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Bioconjugate Chem. 1093, 4, 490-498
Conjugation of Cu,Zn-Superoxide Dismutase with Succinylated Gelatin: Pharmacological Activity and Cell-Lubricating Function Yuichiro Kojima,? Akihiko Haruta,f Teruko Imai,t Masaki Otagiri,t and Hiroshi Maeda*l+ Department of Microbiology, Kumamoto University School of Medicine, and Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto 860, Japan. Received June 1, 1993"
Superoxide dismutase (SOD) and succinylated gelatin (succinyl gelatin) were conjugated to improve i n uivo pharmacological activity of SOD. Lysyl residues of human recombinant Cu,Zn-SOD were crosslinked with carboxyl residues of succinyl gelatin using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Various chemical and pharmacokinetic parameters of the conjugate were determined. Analysis by atomic absorption spectrometry and amino acid composition revealed that the conjugate was composed of about 2.9 mol of succinyl gelatin (with a mean molecular weight of 23 000) to 1 mol of SOD and exhibited an apparent mean molecular weight of 98 000. The conjugate retained almost 100% of ita original activity on a molar basis. When the succinyl gelatin-conjugated Cu,Zn-SOD (Suc-gel-SOD) was administered intravenously to mice, its plasma half-life was prolonged to 29.7 min compared with 4.5 min for native SOD. Tissue distribution analysis revealed that intravenously administered Sucgel-SOD showed a much greater accumulation than native SOD in the liver followed by in decreasing order the kidney, the lung, and the spleen; native SOD was excreted more rapidly into urine before it accumulated in tissues. Furthermore, Suc-gel-SOD exhibited lower antigenicity and immunogenicity than native SOD, and it had a better therapeutic effect against ischemic edema of the foot pad in mice. The conjugate was found to accumulate more than native SOD in the ischemic foot pad. A newly added property of the conjugate is cell-lubricating activity, which facilitated cell passage through micropores and reduced hemolysis during cell passage i n vitro. Thus, Suc-gel-SOD appears to be a promising protein drug with greatly improved pharmacological properties i n uiuo while possessing the same enzyme activity as native SOD.
INTRODUCTION Superoxide dismutase (SOD)' catalyzes the dismutation of highly reactive superoxide anion radical (02'-) to molecular oxygen and hydrogen peroxide, which are then converted to molecular oxygen and water by catalase. SOD has been suggested as a possible therapeutic agent for many diseases associated with Oz-,such as allergy (I), ischemic myocardial damage (2),Crohn's disease (3) and virus infection (4). We previously showed in a model experiment that mice infected with influenza virus are cured to a greater extent with the use of divinyl ethermaleic anhydride [pyran] copolymer-conjugated SOD compared with native SOD, which indicates the impor-
* Corresponding author: Professor Hiroshi Maeda,Department of Microbiology,Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860, Japan. Telephone: (81)-96-3442111 ext. 6321. Fax: (81)-96-362-8362. t Kumamoto University School of Medicine. t Kumamoto University Faculty of Pharmaceutical Sciences. * Abstract published in Advance ACS Abstracts, October 1, 1993. Abbreviations used SOD, superoxide dismutase; 02-,superoxide anion radical, PEG, polyethylene glycol;iv, intravenous(ly);RBCs, red blood cells;succinyl gelatin,succinylated gelatin; Suc-gel-SOD, succinyl gelatin-conjugated Cu,Zn-SOD;DTPA, diethyienetriaminepentaacetic acid; EDCI, l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; FITC, fluorescein isothiocyanate isomer I; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;TNBS, trinitrobenzenesulfonic acid; PAGE, polyacrylamide gel electrophoresis;SDS, sodium dodecyl sulfate; AUC, area under the plasma concentration vs time curve;PBS,0.01 M sodium phosphate-buffered 0.15 M NaCl; F-nativeSOD,FITC-labeled native SOD;F-Suc-gel-SOD, FITClabeled Suc-gel-SOD; FP value, fluorescence polarizationvalue; PCA, passive cutaneous anaphylaxis;IgE, immunoglobulin E; IgG, immunoglobulin G. 1043-1 802/93/2904-0490~04.00/0
tance of pharmacokinetic improvements (4,5). Native Cu,Zn-SOD from bovine red blood cells (RBCs) appears to have little clinical applicability because of its poor pharmacokinetic and immunological properties. A tactic used to circumvent these problems is chemical conjugation with various polymers, on which we have been working for the past 15 years (6-16). Among these polymers, polyethylene glycol (PEG; polyethylene oxide) was most extensively studied, and conjugates of SOD with PEG had a prolonged half-life (17, 18). Conjugates with other polymers such as poly(styrene-co-maleic acidlanhydride (11), divinyl ether-maleic anhydride [pyran] copolymer (4, 5) and poly(viny1 alcohol) (16) showed a similarly prolonged i n uiuo half-life after intravenous (iv) injection, as a consequence of which there were improved therapeutic effects and an elimination of immunological reactivity. Although not described in this article, other critical advantages are removal of toxic Cu2+liberated from Cu,Zn-SOD (191,stabilization of SOD itself, and protection from hydroxyl radical generation by Cu2+(from SOD) via a Fenton-type reaction (ref 19 and unpublished data). In this study, we explored the highly biocompatible natural polymer gelatin for conjugation with SOD. Gelatin is a biocompatible and biodegradable polymer that has been used for a long time in the food, pharmaceutical, and medical fields. It possesses low immunogenicity and thus is used also as a plasma expander (20). We have previously shown that gelatin has a lubricating effect on RBCs and reduces hemolysis when RBCs are passed through micropores (21, 22) under hydrostatic pressure. During ischemic reperfusion, tissue damage is thought to be associated with 02'-generation (23) and may result from increased vascular resistance at the capillary level. It may be possible to decrease the resistance of the capillary and facilitate plasma cell passage by lubrication 0 1993 American Chemical Society
Conjugation of SOD with Succlnylated Gelatin
and to simultaneously remove 02.by use of a gelatinSOD conjugate. Gelatin was converted to a more hydrophilic form by succinylation. The substrate-binding site (lysine) for serine proteases can be blocked, thus producing resistance to proteases. Furthermore, the chemistry of cross-linking is simplified because very few amino groups (0.1 mol per gelatin) are now available in succinylated gelatin (succinyl gelatin); thus only SOD provides most of amino group (24 free amino groups). The purpose of this study was to explore the potential of succinylated gelatin for protein tailoring; to evaluate succinyl gelatin-conjugated Cu,Zn-SOD (Suc-gel-SOD) with regard to plasma half-life, immunogenic potential, and biological activity in uiuo; and to characterize the chemical nature of the conjugate. EXPERIMENTAL PROCEDURES
Animals. Male ddY and A/J mice and Wistar rats were obtained from SLC, Shizuoka, Japan. Chemicals and Reagents. Xanthine and cytochrome c were purchased from Sigma Chemical Co., St. Louis, MO. Diethylenetriaminepentaacetic acid dianhydride (DTPA anhydride), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCI), and fluorescein isothiocyanate isomer I (FITC) were purchased from Dojindo Laboratories, Kumamoto, Japan. Xanthine oxidase was purchased from Boehringer Mannheim GmbH, Mannheim, Germany. 51CrC13was purchased from ICN Biochemicals Inc., Costa Mesa, CA. Human recombinant Cu,Zn-SOD (molecular weight 31 800; specific activity, 4220 units/mg protein) with a purity of more than 99 % ,in which two free NHz-terminal amino group were available, was a generous gift from Nippon Kayaku Co., Ltd., Tokyo, Japan. SOD is composed of a dimeric subunit and contains 22 free eamino groups of lysine and two free NHz-terminal amino groups. Fragmented gelatin with a mean weight of 23 000 was obtained from Seiwa Kasei Co., Ltd., Osaka, Japan and is referred to as gelatin in this report. The molecular weight of gelatin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All other chemicals were of analytical grade commercially available. Preparation of Succinyl Gelatin. To block the free amino groups and increase solubility, gelatin (1.0 g) was succinylated with an excess amount of (500 mg) succinic anhydride at pH 8.3 as described previously (24). The reaction mixture was dialyzed under pressure with distilled water using an Amicon 8050 ultrafiltration system (YM-5 membrane; W. R. Grace & Co., Danvers, MA), and the final volume of 10 mL was lyophilized. This process gave 850 mg of succinyl gelatin, with 98.0 % modification of the amino groups as estimated by quantification of the remaining free amino groups using trinitrobenzenesulfonic acid (TNBS) (25). Conjugation of SOD with Succinyl Gelatin. Succinyl gelatin (160 mg) was dissolved in 5 mL of 0.1 M potassium phosphate buffer (pH 6.0) containing 50 mg of SOD, and then 100 mg of EDCI was added. The reaction continued for 1h at room temperature and for 16 h at 4 "C with stirring. The sample was concentrated to about 2 mL using an Amicon system (YM-30 membrane) and was chromatographed on a column of Sephacryl S-200 (3.5 X 70 cm) (Pharmacia-LKB, Uppsala, Sweden) with 50 mM potassium phosphate buffer (pH 7.0). It was then eluted in 5-mL fractions. Fraction nos. 70-90 were pooled and used as Suc-gel-SOD. Fractions of the conjugates were washed repeatedly with distilled water by ultrafil-
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tration, and the final volume of 5 mL was lyophilized and yielded 64.5 mg of Suc-gel-SOD. Quantitation of the Degree of Conjugation of Succinyl Gelatin to SOD. The amount of succinyl gelatin in the conjugate was calculated on the basis of the content of hydroxyproline, copper, and zinc in comparison with native SOD as determined by amino acid analysis and atomic absorption spectrometry, respectively. The amount of free amino groups in SODSwas determined by TNBS method. Polyacrylamide Gel Electrophoresis (PAGE). PAGE with or without sodium dodecyl sulfate (SDS) at 0.1% was carried out to examine the purity of each preparation in a thin slab (1.0 mm) gel according to the method described by Laemmli (26). The concentration of acrylamide was 14% at pH 8.8 using 0.375 M Tris-HC1 buffer. Enzyme Activity. SOD activity was measured according to the procedure using cytochrome c (27). Briefly, in 3 mL of 50 mM potassium phosphate buffer at pH 7.8 containing 0.1 mM ethylenediaminetetraacetic acid, the reaction mixture was prepared to give final concentrations of lOpM cytochrome c, 50 p M xanthine, and about 10 nM xanthine oxidase. One unit of SOD activity is defined as the amount of SOD required for 50% inhibition of the rate of reduction of cytochrome c, where the rate of reduction of cytochrome c is defined as a decrease of 0.025 absorbance unit per min at 550 nm. Radiolabelingof Proteins. Succinylgelatin and native SOD and Suc-gel-SOD were labeled with 51Crby attaching the chelating agent DTPA anhydride (28-30). Specifically, gelatin was reacted at first with 1 molar equivalent of DTPA over 1 mol gelatin followed by Shephadex G-25 column chromatography and lyophilization. Then, it was succinylated as described above. Chelation of chromium gives greater stability than iodination of tyrosine residues, which frequently yields unreliable values (29),and the association constant of DTPA is on the order of 1023or higher. Pharmacokinetic Experiments. The plasma halflife of the native and conjugated SODS was measured in ddY mice by administering SODS (15 000 unita/kg) via iv injection, and SOD activity was assayed by the above method. The plasma level of native SOD and Suc-gelSOD as a function of time was analyzed by a compartment model, and the plasma half-life was estimated by using the nonlinear least squares program MULTI described by Yamaoka et ai. (31),in which the area under the plasma concentration vs time curve (AUC) was calculated using the trapezoidal rule and extrapolating to infinity. The total body clearance (CL) was calculated as CL (mL/h) = dose (units)/AUC,,
(units.h/mL)
Tissue Distribution of Wr-Labeled Gelatin, Succinyl Gelatin, Native SOD, and Suc-gel-SOD in Mice. 51Cr-labeledproteins were injected into the tail vein of ddY mice at a dose of 12 mg/kg in 0.1 mL of saline. The mice were killed under ether anesthesia at different time intervals after iv injection; various tissues were removed, and radioactivity was counted with use of a y-counter. Each DTPA-conjugated protein had the following specific radioactivity (dpm/mg) after labeling with 51Cr: native gelatin, 8.6 X lo6; succinyl gelatin, 9.2 X lo5; native SOD, 3.0 X lo6; and Suc-gel-SOD, 4.0 X lo6. Fluorescence Polarization. FITC labeling was described previously (32). Briefly, 10 mol of excess FITC over SODSwas reacted for 2 h a t pH 8.3, room temperature, and 0.63 and 1.16 mol of FITC were respectively coupled
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per mol of native SOD and Suc-gel-SOD, which still retained about 21 mol of free amino groups. Measurements were carried out in 0.01 M sodium phosphate-buffered 0.15 M NaCl (PBS) (pH 7.3) by using a Model MAC-2 fluorescence spectropolarimeter (Japan Immunoresearch Laboratories, Ltd., Takasaki, Japan), under strict temperature control at 37.0 f 0.05 "C. A cuvette containing FITC-labeled native SOD (F-native SOD) (2 pg/mL) or FITC-labeled Suc-gel-SOD (F-Suc-gel-SOD) (2 pg/mL, native SOD equivalent) in PBS, in a total volume of 2 mL, was placed in the polarizer. Then, after addition of antinative SOD serum (100 pL) or normal rabbit serum (100 pL) to each cuvette, the fluorescence polarization value (FP value) was determined as described previously (33). Immunological Experiments. The antigenicity of Suc-gel-SOD was examined by use of rabbit anti-native SOD serum. The quantitative precipitin reaction assay between SOD and anti-native SOD serum was performed as described (34). Anti-native SOD serum was obtained from rabbits after immunization three times with 2 mg of native SOD by subcutaneous injection with Freund's complete adjuvant. Immunogenicity of native SOD and Suc-gel-SOD was evaluated by measuring anti-SOD production in rabbits, and in A/J and ddY mice. For the rabbit, anti-Suc-gelSOD serum was obtained from rabbits after immunization as above using 2 mg of SOD equivalent of the conjugate. The quantitative precipitin reaction assay was performed between native SOD and anti-native SOD serum and Sucgel-SOD and rabbit anti-Suc-gel-SOD serum. A / J or ddY mice received an intraperitoneal injection of 0.1 or 1.0 mg of SOD equivalent of the conjugate with Freund's complete adjuvant on days 0 and 14,respectively. Serum samples were collected on day 21. The passive cutaneous anaphylaxis (PCA) reaction to test anti-SOD immunoglobulin E (IgE) in the serum was evaluated with Wistar rats as described (35). Three hours after subcutaneous injection of 4-fold-diluted mouse serum, the degree of the PCA reaction was quantified by measuring the diameter of Evans blue-bound albumin that had entered the skin from the vascular lumen. Ischemic Paw Edema. Paw edema was induced by use of a commercial rubber band (1.8 mm2 cut area of the band; ring diameter = 42 mm), which was tightened to induce ischemia in the leg of mice as described (36). Saline or SOD solutions (20 000 units/kg, 0.2 mL) were injected into the tail vein. The mouse was placed in a plastic cylinder with a slit opening at the right side where the right leg was allowed to come out. Thirty minutes after injection of saline or SOD solutions, the right hind leg was bound (12 turns) with a rubber band just above the articulation. The rubber band was removed after 20 min. The paw thickness was measured at 0,0.33,0.66,1.0,2.0, and 4.0 h after resuming reperfusion, the values were divided by the pretreatment value; and suppression of edema by SOD derivatives was recorded. Vascular Permeability in Reperfused Paw. To determine the change in vascular permeability of the paw, 0.1 mL of Evans blue solution (1% in saline) was iv injected just before the removal of the rubber band. Twenty minutes after the injection, mice were killed by bleeding under ether anesthesia and then the paw was excised. The permeability values were determined spectrophotometrically at 600 nm after extraction of the tissue-associated Evans blue from various tissues immersed with formamide for 24 h at room temperature. Heat-inactivated Suc-gel-SOD was prepared by incubation of 2.4 mg/mL in saline at 100 "C for 1h. Enzyme
activity that remained was less than 0.01% of unheated Suc-gel-SOD. Lubricating Effect on Red Blood Cells. Human blood obtained from healthy volunteers was citrated at 0.1 mM, pH 7.8, and RBCs were separated after washing three times with Alserver's solution (2.26 mM citric acid, 27.2 mM sodium citrate, 71.8 mM NaC1, and 113.9 mM glucose, pH 6.7). A suspension of RBCs at 1%(v/v) was prepared by adding the appropriate volume of PBS (pH 7.3). The filtration apparatus was described by Maeda et al. (22). A cellulose nitrate membrane filter with average pore size of 3 pm was used (Sartorius Membrane Filter GmB., Goettingen, Germany). Each test material dissolved in PBS was added to the RBC suspension and incubated for 30 min at 37 OC. Subsequently, 1mL of the suspension was taken into a plastic syringe that was connected to the filtration system. RBCs were allowed to pass through the membrane at 100 mmHg at 37 OC. Hemolyzed cells and membrane-filtered RBCs after centrifugation at 2000 rpm for 5 min and then lysing with a hypotonic solution were quantified by measuring absorbance at 420 nm. The ratios of the RBCs that passed through the micropores and the number of hemolyzed cells relative to the total number of RBCs applied were calculated. RESULTS
The typical elution profile of Suc-gel-SOD, native gelatin, succinyl gelatin, and native SOD from a Sephacryl 5-200 column is shown in Figure 1A,B. Suc-gel-SOD eluted earlier than native SOD, and its peak width was broadened, indicating that the conjugate has a broader molecular distribution than SOD. Fractions of the conjugates (fractions 70-90) were pooled and used as Sucgel-SOD. About two-thirds of the succinyl gelatin added to the reaction mixture (4.4 molar excess vs SOD) was conjugated to SOD, and three membrane filtrations (cutoff of 30 000) eliminated unreacted succinylgelatin. The elute from the Sephacryl column was concentrated and washed to 1/20 volume using distilled water. Therefore, the contamination of Suc-gel-SOD with unreacted succinyl gelatin was negligible (
