Bringing Biomarkers to the Masses: An Elegant Approach to Discover Potential Biomarkers of Vascular Injury
Toxicology and Safety Assessment, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877
1 For correspondence via. Fax: +203 798 5797. e-mail: rstachle{at}rdg.boehringer-ingelheim.com.
Received August 28, 2007; accepted August 28, 2007
One of the greatest criticisms of scientists is that many do a poor job conveying the importance of their work to the public. My graduate professor used to say that one should be able to take their research project and "tell a story that even your Grandmother could understand." This statement was certainly not meant as a derogatory comment toward all of the grandmothers in the world. This was his "not-so politically correct" way of saying that we all need to think about a concise story, devoid of scientific jargon, to describe our research to the public. We have all had projects where the layperson's description was difficult to develop because of the complexity or proprietary nature of the work. However, the development of biomarkers of toxicity is one of the areas where the complexity of the science can be readily distilled to a concise story highlighting that one of the many areas of toxicological research has a big impact on the public.
A biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention (Biomarkers Definitions Working Group, 2001
). Removing the scientific jargon and applying this definition specifically to toxicology, the concept of a biomarker of toxicity is easy to describe to the public. We are looking for "something" that can be objectively measured that will provide a reliable signal indicating the compound being tested is about to have an adverse effect on a system. Ideally, the biomarker should appear early enough for administration of the compound to be stopped before any adverse effects manifest.
In this issue of Toxicological Sciences, Nicolas Daguès, Valérie Pawlowski, Cécile Sobry, Gilles Hanton, Françoise Borde, Sylvain Soler, Jean-Louis Freslon, and Stephan Chevalier report on their work to discover a potential biomarker for vascular injury seen with phosphodiesterase-4 (PDE4) inhibitors (Daguès et al., 2007). The mechanism of vascular-induced injury caused by PDE4 inhibitors is most likely very complex, and a complete understanding of the mechanism(s) of injury has not been elucidated. In the absence of the exact mechanism, Daguès et al. were able to ask some simple, yet elegant, questions of an acute model of vascular toxicity of PDE4 inhibitors in the rat that they developed to propose a potential biomarker that complements the current understanding of the mechanism of vascular injury caused by PDE4 inhibitors.
To put this work into context, a summary of the potential of PDE4 as a drug target and the pitfalls encountered during the preclinical studies and clinical trials of PDE4 inhibitors is provided. PDE4 inhibitors have been long touted as having great promise for treating a wide variety of inflammatory diseases, improving memory in diseases of cognitive impairment, treating depression, and preventing restenosis of coronary vessels after bypass surgery (Zhang et al., 2005). While there continues to be great hope for oral PDE4 inhibitors, the development of these compounds has been complicated by dose-limiting gastrointestinal (GI) side effects and the development of vascular injury in the mesentery of rodents during preclinical studies. Many oral PDE4 inhibitors described to date have narrow to nonexistent margins of safety for both of these side effects when comparing preclinical exposures to expected efficacious doses.
Emesis and diarrhea have been seen in preclinical and clinical studies, and these GI effects along with the associated nausea are by far the largest reasons for patients dropping out of these trials (Giembycz, 2006). The GI effects are thought to be mediated centrally and are not a direct effect of the inhibitor in the GI tract. There are many hypotheses proposing the mechanism of the GI effects, but the bottom line is that selectivity of the inhibitor, either within the PDE4 isoforms, among different conformational states, or against other proteins, may hold the key to improving the safety margin with respect to these untoward effect, (Dastidar et al., 2007). In any event, emesis and nausea in the clinical trial participants, albeit unpleasant, are nature's biomarkers of the potential for an untoward effect of a compound and are dose limiting for current PDE4 inhibitors.
The vascular toxicity seen in preclinical studies has provided a further challenge for risk assessment by toxicologists and is the subject of tremendous scrutiny. Predominantly, vascular toxicity of PDE4 inhibitors has been restricted to the mesentery of rodents but has also been reported in nonhuman primates with one inhibitor that has other target tissues outside the mesentery (Dietsch et al., 2006; Losco et al., 2004). Vascular injury can occur with some PDE4 inhibitors after just one dose. Most concerning from a safety perspective is that the vascular injury does not appear to be preceded by any changes in parameters typically monitored in toxicology studies. The first outward manifestations of this toxicity in rodents are generic findings in preclinical studies, including diarrhea, reduced food consumption, reduced body weight gain, or weight loss and lethality, but only after vascular lesions have developed. Elevation of circulating neutrophils and reduction of albumin levels have been reported but only after the time when vascular lesions would be expected to be seen histopathologically. Early changes related to the vascular toxicity of PDE4 inhibitors may be reversible, but more prolonged dosing leads to irreversible injury.
In clinical trials with PDE4 inhibitors, vascular injury has not been reported, but this has not alleviated the concern that this finding could manifest in trial participants. In fact, it has been reported that during phase III trials with cilomilast, daily monitoring of all GI events, orthostatic vital signs, and monitoring for blood in the stool was completed to detect bowel infarctions in participants (Giembycz, 2006). Colonoscopies were done when at least one GI adverse event was reported by a participant that also had black or bloody stool or a positive fecal occult blood test. Certainly, a less invasive, less expensive, and predictive biomarker for this toxicity would have been used if it were available and validated. In fact, the need for biomarkers for vascular injury is so great that expert working groups have reviewed the issue (Kerns et al., 2005). More recently, a consortium of pharmaceutical companies with collaboration from the Food and Drug Administration under the organization of the C-Path Institute have formed the Vascular Injury Working Group. The main charge of the consortium is to work together to identify and validate biomarkers of vascular injury for use in preclinical and clinical studies.
Daguès et al. provide compelling evidence that circulating levels of tissue inhibitor of metalloprotease 1 (TIMP-1) in serum may be a suitable biomarker for predicting vascular toxicity of PDE4 inhibitors. The empirical experimental design started with histopathological characterization of the time course of vascular injury in rat mesentery after a single dose of the PDE4 inhibitor CI-1044. From a separate time course experiment, RNA was isolated from mesenteric tissue for microarray analysis, and from this same study, blood samples were harvested for serum and plasma. The forethought of the experimental design shows that Daguès et al. had precisely anticipated the outcome illustrated in the paper. The idea was to first find transcripts from the microarray analysis of the mesenteric tissue that were elevated before histological changes were seen in the mesentery. Next, these signals were validated using by Quantitative Revese Transcription-PCR. At the same time, plasma and serum samples were being analyzed for the presence of a multitude of proteins. Then, the authors asked a simple question and got an almost ideal answer: Do the lists of upregulated RNA transcripts from the mesentery and elevated circulating proteins measured in serum/plasma overlap? Indeed, there is an elevation in the transcript for TIMP-1 in the mesentery and an increase in the circulating levels of TIMP-1 protein in plasma that occurs before histopathological changes in the mesentery. Daguès et al. also make the case for the logic behind TIMP-1 as a biomarker based on their hypothesis of the mechanism behind PDE4 inhibitor–mediated vascular injury in the rat.
Certainly, Daguès et al. have provided important groundwork for the further development of circulating levels of TIMP-1 as a biomarker of PDE4 inhibitor–mediated vascular injury. Their research into potential biomarkers of vascular injury caused by PDE4 inhibitors highlights progress in an important area of toxicology that has a major unmet need. Additionally, this intricacies of the mechanism(s) by which PDE4 inhibitors cause vascular injury is sufficiently complicated to intrigue toxicologists; but the simplicity and elegance of the experimental design of Daguès et al. can be easily reduced to a concise story that the layperson can easily grasp. As toxicologists, we owe it to our discipline to highlight high-quality work in areas of the science that the layperson can understand and appreciate. The need for biomarkers of toxicity is one of the many places where it is simple to construct a concise story, devoid of scientific jargon, to describe toxicological research that has the potential to have a big impact on the public.
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