Nonclinical safety evaluation of biotechnologically derived pharmaceuticals.

The primary objectives of nonclinical safety evaluation for pharmaceutical products are to identify potential target organ toxicity, provide a safe starting dose for clinical trials, and establish dose-response relationships. These objectives do not differ in concept for either small molecular weight compounds or biotechnologically derived pharmaceuticals; they are important for both. The complex structural and biological characteristics of biotechnologically derived pharmaceuticals, however, dictate that different approaches to their safety evaluation are needed. Although their novel mode of production initially raised concerns about their safety, improvements in analytical and manufacturing procedures have largely minimized the perceived risks. It is primarily their exaggerated pharmacodynamic properties that produce the toxicity observed in nonclinical studies. Even though most of these products will require a case-by-case, scientifically based approach, knowledge gained from both nonclinical and clinical evaluation of these novel products have highlighted some general principles with regards to their safety evaluation. These principles include the importance of evaluating species in which the biotechnologically derived pharmaceutical is biologically active, the potential impact of immunogenicity on the interpretation of multiple dose toxicity study results, and the need for both highly sensitive and specific analytical methods to measure their pharmacodynamic properties. An understanding of these principles forms the basis for the development of a scientifically sound nonclinical safety evaluation program.

Preclinical toxicity evaluation of tepoxalin, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, in Sprague-Dawley rats and beagle dogs.

Tepoxalin [5- (4-chlorophenyl)-N-hydroxy-1-(4-methoxyphenyl)-N-methyl-1H-pyrazole -3-propanamide] is an orally active anti-inflammatory agent, which inhibits both cyclooxygenase and 5-lipoxygenase activities. The oral toxicity of tepoxalin was evaluated in 1- and 6-month rat (up to 50 mg/kg/day) and dog (up to 150 mg/kg bid) studies. In rats, increased liver weight, centrilobular hypertrophy, and hepatic necrosis were observed at dosages >/=20 mg/kg/day. Renal changes indicative of analgesic nephropathy syndrome (i.e., papillary edema or necrosis, cortical tubular dilatation) were seen at >/=15 mg/kg. In rats treated for 1 month, these hepatic and renal effects were largely reversible after a 1-month recovery period. Gastrointestinal erosions and ulcers were seen in female rats given 40 mg/kg/day for 6 months. Changes in clinical pathology parameters included decreases in red blood cell count, hemoglobin, and hematocrit mean values; elevation in platelet counts; and an increase in prothrombin and activated partial thromboplastin times. Mild increases in alanine aminotransferase, aspartate aminotransferase, and cholesterol were also noted in rats. Decreased erythrocyte parameters, increased leukocyte counts, and decreased total protein, albumin, and/or calcium were noted in some dogs in the 300 mg/kg/day group following 6 months of dosing. Small pyloric ulcerations were seen at 100 and 300 mg/kg/day dosages for up to 6 months. In both rats and dogs, no accumulation of tepoxalin or its carboxylic acid metabolite was detected in plasma following multiple dosing over a range of 5 to 50 mg/kg/day for rats and 20 to 300 mg/kg/day for dogs. Plasma concentrations of the carboxylic acid metabolite were severalfold higher than those of the parent compound. The no-effect dosages in rats (5 mg/kg/day) and dogs (20 mg/kg/day) were approximately one and six times the ED50 (3.5 mg/kg), respectively, for inhibition of inflammatory effects in the adjuvant arthritic rat without gastric mucosal damage. In terms of severity, the relative lack of gastrointestinal side effects, within the estimated therapeutic dose range, distinguishes tepoxalin from most marketed anti-inflammatory drugs.

Kinetics of granulocytic and erythroid progenitor cells are affected differently by short-term, low-level benzene exposure.

In previous work, we determined that granulocytic (CFU-GM) and erythroid (CFU-E) progenitor cell populations exhibited disparate responses to short-term benzene exposures. We now report on work investigating possible mechanisms for these observed disparate responses. Mice were exposed to either air or 10 ppm benzene for 6 h/d X 5 d. Immediately after the last exposure, mice were injected, i.v., with either saline or hydroxyurea (HU). The dose of HU was sufficient to kill hematopoietic cells in or near S-phase of the cell cycle and sufficient to synchronize the surviving populations of hematopoietic cells. Three days after benzene exposure, CFU-E numbers had declined to 50% of control values while CFU-GM numbers were equal to control values at this time. The benzene exposures were sufficient to double the percentage of CFU-E in S-phase but produced no such increase among CFU-GM. During 3 days of recovery from benzene exposure and HU treatment, the CFU-E population expanded 30-fold while the CFU-GM population expanded less than 3-fold. Following benzene exposure and HU treatment, both progenitor cells produced elevated numbers of their respective progeny. When CFU-E from benzene-exposed mice were cultured with varying concentrations of erythropoietin (EPO), the response at maximal EPO concentration was 66% of the response by control CFU-E. This strongly suggests that the CFU-E populations from benzene-exposed mice had been depleted of cells in or near S-phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Short term benzene exposure provides a growth advantage for granulopoietic progenitor cells over erythroid progenitor cells.

Because chronic benzene exposure is associated with acute myeloblastic leukemia and other myeloproliferative disorders, we sought to determine whether short-term benzene exposure provides a growth advantage for granulopoietic elements over erythropoietic elements. Groups of male DBA/2J mice were exposed to 0, 10, 30, or 100 ppm benzene (6 h/day for 5 days). One day and 5 days after the benzene exposures, the numbers of the two most primitive erythroid progenitor cells (BFU-E and CFU-E) and the numbers of the most primitive granulocytic progenitor cells (GM-CFU-C) were assessed. Additional groups of mice were given hemolytic doses of phenylhydrazine (PHZ) during the 5 days of benzene exposure, while other groups of mice were given PHZ during the 5 days of recovery from benzene exposure. These experiments were designed to determine the effects of benzene exposure on progenitor cell numbers during periods of markedly heightened erythropoiesis. The results demonstrate that short-term benzene exposure does induce a growth advantage for granulocytic cells in both the bone marrow and spleen of exposed mice. Moreover, a benzene-induced shift toward granulopoiesis is observed even in those mice treated with a powerful erythropoietic stimulus. These effects disappear 5 days after cessation of benzene exposure in the bone marrow but persist in the spleen of mice treated with phenylhydrazine.

The ability of the murine erythron to respond to hemolytic doses of phenylhydrazine is significantly impaired by exposures to 10 ppm benzene.

Male C57Bl mice were given 50 exposures (6 h/d x 5 d/wk x 10 wk) to 10 ppm benzene. At regular intervals during the course of the exposures, the numbers of erythroid colony-forming cells (CFU-E) and the numbers of granulocytic colony-forming cells (GM-CFU-C) were assayed. At the end of the benzene exposures, additional groups of mice were given 4 daily injections of phenylhydrazine (PHZ) to induce anemia. During the course of the exposures, the numbers of colony-forming cells from benzene-exposed mice were, with infrequent exceptions, statistically indistinguishable from the numbers of these cells in air-exposed mice. However, in response to the PHZ-induced anemia, the numbers of late erythroid (CFU-E) and granulocytic (GM-CFU-C) progenitor cells were about 30% lower among benzene-exposed mice than among air-exposed mice. These results suggest that concentrations of benzene that induce little or no observable hematopoietic changes may, in fact, greatly alter the hematopoietic capacity of an exposed individual.

The temporal relationship between behavioral and hematological effects of inhaled benzene.

The time-effect relationship of the behavioral and hematological changes caused by inhaled benzene was investigated in C57BL mice. Mice were exposed to air, or 100, 300, 1000, or 3000 ppm benzene in standard inhalation chambers employing dynamic vapor exposure techniques. Mice were exposed for 6 hr/day for the number of days necessary to achieve a minimum concentration X time (Ct) product of 3000 ppm-days. The intermittent exposure regimen of 6 hr/day was employed to simulate occupational exposure. The most sensitive behavioral index (milk-licking) was affected by the lowest concentration tested (100 ppm), while homecage food intake, hindlimb grip strength, and body weight were reduced only at 1000 and 3000 ppm. Some of these previously undocumented behavioral changes occurred as rapidly as hematological changes that have been considered hallmarks of benzene toxicity. A significant decrease in circulating lymphocytes occurred after exposure to all concentrations. Circulating red blood cells were variably affected by benzene, in that anemia resulted after 10 days exposure to 100 ppm and after 3 days exposure to 300 ppm but not after 3 days exposure to 1000 ppm or a single exposure of 3000 ppm. The data indicate a departure from Ct relationships, and suggest that exposure duration as well as daily dose may be an important factor in benzene toxicity.

Behavioral changes in mice following benzene inhalation.

Although benzene is an important occupational health hazard and a carcinogen, the possibility that behavioral changes may forewarn of the later-occurring hematological changes has not been investigated. A time-sampling protocol was used to quantify the occurrence of 7 categories of behavior in the homecage following daily 6-hr exposures to two strains of adult mice (CD1 and C57BL/6J). The behavioral categories were stereotypic behavior, sleeping, resting, eating, grooming, locomotion, and fighting. The inhalation exposures were designed to reflect occupational exposure. Dynamic vapor exposure techniques in standard inhalation chambers were employed. Exposure to 300 or 900 ppm benzene increased the occurrence of eating and grooming and reduced the number of mice that were sleeping or resting. The responses to benzene of both the CD1 and the C57 strains were similar. The positive findings with benzene inhalation indicate the utility of behavioral investigations into the toxicology of inhaled organic solvents. The methods described herein illustrate an objective observation of animal behavior that is capable of documenting toxicity and of guiding detailed follow-up studies aimed at mechanism of action.