing DNA techniques (4, 5). Other applications of therapeutic potential for nonconjugated RIPs lie in the treatment of some cancers (5) and AIDS (6, 7) and as abortifaciens (8). Gelonin, a protein extracted from the seeds of Gelonium multiflorum, is one of the most interesting RIPs and has been widely used in the preparation of conjugates directed against tumor epitopes (9, 10) or as antiviral agent (11, 12). Recently, it has also been suggested that gelonin has DNA-damaging activity, which may be responsible for the elimination of the parasite 6 kb extrachromosomal mitochondrial DNA of Plasmodium falciparum-infected erythrocytes (13-15). Gelonin has been used by our group particularly in the preparation of conjugates with monoclonal antibodies using both new heterobifunctional linkers and noncovalent systems (16-19). Indeed, one favorable aspect of using gelonin is its low systemic toxicity (LD50 40 mg/ Kg body weight) and its low toxicity on intact cells (IC50 > 33 µM on HeLa) due to its incapability of penetrating cell membranes. Furthermore, it is rapidly removed from the bloodstream by kidney filtration (20). Clinical use of RIPs and related immunotoxins may be limited by the host immune response observed in patients after injection of RIPs, as reported in Lambert et al. (21). Allergic reaction and occasional anaphylactic response have, for example, been described for trichosanthin (22, 23), and the same behavior has been found for gelonin (24). Specific antibodies against immunotoxins containing gelonin have been observed after infusion in monkeys;

10.1021/bc015578s CCC: $22.00 © 2002 American Chemical Society Published on Web 06/19/2002

758 Bioconjugate Chem., Vol. 13, No. 4, 2002

high-titer antibody responses developed quickly, and sera from the infused monkeys were able to block the cytotoxicity of the immunotoxin (24). To improve the behavior and thus the therapeutic efficacy of gelonin, alone or conjugated with a targeting moiety, we considered an approach to surface modification using a covalent coupling reaction with poly(ethylene glycol) (PEG) derivatives. PEG is used to modify various proteins. PEG-grafted proteins exhibit increased stability, decreased immunogenicity, and increased circulating lives (25-27). Furthermore, PEG conjugates are more resistant to proteolysis, and in vivo activity can sometimes be enhanced despite a reduction in in vitro activities (28). The major problem of PEG conjugation of RIPs is their sensitivity to lysine modification. Some authors have reported a rapid decrease in the ability of several RIPs, and in particular gelonin, to inhibit protein synthesis after modification with SPDP or other reactives able to modify charge (29-31). To obtain polymer conjugates with almost unaltered gelonin activity, we prepared novel derivatives of mPEG containing a thioimidoester reactive group. This reactive group is able to react with the protein lysyl -amino groups in physiological conditions (pH 7.5), generating an amidinated linkage and thus preserving the positive charge of the protein. This paper describes the synthesis and chemical characterization of mPEG-thioimidates, with different molecular weights and different spacers between mPEG and thioimidate group. These reactives were applied to prepare different conjugates with gelonin. The conjugates were then purified and characterized in terms of molecular mass, in vitro activity, and immunogenicity. The pharmacokinetics and organ distribution were also studied. EXPERIMENTAL PROCEDURES

Materials. mPEG 2000 and 5000 and mPEG succinimidyl succinate with Mr of 5 kDa (SS-5PEG) were purchased from Sigma Chemical Co. (St. Louis, MO). mPEG succinimidyl succinate with Mr of 20 kDa (SS20PEG) was from Shearwaters Corp. (Huntsville, AL). Gelonin was extracted from seeds of Gelonium multiflorum and purified to mass homogenicity (Mr ) 30 kDa) and characterized (32, 33). 2,4,6-trinitrobenzenesulfonic acid (TNBS) was from Serva (Heidelberg, Germany). All other reagents were purchased from Aldrich (Milwaukee, WI). Diethyl ether and tetrahydrofuran (THF) were dried over sodium benzophenone ketyl. General Procedures. 1H NMR spectra were recorded on a Bruker AC 200 instrument, operating at 200 MHz, with tetramethylsilane as internal standard. Ultraviolet spectra were recorded on a Beckman DU-70 spectrophotometer. The reactions were monitored by TLC on F254 silica gel precoated sheets (Merck, Milan, Italy); after development, the sheets were exposed to iodine vapor. Flash column chromatography was performed on 230400 mesh silica gel (Merck). Preparation of Sodium Naphthalene Solution. Naphthalene 2.6 g (20 mmol) was dissolved in 20 mL of dry THF under a nitrogen atmosphere dried by passing it through concentrated sulfuric acid and NaOH platelets in two washing bottles. About 0.5 g (22 mmol) of small clean pieces of sodium was then rapidly added, and the mixture was stirred for 2 h at room temperature under dry nitrogen. Preparation of mPEG(CH2)2CN (1, 2). Five grams of mPEG 2000 (2.5 mmol) or 5000 (1 mmol) was dissolved

Arpicco et al.

in 100 mL of toluene and dried by removal of the watertoluene azeotrope. The solution was cooled to room temperature, concentrated, and then dissolved in 50 mL of dry THF. The sodium naphthalene solution prepared previously was added dropwise to the mPEG solution under dry nitrogen and stirring; addition continued until a persistent pale green solution was obtained. A solution of 3-bromopropionitrile (2 mL, 25 mmol in the case of mPEG 2000 or 830 µL, 10 mmol in the case of mPEG 5000) dissolved in 5 mL of dry THF was slowly added to the solution, and the reaction mixture was allowed to stand overnight at room temperature under dry nitrogen. The reaction mixture was concentrated under reduced pressure and filtered, and the filtrate was added dropwise into 100 mL of diethyl ether under stirring; the resulting solid was collected by filtration and purified. Derivative 1, prepared from mPEG 2000, was purified by flash chromatography with elution in dichloromethane/methanol (96:4). The pure product was obtained with a yield of 60% (3 g): 1H NMR (CDCl3) 3.6 (s, mPEG backbone methylene), 3.3 (s, 3H, terminal mPEG methoxy), 2.6 (t, 2H, -O-CH2-CH2-CN). We obtained 2 g (40%) of derivative 2 after crystallization from diethyl ether; 1H NMR (CDCl3) 3.6 (s, mPEG backbone methylene), 3.3 (s, 3H, terminal mPEG methoxy), 2.5 (t, 2H, -O-CH2CH2-CN). Preparation of mPEG(CH2)6CN (3, 4). The same procedure used to prepare 1 and 2 was followed, using 7-bromoheptanenitrile (25-fold molar excess respect to mPEG). The crude products were purified by flash chromatography (dichoromethane/methanol, 93:7) to give 3 (85%): 1H NMR (CDCl3) 3.55 (s, mPEG backbone methylene), 3.4 (s, 3H, terminal mPEG methoxy), 2.34 (t, 2H, -CH2-CN), 1.59 (m, 4H, -O-CH2-CH2-CH2CH2-CH2-CH2-CN), 1.4 (m, 4H,-O-CH2-CH2-CH2CH2-CH2-CH2-CN). Derivative 4 was purified by flash chromatography with elution in dichloromethane/methanol 92:8 (yield 95%). The NMR spectrum was similar to that of compound 3. Preparation of mPEG-Thioimidate ester hydrochlorides (5-8). The thioimidate ester hydrochlorides 5-8 were obtained from the corresponding nitriles 1-4 by the Pinner synthesis (34). Hydrogen chloride gas, dried by being passed through concentrated sulfuric acid in two washing bottles, was bubbled through ice-cold ethanthiol (560 µL, 7.5 mmol). The nitrile (0.25 mmol) dissolved in anhydrous dichloromethane was quickly added to the cold solution under stirring; the flask was tightly stoppered and left overnight at 0 °C. Anhydrous cold diethyl ether was then added to the reaction mixture, which was left at -20 °C until a crystalline solid formed. The supernatant was decanted, and the precipitate was washed 3 times with anhydrous cold diethyl ether under argon and dried under reduced pressure at room temperature. Compounds 5 (yield 85%) and 6 (yield 75%): 1H NMR (CDCl3) 3.6 (s, mPEG backbone methylene), 3.4 (s, 3H, terminal mPEG methoxy), 3.1 (t, 2H, -O-CH2-CH2CdNH2+), 1.4 (t, 3H, S-CH2-CH3). Compounds 7 and 8 (yield 95%): 1H NMR (CDCl3) 3.64 (s, mPEG backbone methylene), 3.4 (s, 3H, terminal mPEG methoxy), 2.9 (t, 2H, -CH2-CdNH2+), 1.8 (m, 4H, -O-CH2-CH2-CH2-CH2-CH2-CH2-CdNH2+), 1.61.4 (m, 7H -O-CH2-CH2-CH2-CH2-CH2-CH2-Cd NH2+, -S-CH2-CH3). Polymers-Gelonin Conjugate Preparation. A solution of gelonin (5 mg/mL, 500 µL) in PBS-EDTA (100 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.4), deaerated and flushed with argon, was added to

Novel mPEG Derivatives for Protein Derivatization

different molar excesses of the mPEG-thioimidate ester hydrochlorides (5-8). The solution was left for 1 h at room temperature under stirring. The molar ratio of activated mPEG to protein ranged from 2- to 50-fold. For comparison, gelonin was also modified with increasing excesses (from 2- to 50-fold) of the commercially available SS-5PEG, methoxypoly(ethylene glycol) succinimidyl succinate with a Mr of 5 kDa. The unreacted PEG was removed using an Amicon system (Danvers, MA) with YM membranes (cutoff, 10 and 30 kDa). The crude conjugates were separated on a Zorbax Bio Series GF 250 column (250 × 9.4 mm) (Du Pont, Newtown, Connecticut) that was equilibrated and eluted with 0.01 M sodium phosphate, 0.1 M sodium sulfate, pH 7.0, eluting buffer, and flow rate 0.5 mL/min. The chromatographic procedures were performed at 20 °C. All the chromatograms were generated on a MerckHitachi 655A-12 Liquid Chromatographer equipped with a L5000 LC Controller (Merck), and the eluting fractions were monitored at 280 nm using a L4000 UV detector. Peak heights and areas were recorded and processed on a CBM-10A Shimadzu interface (Shimadzu, Milan, Italy). The protein concentration was evaluated spectrophotometrically at 280 nm and also determined by a biuret assay. The extent of protein modification was evaluated colorimetrically following the Habeeb’s method (35). The sample solution (1 mL) at protein concentration between 0.7 and 1 mg/mL was diluted with 1 mL of 4% sodium bicarbonate solution, and 1 mL of 0.1% 2,4,6-trinitrobenzenesulfonic acid (TNBS) solution was added. After 2 h at 40 °C, the reaction was stopped by the addition of sodium dodecyl sulfate and HCl. The absorption at 341 nm was correlated with the number of free amino groups ( ) 12400). Gelonin-PEG conjugates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) using 4-15% precast gels (Bio-Rad, Hercules, CA) in nonreducing conditions. Coomassie blue staining was used to visualize the proteins. Protein Synthesis Inhibition Assay. The inhibitory activity of gelonin-PEG derivatives on cell-free protein synthesis was evaluated with a rabbit reticulocyte lysate prepared as described by Allen and Schweet (36). Gelonin, appropriately diluted, was added to a reaction mixture containing, in a final volume of 62.5 µL: 10 mM Tris/HCl buffer, pH 7.4, 100 mM ammonium acetate, 2 mM magnesium acetate, 1 mM ATP, 0.2 mM GTP, 15 mM phosphocreatine, 3 µg of creatine kinase, 0.05 mM amino acids (minus leucine), 3.3 kBq of L-14C-leucine (Amersham International, Bucks, U.K.), and 25 µL of a rabbit reticulocyte lysate. Incubation was at 28 °C for 5 min. The reaction was arrested with 1 mL of 0.1 M potassium hydroxide, and two drops of hydrogen peroxide and 1 mL of 20% (w/v) of trichloroacetic acid were added. Precipitated proteins were collected on glass-fiber disks, and the radioactivity incorporated was measured with a β-counter (Beckman, Fullerton, CA) after addition of 5 mL of Ready Gel scintillation cocktail (Beckman) containing 0.7% acetic acid. Each experiment was carried out in duplicate. The concentration of RIP causing 50% inhibition of leucine incorporation (IC50) was calculated by linear regression analysis. All tests were repeated three times. Production of Antigelonin mAbs. Antigelonin mAbs were produced according to a standard immunization/ fusion protocol using P3U1 myeloma cells (37). Hybrid cells were selected by culture in HAT (hypoxanthine aminopterin thymidine) medium 18 h after fusion. Hy-

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bridoma supernatants were screened by the enzymelinked immunosorbent assay (ELISA) using gelonincoated microtiter plates. For this study, we mixed three mAbs (B 3.1, A12, E17) that strongly reacted in ELISA assay with native gelonin. The culture medium for hybrids was RPMI 1640 supplemented with 10% FCS and 2 mM L-glutamine. Immunoreactivity. The immunoreactivity was determined using a modification of the ELISA assay (38). Enzyme microtitration plates were coated with antigen (4 µg/well of RIP) solubilized in 50 mM sodium bicarbonate buffer, pH 9.6, and left overnight at 4 °C. The plates were washed 3 times with 0.14 M NaCl/5 mM sodium phosphate buffer, pH 7.5, containing 0.05% (v/v) Tween 20 (PBS-Tween), blocked with 200 µL/well of PBS-Tween containing 0.5 mg/mL bovine serum albumin for 60 min at 37 °C, and washed 3 times as described above. Specific mouse antisera mix was serially diluted in PBS-Tween, added to the antigen coated plates, and incubated for 3 h at 37 °C. Plates were washed 3 times and incubated with 200 µL of appropriately diluted secondary antibody (goat antimouse IgG conjugated with alkaline phosphatase, Sigma) in PBS-Tween for 1 h at 37 °C. Plates were washed 3 times as described above and incubated with 200 µL of 1 mg/mL 4-nitrophenyl phosphate disodium salt in diethanolamine buffer, pH 9.8, for 30 min a 37 °C. Absorbance was measured in a Bio-Rad microtiter plate reader at 405 nm. Pharmacokinetics and Biodistribution. To evaluate the in vivo behavior of gelonin and mPEG modified protein, we prepared different conjugates using 125I gelonin as tracer. The protein was labeled with 125I by the iodogen method (39), and 250 MBq of 125I gelonin (specific activity 166.15 MBq/µg) was added to a solution of unlabeled protein (10 mg/mL). The conjugates were prepared as previously described, reacting gelonin with a 5-fold molar excess of thioimidate derivatives 7 and 8 and with a 3-fold molar excess of SS-20PEG. Pharmacokinetic studies were performed using female Balb/c mice (1 month old, 18-20 g; Charles River Italia, Milan, Italy), the mice drank water plus 0.2% (v/v) Lugol solution 3 days before and during the experiment to block thyroid iodine uptake. The animals were injected intravenously into the tail vein (groups of three animals each) with a single dose of 125I-gelonin and of the mPEG-125Igelonin conjugates ranging from 7.5 to 9.1 MBq. The samples were diluted in a carrier solution of PBS pH 7.4 to a volume of 100 µL. Blood samples were taken from the retroorbital plexus and withdrawn into heparincontaining tubes at various times (0, 10, 15, 20, 30, 60, 90, 120, and 240 min and 24 h for conjugates and 0, 5, 10, 15, 30, and 45 min for gelonin). Experiments were repeated 3 times. The blood samples were precipitated rapidly with 1 mL of cold trichloroacetic acid (12.5%, w/v). The samples were counted directly in a gamma counter (L’ACN, Milan, Italy) and then centrifuged; the resultant pellets were counted and used to determine the pharmacokinetic parameters. Pharmacokinetic parameters were determined from a two-compartmental model analysis using the Software KINETICA 2.00.200 (InnaPhase, Champs Sur Marne, France). Data were fitted using a compartmental open model. Elimination and distribution were represented by the following parameters: mean residence time (MRT), total body clearance (Cl), volume of distribution at steady state (Vss), distribution, and elimination phases (t1/2R, t1/2β). Other groups of five animals each were similarly injected, and the liver, kidney, and spleen, were removed at different times (10, 30, 60, and 120 min, and 24 h).


Arpicco et al.

Scheme 1

Figure 1. Molecular exclusion chromatographic profiles of gelonin and crude conjugates obtained by reaction with 5-fold molar excesses of compounds 7, 8, and SS-20PEG on gelfiltration column Zorbax Bio Series GF 250.

The organs were washed twice in physiological solution, wiped, weighed, and γ-counted. The counting was performed on intact organs and also after mechanical shattering to correctly measure radioactivity. The monitored radioactivity was recorded as concentration, in percent injected dose per gram of organ versus time and represented through histograms. The experiments were repeated 3 times. Care and handling of the animals were in accordance with the provisions of the European Economic Community Council Directive 86/209 recognized and adopted by the Italian Government (approval Decree No. 230/95 B). RESULTS

Chemistry. The mPEG-thioimidate ester hydrochlorides 5-8 were obtained as shown in Scheme 1. These activated polymers were prepared starting from mPEG of molecular weight 2000 and 5000 Da and were characterized by the presence of spacers of different lengths between the polymer chain and the reactive group. Initially, the sodium naphthalene solution was prepared by stirring clean pieces of sodium with a slight excess of naphthalene in dry THF under nitrogen at room temperature (40). Then mPEG 2000 or 5000 was converted to the corresponding nitrile 1 or 2 by titration with sodium naphthalene to generate the alkoxide, followed by treatment with 3-bromopropionitrile. Derivative 1, obtained from mPEG 2000, was easily purified by flash chromatography, but it was not possible to purify the corresponding nitrile 2 prepared from mPEG 5000 in the same way because it was impossible to discriminate the starting product from the obtained nitrile by TLC. Derivative 2 was thus purified by crystallization from diethyl ether. Compounds 3 and 4 were obtained in a similar way, using 7-bromoheptanenitrile instead of 3-bromopropionitrile and were easily purified by flash chromatography. Probably in this case, the presence of a hexamethylenic spacer between the PEG backbone and the nitrile group allowed better discrimination of derivatives 3 and 4 on gel chromatography. The corresponding thioimidates 5-8 were prepared throught the Pinner synthesis by addition of the nitrile to a solution of ethanthiol previously saturated with dry hydrogen chlo-

ride. The yield of activation was usually above 90% and was ascertained by NMR (integration of the peaks corresponding to the terminal mPEG-methoxy and to the methylene in R position to the thioimidate). The thioimidates required storage under very anhydrous conditions and were stored at -20 °C under dry argon. To maintain their reactivity unmodified, it is important to reduce exposure to atmospheric humidity because of the hygroscopicity of the compounds. Under these conditions they are stable for several months. Reaction of mPEG-Thioimidate with Gelonin. The reactivity of the activated polymers 5-8 toward the protein lysyl -amino groups of gelonin, a 30 kDa cytotoxic single-chain glycoprotein, was evaluated. Gelonin was found to be composed of 258 amino acids and contains 21 lysine residues (41). The mPEG-thioimidate was added in various molar excesses (from 2- to 50-fold) to the solution of the protein (5 mg/mL) for 1 h at room temperature; the conjugates were then purified from the excess of mPEG by ultrafiltration. To remove the unreacted gelonin, we submitted the conjugates to molecular exclusion chromatography (Figure 1). This purification is necessary before submitting the samples to characterization assays and to in vitro tests. The concentration of native or PEG-grafted protein was evaluated either spectrophotometrically at 280 nm or using biuret reaction. The derivatization degree was estimated by titration of free amino groups by TNBS method. Figure 2 shows the number of modified amino groups of gelonin versus the different excesses of mPEG-thioimidates. From the figure, it is evident that the derivatization degree increases in proportion to the molar excess of mPEG used. This is particularly evident for derivatives 7 and 8, characterized by the presence of a hexamethylenic spacer between the polymer backbone and the thioimidate reactive group, which was found to be more reactive toward the protein than derivatives 5 and 6, characterized by a dimethylenic spacer. This behavior is more evident when the mPEG-thioimidate excess was increased from 10- to 50-fold. At 50-fold excess of 8, for instance, 12 lysine residues were derivatized with compound 8 and only six with compound 6. We also compared the reactivity of the thioimidates with that of the commercially available SS-5PEG (Mr, 5 kDa) characterized by the presence of a highly reactive succinimidyl ester reactive group, which generates an amidic bond by reaction with protein amino groups. As shown in Figure 2, compound 8 and SS-5PEG demonstrated a similar



Figure 2. Reactivity of different mPEG-thioimidates toward gelonin. The activated polymer was added in molar excesses from 2- to 50-fold to solutions of the protein. The extent of protein modification was evaluated by titration of free amino groups by the TNBS method after purification from excess mPEG. The points represents the arithmetic means of three determinations; SDs (not reported) were 9), and thus direct evaluation of surface charge modification (e.g., by IEF) is not realizable, but as demonstrated in a previous paper (31), a direct relationship between charge modification and loss of cell-free activity exists. Gelonin appeared very sensitive to modification of its RNA N-glycosidase activity; on increasing the derivatization degree over two lysyl groups, activity rapidly decreases. From the results in Table 1, the thioimidate linkage appears able to maintain 50% of native activity for a derivatization degree of three groups, while only 18% was evident using N-hydroxysuccinate analogue under the same conditions. An increase in Mr over 5 kDa is responsible for a rapid fall of gelonin activity: in fact, just one PEG group of 20 kDa can reduce the toxic activity to 12% of initial values. Immunoreactivity is strongly affected by PEG length; a PEG 2 kDa chain is not able, in any of the conditions tested, to reduce antibody recognition by more than 68% (compounds 5, 7), while an increase in Mr to 5 kDa reduces recognition to 37%. A further increase in Mr (SS20 PEG) give not a detectable reaction at any antiserum dilution. Pharmacokinetic Studies and Organ Disposition. The influence of the PEG protein modification on circulation time was studied after intravenous bolus administration of native and modified gelonin to Balb/c mice. The RIP was radiolabeled with 125I and the samples injected i.v. to groups of mice; blood samples were collected at

Table 1. Inhibition of Protein Synthesis in a Cell-Free System and Immunoreactivity of Gelonin and PEG Derivativesa PEG reactive 5 7

SS-5 PEG

modifiedb Lys

IC50c

% immunoreactivityd

2 4 6 2 4 6 10 3 6 10

0.41 0.57 3.73 0.36 1.13 3.82 >5.33 1.73 >5.3 >5.33

76 72 70 76 70 70 68 53 40 38

PEG reactive 6 8

SS-20 PEG

modifiedb Lys

IC50c

% immunoreactivityd

2 3 6 2 3 6 10 1 2 4

0.33 0.89 3.9 0.35 0.66 2.63 >5.33 2.62 4.50 >5.33

60 55 45 64 54 43 37 n.d. n.d. n.d.

The points represent the means of three separate determinations. a n.d. not detectable. b The degree of derivatization was detected by the TNBS method and electrophoresis. c The IC50 (concentration giving 50% inhibition of protein synthesis) expressed in nM was assayed on rabbit reticulocyte lysate. Gelonin has a IC50 value of 0.31 nM. d The relative immunoreactivity (100% for pure gelonin) was obtained using a specific antiserum and assayed on ELISA test.


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of biodistribution confirms the pharmacokinetic profiles; gelonin is rapidly removed from the body by kidney filtration, and an increase in molecular mass proportionally decreases kidney uptake and increases liver and spleen uptake. Specific accumulation in these RES organs is reduced progressively for 5 kDa and for 20 kDa mPEG conjugates, with a proportional increase in blood survival time. DISCUSSION

Figure 4. Pharmacokinetic profiles following an intravenous bolus injection of radiolabeled gelonin and mPEG conjugates in mice. The reported data are the arithmetic means of three determinations, and SDs are indicated. Outline circle, gelonin; solid circle, conjugate obtained with compound 7; solid square, conjugate obtained with compound 8; solid triangle, conjugate obtained with SS-20PEG.

intervals and radioactivity determined. Since similar behavior in in vitro tests of compounds 5 and 6 versus compounds 7 and 8 was found, the analysis on animals were performed only on thioimidate 7 and 8 and with SS-PEG 20 kDa in order to have a comparison for the highest Mr. Figure 4 shows the pharmacokinetic profiles of gelonin and conjugates obtained with compound 7, 8, and SS20PEG. The gelonin rapidly disappears from blood circulation (t1/2R is 2 min and t1/2β is 13 min, see Table 2), while an increase in molecular mass and hydrophilic radius (from 30 kDa for gelonin to 36, 45, and 70 kDa for conjugates with 7, 8, and SS-20PEG, respectively) increases survival time. Following a bicompartmental model for 2 and 5 kDa PEG conjugates, the distribution phase appears rapid (t1/2R of 4 and 7 min), but a progressive decrease in elimination is evident (t1/2β from 70 to 273 min). Different behavior appeared when the conjugate Mr increased above the glomerular sieving threshold: the t1/2R increased to 44 min and the t1/2β up to 25 h. The mean residence time also reflects the slow elimination from the body due to PEGylation; MRT increases in a quadratic mode, from 10 min for gelonin to 94, 324, and 2073 min for the conjugates. The levels of accumulation of native and PEG modified gelonin sampled in the organs involved in toxin removal (liver kidney and spleen) after i.v. administration of conjugates are shown in Figure 5. These values are expressed as percentages of the dose per gram of tissue and were sampled up to 24 h after injection. The pattern

Targeting of toxins is one of the approaches to the selective removal of cancer or viruses. The major problems encountered in the use of toxin as therapeutic agents are due to nonspecific toxicity, low blood-residence time, and immunogenicity induced by the toxin moiety. The main way to solve these problems is to construct recombinant RIPs or in to use soluble hydrophilic masking polymers (42, 43). The present study aimed to evaluate the characteristics of mPEG derivatives able to generate a stable linkage and to increase the immunological and pharmacokinetic properties of the RIP gelonin, chosen as model protein sensitive to surface modification. This RIP has been exploited in therapeutic application both in immunotoxin preparation and as such: several interesting studies have recently been reported (9, 42, 44) as well as other relevant applications of gelonin in the treatment of tumors, involving the use of new techniques such as shock wave permeabilization or photochemical internalization (4547). Several methods of activating PEG to obtain protein conjugates have been described (48), but some of them change the total charge of the modified protein, in some cases reducing its biological activity. In contrast, the imido ester derivatives of mPEG, described in a patent (49), after reaction with the amino groups of proteins, lead to a charged amidine group without altering the positive charge of the protein. Several amidination experiments, using, for example, alcohol dehydrogenase, carbonic anhydrase, trypsin, or lysozime, have demonstrated that retention of the surface charge distribution favors preservation of the tertiary and quaternary structure of proteins, and thus retention of their biological activities (50). By studying the amidination mechanism of proteins, it has been found that the imidic esters lead to the desired amidinated proteins at a pH > 9, while for pH values below 8, the formation of cross-linked products is favored (51). At pH values normally used for the modification of proteins, the imidic ester generated a mixture of reaction products. On the contrary, using a thioimidate derivative such as S-methylthioacetimidate (52), it has been shown that amidination of a standard protein can be obtained even at pH 6 without any side reaction or cross-linking. At this pH, complete modification of all gelonin accessible lysines can be achieved. It has been suggested that reaction of the imidoester RXsC(R)d NH2+ (X ) O or S) with protein amino groups leads to a tetrahedral intermediate. Using imidic esters (X ) O)

Table 2. Pharmacokinetic Parametersa gelonin conjugate with compound 7 conjugate with compound 8 conjugate with SS-20 PEG

t1/2R (min)

t1/2β (min)

MRT (min)

Vss (ml)

Cl (ml h-1)

2.25 ( 0.22 4.40 ( 0.8 7.58 ( 1.5 43.91 ( 4.7

13.27 ( 1.2 70.67 ( 3.5 273.18 ( 15.2 1489.16 ( 23.9

10.16 ( 2 94.05 ( 8.1 324.98 ( 32 2073.06 ( 45

0.129 ( 0.02 0.308 ( 0.11 0.063 ( 0.01 0.017 ( 0.013

0.76 ( 0.12 0.196 ( 0.2 0.0118 ( 0.004 4.9 10-4 ( 3.5 10-5

a Pharmacokinetic parameters (means ( S.D.) of gelonin and conjugates obtained with mPEG of different molecular mass obtained after i.v. administration in mice (n ) 3 each). The values were obtained by adopting a two-compartment open pharmacokinetic model.



Figure 5. Disposition profiles in elimination organs after intravenous administration in mice: native gelonin (panel A); conjugates with compounds 7, 8, and SS-20PEG are shown in panel B, C, and D, respectively. SDs values are reported.

below pH 8, the protonation and elimination of the amino group leading to protein cross-linked derivatives prevails. A better leaving group, such as the thioimidate reagents (X ) S), should favor elimination of the alkanethiolated group even at a low pH (pH 7.5-8), preferentially giving the desired amidinate protein (52). Our thioimidate derivatives of mPEG have shown good reactivity at neutral pH, quite similar to that of commercially available mPEG-SS. This was particularly evident for compounds 7 and 8, which have a hexamethylene spacer group between mPEG and the reactive moiety. The length of this lipophilic and flexible arm is also very important, since the PEG-hexamethylenethioimidate derivative is much more reactive than the dimethylene ones toward the exposed amino groups of the protein. Although PEGylation of proteins usually leads to a decrease in the in vitro activity, due to charge or surface modification, in vivo activity can sometimes be enhanced. This is probably due to the increase in blood circulation and thus the increased diffusion in the perfused organs. This behavior is confirmed by the PEGylation of the RIP trichosanthin (23). In this case, native RIP and mutants were linked by a thioether linkage on specific cystein groups. In all cases, and both with 5 and with 20 kDa mPEGs, a drastic decrease in enzymatic activity appeared (10-20-fold) with a marked decrease in immunogenicity. In our hands, the N-hydroxysuccinimide derivatives 5 kDa PEG also strongly reduce gelonin activity, and this confirms our and other authors’ results (31, 53). Thioimidate reactives, on the contrary, are able

to maintain the activity of gelonin at least three times better than N-hydroxysuccinimide reactives. The results also show that a polymeric chain of high Mr is not suitable because, even at lower derivatization degree, the length of mPEG 20 kDa is sufficient to completely inhibit accessibility to the catalytic site. The hindrance induced by this chain toward specific antiserum is complete in all conditions, and the pharmacokinetics and tissue distribution confirm the longest retention in the blood circulation. The better candidate appears to have a 5 kDa PEG chain that, as reviewed by the authors (54), has been extensively used for many proteins of therapeutic interest. A mean of three groups of compound 8 allows significant biological activity to be maintained, reduces immunogenicity to one-half of the initial value, and greatly extends circulatory life (MRT of 5.4 h vs 10 min for gelonin). This value is confirmed by a lower RES organs uptake. In conclusion, we have shown that novel PEG derivatives are able to increase the behavior of RIPs, maintaining RNA N-glycosidase activity at high levels. This may make gelonin a better therapeutic candidate; in particular, the improvement in immunological properties makes it possible for a panel of toxin/immunotoxin to be administered. This approach has been applied in treating both tumors and HIV, with an evident increase in therapeutic results (21, 55-57). In order not to reduce the activity of gelonin with other derivatization steps, conjugation with targeting molecules could utilize the noncovalent interaction, as described by the author elsewhere (18).

764 Bioconjugate Chem., Vol. 13, No. 4, 2002 ACKNOWLEDGMENT

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Novel Poly(ethylene glycol) Derivatives for Preparation of Ribosome

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