2
Investigative Toxicology: The State of the Art1

As context for the discussion of emerging safety science, it is useful to review the current state of the art in investigative toxicology. To this end, Dr. Frazier described the work of his group at GlaxoSmithKline (GSK) on a program no longer being pursued, aimed at identifying a TGF-beta (transforming growth factor) receptor kinase inhibitor with specific activity against ALK5 (activin receptor-like kinase 5) but not other ALK (activin receptor-like kinase) receptors. According to Frazier, this work illustrates how leveraging existing tools and techniques with recent advances can make it possible to achieve the potential of cutting-edge safety science. Frazier also explained that, instead of asking the classic mechanistic questions that many people try to answer using toxicology, his group first tries to decide whether a particular toxicologic liability is a class-wide pharmacological phenomenon or is due to some individual, off-target liability. This is information can help in making decisions about lead optimization, and about whether to take a program forward or revert to a backup program and start over.

ALK5 is a transmembrane TGF-beta receptor that signals through the Smad pathways and results in nuclear translocation and activation of TGF-beta-responsive genes. Research has shown that overexpression of TGF can lead to renal fibrosis and that using antagonists to ALK5 can stop and in some cases reverse the effects of the fibrosis. During the ALK5 program,

1

This chapter is based on the presentation of Kendall Frazier, Director of Cellular and Molecular Pathology, GlaxoSmithKline.



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2 Investigative Toxicology: The State of the Art1 A s context for the discussion of emerging safety science, it is use- ful to review the current state of the art in investigative toxicol- ogy. To this end, Dr. Frazier described the work of his group at GlaxoSmithKline (GSK) on a program no longer being pursued, aimed at identifying a TGF-beta (transforming growth factor) receptor kinase inhibitor with specific activity against ALK5 (activin receptor-like kinase 5) but not other ALK (activin receptor-like kinase) receptors. According to Frazier, this work illustrates how leveraging existing tools and techniques with recent advances can make it possible to achieve the potential of cutting-edge safety science. Frazier also explained that, instead of asking the classic mechanistic questions that many people try to answer using toxicology, his group first tries to decide whether a particular toxicologic liability is a class-wide pharmacological phenomenon or is due to some individual, off-target liability. This is information can help in making decisions about lead optimization, and about whether to take a program forward or revert to a backup program and start over. ALK5 is a transmembrane TGF-beta receptor that signals through the Smad pathways and results in nuclear translocation and activation of TGF- beta-responsive genes. Research has shown that overexpression of TGF can lead to renal fibrosis and that using antagonists to ALK5 can stop and in some cases reverse the effects of the fibrosis. During the ALK5 program, 1This chapter is based on the presentation of Kendall Frazier, Director of Cellular and Molecular Pathology, GlaxoSmithKline. 

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 EMERGING SAFETY SCIENCE three major toxicities were encountered at various stages of development: pulmonary hemorrhage (exhibited during early-stage development), bone physeal abnormalities (exhibited during first-time-in-human studies), and heart valve lesions (exhibited during 10-day dose range–finding studies prior to first-time-in-human studies). The investigation of each posed dif- ferent challenges and demanded different techniques. PuLMONARy HEMORRHAgE The first problem encountered in the ALK5 program was pulmonary hemorrhage. Histopathology of lung tissue in treated rats showed diffuse alveolar damage characterized by fibrin exudation, alveolar septal necro- sis, and inflammatory cells; the damage was present with a number of test compounds. Because the researchers were looking at several compounds from a series, they wanted to determine whether they would encounter this problem with every compound. Frazier’s group hypothesized that the lung damage was being caused by free radical production and reactive oxygen species. Therefore, they decided to look at the different compounds, see which ones caused free radical production, and then determine whether this superoxide production correlated with the alveolar damage. Using an in itro model that employed an A549 lung adenocarcinoma cell line, they incubated the cells with either the compound or a control for 4 hours, exposed the cells to a hydroethidine dye, and then ran them through a flow cytometer with a 488 nm argon laser. When reactive oxygen species are present, hydroethidine dye turns into ethidium bromide, which fluoresces when exposed to 488 nm light. Therefore, this approach made it possible to determine quickly whether free oxygen radicals were present in a given set of lung cells. The group looked at 150 compounds originating from three sepa- rate programs at GSK, all of which had encountered the same kind of pulmonary hemorrhage. A clear dose–response relationship was found, with higher doses leading to greater superoxide production. Some com- pounds led to much greater superoxide production than others, while some showed few reactive oxygen species at all (see Figure 2-1). Fur- thermore, the superoxide production correlated with the histopathology results: the compounds that showed increased superoxide production were the same as those that showed increased lung damage. Finally, after determining which compounds were causing superoxide production, the group reexamined those compounds’ biochemical structures and found that most had a similar side chain. Frequently, one chemical series tends to be highly prone to reactive oxygen, and in this case the effects of the reactive oxygen had nothing to do with the fact that the ALK5 signal was occurring. Thus the group concluded that the lung damage was not due

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Check against original color printout.  INVESTIGATIVE TOXICOLOGY Compound 1 Compound 2 100 Compound 3 Compound 4 % Increase in Superoxide Production 90 Compound 5 80 Compound 6 Compound 7 70 Compound 8 60 50 40 30 20 10 0 –10 10 25 50 100 Concentration [µM] FIguRE 2-1 ALK5 (activin receptor-like kinase 5) inhibitors: the relationship between dose and intracellular superoxide production. The figure depicts the relationship between increasing doses of eight candidate compounds and the per- centage of superoxide production. A clear dose–response relationship was found, with higher doses leading to greater superoxide production. It was further found that the increased superoxide production correlated with histopathology results: the compounds that induced superoxide production also exhibited increased lung Figure 2 -1 damage. SOURCE: Frazier, 2007. Revised to a class-wide pharmacological phenomenon, but was explained by a particular structure–activity relationship. Frazier’s group ranked 10 candidate compounds according to their superoxide generation; concurrently, the group also ran 10-day toxicol- ogy tests of the compounds. The results correlated remarkably well: the candidates that generated large amounts of superoxide showed lung lesions after 10 days, whereas those that induced limited superoxide production did not show lesions. The candidate with the least superox- ide production, which was selected for moving forward, failed to elicit pulmonary hemorrhaging even after a 28-day toxicology study. BONE PHySEAL ABNORMALITIES When the GSK researchers examined the femorotibial joint in animals treated with various ALK5 inhibitors, they discovered a second problem

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 EMERGING SAFETY SCIENCE with the ALK5 program: they repeatedly observed a particular type of bone lesion involving hypertrophy of the physes, or growth plates. The plates were much wider; the chondrocytes (cartilage cells) had a com- pletely different appearance; and there was a large number of cells in the zone of proliferation, the part of the growth plate where the chondrocytes divide rapidly. To understand the drug safety implications of this phenomenon, the group had to determine whether the lesions had a pharmacological basis—which would mean that every compound that inhibited ALK5 would have the same effect—and determine the clinical implications of the lesions. However, they hypothesized that because the target popula- tion for these drugs is adults, and adults have closed growth plates, the presence of the lesions in test animals might not be problematic. Ten-day toxicological studies in rats showed a clear dose–response relationship between the various compounds and hypertrophy in the growth plates. Furthermore, the compounds that were most potent in inhibiting ALK5 had a greater effect on the physes, which implied that formation of the lesions was due to a pharmacological mechanism. When 10-week-old and 9-month-old rats were compared, the former were found to be more susceptible to the effect, implying that, as hypothesized, the clinical target population might not be affected even if there was a phar- macological basis for the effect. In a normal growth plate, there are a number of zones: a resting zone, a zone of proliferation, a zone of pre- hypertrophy, a zone of hypertrophy, and finally, a mineralization front. Studies conducted over the past few decades have revealed that each of these zones has a different population of chondrocytes, the cells that produce and maintain the cartilaginous matrix. The different popula- tions have completely different cytokine profiles, gene expression, and protein expression. In short, the cartilage cells in the growth plate make up a highly heterogeneous population. It is important, then, to be able to examine the cells in each of these populations individually. Thus while recognizing the essential role of gene arrays and metabolomics, one must be sure to look at the correct cell population. Frazier’s group therefore attempts to isolate individual cell populations on which to perform either transcriptomics or other profiling. To isolate cells from the various zones, Frazier’s group used a bat- tery of special stains and immunohistochemical approaches. Using these advanced techniques, they were able to isolate and gain additional infor- mation from these different cell populations: • Confocal microscopy allowed them to obtain a reliable count of the number of cells in various sections of the physis.

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 INVESTIGATIVE TOXICOLOGY • Immunohistochemistry showed increased physeal proliferation and decreased physeal apoptosis. • Movat staining procedures revealed increased physeal proteogly- can deposition in the hypertrophic zone. • Studies on Von Kossa–stained frozen whole-leg preparations yielded mineralization information indicating that the bone changes were limited to the area right at the growth plate, with very minimal changes in the subphyseal area. This finding had great clinical relevance because it implied that the only changes caused by the compounds were associated with an actively growing growth plate, which would not be expected in individuals much older than about age 16–17. • In situ zymography showed that there was a loss of MMP9 and MMP13 activity in the growth plate, which indicated a decreased matrix turnover and altered chondrocyte proliferation. These outcomes are asso- ciated with TGF-beta signaling; thus an ALK5 inhibitor, which disrupts TGF-beta signaling, would be expected to cause changes in MMP activity, as well as the other changes. All of this evidence was indirect, but it led the group to believe that the physeal effect was probably pharmacological. To confirm the mechanism involved, they performed laser-capture microdissection of the growth plate. While this procedure is difficult, Frazier’s group has developed techniques that make it possible to extract individual cell populations, which has opened up many new opportunities. The group also has techniques for amplifying and successfully isolating RNA from less than 1 ng and fewer than 150 cells. This capability makes it possible as well to conduct proteomic and transcriptomic analyses of small groups of cells from archives of paraffin-embedded, formalin-fixed tissue and to determine retrospectively what was occurring in some earlier toxicologi- cal studies. In the ALK5 studies, Frazier’s group used this capability to isolate groups of cells from the various zones in the physes for gene expression analyses. After 3 days of treatment with an ALK5 inhibitor, they observed marked changes in the gene expression profile, particularly in those genes already known to be associated with TGF-beta. Tapping their knowledge of ALK5’s impact on each of the downstream mediators, the group was able to correlate the genes with what would be expected if ALK5 were inhibited. To summarize, Frazier’s group found that ALK5 inhibitors—at high doses—result in • the dysregulation of a number of ALK5-related cytokines involved in chondrocyte maturation at the growth plate;

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0 EMERGING SAFETY SCIENCE • increased proliferation and decreased apoptosis of chondrocytes; • decreased MMP activity; and • alteration of the proteoglycan. Furthermore, the physeal lesions caused by the ALK5 inhibitors are simi- lar to those seen in previous studies when some of the same ALK5-related cytokines were inhibited or knocked out in experimental animals: there is a very limited effect in the surrounding bone. The researchers concluded that the physeal hypertrophy observed in the ALK5 studies is a supra- pharmacologic effect of the inhibition of TGF-beta receptors at high doses. Even though suprapharmacologic doses were being used, this conclusion indicated a target liability that would need to be addressed if the program moved forward. Expanding on the reasons for identifying the pharmacology so thor- oughly, Frazier noted that TGF-beta inhibition would be expected to cause such physeal lesions given what was already known about the system, but that his group also wanted to understand why disruption of PTHRP (parathyroid hormone-related protein), VEGF (vascular endo- thelial growth factor), FGF (fibroblast growth factor), and several other disparate cytokines causes similar physeal lesions. They discovered that at the zone level—the cellular level of the chondrocyte—it was not one knockout but the synergy and interaction of many factors at once that caused the physeal dystrophy. Thus it is not FGF that is causing the problem or TGF-beta or PTHRP, because in each zone they are much dif- ferent; it is their interaction. If they are not turned on at the right time in the right amounts, the chondrocytes do not know when to expand, when to divide, and when to undergo apoptosis. If these things do not occur in exactly the right order, the result is the physeal hypertrophy the research- ers were observing. In short, the detailed investigation was done not so much to understand what was happening with ALK5, but to understand the details for application to other drug programs that might see a similar effect. Finally, the effect of the ALK5 compounds was found to be dose- and time-dependent; it was also dependent on the age of the experimental animals, as the older rats were somewhat resistant to it. Thus the group was able to conclude that the risk to the target population should be fairly limited, since those who would be given the drug would be old enough that they would have closed physes. HEART vALvE LESIONS The third pathology associated with the ALK5 inhibitors was heart valve lesions. The lead ALK5 inhibitor demonstrated an incidence of hem-

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 INVESTIGATIVE TOXICOLOGY orrhagic, degenerative, and inflammatory lesions in heart valves, which occurred during 10-day dose range–finding studies as soon as 2 days and no later than 10 days after dosing. The lesions appeared at doses that were far above clinically relevant levels: clinically relevant doses for most of the ALK5 inhibitors are in the range of 3–10 mg/kg, but the laboratory animals were given doses in the range of 100–1,000 mg/kg. A few previous ALK5 inhibitors had produced similar lesions, so the researchers wanted to determine whether these lesions represented a class-wide pathology that would be a liability for the entire ALK5 pro- gram. To answer this question, the group performed 10-day toxicological studies of another six ALK5 inhibitors at high doses, and they found heart valve lesions in virtually every study. Indeed, they failed to find lesions only when there was inadequate exposure or when they used an ALK5 compound that had no pharmacological activity. Thus they con- cluded that this was a class-wide pharmacological effect. The mechanism appeared to be related to effects of the compounds on the endothelium and the basal lamina. This class of compounds exhibited a variety of vas- cular effects; because of the stress associated with the turbulence of blood flow, however, the heart valves would likely be the first place lesions would manifest. The heart valve lesions were novel. They had very rapid onset and caused potentially irreversible functional damage, and even though they appeared only at doses much higher than clinical levels, they were considered problematic. In addition, there is currently no toxicological biomarker for such a heart valve lesion, especially one that would be observable in 2–3 days, and the researchers had no reason to believe that the lesions were rodent-specific. The group performed laser-capture microdissection on valves from rats, dogs, and monkeys, and found that ALK5 was expressed in the heart valves of all three species. While the researchers did not see the lesions in dogs, they had no basis for assuming that the lesions would fail to appear in longer-term studies or at higher doses. Given the rapid onset, lethality, potential irreversibility, and lack of a biomarker for the heart valve lesions, Frazier’s group recommended termination of the ALK5 program. This decision was validated by another company’s recent findings of similar heart valve lesions with an ALK5 inhibitor and observation of heart valve lesions in dogs. SuMMARy The investigative studies in the ALK5 program had three distinct purposes. First, in the case of pulmonary hemorrhage, they were used to identify a potential lead that was lacking this specific toxicity. Second,

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 EMERGING SAFETY SCIENCE with the physeal abnormalities, they were used to examine a mechanism that would put a particular finding into proper clinical perspective. And third, with both the physeal abnormalities and the heart valve lesions, they were used to determine whether a particular finding was structur- ally based or a class-wide pharmacological effect. Such a determination is not always straightforward, since finding that multiple compounds in a class cause the same problem is not the same as showing that the problem is class-wide. To elaborate, a number of compounds caused pulmonary hemorrhage, and it would have been easy to conclude that the effect must be class-wide. Yet it turned out that this was probably not a pharmacologi- cal effect, but was associated with a structure–activity relationship. Thus it is important to explore the mechanism behind an effect, particularly if one is trying to answer a class-wide pharmacological question. Further- more, it is important to sample the target populations cleanly, as there are multiple cell populations within every organ, and confocal imagery or laser capture microdissection (LCM) can be used to identify and isolate the individual cell populations of interest.