Biomedical subjects

S Walbridge

Publications and source records attributed to S Walbridge.

26 records · Page 2Linked to original sources

PubMed1993

Pharmacokinetics and toxicology of immunotoxins administered into the subarachnoid space in nonhuman primates and rodents.

Immunotoxins have been suggested as possible therapeutic agents in patients with leptomeningeal carcinomatosis. The pharmacokinetics, stability, and toxicity of immunotoxins injected into the i.t. space were examined in rats and rhesus monkeys. Monoclonal antibodies specific for the human (454A12 and J1) and rat (OX26) transferrin receptors were coupled 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 10(-7) M. In rats, the 10% lethal dose (LD10) of the anti-human transferrin receptor immunotoxin was a dose yielding a nominal CSF concentration of 8.8 x 10(-7) M whereas the LD10 of the anti-rat transferrin receptor immunotoxin (OX26-rRA) was a dose yielding a nominal CSF concentration of 1.2 x 10(-7) 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 concentration curve at the LD10 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 irrelevant immunotoxin. The pharmacokinetics of the 454A12-rRA immunotoxin 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 immunoblot analysis showed that the immunotoxin was stable for up to 24 h after administration. Possible losses in addition to sampling include diffusion into brain tissue and transcapillary permeation. The apparent volume 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. Immunotoxins injected into the subarachnoid space may have potential for treatment of leptomeningeal carcinomatosis.

Animals↗

PubMed1993

Stability, clearance, and disposition of intraventricularly administered oligodeoxynucleotides: implications for therapeutic application within the central nervous system.

We report experiments in the rat demonstrating the feasibility of intraventricular administration of oligodeoxynucleotides (ODNs) as a regional treatment approach to disorders within the central nervous system (CNS). Although we find little intrinsic nuclease activity in cerebrospinal fluid (CSF), phosphodiester ODNs are rapidly degraded by brain-associated alpha-exonuclease activity. Phosphorothioate ODNs, however, appear resistant to degradation in the CNS and, after intraventricular administration, we find they are cleared in a manner consistent with CSF bulk flow. Continuous infusion of ODN at 1.5 nmol/hr by miniosmotic pump can maintain micromolar concentrations of intact phosphorothioate ODN in CSF for at least 1 week without obvious neurologic or systemic toxicity. After infusion, extensive brain penetration and marked cellular uptake, especially by astrocytic cells, is demonstrated.

Animals↗

PubMed1993

In situ retroviral-mediated gene transfer for the treatment of brain tumors in rats.

Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. This suggests that retroviral-mediated gene transfer might permit targeting of gene integration into malignant cells in organs composed mainly of quiescent nonproliferating cells, such as in the brain. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs ("suicide" genes) into proliferating brain tumors may be used to treat this cancer. We investigated the efficacy and dynamics of in vivo transduction of growing brain tumors with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir. Ganciclovir is phosphorylated by thymidine kinase to toxic triphosphates that interfere with DNA synthesis, resulting in the preferential death of the transduced tumor cells. Rats inoculated with 4 x 10(4) 9L gliosarcoma cells into the frontal lobe were treated 7 days later with an intratumoral stereotaxic injection of murine fibroblasts (NIH 3T3 cells) that were producing a retroviral vector containing the herpes simplex-thymidine kinase gene. Controls received vector producer and nonproducer NIH 3T3 cell lines containing the Escherichia coli lacZ (beta-galactosidase) gene as well as nonproducer NIH 3T3 cells containing the thymidine kinase gene. The animals were rested for 7 days to allow time for in situ transduction of the proliferating tumor cells with the herpes-thymidine kinase retroviral vector. The animals were then treated with ganciclovir, 15 mg/kg i.p. twice a day for 14 days. Gliomas receiving an injection of 3-5 x 10(6) thymidine kinase producer cells regressed completely in 23 of 30 rats given ganciclovir therapy, while 25 of 26 control rats developed large tumors. Intratumoral injection of a lower concentration of thymidine kinase vector producer cells (1.8 x 10(6)) resulted in a lower frequency of tumor regression (5 of 13 rats). To estimate the efficiency of in vivo gene transfer, 9L brain tumors were given injections of 5 x 10(6) beta-galactosidase vector producer cells. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranaside staining revealed maximal staining of beta-galactosidase within the tumor 7-14 days after injection of the vector producer cells. In vivo transduction rates in harvested tumors ranged from 10 to 70%. There was no evidence of transduction of the surrounding normal neural tissue. Occasional blood vessel endothelial cells within or adjacent to the tumor were observed to be 5-bromo-4- chloro-3-indolyl-beta-D-galactopyranaside positive.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗

PubMed1993

Toxicity studies of retroviral-mediated gene transfer for the treatment of brain tumors.

Retroviral-mediated transfer of the herpes simplex virus thymidine kinase (HSVtk) gene into malignant tumors confers drug susceptibility to the antiviral drug ganciclovir. The authors have recently shown that in situ transduction of the rat 9L brain tumor following HSVtk-producer cell implantation led to tumor regression after ganciclovir administration in treated rats. A wide spectrum of potential adverse effects may, however, be associated with the application of this approach to treat brain tumors, including dissemination of the retroviral vector to nontumoral tissues within or outside the central nervous system, proliferation of the injected murine vector-producer cells at the injection site, immune-mediated responses to the implantation of xenogeneic cells, and damage to the brain from toxic by-products of the HSVtk-ganciclovir interaction. These possibilities were investigated using intracerebral and systemic injections of retroviral vector-producer cells carrying the HSVtk or the lacZ gene in mice, rats, and nonhuman primates. Using the lacZ gene as a reporter gene, no evidence of beta-galactosidase activity consistent with vector transduction was detected in any major body organ in the treated mice or rats. Similarly, the HSVtk gene transfer did not result in toxicity, with or without ganciclovir administration. In studies using rat and monkey models, no proliferation of the vector-producer cells occurred after intracerebral injection. Vector-producer cell survival was limited to 7 to 14 days. High-dose steroid therapy did not appear to extend the survival of these xenogeneic cells in rats. No significant inflammatory response was observed in the meninges or brain parenchyma. Endothelial cells were occasionally transduced in brain capillaries adjacent to the injected site of the vector-producer cells. Injection of producer cells into brain tissue elicited mild edema and reactive gliosis surrounding the injection site, which were probably the cause of a transient toxic response arising 4 to 5 days following injection of the producer cells; short-term administration of dexamethasone eliminated that response. No neurological deficits were observed in the rats or primates treated with the HSVtk vector-producer cells, with or without ganciclovir. In primates injected with producer cells, magnetic resonance imaging before, during, and after ganciclovir administration showed minimal and localized breakdown of the blood-brain barrier without significant edema or mass effect. Similarly, histological examination of the monkeys' brains showed no damage to neurons, astroglia, or myelin. Long-term clinical (> 9 months) and radiological (3 months) assessment of the primates has revealed no evidence of toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.

Animals↗

PubMed1992

Treatment of murine primary brain tumors with systemic interleukin-2 and tumor-infiltrating lymphocytes.

Methods have recently been described for the isolation and expansion of lymphocytes that have trafficked into animal and human tumors. These CD8-positive tumor-infiltrating lymphocytes (TIL's) have exceptional trafficking ability to, and efficacy against, tumor targets in extracranial sites. Prior to Phase I clinical trials for patients with gliomas, adoptive immunotherapy with TIL's was studied in a mouse model of primary brain tumors to determine if intracerebral tumors have a similar response. Glioma 261 (GL261) tumors were grown in the subcutaneous space of C57BL/6 mice. After enzymatic digestion, the cells were incubated in vitro with interleukin-2 (IL-2) until a confluent population of T lymphocytes was present. The in vitro efficacy of these TIL's was tested against fresh GL261 targets with a chromium release assay; the in vivo efficacy was tested against GL261 tumors in the liver and against irradiated and nonirradiated GL261 tumors in the brain. Mice received one of the following: intraperitoneal saline; intraperitoneal IL-2 (7500 to 50,000 U three times daily for 5 days); IL-2 plus intravenous TIL's (1 to 3 x 10(7) cells); 10 Gy cranial irradiation; irradiation plus IL-2; or irradiation plus IL-2 plus TIL's. The TIL preparation killed 77% of tumor targets in 4 hours at an effector:target ratio of 100:1. In animals with GL261 tumors in the liver, at 2 weeks there were 93 +/- 37, 128 +/- 45, and 21 +/- 14 liver metastases in the control, IL-2, and IL-2 plus TIL groups, respectively. However, in animals with GL261 tumors in the brain, no treatment group had an increased survival rate compared to the control group. It is concluded that, although TIL and IL-2 immunotherapy can be used effectively to treat brain tumors in vitro and at sites outside the central nervous system, it is ineffective against the same type of tumor in the brain. Different methods of delivery or different combinations of these immunomodulators may be more effective; however, based on these findings, treatment of patients with IL-2 and TIL cannot be recommended until efficacy has been demonstrated in an animal model.

Animals↗

PubMed1990

Epidermal growth factor receptors on ependymomas and other brain tumors.

Epidermal growth factor receptor (EGFR) and transferrin receptor levels were determined in 14 intracranial neoplasms (four glioblastomas multiforme, four medulloblastomas, four ependymomas, one cerebellar astrocytoma, and one acoustic neurinoma) and in four samples of "normal" brain tissue. A competitive radioreceptor assay with 125I-epidermal growth factor and 125I-transferrin was performed using the primitive neuroectodermal tumor-derived TE-671 tissue-culture cell line as a standard. Epidermal growth factor receptors were present on TE-671 cells, all four ependymomas, and two of the four glioblastomas multiforme. The number of EGFR's per cell for ependymomas were estimated to range from 1000 to 6000. Transferrin receptors were detected on TE-671 cells, two of the four medulloblastomas, and one of the four glioblastomas multiforme. A cell surface binding assay, performed directly on the rat ependymal cell monolayer, was also analyzed. The identification of EGFR's on ependymomas and TR's on medulloblastomas suggests that malignant central nervous system tumors that spread by cerebrospinal fluid pathways may be treatable by intrathecal antibody-toxin conjugates. The presence of EGFR's on all of the ependymomas may reflect a role of the receptor in the malignant phenotype of this tumor.

Animals↗

PubMedDate not supplied

Neurotoxicity of immunotoxins.

Immunotoxins are being tested for the therapy of systemic cancer and brain tumors. Neurotoxicity has been dose limiting for several of these antibody conjugates given systemically. We review the animal and clinical data related to the neurotoxicity of immunotoxins in attempt to understand the molecular basis for the unexpected neural involvement and to prevent or minimize this toxicity in future clinical trials.

Animals↗