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International Clinical Trials

Preclinical Targets


Bioluminescent imaging can be effectively employed for enhanced accuracy and improved success in cancer drug development, as Rajendra Kumari at PRECOS explains

Oncology therapeutics R&D represents the greatest opportunity today for pharmaceutical companies to significantly affect cancer-related morbidity and mortality in the developed world. Unfortunately, attrition rates for cancer drugs which enter into clinical trials are much higher than in other therapeutic areas. According to a recent study, it was shown that between 1995 and 2007, 77 per cent of 800 cancer drugs entering Phase 1 clinical trials failed to be released to the market (1). In the US, only eight per cent of oncology drugs that entered into drug development between the early 1990s and mid-2000s received approval from the FDA (1).

As a consequence, there is an increasing demand within the pharmaceutical and biotech industry for more ‘challenging’ and accurate preclinical modelling for oncology drug development. It is essential to use patient-relevant models to ensure that preclinical efficacy assessment is predictive of how the drug is likely to behave in clinical trials with cancer patients. In response to these requirements, innovative new technology is being developed in order to fast-track new agents into the clinic. It is vital to evaluate potential therapeutic agents or regimens and conduct research into tumour initiation and development. Threedimensional models fully recapitulate and more accurately reflect the tumour micro-environment. Bioluminescencebased models allow real-time read-outs of drug responses from individual cell types or xenografts in which the human stromal element is retained. This article highlights different preclinical models and discusses three recently developed models for prostate cancer, breast cancer and brain cancer. T

HE CHALLENGE OF INEFFICIENT PRECLINICAL MODELS

Thirty per cent of failed oncology drugs in development phases are attributed to a lack of efficacy or unpredictable toxicity. Preclinical animal models are used primarily to predict the safety and efficacy of compounds prior to their use in humans. A fundamental problem is that the majority of major pharmaceutical and biotech companies developing products in oncology only employ a limited and basic portfolio of cancer models, most of which are based on the use of animal cells or tissue.

In general, oncology is one area of drug development in which animal models are not particularly predictive of the true human pathophysiology as they have little relevance to the patient’s tumour. For example, one modelling type that has been extensively used by pharmaceutical companies is basic xenograft models, in which a tumour cell line is injected into a non-human model. This offers no real resemblance to what happens in man during disease progression. The standard models that are currently used, such as the xenograft, do not provide the capacity for continuous measurement of response and are not capable of optimally modelling the biology of the cancer. The molecular profile of these cell lines can be considerably different from primary tumour cells, and standard models will not exhibit optimal paracrine signalling due to the lack of human tissue, potentially misguiding the determination of efficacy of novel therapeutic agents.

In order to overcome the limitations of standard models, new and innovative technologies are now being offered by some specialist service providers. These technologies have been specifically designed to create patient-relevant models which enable reliable preclinical efficacy assessment, thereby increasing drug development efficiency and reducing attrition rates. With increasing regulatory and scientific rigour, coupled with the pressure to reduce everincreasing cancer drug attrition rates and R&D costs through outsourcing, there is an increasing demand for specialist service providers who can offer innovative techniques in order to improve the efficiency and success of oncology drug development.

RECENT INNOVATIONS

Preclinical oncology research specialists are working to develop cutting-edge technology and innovative approaches to more fully recapitulate the tumour micro-environment both for in vitro and in vivo modelling. A key area of research is bioluminescent imaging. This approach, which is vital to oncology R&D, allows the real-time visualisation and monitoring of the tumour micro-environment and allows for biological changes to be observed within cells in the presence of a cancer drug.

Bioluminescent preclinical models can be evaluated in real-time using the Xenogen biophotonic spectrum IVIS 100 imaging systems. Tumour cell lines transfected with the report gene firefly luciferase emit detectable levels of light in the presence of the substrate D-luciferin, which rapidly penetrates all tissue including the blood-brain barrier and placenta. The amount of light emitted is proportional to the number of live cells present, enabling cell proliferation to be monitored regularly in real-time in the same subject throughout the course of the experiment. A standard cytotoxic agent is administered as a positive control at a dose and regimen based on the patient rather than the maximum tolerated dose for the animal in order to better facilitate translation of the results to the clinical scenario.

This method is facilitated by the innovative development of biological reporters, which are expressed in human cancer cells so that they emit light or fl uorescence in response to different environmental stimuli. The reporters also emit light in response to changes and progression of the disease in response to drugs. For example, it is possible to monitor the presence of hypoxia (low oxygen levels), blood vessel formation, as well as cell proliferation and cell death.

This approach can be used to evaluate genes up-regulated in response to radiotherapy or similar insult, intra-cellular signally activated by ligand binding to cell surface receptors, and cells with cancer stem cell-like properties and that are undergoing epithelial-mesenchymal transition, a phenotype linked into cell invasion and metastasis. These reporter systems therefore cover a number of key tumour properties, including predictivity of secondary spread and resistance to standard of care treatments.

PROSTATE CANCER MODELS

A range of new models have now been developed that can be evaluated using bioluminescent imaging technology to more closely model the patient disease in terms of stromal (supportive tissue) interactions, angiogenic response (the creation of new blood vessels) and chemotherapeutic sensitivity. Multiple prostate cancer models are currently available, including a bioluminescent variant of the hormone sensitive LNCaP, which can also be modelled orthotopically.

In addition, scientists have developed and optimised a panel of bioluminescent prostate cancer cell lines for orthotopic and mestastatic modelling. A recently validated model is the orthotopic prostate model, involving the bioluminescent cell line, PC3M, in which cells are injected directly into the prostate of nude mice, and growth is monitored in real-time by whole body visualisation using patented technology. At termination the prostate is removed and examined macroscopically and histologically. Bioluminescent imaging clearly indicates the location of the tumour and confi rms that the tumour occupies the cuboid prostate tissue region.

BREAST CANCER MODELS

A broad panel of new xenograft breast cancer models has been developed, which can be implanted either under the skin (subcutaneous), in the mammary fat pad (MFP) or directly into the heart muscles or ventricles (intracardiac). Research has been undertaken involving bioluminescent imaging of the MDA-MB-231 bone metastasis model in nude mice. MDA-MB-231 expressing firefly luciferase was used experimentally to model bone metastasis following intracardiac injection of a cell inoculum. Cells rapidly lodged in the end plates of the long bones, jaw, ribs and spine, and bioluminescence could be detected using an imaging system from day seven and monitored throughout the study. Lytic lesions were formed, copying clinical presentation and these could be detected and quantified post-mortem by X-ray, histology and tomographic imaging modalities such as MRI. The standard of care agent pamidronate was used as a positive control comparison and has been shown to significantly reduce lytic lesions.

Bioluminescent imaging of the MDA-MB-231 orthotopic breast cancer xenograft model was also performed, whereby bioluminescent MDA-MB-231 was injected in the mammary fat pad metastasize to the lungs via the lymphs. The growth of the primary tumour could be monitored in real-time using an imaging system from day seven, whereas metastatic lesions could be detected and quantifi ed post-mortem by imaging or histology. The standard of care agent taxotore was used as a positive control comparison and has been shown to signifi cantly reduce tumour size.

BRAIN CANCER MODELS

A number of subcutaneous and orthotopic intra-cranial models of brain cancer have been introduced recently. Bioluminescent variants of U87MG and U373MG glioblastomas allow real-time imaging of internal tumours and treatment effects. These models have been developed with sensitivity to clinically relevant doses of chemotherapeutics which are used as positive control, such as Temozolomide and Lamusine. U87MG and U373MG can also be enriched for stem cells and neurospheres transplanted orthotopically. Development of specifi c therapies targeted at cancer stem cells is crucial for improvement of survival and quality of life of cancer patients, especially those with metastatic disease. In addition, metastatic brain tumour variants from breast or melanoma primaries are being pursued for the development of relevant metastasis models. Metastasis in the brain has been seen in some mice following intracardiac injection of cells. By serial transplantation, it is aimed to select a cell line with increased metastatic ability. The development can be accelerated using an imaging system, as it has been established that the substrate will pass the blood-brain barrier. Spontaneous metastasis variant of human breast cancer is also under development, whereby bioluminescent cells are injected into the mammary fat pad followed by resection of the primary tumour. Additionally, research is being undertaken to develop spontaneous metastasis from murine melanoma.

CONCLUSION

Due to the level of cancer drug attrition in the clinic, drug discovery programmes are looking to the early preclinical phase to optimally challenge new targets in terms of their potential efficacy, prior to initiating a full drug discovery cascade. A broad range of new models have now been developed using innovative technology to more closely model the patient disease in terms of stromal interactions, angiogenic response and chemotherapeutic sensitivity. These newly established models can fully recapitulate the TME both in vitro and in vivo by use of either human mesenchymal stem cells (MSCs) or patient-derived cancer-associated fi broblasts (CAFs). These can be fluorescent/bioluminescent-based to allow realtime read-outs of the drug responses of individual cell types or xenografts in which a human stromal element is retained. With the use of this cutting edge technology, the development of oncology therapeutics can be conducted in a more effi cient, cost-effective and ultimately successful manner, benefi tting the wider scientifi c community and, vitally, enabling new cancer drugs to reach patients sooner.

Reference

1. 2nd Annual Cancer Drug Discovery Symposium, http://www.hrsrh. on.ca/portalEn/LinkClick.aspx?fi leticket=zGcnhC8YIRw%3D&tabid= 114&mid=1985

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Rajendra Kumari is Chief Operating Offi cer and Co-founder of PRECOS. Rajendra’s expertise in cancer cell biology and model development has enabled her to implement the technology in the cancer models offered by PRECOS, through robust protocol management, validation of in vitro/ in vivo testing and optimisation, data and statistical analysis of models and dissemination of scientifi c information. She manages the commercial projects which utilise these models, interfacing with customers and the PRECOS team to ensure optimal project design and implementation as well as scientific analyses, interpretation and reporting of the evaluation of novel anti-cancer therapeutics. Email: enquiries@precos.co.uk
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