20.109(F11): Laura and Shelley

Research Proposal

Overview

Recent studies have shown the importance and potential in developing inhibitors of signaling cascades involved in cancer. Although there are currently drugs on the market that are kinase inhibitors, they do not always prove effective, especially in the presence of a larger tumor. To fill this gap in therapies, we have developed a two-part proposal. Novel therapeutics need to be designed that target multiple components within a signal cascade, both upstream and downstream. Such a drug compound should then be formulated with other drugs on the market already known to efficiently inhibit certain kinase proteins. For optimal delivery, a hydrogel system with pH sensitivity will be used. Cancers resulting in large tumors are often treated surgically; after a large portion of the tumor is removed, hydrogels encapsulating the drug formulation should be inserted around the remaining parts of the tumor. Use of the hydrogel will result in higher specificity and localization of the drug therapy on the tumor. The pH of the outside of a tumor is lower than physiological pH, so pH sensitivity can be used to ensure the drug is only released on the tumor, the desired target. The gradual degration of the hydrogel allows for longer lasting time release of the drug compound with higher specificity than found in drugs orally delivered.

Content Outline

1. Background

• Cancer and why need novel therapies and alternative drug delivery
• Signaling Cascades
• Ras/Bad pathway and possible targets
• References

2. Identifying novel compounds for further testing

• Which library to use and screen (look at what was used for past drugs that exist that inhibit RAF (eg Sorafenib)
• How will we screen? Affinity testing, specificity, pH stability, half life (must have very long half life – possibly look into hydrogel release resulting in conformational change to the active form of the drug)
• References
  • A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays (10) - The article first gives an overview on the use and importance of high throughput screening and then proposes more rigorous statistics to use when determining hits. HTS requires sensitivity, reproducibility and accuracy which current measures of hits don't always account for. Signal to noise and signal to background are ratios used in determining hits, but the ratios do not take into account variability of sample and signal.
  • Discovery and development of sorafenib: a multikinase inhibitor for treating cancer (17) - This paper is a case study on the discovery and development of Sorafenid, a kinase inhibitor used for cancer treatment. The discovery process started with a high throughput screen using a scintillation proximity assay. Close to 200,000 compounds were found and then tested further. Combinatorial and medicial chemists then worked on optimizing the structure of key compound 3-thienyl urea. This paper also details clinical studies done prior to FDA approval.

3. Methods for testing compound

• In Vitro Tests
  • Introduce basic signaling cascade into bacteria to see how compound affects BAD signaling (look at yeast as well?)
  • IC50, CC50, EC50
  • Run gels for protein presence (purify proteins)
  • Measure phosphorylation rates since we are working with a kinase
• In Vivo Tests
  • Assay measuring rates of apoptosis and rate of proliferation
• Resources
  • The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity (15) - Researchers tested the effectiveness of using ATP bioluminescence assays and suggested its use as a measure of cytokine activity. Studies showed that there was significant correlation between increased cell number and ATP. Note: the original paper describing this method should be looked at further (cited as: Higashi et. al 1985 and Stanley 1986).
  • The conduct of in vitro and in vivo drug-drug interaction studies: a pharmaceutical research and manufacturers of America (PhRMA) perspective (18) - This paper is an overview of in vitro and in vivo studies on drug-drug interactions. In vitro assays are important in determining whether one of the drugs is a cytochrome p450 inhibitor, blocking metabolism of the other drug. Both drugs should have different modes of interaction and metabolism. The paper outlines thresholds for needs to do further DDI studies. It also details in vivo studies performed when in vitro studies indicate possible negative drug-drug interaction.

4. Delivery of compound – Hydrogels

• Introduction of Hydrogels
• pH sensitive Hydrogels
• How long the drug can sit in there, release timing of the drug, active vs. inactive form

5. Surgical Applications

• Insert hydrogel once majority of tumor is removed via surgical methods
• Release of drug attacks all of remaining surfaces of tumor (tumor ideally is shallow for this to occur effectively)
• How many are inserted? What size?

6. Future Research

• Synthetic transporter built into the hydrogel that can be controlled by laser, gets moved to inside of tumor, then releases drug content