Susan J. Donohue

Human hydroxyindole-O-methyltransferase in pineal gland, retina and Y79 retinoblastoma cells.

Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) was studied in extracts of human pineal gland, retina and Y79 retinoblastoma cells. HIOMT enzyme activity and immunoreactive protein (approximately 42 kDa) were undetectable in the human retina; very low levels of HIOMT mRNA were detected using a highly sensitive RT-PCR/Southern blot method, as has been reported. Analysis of extracts of Y79 cells indicated that HIOMT enzyme activity, immunoreactivity (approximately 42 kDa) and mRNA (approximately 1.3 kb) were detectable at approximately 1/5-1/40 the levels found in the pineal gland. This unambiguously establishes that the HIOMT gene is expressed in Y79 cells. Kinetic analysis of Y79- and pineal-derived HIOMT indicates that the enzyme is generally similar in both tissues; one difference, however, is that substrate inhibition by N-acetylserotonin is greater with the Y79-derived enzyme. These studies show that Y79 cells represent a valid model to study the regulation of human HIOMT protein and mRNA; the differences detected may reflect the existence of tissue-specific regulatory mechanisms or differential patterns of expression of HIOMT isoforms.

Toxicity of dolastatin 10 in mice, rats and dogs and its clinical relevance

Purpose: Dolastatin 10 (DOL 10), an oligopeptide isolated from the sea hare Dolabella auricularia, has been shown to be a highly potent cytotoxic agent in a variety of human tumor cell lines. The purpose of this study was to conduct preclinical toxicity evaluations to determine the target organ(s) of toxicity and its reversibility, the dose-limiting toxicity and the maximum tolerated dose (MTD), and to use this information for arriving at a safe starting dose and dose schedule for phase I clinical trails. Methods: DOL10 was administered as a single intravenous bolus dose to CD2F1 mice, Fischer-344 rats and beagle dogs. Endpoints evaluated included clinical observations, body weights, hematology, serum clinical chemistry, and microscopic pathology of tissues. Results: The MTD (i.e. the highest dose that did not cause lethality but produced substantial toxicity) was approximately 1350 μg/m2 body surface area (450 μg/kg) in mice, 450 μg/m2 (75 μg/kg) in rats and ≤400 μg/m2 (≤20 μg/kg) in dogs. Adverse signs were observed at doses ≥1350 μg/m2 in mice, ≥150 μg/m2 in rats and ≥400 μg/m2 in dogs. Decreased weight gain or actual weight loss was observed at doses ≥1350 μg/m2 in mice, ≥600 μg/m2 in rats and ≥450 μg/m2 in dogs. In all three species, the primary target organ of toxicity was the bone marrow, as indicated by decreases in the numbers of erythroid cells, myeloid cells, and megakaryocytes in the femoral bone marrow and by decreased white blood cell (WBC) and reticulocyte counts in peripheral blood. Marked neutropenia (i.e. >50% decrease compared to control animal or baseline values) was the principal effect on WBCs and occurred within a week of dosing. A mild anemia was evident 1 week after administering the drug to rats and dogs. The hematologic effects were transient and reversed by study termination. Other lesions at the MTD levels were cellular depletion and necrosis in lymphoid organs (rats and dogs), marked depletion of extramedullary hematopoietic cellular elements in the spleen (rats), thymic atrophy (mice and dogs), and minimal cellular necrosis in the ileum (rats). More extensive and severe pathology was observed in animals sacrificed in a moribund condition or found dead. Conclusions: Myelotoxicity was dose-limiting in all three species with mice being the least sensitive. In a phase I clinical trial, granulocytopenia was dose-limiting. Moreover, the MTD of DOL10 for rats and dogs is comparable to the human MTD. Therefore, the results from the preclinical toxicology studies correctly predicted a safe starting dose, the dose-limiting toxicity, and the MTD in humans.

CHAPTER 17 – RELEVANCE OF PRECLINICAL PHARMACOLOGY AND TOXICOLOGY TO PHASE I TRIAL EXTRAPOLATION TECHNIQUES: RELEVANCE OF ANIMAL TOXICOLOGY

The chapter reviews the studies of a wide variety of anticancer drugs investigated by the U.S. National Cancer Institute (NCI) to prove the relevance of preclinical pharmacology and toxicology to Phase I trial extrapolation techniques. The development of drugs for the treatment of patients with cancer is perhaps more difficult than that of drugs for other non-life-threatening indications as cancer drugs tend to be the most toxic agents intentionally administered to man. This is further complicated by the fact that the typical patient in a Phase I trial is not a healthy human volunteer but a very ill, terminal cancer patient for whom all other therapeutic interventions have failed. Thus, the starting dose (SD) that is selected for the first human trial of cancer drugs must not only be safe but also offer some hope of benefit to the patient. However, it can also be extremely difficult to select a high enough dose so that the number of dose escalations is kept to a minimum to achieve either a maximum tolerated dose (MTD) or a biologically effective dose in humans. The results of the NCI studies that used agent directed techniques for the pharmacologic and toxicologic evaluation of antitumor agents in preclinical animal models are impressive in their prediction of human MTDs.

Vaccination with a recombinant vaccinia vaccine containing the B7-1 co-stimulatory molecule causes no significant toxicity and enhances T cell-mediated cytotoxicity

B7‐1 is a co‐stimulatory molecule that provides a second signal for T‐cell activation. Several studies have demonstrated that vaccination with a vector containing genes encoding B7‐1 and an antigen appears to be efficacious at promoting immune responsiveness to the antigen. To evaluate the safety of such a protocol and determine the effect of the B7‐1 vector on immune responsiveness, female C57BL/6 mice were administered Wyeth wild‐type vaccinia virus (V‐WT) or V‐WT containing the gene for B7‐1 (rV‐B7‐1) as a single s.c. injection or 3 monthly s.c. injections. Immunologic parameters were evaluated in half of the mice and general toxicity in the other half. Immunologic end points included determination of splenic lymphocyte phenotypes, mitogen‐induced T‐ and B‐cell proliferation, T‐cell proliferation in response to alloantigens, cell‐mediated cytotoxicity (CMC), natural killer cell activity and serum anti‐nuclear antibody (ANA) titers. No significant signs of general toxicity were noted. The primary immunologic effect was an increase in the ability of spleen cells to lyse allogeneic targets and to proliferate in response to allogeneic stimulation. Numbers of splenic CD8+ cells were also increased. These effects were more pronounced after 3 vaccinations than after a single vaccination. Minimal differences in ANA were observed between mice immunized with V‐WT and rV‐B7‐1. In addition, no serum antibodies against B7‐1 were detected in any mice. The data suggest that vaccination with rV‐B7‐1 augments CMC with minimal toxicity. Int. J. Cancer 85:508–517, 2000.