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European Biopharmaceutical Review

A Marked Difference


Biomarkers carry the promise of allowing better decision-making in drug discovery, enabling researchers to identify unfavourable properties and confirm the expected efficacy profile of new drugs earlier in the development process

The pharmaceutical industry is facing high preclinical and clinical development costs and declining drug discovery success rates. To stay competitive, companies are re-evaluating their drug development process in order to address the issue of high attrition rates for many drug candidates. The use of biomarkers at each stage of R&D can improve decision-making and productivity, and improve clinical trial success rates. An increased emphasis is being placed on the development of predictive biomarkers that can provide early evidence of safety and efficacy while a molecule is still in preclinical development, and can provide assurance that lead candidates will have a high probability of success at subsequent milestones, potentially reducing the time and cost of drug development (1).

The FDA has issued guidance on bioanalytical method validation for assays that support pharmacokinetic studies specific to small-molecule drugs. However, there is little regulatory guidance on biomarker assay validation. In 2003, the American Association of Pharmaceutical Scientists participated in a Biomarker Method Validation Workshop to address the validation challenges of biomarker assays in support of drug development; they concluded that a ‘fit-for-purpose’ approach should be recommended and that assay validation should be tailored to meet the intended purpose of the biomarker study (2).

The history regarding the development of the techniques used for biomarker analysis, both for genomics and proteomics or bio-humoral marker assessment, has developed from the use of single test tubes through to immunoassays, and finally to new technologies including platforms for genomics, proteomics and multiplex ligand-binding assays. The use of multiplexing technologies is constantly increasing in laboratories, allowing scientists to obtain more data from the same, smaller sample volumes (3). This article focuses mainly on biohumoral markers, and looks at how biomarker analysis can support both drug discovery and drug safety.

Biomarkers Analysis in Support of Drug Discovery


Biomarker analysis can support the drug discovery process in various disease areas in several ways, by providing: in vivo pharmacodynamic assays for the robust characterisation of lead molecules and candidates; disciplined support to target early lead optimisation and clinical projects; and by offering biochemical assays for the definition of appropriate PK/PD models to assist the design of first-time-in-human studies, and to reinforce the potential link between preclinical and clinical studies. Depending on the disease, a single biomarker or multiple biomarkers approach can be adopted. In particular, biomarker panels are strictly driven by the disease, since there is mounting evidence of multiple deregulated contributing factors in some diseases (for example, in major depressive disorder).

The use of immunoassays such as ELISAs, EIAs and radioactive immunoassays are suitable for a single biomarker approach. In the case of multiple factors contributing to some disease, the most appropriate disease-driven panel can be evaluated exploiting available platforms, such as Luminex and Meso Scale Discovery.

When a biomarker study is designed, the circadian rhythmicity of the biomarker of interest should be taken into account in order to select the appropriate period within the light-dark cycle in which to perform the experiments. This is decided on the basis of the expected drug effect on its levels. For example, rat corticosterone, prolactin, insulin and leptin plasma levels show a peculiar circadian rhythm (see Figure 1) (4). It is also preferable to work in a stress-controlled situation and to avoid, or minimise, the stress imposed on animals due to handling, treatments and blood collection. Manipulation of laboratory animals can be stressful and may introduce variability of data, leading to confounding results. To overcome this problem, automated blood sampling apparatus can be used to perform serial blood sampling in cannulated rats (5). The use of this technology ameliorates the quality of data generated, reducing variability due to serial sampling from the same animals, minimising the stress derived from conventional sampling and drastically reducing the number of animals used, thus satisfying two of the 3Rs ethical framework: refinement and reduction (6).

Arginine-8-vasopressininduced ACTH Release

Results of corticosterone and ACTH basal levels were compared to that obtained by conventional blood sampling techniques. Figure 2 presents a comparison of corticosterone timecourse levels in rats, where blood was collected by manual acute sampling or by serial automatic blood sampling. High corticosterone levels due to acute sampling were avoided when performing serial automated blood sampling in cannulated rats. Rat connection to the Accusampler apparatus gave a transient increase in corticosterone levels due to cage change that was normalised within one hour (see Figure 3). For this reason, rats were routinely connected at least one hour before starting blood sampling.

The application of this apparatus to the development of a PK/PD model to test Vasopressin1b (V1b) receptor antagonist compounds is described; the model was based on the increase of plasma ACTH released in the rat following intravenous treatment with arginine-8-vasopressin (AVP). The effects of V1b receptor antagonist compounds in blocking the increase of ACTH due to AVP injection was tested. Figure 4a shows data generated from terminal sampling. A study (n=6 rats per group) was performed to assess the AVP dose (0.3 and 1μg/kg, intravenous administration) able to induce a statistically significant increase in plasma ACTH level with respect to the basal, and to assess terminal endpoint (10 and 20 minutes). AVP at 1μg/ kg was able to induce a significant increase in ACTH plasma level following 10 minutes from injection with respect to vehicle-treated rats. It is worth noting that vehicle - treated rats showed high ACTH basal levels.

Under these conditions, a V1b receptor antagonist was tested at 10mg/ kg by intraperitoneal (ip) pre-treatment 30 minutes before AVP injection. To achieve a significant effect in blocking AVP-induced ACTH release, the results from two experiments were considered (n=20 rats per group). In Figure 4b, data generated using Accusampler apparatus are shown. By using a lower number of rats than in the previous experiment (16 versus 36), a timecourse of ACTH plasma level was generated by serial blood sampling before 10, 20 and 45 minutes following AVP intravenous administration at both 0.3 and 1μg/kg. A very low basal ACTH level in vehicle treated rats led to a statistically significant effect on both AVP doses tested following 10 and 20 minutes from administration. The dose of 1μg/kg of AVP and the timepoint of 10 minutes following administration were selected as before. Under these conditions, a V1b receptor antagonist compound was tested at 3, 10 and 30mg/kg by ip pre-treatment 30 minutes before AVP injection. Exploiting the serial blood sampling, blood was collected before and 10 minutes after AVP challenge in order to see the effect of compound treatment on ACTH.

The compound tested showed a significant effect in blocking AVPinduced ACTH release at both doses and also showed an effect in increasing basal ACTH plasma levels at the highest dose tested. More information was obtained by using twice 20 rats, compared to the experiment performed by terminal sampling.

Table 1 summarises the potential application of automated blood sampling apparatus to preclinical studies: physiological levels of ACTH were observed; blood sampling was performed on the same animals, reducing data variability; and a dramatic reduction in the number of the animals used (36 versus 96) was possible to finally obtain more information on the compound activity.




Safety Biomarkers

Among the numerous drug candidates that enter the drug development phases, only a few reach FDA approval: the high levels of attrition are mainly due to preclinical or clinical safety issues. It is therefore instrumental to have tools such as safety biomarkers that provide the means for monitoring onset and/ or progression of potential side-effects. This process enables a more robust risk assessment and allows more molecules to enter clinical trials, leading to the development of valuable medicines.

Biomarkers of toxicity or safety biomarkers are used in both preclinical and clinical environment to characterise specific safety issues during drug development. An ideal biomarker should predict rather than report a biological effect both in terms of dose, meaning it is detectable at doses lower than those causing damage, and in terms of time, for example in cases where the biomarker is detectable after one dose when the damage requires repeating dosing. However, biomarkers that report rather than predict can still be useful if they prevent further damage or permit cessation of treatment and subsequent recovery (7).

Safety biomarkers are usually measured together with other study end-points such as clinical signs, TK data, histopathology and, when available, with other tools. The identification of preclinical biomarkers of the adverse response of animals to potential new drugs and the evaluation of their relevance and applicability to humans form part of an integrated assessment of safety and efficacy that can inform clinical decisions.

Testing Cardiotoxicitiy Levels with cTnI and NT-proBNP

A case study on the analysis of cardiac troponin I (cTnI) and aminoterminal pro-brain natriuretic peptide (NTproBNP) as markers of cardiotoxicity and cardiac hypertrophy in a toxicological study is presented. Cardiac troponin I is a biomarker of cardiac toxicity well recognised in the clinic, with animal studies having demonstrated it can be used as reverse translational biomarker (8). Measurements of N-terminal fragment of the prohormone B-type natriuretic peptide (NT-proBNP) – an endogenously produced neurohormone primarily secreted from the cardiac ventricular myocytes in response to cardiac stress – are now used in clinical settings in the diagnosis of left ventricular systolic and diastolic dysfunction (cardiac stretch/stress), as well as prognostically in a variety of cardiac disease states including heart failure. NT-proBNP can also be used as a reverse translational biomarker, particularly in dogs (9).

A time course study over a 26-week treatment, followed by a recovery period, was set up to investigate cardiac changes observed in dogs treated with a NK-1 antagonist. Dogs were assigned to two oral treatment groups, one receiving vehicle alone and one receiving the test compound. Blood was collected every two weeks and in terminal samples on weeks six, 13, 20, 26 or following completion of a 22-week recovery period, from control and treated animals for the analysis of cTnI and NT-proBNP levels. In a subgroup of animals, the dosing was stopped after 13 weeks, followed by a 22-week recovery period, and the same investigations were performed.

Cardiac biomarker analysis data were evaluated in integration with several other cardiac morphological and functional end-points: histopathology; immunohistochemistry; morphometry; transmission electron microscopy (TEM); and echocardiography.

The cTnI data correlated well with TEM and histopathology findings along different time points of the study, with increased cTnI values observed in treated dogs, which corresponded with their respective controls starting from week six onwards, as indicated by the red values reported at the top of Figure 5. From week six, some ultrastructural changes in the heart also became evident (intracytoplasmic multilamellar bodies in the sarcoplasm of myofibers, and in endothelial or smooth muscle cells of blood vessels) which increased in severity in the following timepoints showing a time and test article-relationship. On the contrary, histopathological myofiber degeneration/necrosis in the heart was evident from week 20, as illustrated by Figure 5.

In Figure 6 on page 42, NT-proBNP data are shown to correlate well with echocardiography and morphometry findings along different timepoints of the study. In the figure, the red line on the left-hand side represents NT-proBNP values in treated dogs, while the blue line represents control animals. Statistically significant higher values were observed in treated dogs starting from week two onwards, as indicated by the green arrow, and they were persistently elevated up to the end the of recovery period. On analysis of the echocardiography data, as illustrated by the picture in the right-hand side, an increase in the left ventricular mass (due to an increased left ventricular wall thickness) was observed from week six onwards. The anatomical effects noted by echocardiography were consistent with those observed at study termination at heart weight analysis, in which an increase in the absolute heart weight was observed in dogs treated with the NK-1 antagonist. Morphometric evaluation of the heart showed an increase in heart volume in treated animals in comparison with controls in both ventricle muscle and chambers.

The integrated approach used in this study helped in the characterisation of the lesion and the functional impairment observed in the heart. Determinations of cTnI in conjunction with histopathology and electron microscopy, were essential in the characterisation of the onset of the lesion and its slow evolution. NTproBNP determination, in conjunction with echocardiography and morphometry, acted as an early marker of heart hypertrophy, anticipating echocardiography changes as well as morphometry. The study demonstrated that both cTnI and NT-proBNP are reliable, sensitive and non-invasive markers of drug-induced cardiac changes in the male beagle dog.

Conclusion

The use of high throughput technologies, such as multiplex biomarker analysis and innovative blood collection techniques, can help generate high-quality data in a more efficient and cost-effective way. Biomarkers are becoming an invaluable tool to drug discovery and development, allowing better decision making by identifying unfavourable properties or confirming the expected efficacy profile of new drugs early in the development, reducing attrition and accelerating the progression to the clinic.


References

  1. Colburn WA, Biomarkers in drug discovery and development: from target identification through drug marketing, J Clin Pharmacol 43: pp329-341, 2003
  2. Lee JW, Devanarayan V, Barrett YC, Weiner R, Allinson J, Fountain S, Keller S, Weinryb I, Green M, Duan L, Rogers JA, Millham R, O’Brien PJ, Sailstad J, Khan M, Ray C and Wagner JA, Fit-for-purpose method development and validation for successful biomarker measurement, Pharm Res 23(2): pp312-328, 2006
  3. Chau CH, Rixe O, McLeod H and Figg WD, Validation of analytical methods for biomarkers used in drug development, Clin Cancer Res 14: pp5,967-5,976, 2008
  4. Bertani S, Carboni C, Criado A, Michielin F, Mangiarini L and Vicentini E, Circadian profile of peripheral hormone levels in Sprague-Dawley rats and in Common Marmosets (Callithrix jacchus), In Vivo 24: pp827-836, 2010
  5. Royo F, Bjork N, Carlsson HE, Mayo S and Hau J, Impact of chronic catheterisation and automated blood sampling (Accusampler) on serum corticosterone and fecal immunoreactive corticosterone metabolites and Immunoglobulin A in male rats, J Endocrinol 180: pp145-152, 2004
  6. Russel WMS and Burch R, The Principles of Humane Experimental Technique, 1959
  7. Duffy PA, Betton G, Horner S, Horner J, Cotton P, McMahon N, Lawrence C, Prior H, Armstrong D, Philp K and Roberts RA, Biomarkers for safety and toxicology: drug induced cardiac injury and dysfunction, Eur J Cancer Suppl 5(5): pp143-151, 2007

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Federica Crivellente is a Research Investigator in the Biomarkers and Applied Immunology Laboratory at Aptuit, Italy. Federica holds a BSc in Biology from the University of Padua and a PhD from the Department of Public Health and Community Medicine in the Unit of Forensic Medicine at the University of Verona. Prior to joining Aptuit in 2010, Federica was Clinical Pathology Manager at GlaxoSmithKline. She also acted as Safety Assessment Project Representative and was involved in many GSK worldwide projects on safety issues. Federica is a member of the American Society of Veterinary Clinical Pathology and is co-author of about 30 full publications in international journals and books.

Elena Vicentini
is a scientist working in the Biomarkers and Applied Immunology Laboratory at Aptuit. She has a degree from the Agro-Industrial Biotechnology at Verona University. Elena joined GlaxoSmithKline in 2000 working on the generation of transgenic mouse models for the study of mood disorders and on the biochemical characterisation of preclinical stress models. She has driven and progressed preclinical programmes to the screening phase of drug discovery, working in the Translation Biology group and acquiring deep experience in biomarker analysis.
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