A critical review of the effectiveness of rodent pharmaceutical carcinogenesis testing in predicting for human risk.

The pharmaceutical industry and regulatory agency toxicology testing paradigms in the United States currently appear successful, in part because of the continuously increasing life expectancy and the declining age-adjusted cancer rates in the United States. Although drugs likely have a minimal impact on the population statistics for cancer rates, pharmaceutical pathologists and toxicologists must focus on the individual risk for pharmaceutical carcinogenesis. As our understanding of carcinogenesis increases exponentially, and after hundreds if not thousands of rodent cancer tests, significant improvement in the precision of human pharmaceutical carcinogenesis hazard identification should now be possible and would enable a reduction in the substantial false-negative and false positive-rates reported herein. The appropriate use of acute, subchronic, chronic, and special toxicology tests to identify the major associated cancer risk factors, specifically, hormonal modulation, immunosuppression, genetic toxicity, and chronic toxicity, can be recognized through this review of pharmaceutical carcinogens. Significant opportunities exist for improving the effectiveness and efficiency of the current cancer risk assessment paradigm.

Effect of proteasome inhibitors with different chemical structures on the ubiquitin-proteasome system in vitro.

Proteasome inhibitor therapeutics (PITs) have the potential to cause peripheral neuropathy. In a mouse model of PIT-induced peripheral neuropathy, the authors demonstrated that ubiquitin-positive multifocal protein aggregates with nuclear displacement appear in dorsal root ganglion cells of animals that subsequently develop nerve injuries. This peripheral-nerve effect in nonclinical models has generally been recognized as the correlate of grade 3 neuropathy in clinical testing. In differentiated PC12 cells, the authors demonstrated perturbations correlative with the development of neuropathy in vivo, including ubiquitinated protein aggregate (UPA) formation and/or nuclear displacement associated with the degree of proteasome inhibition. They compared 7 proteasome inhibitors of 3 chemical scaffolds (peptide boronate, peptide epoxyketone, and lactacystin analog) to determine if PIT-induced peripheral neuropathy is modulated by inhibition of the proteasome (ie, a mechanism-based effect) or due to effects independent of proteasome inhibition (ie, an off target or chemical-structure-based effect). The appearance of UPAs was assayed at IC(90) +/- 5% (90% inhibition concentration +/- 5%) for 20S proteasome inhibition. Results show that each of the investigated proteasome inhibitors induced identical proteasome-inhibitor-specific ubiquitin-positive immunostaining and nuclear displacement in PC12 cells. Other agents--such as paclitaxel, cisplatin, and thalidomide, which cause neuropathy by other mechanisms--did not cause UPAs or nuclear displacement, demonstrating that the effect was specific to proteasome inhibitors. In conclusion, PIT-induced neuronal cell UPA formation and nuclear displacement are mechanism based and independent of the proteasome inhibitor scaffold. These data indicate that attempts to modulate the neuropathy associated with PIT may not benefit from changing scaffolds.

Predictive toxicology approaches for small molecule oncology drugs.

A daunting, unmet medical need exists for effective oncology chemotherapies, with cancer deaths in 2009 to exceed 560,000 in the United States alone. Because of the rapid demise of the majority of cancer patients with metastatic disease, oncology drug development must follow a much different paradigm than therapeutic candidates for less onerous diseases. The majority of drug candidates in development today are targeted at cancer therapy. Many of these candidate chemotherapeutic agents are active against novel targets, often presenting unique toxicological profiles. Since many of these novel targets are not unique to cancer cells, therapeutic margins may not exist. Decision making, in this event, is among the most challenging that any pharmaceutical toxicologist/pathologist or regulator will face. Nonclinical development scientists must compress timelines to present therapeutic options for cancer patients who have failed conventional therapy. In support of this goal, the U. S. Food and Drug Administration has created an oncology-specific paradigm for nonclinical testing and has introduced strategies to accelerate development and approval of successful candidates. Pharmaceutical toxicology testing strategies must not only satisfy regulation as the minimal expectation, but also attempt to reduce the current high attrition rates for oncologic candidates. A successful toxicology testing strategy represents the substance of this treatise.

Testing paradigm for prediction of development-limiting barriers and human drug toxicity.

The financial investment grows exponentially as a new chemical entity advances through each stage of discovery and development. The opportunity exists for the modern toxicologist to significantly impact expenditures by the early prediction of potential toxicity/side effect barriers to development by aggressive evaluation of development-limiting liabilities early in drug discovery. Improved efficiency in pharmaceutical research and development lies both in leveraging "best in class" technology and integration with pharmacologic activities during hit-to-lead and early lead optimization stages. To meet this challenge, a discovery assay by stage (DABS) paradigm should be adopted. The DABS clearly delineates to discovery project teams the timing and type of assay required for advancement of compounds to each subsequent level of discovery and development. An integrative core pathology function unifying Drug Safety Evaluation, Molecular Technologies and Clinical Research groups that effectively spans all phases of drug discovery and development is encouraged to drive the DABS. The ultimate goal of such improved efficiency being the accurate prediction of toxicity and side effects that would occur in development before commitment of the large prerequisite resource. Good justification of this approach is that every reduction of development attrition by 10% results in an estimated increase in net present value by $100 million.

Morphological transformation of Syrian hamster embryo cells at pH 6.7 by bemitradine, a nongenotoxic carcinogen.

Bemitradine is a compound that was intended for use as a diuretic antihypertensive drug. In the preclinical safety assays, it was found to be nongenotoxic in five in vitro assays (Ames, mouse lymphoma, CHO/HGPRT, CHO chromosome aberration, and UDS) and in one in vivo assay (mouse bone marrow micronucleus). In a subsequent long-term bioassay using Sprague-Dawley rats, this compound was found to be a rodent carcinogen at multiple sites. Because of the carcinogenicity, further development of this compound as a drug was halted. Because the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7 has been demonstrated to be a good predictor of carcinogenic activity in animals, this nongenotoxic compound was used to determine if this in vitro assay system could be utilized to predict the potential for the carcinogenicity in rodents. The SHE cell transformation assay was validated initially for use in this laboratory using genotoxic and nongenotoxic carcinogens including benzo(a)pyrene, 20-methylcholanthrene, 2-acetylaminofluorene, methapyrilene, and phenobarbital. Each of these chemicals induced a statistically significant increase in morphological transformation frequency. Bemitradine was initially tested in a range-finding cytotoxicity assay at 10-250 microg/mL for treatment periods of 7 days. Doses used in the 7-day treatment transformation assay were 1.25, 2.5, 5.0, 7.5, and 10.0 microg/mL. Statistically significant increases in morphological transformation frequencies were observed at 1.25, 2.5, and 7.5 microg/mL, indicating a positive response. The experiment was repeated with similar results confirming the previous conclusion. These data provide additional evidence that the pH 6.7 SHE cell transformation assay may be a valuable in vitro tool to detect nongenotoxic rodent carcinogens.

Xpa and Xpa/p53+/- knockout mice: overview of available data.

DNA repair deficient Xpa-/- and Xpa-/-/p53+/- knock-out mice in a C57BL/6 genetic background, referred to as respectively the XPA and XPA/p53 model, were investigated in the international collaborative research program coordinated by International Life Sciences Institute (ILSI)/Health and Environmental Science Institute. From the selected list of 21 ILSI compounds, 13 were tested in the XPA model, and 10 in the XPA/p53 model. With one exception, all studies had a duration of 9 months (39 weeks). The observed spontaneous tumor incidence for the XPA model after 9 months was comparable to that of wild-type mice (total 6%). For the XPA/p53 model, this was somewhat higher (9%/13% for males/females). The 3 positive control compounds used, B[a]P, p-cresidine, and 2-AAF, gave positive and consistent tumor responses in both the XPA and XPA/p53 model, but no or lower responses in wild-type mice. From the 13 ILSI compounds tested, the single genotoxic carcinogen (phenacetin) was negative in both the XPA and XPA/p53 model. Positive tumor responses were observed for 4 compounds, the immunosuppressant cyclosporin A, the hormone carcinogens DES and estradiol, and the peroxisome proliferator WY-14,643. Negative results were obtained with 5 other nongenotoxic rodent carcinogens, and 2 noncarcinogens tested. As expected, both DNA repair deficient models respond to genotoxic carcinogens. Combined with previous results, 6 out of 7 (86%) of the genotoxic human and/or rodent carcinogens tested are positive in the XPA model. The positive results obtained with the 4 mentioned nongenotoxic ILSI compounds may point to other carcinogenic mechanisms involved, or may raise some doubts about their true nongenotoxic nature. In general. the XPA/p53 model appears to be more sensitive to carcinogens than the XPA model.

Safety assessment for non-genotoxic rodent carcinogens: curves, low-dose extrapolations, and mechanisms in carcinogenesis.

The most frequent biologic events associated with multispecies multisite rodent non-genotoxic carcinogenesis include robust liver P450 enzyme induction without liver toxicity. This cancer process is not relevant for humans as exemplified by phenobarbital. A host of other pharmaceuticals, including loratidine and atorvastatin, with current sales in the billions of dollars attests to the lack of relevance of our rodent models acting via this specific process. Hopefully, the mechanism of phenobarbital will no longer draw research money in further probing the irrelevant in human carcinogenesis. The known human carcinogens not linked with genetic injury all perturb tissue growth/replication in humans as well as in rodent short-term tests at doses ultimately linked with cancer. Clearly identifying exposures perturbing growth/replication versus exposures not perturbing growth provides an effective tool for demonstrating the threshold for cancer risk in refining the risk assessment as capably described by Dr. Klaunig. Hopefully, we can begin to increasingly use data-based decisions in risk assessment versus hypothetical low- dose extrapolations and mathematical modeling, building on the wealth of current understanding in human and rodent cancer processes in arenas beyond pharmaceutics.

The pathologist and toxicologist in pharmaceutical product discovery.

Significant change is occurring in the drug discovery paradigm; many companies are utilizing dedicated groups from the toxicology/ pathology disciplines to support early stage activities. The goal is to improve the efficiency of the discovery process for selecting a successful clinical candidate. Toxicity can be predicted by leveraging molecular techniques via rapid high-throughput, low-resource in vitro and in vivo test systems. Several important activities help create a platform to support rapid development of a new molecular entity. The proceedings of this symposium provide excellent examples of these applied concepts in pharmaceutical research and development. Leading biopharmaceutical companies recognize that a competitive advantage can be maintained via rapid characterization of animal models, the cellular identification of therapeutic targets, and improved sensitivity of efficacy assessment. The participation of the molecular pathologist in this quest is evolving rapidly, as evidenced by the growing number of pathologists that interact with drug discovery organizations.

Revolution through genomics in investigative and discovery toxicology.

The remarkable technologic and methodologic advances spurred on by the Human Genome Project are being applied throughout the life sciences. In the field of toxicology, high-resolution assays now make it possible to discover virtually all the differences in gene expression brought on by exposure to a particular xenobiotic. There are 2 principal approaches used to build a catalog of changes in gene expression: hybridization microarrays and gel-based methods, such as differential display and AFLP-based mRNA finger-printing. The power of such approaches is exemplified by the identification of more than 300 genes that differ in expression level by at least 2-fold in response to the nongenotoxic rodent liver carcinogen phenobarbital.

Interspecies differences in renal localization of cyclooxygenase isoforms: implications in nonsteroidal antiinflammatory drug-related nephrotoxicity.

Cyclooxygenase (COX) exists in 2 related but unique isoforms: one is constitutive (COX-1) and functions in normal cell physiology, and the other is inducible (COX-2) and is expressed in response to inflammatory stimuli. Nonsteroidal antiinflammatory drugs (NSAIDs) cause renal toxicity following inhibition of renal cyclooxygenases. Humans and animals exhibit differences in susceptibility to NSAID-related renal toxicity, which may be associated with differences in expression of 1 or both isoforms of COX in the kidney. In this study, we evaluated COX-1 and COX-2 expression in the kidneys of mixed-breed dogs, Sprague-Dawley rats, cynomolgus monkeys, and humans. In addition, the effect of volume depletion on renal COX expression was investigated in rats, dogs, and monkeys. COX expression was evaluated using 1 or more of the following procedures: reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. We demonstrated that both COX isoforms are expressed in the kidneys of all species examined, with differences in the localization and level of basal expression. COX-1 is expressed at high levels in the collecting ducts and renal vasculature of all species and in a small number of papillary interstitial cells in rats, monkeys, and humans. Basal levels of COX-2 are present in the maculae densa, thick ascending limbs, and papillary interstitial cells in rats and dogs and in glomerular podocytes and small blood vessels in monkeys and humans. COX-2 expression is markedly increased in volume-depleted rats and dogs but not monkeys. These results indicate that significant interspecies differences exist in the presence and distribution of COX isoforms, which may help explain the difference in species susceptibility to NSAID-related renal toxicity.

Phenobarbital does not promote hepatic tumorigenesis in a twenty-six-week bioassay in p53 heterozygous mice.

The tumorigenic potential of phenobarbital was examined in a 26-wk carcinogenesis bioassay using p53 heterozygous mice and wild-type controls. Fifteen mice/sex/genotype were exposed to either 500 or 1,000 ppm phenobarbital in the diet. Dietary administration of 3,750 ppm p-cresidine, a transspecies mutagenic carcinogen, to both heterozygous and wild-type mice served as a positive control. Phenobarbital treatment caused increases in liver:body weight ratios and histologic evidence of centrilobular hepatocellular hypertrophy. No tumors were observed in any phenobarbital-treated mice. Mice given p-cresidine exhibited a moderate reduction in body weight gain over the course of the study. Heterozygous mice treated with p-cresidine exhibited a high incidence of urinary bladder tumors. Similar tumors were also present in a small number of p-cresidine-treated wild-type mice. Our results demonstrate the lack of a hepatic tumor response to phenobarbital, a compound that is a potent and potent and prototypic hepatic microsomal enzyme inducer, a nongenotoxic rodent carcinogen, and a human noncarcinogen. This finding supports the continued utility of this model as an alternative to the mouse bioassay for human carcinogenic safety assessment of potentially genotoxic carcinogenes because it did not produce a false-positive response to this potent nongenotoxic agent.

Exploration of the transformation potential of a unique male rat protein alpha2u-globulin using hamster embryonic cells.

Several environmentally and socially important chemicals such as d-limonene and unleaded gasoline have been demonstrated to induce alpha2u-globulin (alpha 2u) nephropathy in male rats. Substantial progress has been made in characterizing the biological effects of these chemicals on the kidney and in further defining prerequisite events in the pathogenesis of this syndrome. The alpha 2u increase in the kidney is hypothesized to be the proximal event in the toxicologic and tumorigenic sequelae associated with administration of these xenobiotics over the male rat's lifetime rather than a direct effect of the administered chemical. The administered chemical appears to simply mediate the increase in alpha 2u concentration in the kidney. To further investigate the properties of alpha 2u, this protein was tested in the pH 6.7 Syrian hamster embryo (SHE) cell transformation assay. The alpha 2u caused morphological transformation in these cells, whereas another protein, bovine serum albumin, did not induce transformation at equimolar concentrations, suggesting a protein-specific phenomenon. Neither d-limonene nor trimethylpentane (a causal component in unleaded gasoline) induced SHE cell transformation. These results support the hypothesis that alpha 2u increase in proximal convoluted tubules may directly cause renal tumorigenesis in male rats. The SHE cell transformation assay may be a useful tool for mechanistic studies of this syndrome.

Effect of papillotoxic agents on expression of cyclooxygenase isoforms in the rat kidney.

Inhibition of renal vasodilatory prostaglandins (PGs) and secondary ischemia due to inhibition of cyclooxygenase (COX) activity has been suggested as a possible mechanism for development of analgesic-related renal papillary necrosis (RPN) in rats. Recently, it has been shown that COX exists in two related but unique isoforms, COX-1 and COX-2. It is unclear what potential roles these isoforms play in the maintenance of blood flow in the renal papilla or genesis of RPN. We evaluated the effect of 2 papillotoxic agents, including a nonsteroidal anti-inflammatory drug, indomethacin, and a chemical agent, 2-bromoethanamine hydrobromide (2-BEA), on COX-1 and COX-2 in the renal papilla as a means of assessing what changes occur in the expression of these isoforms during the development of RPN. Female Wistar rats approximately 10-17 wk old were treated with either indomethacin (75 mg/kg, single dose, or 10 mg/kg/day for 5 days) or 2-BEA (100 mg/kg/day for 4 days) to create lesions of RPN. In this study, a single 75-mg/kg dose of indomethacin did not cause light microscopic changes of RPN. However, RPN was observed in animals administered indomethacin at 10 mg/kg/day for 1 wk or 2-BEA for 5 days. The immunohistochemical analyses of kidneys showed that both COX-1 and COX-2 were present in the renal papilla of control rats. In animals treated with indomethacin (75 mg/kg), a slight to moderate decrease in both isoforms was observed in essentially normal renal papillary cells within 2 hr, that was followed by an increase in COX-2 immunoreactivity in the renal papilla, macula densa, and thick ascending limbs (both 10- and 75-mg/kg animals). This COX-2 immunoreactivity was greatest in animals with concomitant indomethacin-induced gastrointestinal injury, suggesting a possible role of inflammatory cytokines in COX-2 induction. No changes in the expression of COX isoforms in the intact papilla occurred as a result of 2-BEA; however, cells undergoing degeneration and necrosis lost immunoreactivity to both COX isoforms. The possible mechanism that leads to an initial decrease in COX immunoreactivity in indomethacin-treated animals is not known; however, a reversible ultrastructural change in the papillary cells cannot be ruled out. This decrease in COX isoforms in the renal papilla may contribute to the development of RPN through the loss of vasodilatory PGs.

A critical appraisal of the value of the mouse cancer bioassay in safety assessment.

Substantial progress in understanding nongenotoxic or secondary mechanisms of tumorigenesis has been made over the past decade. However, the methods used in regulated studies for assessment of carcinogenic potential in chemicals in development have not evolved significantly. Based on the experience of over 30 yr of testing and the societal need to control costs, reevaluation of standard cancer rodent bioassay protocols is being done. Expert consensus after evaluating the results of full-scale rodent bioassays of both sexes in 2 species is that a reduced protocol is acceptable. After review of relevant data, it is our opinion that cancer hazard assessment in male and female rats only would be sufficient and that, in the future, mouse bioassays will not add significant value on a routine basis. We are unable to find an example of a mouse tumorigenic finding that predicts a confirmed or probable human response with negative findings in a rat bioassay. The savings realized by eliminating mouse testing from routine protocols would be substantial and better spent in expanding short-term studies to add to our understanding of chemical carcinogenesis.

Pathology of perbutylated-N-butyl-1-deoxynojiromycin (an alpha-glucosidase-1 inhibitor) in Sprague-Dawley rats.

Perbutylated-N-butyl-1-deoxynojiromycin (p-N-butyl-DNJ, SC-49483), an alpha-glucosidase-1 inhibitor, is a candidate anti-HIV agent targeted against viral glycoprotein processing in host cell endoplasmic reticulum. The potential toxicity of this compound was evaluated in Sprague-Dawley rats after 4, 13, or 26 wk of oral administration at doses ranging from 300 to 3,670 mg/kg/day. In these studies, the target organs of p-N-butyl-DNJ effects were thyroid gland, salivary gland, stomach, and pancreas. The most prominent histologic change in these organs was the presence of clear or lightly eosinophilic vacuoles in the cytoplasm of thyroid follicular cells, gastric chief cells, salivary gland acinar cells, and exocrine pancreatic acinar cells. Ultrastructurally, these vacuoles were consistent with dilated rough endoplasmic reticulum, which sometimes contained homogeneously stained, moderately electron-dense material. The vacuoles in thyroid follicular cells contained pale eosinophilic colloidlike material consistent with accumulated thyroglobulin, as shown by immunohistochemical staining methods. The biological functions of these organs were not adversely affected as evidenced by the absence of clinical signs and the results of selected hormonal analyses. The morphologic changes were completely reversed after a 4-wk recovery period. The incidence and severity of histologic changes were decreased after 13 and 26 wk of treatment compared to 4 wk of treatment, indicating an attenuation of the host response or adaptation to the prolonged p-N-butyl-DNJ administration. We believe that morphologic changes in thyroid follicular cells, salivary gland acinar cells, pancreatic acinar cells, and gastric chief cells were the result of nonspecific inhibition of host alpha-glucosidase(s) by p-N-butyl-DNJ, causing clinically silent perturbation in host cell glycoprotein processing and/or glycoprotein transport.

Recombinant human interleukin-3 induces extramedullary hematopoiesis at subcutaneous injection sites in cynomolgus monkeys.

Parenteral administration of recombinant hematopoietic growth factors has been sporadically associated with cutaneous complications, including injection site reactions in humans and nonhuman primates. In this study, subcutaneous injection sites were evaluated from 12 cynomolgus monkeys administered a recombinant human interleukin-3 (rhIL-3) at dose levels of 0, 70, or 700 micrograms/kg daily for 18 days. Monkeys administered rhIL-3 developed small (0.5-1-cm-diameter), firm nodules at the subcutaneous injection sites. Histologically, these nodules from 4 of 8 rhIL-3-treated monkeys contained trilineage extramedullary hematopoiesis (EMH) represented by precursors of myeloid, erythroid, and megakaryocytic series cells. The lineage of hematopoietic cells was confirmed by histochemical and immunohistochemical methods. Hematopoietic cells of myeloid and megakaryocytic lineages were more common than erythroid cells. Of myeloid cells, immature eosinophils were more common, which usually formed small sheets or clusters in the panniculus and deep dermis. This report describes, for the first time, the occurrence of cutaneous EMH at the injection sites of recombinant hematopoietic growth factors, which should be differentiated from inflammation. We believe the cutaneous EMH was the exaggerated pharmacologic effect of rhIL-3.