[Show abstract][Hide abstract] ABSTRACT: Recombinant human erythropoietin (rhEPO) is widely used for the treatment of patients with anaemia and its loss of patent protection has stimulated the development of cheaper biosimilar products. However, the quality and comparability of rhEPO products recently marketed in several developing countries is questionable. Paying attention to quality in its isolation, purification and analytical characterization, it has been possible to produce a biosimilar rhEPO that is comparable with the originator product. Non-clinical safety testing was initially carried out in the absence of a regulatory framework and contributed to the receipt of marketing approval for biosimilar rhEPO in Eastern Europe. Subsequently, this non-clinical testing was extended to take into account the recent guidelines for similar biological medicinal products published by the European regulatory authorities, which were markedly influenced by the intervening occurrence of pure red cell aplasia in patients taking what proved to be an impure rhEPO product. This Mini Review discusses the challenges faced, approaches taken and lessons learned in developing a biosimilar rhEPO product, both before and after the publication of the regulatory guidelines.
[Show abstract][Hide abstract] ABSTRACT: [(18)F]1-(2-Fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine ([(18)F]SFE) is a novel, selective, high-affinity sigma-1 receptor radioligand that has been preclinically well characterized in rodents. To support an investigational new drug (IND) application for the first evaluation of [(18)F]SFE in humans, single-organ and whole-body radiation adsorbed doses associated with [(18)F]SFE injection were estimated from rat distribution data. In addition, single- and multiple-dose toxicity studies were conducted in rabbits and in dogs. Multiple-dose toxicity studies in rabbits and single-dose toxicity studies in beagles suggest at least a 100-fold safety margin for humans studies at a mass dose limit of 4.0 mug per intravenous injection, based on the combined no observable adverse effect levels (NOAEL, mg/m(2)) measured in these species. Radiation dosimetry estimates obtained from rat biodistribution analyses of [(18)F]SFE suggest that most tissues would receive about 0.010-0.020 mGy/MBq, while the adrenal glands, brain, bone, liver, lungs, and spleen would receive slightly higher doses (0.024-0.044 mGy/MBq). The adrenal glands were identified as the critical organ, because they received the highest adsorbed radiation dose. The total exposure resulting from a 5 mCi administration of [(18)F]SFE is well below the FDA-defined limits for yearly cumulative and per-study exposures to research participants. These combined results support the expectation that [(18)F]SFE will be safe for use in human positron emission tomography (PET) imaging studies with the administration of 5 mCi and a mass dose equal to or less than 4.0 mug SFE per injection.
[Show abstract][Hide abstract] ABSTRACT: Quinocarmycin analog DX-52-1 is a cyanated derivative of quinocarmycin, a compound isolated from cultures of Streptomyces melanovinaceus. DX-52-1 was selected for preclinical development because it showed efficacy against melanoma cell lines in the NCI human tumor cell screen and melanoma xenografts in mice. This report describes studies in rats and dogs to determine the maximum tolerated dose (MTD) and identify dose-limiting toxicities (DLT) in each species in different regimens to establish a safe starting dose and potential target organs of DX-52-1 for phase I clinical trials.
DX-52-1 was administered to Fischer 344 rats using repeated intravenous (i.v.) slow bolus injections following q3hx3 and q3hx3,q7dx3 regimens, and to beagle dogs using a single injection, 6-h continuous i.v. infusion (c.i.v.) and weekly 6-h c.i.v. for 3 weeks. Endpoints evaluated included clinical observations, body weights, hematology, serum clinical chemistry, and microscopic pathology of tissues.
The MTD of DX-52-1 was a total dose of 18 mg/m(2) body surface area for q3hx3 administration in rats and 30 mg/m(2) for a single c.i.v. administration in dogs. The total dose MTD for rats on a weekly (q3hx3,q7dx3) regimen was 54 mg/m(2), and for dogs on the weekly x3 (6-h c.i.v.) infusion was 60 mg/m(2). In rats, significant elevations in blood urea nitrogen and creatinine were observed together with acute renal tubular necrosis histologically. Modest increases in liver enzymes were also observed, as were decreases in reticulocytes that were unaccompanied by histologic changes in liver and bone marrow. In dogs, adverse signs included vomiting/retching, diarrhea, and transient hypothermia; also red blood cells, hemoglobin, hematocrit, and lymphocytes were decreased. Histologic evaluation of tissues from dogs revealed necrosis and cellular depletion of the bone marrow, and extensive damage to the entire gastrointestinal tract, including marked cellular necrosis of the mucosa and lymphoid necrosis of the gastrointestinal associated lymphoid tissue. Destruction of the mucosal lining of the intestinal tract was likely responsible for dehydration, toxemia, septicemia, and shock seen in moribund dogs.
The MTD values were comparable between rats and dogs given roughly similar dose regimens (single dose or weekly) and both species tolerated a higher total dose with weekly administration. However, the principal target organ responsible for DLT in rats was the kidney, whereas in dogs, the most severe effects were on the gastrointestinal tract and bone marrow. Both renal and gastrointestinal toxicities were reported in patients after 6-h c.i.v. infusions in a limited phase I clinical trial, indicating that neither animal model alone was predictive of DX-52-1-induced toxicity in humans, and that both species were required to define human toxicity.
No preview · Article · Apr 2003 · Cancer Chemotherapy and Pharmacology
[Show abstract][Hide abstract] ABSTRACT: We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Mar 2000 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
The MTD (i. e. the highest dose that did not cause lethality but produced substantial toxicity) was approximately 1350 microg/m(2) body surface area (450 microg/kg) in mice, 450 microg/m(2) (75 microg/kg) in rats and </=400 microg/m(2) (</=20 microg/kg) in dogs. Adverse signs were observed at doses >/=1350 microg/m(2) in mice, >/=150 microg/m(2) in rats and >/=400 microg/m(2) in dogs. Decreased weight gain or actual weight loss was observed at doses >/=1350 microg/m(2) in mice, >/=600 microg/m(2) in rats and >/=450 microg/m(2) 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.
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.
No preview · Article · Feb 1999 · Cancer Chemotherapy and Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Megakaryocyte growth and development factor (MGDF) stimulates megakaryopoiesis and thrombopoiesis in vivo. Previous studies indicate that administration of pegylated recombinant human (PEG-rHu) MGDF in combination with recombinant murine granulocyte colony-stimulating factor (rMuG-CSF) prevented lethality and reduced hematotoxicity in carboplatin-treated/irradiated mice, a disease-state animal model of radio-chemotherapy. In the current study we have further characterized the effects of PEG-rHuMGDF in combination with rMuG-CSF with respect to clinical chemistry, hematology variables, and histologic evaluations to determine whether any potential toxicological interaction exists both in normal and myelosuppressed mice. Myelosuppression and subsequent thrombocytopenia in mice was induced with a combination of a single intraperitoneal injection of 1.25 mg carboplatin followed 4 h later with sublethal gamma irradiation exposure of 500 rad. Both normal and carboplatin-treated/irradiated mice were administered daily subcutaneous injections of 50 micrograms/kg PEG-rHuMGDF alone and in combination with 10 micrograms/kg rMuG-CSF for 21 consecutive days. Administration of PEG-rHuMGDF alone or in combination with rMuG-CSF to carboplatin-treated/irradiated mice increased survival 70 and 100%, respectively, and accelerated platelet recovery. Microscopic examination of nonhematopoietic organs showed no evidence of any morphological changes in normal and carboplatin-treated/irradiated animals. In hematopoietic organs clinically significantly increased granulopoiesis and megakaryopoiesis, as well as extramedullary granulopoiesis within the mandibular and mesenteric lymph nodes, were present. The erythroid line was unaffected by cytokine treatment. In normal, non-carboplatin-treated/irradiated mice, platelet counts increased 6 and 12-fold above baseline in the groups administered PEG-rHuMGDF alone or in combination with rMuG-CSF, respectively. The results of this study provide a basis for coadministration of PEG-rHuMGDF with Filgrastim (rHuG-CSF) in the clinical treatment of myelosuppression induced by radiation and chemotherapy.
Preview · Article · Oct 1998 · Toxicological Sciences