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Comparison of the Pharmacokinetics and Hepatotoxic Effects of Saporin and Ricin A-Chain Immunotoxins on Murine Liver Parenchymal Cells David C

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Comparison of the Pharmacokinetics and Hepatotoxic Effects of Saporin and Ricin A-Chain Immunotoxins on Murine Liver Parenchymal Cells David C (CANCER RESEARCH 48. 7072-7078. December 15. 1988] Comparison of the Pharmacokinetics and Hepatotoxic Effects of Saporin and Ricin A-Chain Immunotoxins on Murine Liver Parenchymal Cells David C. Blakey,1 David N. Skilleter, Roger J. Price, Graham J. Watson, Leigh I. Hart, David R. Newell, and Philip E. Thorpe2 Drug Targeting Laboratory, Imperial Cancer Research Fund, Lincoln 's Inn Fields, London WC2A 3PX [D. C. B., G. J. W., P. E. TJ; MRC Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey SMS 4EF [D. N. S., R. J. P.]; and Drug Development Section, Institute of Cancer Research, Clifton Avenue, Sulton, Surrey SM2 5PX fL. I. H., D. R. N.], England ABSTRACT of Thy-1.1-expressing AKR-A lymphoma cells (22). A single i.v. injection of IT-sap eliminated approximately 99.999% of Immunotoxins containing the ribosome-inactivating protein, saporin, the lymphoma cells growing in the peritoneal cavity of the mice, are very effective antitumor agents but are highly toxic to mice. They whereas an equivalent dose of IT-A eliminated only 99% of the induce severe necrotic lesions in the liver parenchyma of the recipients. cells. Unfortunately, the IT-sap was 5 times more toxic to mice. Such extensive damage to the liver parenchyma is not observed with ricin A-chain immunotoxins even at 5-fold higher dosage. The hepatotoxicity It produced necrotic lesions in the liver parenchymal cells of of the saporin immunotoxins was found in the present study to arise from the mice which were severe enough to account for death (22, a combination of two effects. First, saporin and saporin immunotoxins 23). In contrast, the IT-A caused only minor damage to the were 30- and 6-fold more toxic to primary cultures of mouse liver liver parenchymal cells, even at doses approaching the median parenchymal cells than were ricin A-chain and ricin A-chain ¡mmunotox- lethal dose (Table 1). The hepatotoxic effects of the IT-sap were ins, respectively. This was despite the fact that the cells bound 4- to 5- not due to its binding to liver cells via the antigen binding sites fold less saporin or saporin immunotoxins than ricin A-chain or ricin A- or the Fc portion of the antibody or to hepatic recognition of chain immunotoxins. The binding of ricin A-chain and its immunotoxin the carbohydrate in the antibody component (22, 23). to the cells was mediated through the carbohydrate residues present on In the present study, we investigated the cause of the hepa the A-chain whereas saporin is not glycosylated and thus must bind to totoxicity of IT-sap. We found that the toxicity appears to arise other sites on the cell surface which result in transport of saporin from a combination of two effects, (a) Isolated mouse liver relatively efficiently to the cytosol. The second reason for the hepatotoxic action of the saporin immunotoxin was that it had a longer blood half- parenchymal cells are about 30-fold more sensitive to saporin life (t:,n = 1.1 h; tifß=17.1 h) than the ricin A-chain immunotoxin (Iv, and 6-fold more sensitive to IT-sap than to ricin A-chain and = 0.52 h; r.,/i = 9.7 h). Analyses using a two-compartment pharmacoki- IT-A, respectively, (b) IT-sap is not subject to clearance by the netic model showed that the two immunotoxins broke down in vivo to carbohydrate recognition systems of the liver. It therefore per give free antibody at a similar rate (r... = 10-12 h) but that the ricin A- sists for longer periods in the mice giving it more time to chain immunotoxin was eliminated 11 times more rapidly than the saporin interact with the parenchymal cells and kill them. The slower immunotoxin by routes other than breakdown. It was calculated that, in clearance of the IT-sap in vivo may also explain its better mice given a median lethal dose of saporin immunotoxin, the blood levels antitumor activity in the AKR-A tumor model. of immunotoxin remained above the concentration that killed 50% of parenchymal cells in vitro for more than 48 h. In mice given a median lethal dose of ricin A-chain immunotoxin, the blood levels fell below the MATERIALS AND METHODS concentration that was toxic to parenchymal cells in vitro within 4 h. The longer blood half-life of the saporin immunotoxin may also explain Materials. Saporin was kindly supplied by Drs. F. Stirpe and L. our previous finding that it had antitumor activity superior to that of a Barbieri (Istituto di Patologia Generale, Università degli Studi di Bo ricin A-chain immunotoxin in mice. logna, Bologna, Italy). It had been isolated from an aqueous extract of the seeds of S. officinalis by ion exchange chromatography on carbox- ymethylcellulose (1) and further purified on a column of Sephacryl S- INTRODUCTION 200(10). Ricin was purified from an aqueous extract of defatted castor bean Saporin is a protein from the seeds of Saponaria officinalis cake as described by Nicolson and Blaustein (24). Highly purified ricin (soapwort) which inactivates eukaryotic ribosomes in a manner A-chain was prepared from the ricin as described previously (25). similar to that of the A-chain of ricin and other plant toxins The hybridoma cell line, MRC OX7, secreting a mouse IgGl anti- (1, 2). Novel antitumor agents (ITs)3 have been synthesized by Thy 1.1 antibody, was kindly provided by Dr. A. F. Williams (MRC attaching these and other ribosome-inactivating proteins to Cellular Immunology Unit, University of Oxford). The antibody was monoclonal antibodies directed against tumor-associated anti purified from the blood and ascitic fluid of hybridoma-bearing mice as gens (3-21). We previously showed that an IT prepared from described by Mason and Williams (26). The hybridoma cell line, LICR- LON-R10, secreting a mouse IgGl subclass antibody to human glyco- saporin and monoclonal anti-Thy-1.1 antibody (OX7) produced phorin, was kindly supplied by Dr. P. A. W. Edwards (Ludwig Institute, an antitumor effect superior to that of an analogous IT prepared Sutton, England). from ricin A-chain when administered to mice bearing allografts Williams E tissue culture medium and fetal calf serum were pur chased from Gibco-Biocult, Ltd. (Paisley, Scotland). SPDP was pur Received 6/2/88; revised 8/31/88; accepted 9/16/88. chased from Pharmacia (Uppsala, Sweden). Na'"I (IMS 30), L-[4,5- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in 3H]leucine (TRK 170) and L-[{/-14C]leucine (CFB 67) were obtained accordance with 18 U.S.C. Section 1734 solely to indicate this fact. from Amersham International (Amersham, England). The lodo-Gen 1Present address: ICI Pharmaceuticals, CTL Building, Alderley Park, Mac- reagent for protein iodination was from Pierce, Ltd., (Chester, Eng clesfield, Cheshire SK10 4TJ, England. 2To whom requests for reprints should be addressed. land). Collagenase type 1A was from Sigma (Poole, England). All other ' The abbreviations used are; IT, immunotoxin; SPDP, A'-succinimidyl-3-(2- reagents were of analytical grade. pyridyldithio)propionate; SDS, sodium dodecyl sulfate; K ',,,. 50% inhibitory Preparation of ITs. Saporin (M, 30,000) and ricin A-chain (M, 30,000 concentration; OX7, monoclonal antibody against Thy-1.1; IT-A, immunotoxin containing ricin A-chain; IT-sap, immunotoxin containing saporin; AB, antibody and 32,000) were coupled to OX7 antibody (M, 150,000). Where released from IT by breakdown in vivo; AUC, area under plasma concentration specified in the text, analogous ITs prepared with RIO antibody, a versus time curve. monoclonal antibody against human glycophorin were used. The cou- 7072 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. HEPATOXICITY OF SAPORIN IMMUNOTOXINS Table 1 Toxic lesions caused by sapori«,ricin A-chain, and ITs and oxidative phosphorylation prevent endocytosis (33). As shown LDso" Oig ricin A or previously, they reduce the uptake of ricin by 85% in rat liver cells and lesions'SaporinIT-sapRicinsaporin/25 g mouse) Major histológica! the toxin associated with the cells can be almost completely removed 70 Damagetubulesof to convoluted with galactose (0.1 M) showing that it is attached to the cell surface kidney.25 (32). Severe necrosis of hepatic pa For toxicity measurements, the hepatocyte cultures were incubated renchyma!tored cells. Damage with saporin, ricin A-chain, or ITs for 24 h in serum free medium or spleen.710 pulp of AIT-A1 Destructionnon-parenchymal of hepatic in medium containing 5% (v/v) autologous mouse serum. The cells cells and ger were then washed twice with serum-free medium and 1 ¿¿CiofL-[4,5- Lie-berkuhn.1 minal cells in crypts of 3H]leucine was added to each culture. They were then incubated for a further 2-h period after which the radioactivity incorporated by the cells 20 Destructionnon-parenchyma) of hepatic cells and ger was measured as described by Villa et al. (34). Lie-berkuhnminal cells in crypts of Blood Clearance Measurements. Groups of 3 or 4 adult male HAI .H/ c mice weighing about 25 g each were given i.v. injections of radioio * The median lethal doses (LDsos) for the immunotoxins refer to the quantities dinated saporin, ricin A-chain, ITs, or antibody (10 ng of protein of ricin A-chain or saporin that they contained to facilitate comparison with containing I x 10* cpm). Samples of blood were drawn from the tail unconjugated ricin A-chain or saporin. The LDwS of the immunotoxins in terms of total protein were approximately 6-fold greater.
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