Nonclinical aspects of biopharmaceutical development: discussion of case studies at a PhRMA-FDA workshop.

Robust assessments of the nonclinical safety profile of biopharmaceuticals are best developed on a scientifically justified, case-by-case basis, with consideration of the therapeutic molecule, molecular target, and differences/similarities between nonclinical species and humans (ICH S6). Significant experience has been gained in the 10 years ensuing since publication of the ICH S6 guidance. In a PhRMA-FDA-sponsored workshop, "Nonclinical Aspects of Biopharmaceutical Development," industry and US regulatory representatives engaged in exploration of current scientific and regulatory issues relating to the nonclinical development of biopharmaceuticals in order to share scientific learning and experience and to work towards establishing consistency in application of general principles and approaches. The proceedings and discussions of this workshop confirm general alignment of strategy and tactics in development of biopharmaceuticals with regard to such areas as species selection, selection of high doses in toxicology studies, selection of clinical doses, the conduct of developmental and reproductive toxicity (DART) studies, and assessment of carcinogenic potential. However, several important aspects, including, for example, appropriate use of homologues, nonhuman primates, and/or in vitro models in the assessment of risk for potential developmental and carcinogenic effects, were identified as requiring further scientific exploration and discussion.

Radiotherapy dosimetry using a commercial OSL system.

A commercial optically stimulated luminescence (OSL) system developed for radiation protection dosimetry by Landauer, Inc., the InLight microStar reader, was tested for dosimetry procedures in radiotherapy. The system uses carbon-doped aluminum oxide, Al2O3:C, as a radiation detector material. Using this OSL system, a percent depth dose curve for 60Co gamma radiation was measured in solid water. Field size and SSD dependences of the detector response were also evaluated. The dose response relationship was investigated between 25 and 400 cGy. The decay of the response with time following irradiation and the energy dependence of the Al2O3:C OSL detectors were also measured. The results obtained using OSL dosimeters show good agreement with ionization chamber and diode measurements carried out under the same conditions. Reproducibility studies show that the response of the OSL system to repeated exposures is 2.5% (1sd), indicating a real possibility of applying the Landauer OSL commercial system for radiotherapy dosimetric procedures.

The water-equivalence of phantom materials for 90Sr-90Y beta particles.

Intravascular brachytherapy requires that the dose be specified within millimeters of the source. High dose gradients near brachytherapy sources require that the source-detector distance be accurately known for dosimetry purposes. Solid phantoms can be designed to accommodate these stringent requirements. This study reports dosimeter readings from 90Sr-90Y sources measured in water, A150, polystyrene and in an epoxy-based water-equivalent plastic. Measurements showed that while A150 and the epoxy-based plastic agreed well with water when the surface of the source contacted the detector housing, the relative response in the phantoms decreased with increasing depth in phantom, falling to approximately 0.55 those of water at a depth of 5 mm. Readings in polystyrene were within 4% of those in water between 1 and 2 mm depth. However, while polystyrene followed water more closely than the other two materials, at greater depths the relative response in polystyrene to water varied from 0.65 to 1.34. When the density of the materials is accounted for, the relative response in A150 is nearly constant with increasing areal density. Furthermore, the response in A150 shows the closest agreement with that in water of any of the solid materials for higher areal densities. For values below 0.3 g/cm2, polystyrene shows the closest agreement with water.

Identification of urinary metabolites of isoprene in rats and comparison with mouse urinary metabolites.

Isoprene, a major commodity chemical used in production of polyisoprene elastomers, has been shown to be carcinogenic in rodents. Similar to findings for the structurally related compound butadiene, mice are more susceptible than rats to isoprene-induced toxicity and carcinogenicity. Although differences in uptake, and disposition of isoprene in rats and mice have been described, its in vivo biotransformation products have not been characterized in either species. The purpose of these studies was to identify the urinary metabolites of isoprene in Fischer 344 rats and compare these metabolites with those formed in male B6C3F1 mice. After i.p. administration of 64 mg [14C]isoprene/kg to rats and mice, isoprene was excreted unchanged in breath ( approximately 50%) or as urinary metabolites ( approximately 32%). In rats isoprene was primarily excreted in urine as 2-hydroxy-2-methyl-3-butenoic acid (53%), 2-methyl-3-buten-1,2-diol (23%), and the C-1 glucuronide conjugate of 2-methyl-3-buten-1,2-diol (13%). These metabolites are consistent with preferential oxidation of isoprene's methyl-substituted vinyl group. No oxidation of the unsubstituted vinyl group was observed. In addition to the isoprene metabolites found in rat urine, mouse urine contained numerous other isoprene metabolites with a larger percentage (25%) of total urinary radioactivity associated with an unidentified, polar fraction than in the rat (7%). Unlike butadiene, there was no evidence that glutathione conjugation played a significant role in the metabolism of isoprene in rats. Because of the unidentified metabolites in mouse urine, involvement of glutathione in the metabolism of isoprene in mice cannot be delineated.

Carcinogenicity of 2',3'-dideoxycytidine in mice.

2',3'-dideoxycytidine (ddC) is a synthetic pyrimidine nucleoside analogue approved for treatment of HIV-positive patients. Previous studies indicated that ddC has the potential to cause thymic lymphoma in C57BL/6 x C3H F1 (hereafter called B6C3F1) mice. In this study, we evaluated the carcinogenic potential of ddC in two different mouse models. B6C3F1 hybrid mice carry ecotropic endogenous proviral sequences that may be activated to cause lymphoma, whereas NIH Swiss mice lack proviral sequences that can be expressed. The mice were treated with ddC by gavage at 500 and 1000 mg/kg/day for up to 6 months (human dose, 2.25 mg/day) and evaluated for toxicity, plasma levels of ddC, and pathological changes. Lymphocyte cell markers from the thymic lymphomas were assessed by immunophenotyping. Expression of p53 protein was evaluated using immunohistochemical staining. Treatment-related thymic lymphomas were present in both mouse models with a higher incidence in NIH Swiss than in B6C3F1 mice. The lymphomas were more prevalent in females than in males of both mouse models. Most mice with thymic lymphoma died during the course of the study. In addition to the thymus, lymphoma was often present in lymph nodes, spleen, and other organs. Lymphomas arose more frequently in mice that lack endogenous ecotropic retroviral sequences and thus were not due to activation of endogenous provirus. During the third month of the study, a few NIH Swiss mice that died had granulosa cell tumors of the ovary. Treatment-related but reversible thymic atrophy was observed in both mouse models. There was a very high correlation between the internal dose of ddC and the incidence of thymic lymphoma in both mouse models. Most of the lymphocytes from control thymuses and ddC-induced lymphomas were positive for Thy-1.2 (pan-T), heat stable antigen, and CD4 and CD8 markers, with no marked differences in the lymphocyte markers of the tumors between sexes or dose groups. p53 protein was detected in only 20% (23/115) of the ddC-induced lymphomas with mostly minimal expression in scattered cells. Because ddC induced lymphomas in two different mouse models, the potential carcinogenic risk should be considered in long-term treatment of HIV-positive patients, especially children and adolescent patients treated with ddC.

Photochemistry of DNA using 193 nm excimer laser radiation.

Photoproducts in double-stranded DNA induced by 193 nm radiation have been investigated. Double-stranded, supercoiled pBR322 DNA in buffered aqueous solution was exposed to varying fluences of 193 nm radiation from an ArF excimer laser. The quantum yields for formation of cyclobutylpyrimidine dimers, frank strand breaks and alkali labile sites were calculated from the conversion of supercoiled (Form I) DNA to relaxed (Form II) DNA after treatment with Micrococcus luteus dimer-specific endonuclease, no treatment, or treatment with alkali and heat, respectively. The quantum yields were 1.65 (+/- 0.03) X 10(-3) for pyrimidine dimers, 9.4 (+/- 3.2) X 10(-5) for frank strand breaks and 9.6 (+/- 3.6) X 10(-5) for alkali labile sites. The quantum yields for pyrimidine dimers and strand breaks and alkali labile sites were not affected by 10 nM mannitol. The relative quantum yields for these DNA photoproducts induced by 193 nm radiation differed markedly from those produced by 254 nm radiation.

The toxicity of dimethylamine in F-344 rats and B6C3F1 mice following a 1-year inhalation exposure.

Dimethylamine is a widely used commodity chemical, for which there are few chronic toxicity data. Male and female F-344 rats and B6C3F1 mice were exposed by inhalation to 0, 10, 50, or 175 ppm dimethylamine (DMA) for 6 hr/day, 5 days/week for 12 months. Groups of 9-10 male and female rats and mice were necropsied after 6 and 12 months of exposure. No male mice were sacrificed at 12 months due to a high incidence of early deaths in that group. The mean body weight gain of rats and mice exposed to 175 ppm DMA was depressed to approximately 90% of control after 3 weeks of exposure. The only other treatment-related changes were concentration-related lesions in the nasal passages. Two distinct locations in the nose were affected: the respiratory epithelium in the anterior nasal passages, and the olfactory epithelium, especially that lining the anterior dorsal meatus. There was focal destruction of the anterior nasoturbinate and nasal septum, local inflammation, and focal squamous metaplasia of the respiratory epithelium in rats and mice. Mild goblet cell hyperplasia was observed only in rats. The olfactory epithelium exhibited extensive loss of sensory cells with less damage to sustentacular cells. There was also loss of olfactory nerves, hypertrophy of Bowman's glands, and distension of the ducts of these glands by serocellular debris in regions underlying degenerating olfactory epithelium. At the 175-ppm exposure level, rats had more extensive olfactory lesions than mice, with hyperplasia of small basophilic cells adjacent to the basement membrane being present in rats but not mice. After 12 months of exposure to 10 ppm DMA, minimal loss of olfactory sensory cells and their axons in olfactory nerve bundles was observed in the nasal passages of a few rats and mice. These results indicate that the olfactory sensory cell is highly sensitive to the toxic effects of DMA, with minor lesions being produced in rodents even at the current threshold limit value of 10 ppm.

Pathology of toxic responses to the RD50 concentration of chlorine gas in the nasal passages of rats and mice.

Male Swiss-Webster mice and Fischer-344 rats were exposed to chlorine gas at their respective RD50 concentrations (ca. 9 to 11 ppm). The RD50 concentration is that concentration which reduces respiratory rate by 50%. The exposures were carried out for 6 hr per day for 1, 3, or 5 days, and the animals were killed immediately at the end of the last exposure. The nasal passages were examined by light and scanning electron microscopy. Lesions were observed in all exposed groups and were of similar severity and character in rats and mice. The most severe changes were found in the olfactory mucosa of the anterior portion of the dorsal meatus and consisted of partial to complete degeneration of olfactory sensory cells, with olfactory sustentacular cells being more resistant to chlorine exposure. Lesions in the respiratory epithelium were located primarily on the free margins of the naso- and maxilloturbinates and adjacent nasal septum. Scanning electron microscopy, using large size specimens, demonstrated loss of olfactory cilia in areas of the olfactory epithelium which appeared unaffected by light microscopy. Scanning electron microscopy was also helpful in locating areas of respiratory epithelium exhibiting loss of cilia and cellular exfoliation, which occurred primarily on naso- and maxilloturbinates. Therefore, chlorine-induced severe lesions in specific locations in both the olfactory and respiratory epithelia of the nasal passages with more widespread loss of respiratory and olfactory cilia.