ICANCER RESEARCH 53.3752-3757. August 15,1993] Pharmacokinetics and Toxicology of Immunotoxins Administered into the Subarachnoid Space in Nonhuman Primates and Rodents Karin Muraszko, Cynthia Sung, Stuart Walbridge, Larry Greenfield, Robert L. Dedrick, Edward H. Oldfield, and Richard J. Youle Biochemistry Section, Surgical Neurology Branch, National Inslilule of Neurological Diseases ami Stroke /K. M., S. W., E. H. O., R. J. Y.¡,and Biomedicai Engineering and Instrumentation Program, Intramural Research Resources, National Center for Research Resources ¡C.S., R. D.], N1H. Bethesda, Maryland 20892, and Cetus Corp., Emeryville, California "460ft /L. G.J ABSTRACT Immunotoxins have been suggested as possible therapeutic agents in patients with leptomeningeal carcinomatosis. The pharmacokinetics, sta bility, and tuxicity of immunotoxins injected into the i.t. space were ex amined in rats and rhesus monkeys. Monoclunal antibodies specific for the human (454A12 and Jl) and rat (OX26) transferrin receptors were cou pled to recombinant ricin A chain. In monkeys, the maximally tolerated dose of the anti-human transferrin receptor immunotoxin (454A12-rRA) was a dose that yielded a nominal cerebrospinal fluid (CSF) concentration of approximately 1.2 x IO"7 M. In rats, the 10% lethal dose (LD10) of the anti-human transferrin receptor immunotoxin was a dose yielding a nom inal CSF concentration of 8.8 x III M whereas the LDIO of the anti-rat transferrin receptor immunotoxin (OX26-rRA) was a dose yielding a nom inal (SI concentration of 1.2 x II) M.Thus, the species-relevant antibody resulted in toxicity at a concentration one-seventh that of the immunotoxin with the irrelevant antibody. A comparison of the area under the concen tration curve at the 1.l>m for rats with the area under the concentration curve at the maximally tolerated dose in monkeys and humans shows that the species-relevant immunotoxin was a better predictor of the toxic dose of the anti-transferrin receptor immunotoxin in humans than the irrele vant immunotoxin. The pharmacokinetics of the 454A12-rRA immuno toxin within the CSF of monkeys showed a biphasic clearance with an early-phase half-life of 1.4 h and a late phase half-life of 10.9 h. The clearance was 4.4 ml/h or approximately twice the estimated clearance due to bulk flow of CSF. Loss by degradation was ruled out because iminii- noblot analysis showed that the immunotoxin was stable for up to 24 h after administration. Possible losses in addition to sampling include dif fusion into brain tissue and transcapillary permeation. The apparent vol ume of distribution was 10.1 ml or approximately three-fourths the total CSF volume of the monkey. Dose limiting toxicity corresponded with the selective elimination of Purkinje cells in both rats and monkeys and was manifested clinically as ataxia and lack of coordination. The onset of ataxia in monkeys occurred within 5 days and, in the more mild form, was reversible with time. There was evidence of only minimal inflammation within the CSF, and there were no signs of systemic toxicity. Immunotox ins injected into the subarachnoid space may have potential for treatment of leptomeningeal carcinomatosis. INTRODUCTION Meningea! carcinomatosis occurs in 5-20% of all cancer patients, the majority caused either by breast carcinoma or lung carcinoma (1). There are recent indications that the incidence of meningea! carcino matosis is increasing as patients survive longer with improved sys temic therapy. Yet, in two-thirds of cases, it occurs when systemic disease is stable or in complete remission (1-7). These patients have an exceedingly poor prognosis. Patients able to tolerate maximal therapy (i.t. methotrexate and whole-brain irradiation) have a 6-7- month mean survival and less than 15% are alive after 1 year, even with aggressive therapy (7-15). In children, the incidence of CSF1 Received 12/30/92; accepted 6/8/93. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1The abbreviations used are: CSF, cerebrospinal fluid; MTD. maximally tolerated dose; LDu>. 10% lethal dose; AUC. area under the concentration curve; CNS. central spread of primary CNS tumors such as medulloblastomas and ependy- momas can be quite high. As many as 44% of children with medullo- blastoma have evidence of spread via CSF pathways, and 31% of children who die from ependymomas have evidence of CSF métas tases (16). Malignant gliomas, particularly in the pediatrie population, may also have CSF spread. Thus, CSF spread of cancer causes sig nificant morbidity, and the choice of treatments now available is limited. Monoclonal antibodies coupled to protein toxins such as ricin, called immunotoxins, represent a potent and cell type-selective new class of chemotherapeutic agents (17-19). Immunotoxins can specif ically kill a variety of tumor cell types in vitro and in vivo, however, systemic delivery of immunotoxins is hampered by rapid clearance into nontarget tissues and slow transport into tumor tissue. Regional delivery may overcome some of these limitations to immunotoxin delivery, particularly for therapy of CNS cancer, where the blood- brain barrier additionally impedes macromolecule delivery (8, 9, 20- 22). In animal models of leptomeningeal carcinomatosis, immunotox ins delivered directly into the CSF prolong survival in tumor-bearing animals (20, 23). These and other studies have demonstrated a poten tial therapeutic benefit of administration of immunotoxins to the CSF in patients with leptomeningeal carcinomatosis (20, 24, 25). Our study assessed the pharmacokinetics, stability, and toxicity of immunotoxins in the CSF of rats and rhesus monkeys. To address the nonspecific toxicity of immunotoxins in animals and to thoroughly identify the potential of species-specific, antibody-mediated toxicity in an animal model we constructed and studied a human-specific immunotoxin as well as an analogous rat-specific immunotoxin. MATERIALS AND METHODS The immunotoxins studied were: (a) 454A12-rRA, an anti-human transfer rin receptor monoclonal antibody of the IgGl isotype (454A12) (2ft) conju gated to recombinant ricin A chain (rRA); (b) Jl-rRA, an IgG2a monoclonal antibody to the human transferrin receptor coupled to rRA (26); and (c) OX26-rRA. an IgG2a monoclonal antibody to the rat transferrin receptor (27) conjugated tu rRA. Conjugation of rRA to the antibodies was carried out as described previously (28). Protein Synthesis Assay The cytotoxic effects and potency of the anti-rat transferrin receptor immu notoxin were tested on a rat myeloma cell line, Y3-Agl.2.3 (29) and a rat bladder carcinoma cell line. NBT II (30). The myeloma cell line was main tained in RPMI 1640 containing 10% fetal calf serum, 10 HIM4-(2-hydroxy- ethyl)-l-piperazineethanesulfonic acid, 20 fig/ml gentamicin, and 2 RIM glutamine; the bladder cancer cell line was maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 2 ITIMglutamine, 1 IBM sodium pyruvate, 0.1 IBM nonessential amino acids, and 10 /xg/ml gentamicin. The protein synthesis rate was assayed as previously described (24). nervous system; PBS. phosphate-buffered saline; i.t., intrathecal; p.c., percutaneous; TfR, transferrin receptor; In-DTPA. indium diethylenetriaminepentaacetic acid. 3752 Research. on December 18, 2021. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from