William R. Foster

Interlaboratory Evaluation of Genomic Signatures for Predicting Carcinogenicity in the Rat

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.

Drug safety is a barrier to the discovery and development of new androgen receptor antagonists

Androgen receptor (AR) antagonists are part of the standard of care for prostate cancer. Despite the almost inevitable development of resistance in prostate tumors to AR antagonists, no new AR antagonists have been approved for over a decade. Treatment failure is due in part to mutations that increase activity of AR in response to lower ligand concentrations as well as to mutations that result in AR response to a broader range of ligands. The failure to discover new AR antagonists has occurred in the face of continued research; to enable progress, a clear understanding of the reasons for failure is required.

A Retrospective Analysis of Toxicogenomics in the Safety Assessment of Drug Candidates

Toxicogenomics is considered a valuable tool for reducing pharmaceutical candidate attrition by facilitating earlier identification, prediction and understanding of toxicities. A retrospective evaluation of 3 years of routine transcriptional profiling in non-clinical safety studies was undertaken to assess the utility of toxicogenomics in drug safety assessment. Based on the analysis of studies with 33 compounds, marked global transcriptional changes (>4% transcripts at p < 0.01) were shown to be a robust biomarker for dosages considered to be toxic. In general, there was an inconsistent correlation between transcription and histopathology, most likely due to differences in sensitivity to focal microscopic lesions, to secondary effects, and to events that precede structural tissue changes. For 60% of toxicities investigated with multiple time-point data, transcriptional changes were observed prior to changes in traditional study endpoints. Candidate transcriptional markers of pharmacologic effects were detected in 40% of targets profiled. Mechanistic classification of toxicity was obtained for 30% of targets. Furthermore, data comparison to compendia of transcriptional changes provided assessments of the specificity of transcriptional responses. Overall, our experience suggests that toxicogenomics has contributed to a greater understanding of mechanisms of toxicity and to reducing drug attrition by empiric analysis where safety assessment combines toxicogenomic and traditional evaluations.

Discovery of BMS-641988, a Novel and Potent Inhibitor of Androgen Receptor Signaling for the Treatment of Prostate Cancer

Despite an excellent initial response to first-line hormonal treatment, most patients with metastatic prostate cancer will succumb to a hormone-refractory form of the disease. Because these tumors are still dependent on a functional androgen receptor (AR), there is a need to find novel and more potent antiandrogens. While searching for small molecules that bind to the AR and inhibit its transcriptional activity, BMS-641988 was discovered. This novel antiandrogen showed an increased (>1 log) potency compared with the standard antiandrogen, bicalutamide, in both binding affinity to the AR and inhibition of AR-mediated transactivation in cell-based reporter assays. In mature rats, BMS-641988 strongly inhibited androgen-dependent growth of the ventral prostate and seminal vesicles. In the CWR-22-BMSLD1 human prostate cancer xenograft model, BMS-641988 showed increased efficacy over bicalutamide (average percent tumor growth inhibition >90% versus <50%), even at exposure levels of bicalutamide 3-fold greater than what can be attained in humans. Furthermore, BMS-641988 was efficacious in CWR-22-BMSLD1 tumors initially refractory to treatment with bicalutamide. BMS-641988 was highly efficacious in the LuCaP 23.1 human prostate xenograft model, inducing stasis throughout the approximately 30-day dosing. To explore the functional mechanisms of BMS-641988, gene expression profiling analysis was done on CWR-22-BMSLD1 xenograft models in mice. Treatment with BMS-641988 resulted in a global gene expression profile more similar to castration compared with that of bicalutamide. Overall, these data highlight that the unique preclinical profile of BMS-641988 may provide additional understanding for the hormonal treatment of prostate cancer.

Discovery of a small molecule antagonist of the parathyroid hormone receptor by using an N-terminal parathyroid hormone peptide probe

Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1–84) or recombinant human PTH-(1–34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1–34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.

Discovery of the CCR1 antagonist, BMS-817399, for the treatment of rheumatoid arthritis.

High-affinity, functionally potent, urea-based antagonists of CCR1 have been discovered. Modulation of PXR transactivation has revealed the selective and orally bioavailable CCR1 antagonist BMS-817399 (29), which entered clinical trials for the treatment of rheumatoid arthritis.

The discovery of BMS-457, a potent and selective CCR1 antagonist

A series of compounds which exhibited good human CCR1 binding and functional potency was modified resulting in the discovery of a novel series of high affinity, functionally potent antagonists of the CCR1 receptor. Issues of PXR activity, ion-channel potency, and poor metabolic stability were addressed by the addition of a hydroxyl group to an otherwise lipophilic area in the molecule resulting in the discovery of preclinical candidate BMS-457 for the treatment of rheumatoid arthritis.

Hormone replacement therapy and cardiovascular disease: does route of administration and formulation matter?

Hormone replacement therapy (HRT) was (until recently) one of the most commonly prescribed drugs in the developed world, with the perceived benefits on a wide range of possible adverse events including general well-being, osteoporosis and cardiovascular morbidity and mortality, leading to an explosion in use of HRT. Furthermore, despite the increased risk of breast cancer with HRT, the early observational studies and meta-analyses actually suggested a reduction in mortality, cardiovascular disease and osteoporosis risk among users of HRT compared with non-users (Grady et al, 1992; Edmunds & Lip, 2000), leading to the widespread belief among patients and health care professionals that HRT was a new drug for the prevention and treatment of cardiovascular disease. The observation that many of the biochemical and physiological markers of cardiovascular risk (for example, lipid profile, insulin resistance, blood pressure, clotting, etc.) were modulated in a beneficial way by HRT lent further support to the idea that HRT could be useful for patients at risk of cardiovascular disease [The Writing Group for the postmenopausal oestrogen/progestin interventions (PEPI) trial 1995, Lieberman et al, 1994]. Interestingly, more recent metaanalyses that adjusted for factors such as socioeconomic class and education, or included only good quality observational data, found no significant beneficial cardiovascular effect (Humphrey et al, 2002). The Heart and Estrogen/Progestin Replacement Study (HERS) was the first randomized controlled trial to show a significant increase in cardiovascular events in HRT users compared with non-HRT users (Grady et al, 2002). Similar findings have since been reported in several good studies, most notably the Women’s Health Initiative (WHI) study – which was stopped early due to an increase in breast cancer rates which exceeded a predetermined level (Manson et al, 2003). Over the follow-up period (mean 5Æ2 years) there was also a significant increase in coronary heart disease events (acute myocardial infarction, silent myocardial infarction, coronary heart disease death) and stroke in users of HRT compared with controls (Wassertheil-Smoller et al, 2003). Importantly, these studies have used oral preparations of HRT. The possible reasons behind the difference in observational studies and randomized controlled trial data are many and have been previously debated. Briefly, HRT users tended to be more affluent, healthier, have better access to health care services, exercise more and are thinner, and are more likely to be compliant with medication than non-users (Lee et al, 2002). In addition, it is likely that physicians are more likely to treat women perceived as being healthy with HRT (Humphrey et al, 2002), and many of the traditional risk factors for coronary artery disease have been previously seen as contra-indications for HRT use. Also, many of the early studies, on which the first meta-analyses were based, did not correct for potential confounding factors such as lower socio-economic class, especially since it is clear that awareness and attitudes to HRT are dependent upon a number of factors, including ethnicity and education (Lydakis et al, 1998). More recent attention has focussed on the different formulations of HRT that are available. It is clear that the different routes of administration result in many different effects, possibly due to the relatively constant drug level with transdermal patches compared with oral formulations, and the possible effects of first pass metabolism with oral administration. For example, the increase in high-sensitivity C-reactive protein (hsCRP) seen with oral HRT is not found in transdermal administration (Vongpatanasin et al, 2003). In addition, one recently published case–control study showed that venous thromboembolism was a risk associated with oral oestrogen replacement therapy, but not transdermal patches (Scarabin et al, 2003). Certainly, the relative plasma proportions of the different sex steroids vary according to the route of administration and precise formulation used. These observations have therefore opened a debate regarding the effect of the route of administration of the drug. One randomized (but not blinded) trial (Strandberg et al, 2003) evaluated the effect of oral versus transdermal HRT on several markers of cardiovascular disease, and found that the transdermal route resulted in neutral or beneficial effects on several markers when compared with the oral route. Examples of the differing effects of oral and transdermal HRT are summarized in Table 1. As previously mentioned, HRT has been shown to exert a number of advantageous biochemical and physiological effects on users. In particular, HRT lowers total cholesterol, low-density lipoprotein (LDL)cholesterol and systolic blood pressure and raises high density lipoprotein (HDL)-cholesterol (The Writing Group for the PEPI Trial, 1995). However, it also has other effects that may impact in a less favourable way on the progression of atherosclerosis and thrombosis (Lip & Felmeden, 2001); in particular, oral HRT has been associated with increased levels of hsCRP (Cushman et al, 1999) and increased haemostasis (Caine et al, 1992), possibly predisposing to venous thromcorrespondence

Cloning, expression and characterization of monkey (Macaca fascicularis) CD137

CD137 plays an important role as a co-stimulatory molecule in activated T cells. Agonistic CD137 specific antibodies have been investigated as therapeutic agents to promote tumor-specific immune responses by direct activation of T cells. As part of the pre-clinical pharmacological evaluation of cynomolgus monkeys, monkey CD137 was cloned and characterized. The deduced amino acid sequence encoded a full-length gene of 254 amino acids 95% identical to human CD137. Sequence variants identified in monkey CD137 include four splice variants lacking the transmembrane domain. These variants were detectable in human including two previously unreported variants. Two missense single nucleotide polymorphisms were detected present in 42 and 50% of 36 monkeys tested. In both monkey and human, mRNA expression of full-length CD137 and splice variants were significantly increased in peripheral blood mononuclear cells (PBMCs) upon stimulation by anti-CD3 antibodies. Recombinant monkey CD137 protein was bound with high affinity by an agonistic anti-human CD137 antibody but not by an anti-mouse CD137 antibody. In summary, compared to human, monkey CD137 showed distinct extracellular domain amino acid sequence and sequence polymorphisms. Thus, antibodies directed against epitopes in this extracellular domain could have differences in pharmacologic activity between cynomolgus monkeys and human or across individual cynomolgus monkeys.

Chapter 27 Nonclinical Toxicogenomics in the Pharmaceutical Environment

Publisher Summary Toxicogenomics is a maturing research tool in its application to toxicology. This chapter provides an experimentally, rather than theoretically, grounded projection of the applications and potential impact of the new technology of toxicogenomics. While prudent use of toxicogenomics is able to focus on mechanistic investigations and indirectly provide potential counter-screens for toxicity and secondary pharmacology, the direct predictive application of toxicogenomics must be carefully validated for specific toxicities. Before embarking on extensive toxicogenomics studies, careful consideration should be given to the appropriateness of toxicogenomics for the evaluation of the findings of interest. Factors that complicate mechanistic investigations include the presence of general toxicity as manifested, for example, in overt clinical signs, substantial reduction in food intake, and body weight loss. The presence of profound pathological changes or excessive amounts of transcriptional changes also confounds the ability to meaningfully interpret transcriptomic data sets. In addition, the sensitivity of this tool to environment and other influences in its application to animal models as well as toxicology studies must always be considered in the interpretation of data.