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
  • "The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation" (4) - Describes the pathway overlap between these two paths that control cell proliferation, migration, and death and are commonly mutated in cancers. It identifies some common substrates including FOXO, GSK3, Mad, and BAD.
  • "Roles of the Ras/Raf/MEK/ERK pathway in leukemia therapy" (5) - Review article of the pathway that controls cell proliferation, migration, and death. Includes a MAP kinase pathway and other involved molecules including BAD that are potential targets. Shows 15 existing drugs that target different points in the pathway, and discusses a few other possible targets. Although targeted specifically at how the drugs work for leukemia, the same pathway is used in many types of cancers.
  • "Bad, a Heterodimeric Partner for Bcl-XL and Bcl-2, Displaces Bax and Promotes Cell Death" (9) - Bad is a protein fairly downstream in the Ras/Raf/MEK/ERK pathway. It promotes apoptosis (cell death) by forming a dimer with Bcl-XL and Bcl-2. It competes with Bax, an anti-apoptosis protein, for binding with Bcl-XL.
  • "BAD Contributes to RAF-mediated Proliferation and Cooperates with B-RAF-V600E in Cancer Signaling" (7) - defines Bcl-2 classes and shows that increasing BAD leads to increased cell death and shows targets for increasing BAD's activity through phosphorylation.

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.
  • "THe Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation" (4) - Describes the pathway overlap between these two paths that control cell proliferation, migration, and death and are commonly mutated in cancers. It identifies some common substrates including FOXO, GSK3, Mad, and BAD.

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.
  • Comparison of 5 microplate colorimetric assays for in vitro cytotoxicity testing and cell proliferation assays (11) - Many assays are no available to measure cytotoxicity and cell proliferation. However, some are more popular than others; the MTT assay is one of the most commonly used. The authors of the article tested and compared five assays: MTT, AP, CVDE, NR, and SRB. Results showed a few key points when considering use of a given assay. More sensitive assays showed shorter linear ranges. Staying within the linear range for the assay is important when measuring for an accurate IC50 value. The assays with the most sensitivity were AP, SRB and CVDE. None of the assays showed much difference in measured toxicity profiles.
  • A comparison of three assays used for the in vitro chemosensitivity testing of human tumours (12) - This paper discusses the differences in three assays used to measure chemosensitivity of tumours. Such assays have showed "good correlation between in vitro results" and responses seen in the clinic. The B (Biochemical) assay, C (clonogenic) assay, and M (Monolayer) assay were tested using multiple cell lines and drugs to test efficiency in measuring chemosensitivity. Results showed that accurate predictions given each assay depended on the drug and cell line tested. The M and C assays were most comparable in results; The B assay showed lower sensitivity, but higher cut-off points.
  • A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity (13) - Researchers tested variables such as solvents used to optimize the MTT assay. The paper first details the importance and use of the MTT assay, followed by data on changing solvents affected chemosensitivity measurements. Results concluded that DMSO would be a better universal solvent to use rather than the originally proposed use of acidified isopropanol.
  • Preclinical in vitro screening assays for drug-like properties (14) - All ADMET properties should be taken into account when designing a drug during the preclinical in vitro and in vivo testing. The author, Li, details each of the following properties: Absorption, Distribution, Metabolism, Excretion, and Toxicity. He also details the importance of testing for drug-drug interactions. Within each sub-section of the article, in vitro assays and in vivo tests are described for measuring the efficacy and safety of the drug.
  • Some Practical Considerations and Applications of the National Cancer Institute in Vitro Anticancer Drug Discovery Screen (16) - The National Cancer Institute developed a preliminary in vitro screen testing the effectiveness of an anti-cancer drug on 60 human tumor cell lines. The drug compound is tested over a range of concentrations and if it shows successful results, dose response curves are made for 60 different concentrations. From this, the percentage growth term, 50% growth inhibition parameter, and lethal concentration of drug are measured.

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
• References
  • "Hydrogels in drug delivery: Progress and challenges" (19) - Review article focusing on temperature controlled hydrogels and the possibility of making them injectable so they form in situ. Shows that hydrogels have been used for release as long as 60 days and many have initial burst phase.
  • "Environment-sensitive hydrogels for drug delivery" (20) - Review article covering many types of regulation including temperature, pH, insulin, electric field, and light. Adresses challenges of slow response time and biocompatibility problems.
  • "Preparation of Thermo-Responsive and Injectable Hydrogels Based on Hyaluronic Acid and Poly(N-isopropylcrylamide) and Their Drug Release Behaviors" (21) - describes preparation and testing of a thermo responsive injectable hydrogel. The drug shows a 12 hour burst phase then a sustained release over more then 60 hours.

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?
• Refrences
  • "Cellular pH Gradient in Tumor versus Normal Tissue: Potential Exploitation for the Treatment of Cancer" (22) - Shows that over several studies by several people, that the extracellular pH inside a tumor is slightly more acidic then the surrounding tissue. Additionally, the pH within the tumor cells is not higher so the pH gradient between the inside and outside of a cell is different for tumor cells then other cells. It sugests the use of slightly acidic drugs to enhance uptake in tumor cells compared to normal cells.

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