|Date of Web Publication||14-Nov-2013|
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. Abstracts. Emerg Sci 2013;2, Suppl S1:11-4
Overview of oncology drug development
Graduate School of Biomedical Sciences, University of North Texas HSC, Fort Worth, TX, USA
Drug discovery and development processes are very complex, time consuming, expensive and involve multiple disciplines of science and medicine. From thousands of candidate drug molecules screened in vitro, one or two may make it as an approved drug. The drug development process occurs mainly in two stages-preclinical and clinical development. The preclinical studies involve animals while clinical studies involve humans. The clinical development is further classified into Phase I, Phase II and Phase III trials. Due to the complexity of the processes involved, it often takes a decade or more and costs approximately a billion dollars to bring a drug to the market. Oncology drug development is distinct and different in many ways. Due to the nature of the disease, the drug regulatory agencies around the world allow for accelerated development of oncology drugs. The complexity of the drug development process can be appreciated from the interdependent and often conflicting events involved in the clinical development process, as can be seen as can be seen in the Figure 1.
Translational oncology research: Molecular profiling in breast cancer
Department of Medicine, Division of Hematology/Oncology, Sylvester Comprehensive Cancer Centre, University of Miami, Leonard M. Miller School of Medicine, USA
Gene expression profiling is among the most exciting fields of current clinical research in oncology. Ongoing trials will answer the question whether genetic fingerprints of tumors can contribute to stratify patients with regard to adjuvant therapy. A bit further ahead is the vision to predict therapy response with the help of genetic tumor analysis.
The basic idea behind gene expression profiling in oncology is that there are many genes that may have an influence on cancer growth, progression, metastatic potential and other clinical features and that the degree to which these "oncogenes" are present or not determines the risk of patients for rapid progression or recurrence.
This research has led to commercially available polymerase chain reaction (OncoType) and microarray tests (PAM 50 and Symphony) that have begun to fundamentally change the way medical oncologists quantify recurrence risk in early stage breast cancer patients. And, it has altered the clinic-pathologic paradigm of selecting patients for adjuvant cytotoxic chemotherapy. Sufficiently powered prospective studies are underway that may establish these initial molecular assays as elements of standard clinical practice in breast cancer treatment.
There are different ways to select meaningful genes for such "genetic fingerprints". Researchers at the Netherlands Cancer Institute for example, took a look at all 25,000 genes in patients with early breast cancer and identified the 70 most relevant genes that differed between patients with good and patients with bad prognosis.
Currently this 70-gene-signature is being evaluated in a randomized prospective trial, the international EORTC MINDACT trial that aims at analyzing whether this "genetic fingerprint" can be used as an additional marker to stratify patients with respect to adjuvant therapy. Results are to be expected in a few years.
A genetic test that helps to decide whether adjuvant therapy is advisable or not can certainly be of clinical relevance. But it falls short of fulfilling the most ambitious vision of a personalized oncology that can offer therapies to individual patients from which the doctor knows in advance that they will work. This is the realm not of prognostic but predictive tests. Predictive tests have to take into account genes that show a correlation with response or resistance to individual therapies.
This is a field of intense research at the moment. First predictive genome assays are currently being able to analyze many genes (Caris, Foundation Medicine, TheraPrint and others).
The first prospective studies of this kind are underway. The i-Spy trials, for example, work with adaptive randomization. TAILORx and RESPONDER in the adjuvant setting and SideOut in the metastatic setting. At the end, oncology might look very different from what it looks today with individualized molecular proofing independent of stage, histological and anatomic diagnosis. The era of precision medicine is just beginning.
Developing vaccines against cancers
Department of Urology, Roswell Park Cancer Institute, USA
Dendritic Cell (DC) are the most powerful antigen presenting cells. This has made them a prime target of investigation for cancer immunotherapy. However, clinical results of previous DC vaccine trials have failed for a variety of reasons. We here present a systematic approach to designing a feasible, cost-efficient and immunologically well-defined DC vaccine for urologic patients expressing the cancer/testis antigen NY-ESO-1.
For all experiments, monocytes were isolated via cold agglutination or ELUTRA from blood products from healthy donors. Monocytes were cultured in GM-CSF and IL-4 in culture flasks or gas-permeable culture bags. The impact of a cell culture gell-surface (0.5% polyHEMA) was assessed as alternative to culture flasks or gas-permeable culture bags. Maturation cocktails consisted of either TNF-a alone or TNF-a, IL-6, IL1 beta and PGE-2. DC were pulsed with flu peptide or NY-ESO-1 peptide alone or with a DEC205/NY-ESO-1 full length fusion protein. DC phenotype was analyzed using comprehensive flow cytometry and DC function was assessed using CD8+ NY-ESO-1-specific tetramers in a CD107/IFN-g double stain.
DC phenotype was improved when DC were grown in culture bags or on the gel matric and matured with the maturation cocktail. Matured DC pulsed with DEC/NY-ESO-1 fusion protein induced IFN-g production in a dose-dependent manner. Anti-DEC205 staining after loading with the fusion protein demonstrated complete saturation of the DEC205 surface marker with the fusion protein at the therapeutic dose of 50 mg. Timing of loading of the fusion protein to DC was found to be critical and highly matured DC demonstrated not only highest DEC205 expression, but also highest CD8+ T cell IFN-g production. When compared with a number of well-described immunogenic NY-ESO-1 peptides, DCs pulsed with peptides were unable to induce CD4+ T cell responses, while DC pulsed with the fusion product elicited strong CD4+ T-cell responses. In intracellular staining assays, it became evident that the DEC205/NY-ESO-1 fusion protein was taken up intra-cellularly within 15 minutes. It co-localized with HLA-Class I, but not class II molecules.
We demonstrate a systematic approach to the design of a clinical antigen-specific DC vaccine. These results are provocative and challenge current DC biology. This DC vaccine approach deserves further investigation responses and may play a significant role for patient undergoing immunotherapy.
Drug delivery using nanotechnology. Various nanoparticle based platforms for therapy of cancer
Jamboor K Vishwanatha, Anindita Mukerjee, Andrew Godowski 1 , Jessica Castaneda-Gill, Amalendu P Ranjan
Department of Genetics and Molecular Biology, Institute of Cancer Research, 1 Texas College of Osteopathic Medicine, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, Texas-76107, USA
Nanotechnology when engineered together with biotechnology opens a fascinating field with applications in diverse areas such as drug targeting and delivery, medical imaging, biosensing, biomaterials and nanotechnology. Recent advances in nanoscience and nanotechnology have led to the development of combinatorial nanosystems. It is highly desirable that nanoparticles can not only provide sensitive imaging and selectively deliver anticancer drugs to tumor sites but also specific targeting. Targeting anticancer drugs to their specific molecular targets is a major challenge in cancer therapy. In our studies, we used a novel non-covalent insertion of a homo-bifunctional spacer for targeted delivery of curcumin to various cancer cells. Curcumin has been found to be very efficacious against various cancer cells. Functionalized nanoparticles for antibody/targeting agent conjugation was prepared using a cross-linking ligand, bis(sulfosuccinimidyl) suberate (BS3), which has reactive carboxyl group to conjugate efficiently to the primary amino groups of the targeting agents. In our studies, we demonstrated successful conjugation of antibodies, Annexin A2 or PSMA, to curcumin loaded PLGA nanoparticles for targeting to prostate and breast cancer cells respectively [Figure 1]. The percent antibody attachment to PLGA nanoparticles was found to be 92.8%. Robust intra-cellular uptake of the targeted nanoparticles was observed in the cancer cells. Cell viability studies revealed that these curcumin loaded nanoparticles resulted in less cell viability for the cancer cells as compared to normal cell line.
Theranostic nanomedicine is emerging as a promising therapeutic strategy which combines therapy with diagnosis. These nanosystems are capable of diagnosis, drug delivery and monitoring of therapeutic response, are expected to play a significant role in the genesis of the era of personalized medicine. A schematic representation of various types of combinatorial nanoparticle-systems is illustrated in Figure 2.
Bone is a major site of metastasis in several type of cancer including breast, prostate, lung cancer and multiple myeloma. Metastasis of cancer to the bone could results in both osteolytic and osteoblastic lesions. The current standard therapeutic interventions such as radiation, surgery, chemotherapy, bone marrow replacement are limited by toxic side effects, drug resistance lack of efficacy. Our multifunctional biodegradable nanoparticle technology is an innovative tool to deliver chemotherapeutic drugs such as curcumin and antibody drug conjugates (ADC) to the bone. Antibody drug conjugates are gaining momentum as an important drug category with the ability to specifically target potent cytotoxic drugs to cancer cells. Several general considerations must be taken into account when developing these drugs. The first concern is the monoclonal antibody and target which are key to the specificity of this system. This target must either be exclusive to the cancer cell or highly up regulated compared to other healthy tissues in the body. Also, the antibody must have the ability to be internalized rapidly. The next important aspect that must be taken into account is the linker technique used to conjugate the chemotherapeutic drug to the antibody. The linker must be stable enough not to release the drug in the blood stream but capable of cleavage once internalized within the cancer cell. The last important aspect of an ADC is selection of a drug that will be attached to the antibody. This drug must be sufficiently potent to cause cytotoxicity with a limited number of molecules. If antibodies are overloaded with conjugated drug their functioning and binding capacity will be diminished. ADCs hold great promise for delivering new treatment options for cancer patients while minimizing many of the potential toxic side effects often seen with tradition chemotherapeutics.
In another of our studies, PLGA nanoparticles were used for glioblastoma therapy. Methylene blue (MB) has received increased attention as a therapeutic molecule for enhanced protection and improved function of mitochondria. These studies showed that MB utilization reduced neurodegeneration by increasing oxygen consumption, reduce lactate production, and inhibit glioblastoma cell proliferation. Although MB is to pass through the blood-brain barrier, delivery via its traditional oral route results in the majority being eliminated by first-pass metabolism. While MB may also be administered IV, the improvement in uptake/delivery to the brain is minimal. Formulating MB within PLGA nanoparticles improves the low bioavailability of MB.
These developments and results have emphasized the potential of our multifunctional nanoparticles to improve the clinical efficacy of nanoparticle based therapy in patients with cancer.
Micro ribonucleic acid biology and applications in drug development
Department of Thoracic Surgery, Thoracic Surgery Research Laboratory, Roswell Park Cancer Institute, USA
The last few years have witnessed an explosion of studies focusing on the biology and clinical application of microRNAs. These small regulatory non-coding RNAs are now known to influence almost all aspects of cellular behavior in both physiological and pathological contexts. This presentation provides a quick overview of microRNA biology, mechanism of action, assays and proposed clinical applications. MicroRNAs are generated from genes, just like coding RNAs. The transcripts, known as pri-miRNAs are processed by DROSHA into pre-microRNAs. These pre-miRs are translocated from the nucleus to the cytoplasm where they are further processed by DICER into a duplex structure that consists of two effector molecules. Each effector molecule is considered a mature microRNA and can be paired to target mRNAs either perfectly or imperfectly via a RISC complex to either degrade or decrease translation of the target mRNA respectively [Figure 1]. It is the possibility of imperfect pairing that enables one miRNA to potentially control many mRNAs. The rational prediction of miRNA targets, an essential step in understanding their function, is currently started with a bio-informatics analysis based on certain ground rules. However, there is often lack of agreement among different prediction algorithms and the overall accuracy of these predictions is about ~60%. Therefore, the laboratory validation of these predicted targets is very important. The presentation goes through various modes of validation and pitfalls in each method. The various methods of assaying microRNAs are presented with the benefits and pitfalls of each method. We have seen only modest correlation between platforms in our experiments and caution extrapolation of one finding to other platforms.
A brief history of how the link between microRNAs and cancer was elucidated is presented. A basic model of how microRNAs are thought to be involved in cancer is also presented. MicroRNAs can serve different roles based the cellular context. Therefore, the same microRNA that functions as a tumor suppressor can be an oncogene in a different cellular system. MicroRNAs have been studied as potential biomarkers for early diagnosis, prognostication and biomarkers of response. This is because microRNAs are stable in body fluids and paraffin embedded specimens, and are easily assayed. Using lung cancer as a model, some typical experiments conducted in this area are described, including pitfalls and promises. Developing microRNA biomarkers for response is emerging as an important component of drug development.
As microRNAs are essentially all "druggable", it is tempting to attempt the use of this knowledge and technology to treat cancer. While enough good candidate microRNAs for this use have been found in most cancer, like all gene therapy, the difficulty is more with drug delivery rather than the target gene itself. Several stragies are being develop to maximize delivery of microRNAs including liposomes, nanoparticles and nucleotide modifications. The last part of the presentation summarizes current ongoing trials in this sphere and emerging technologies aimed at making this dream a reality.
Cyclins as effective biomarkers for targeting oral cancer
Molecular Oncology Lab, Centre for Life Sciences, Central University of Jharkhand, Brambe, Ranchi, Jharkhand, India
Oral cancer is the one of the most common form of cancer worldwide, and the majority of cases occur in India and Southeast Asia. Little is known about this type of cancer despite recent advances in cancer research. The generally asymptomatic nature of the early oral lesions causes them to remain unnoticed and undetected in majority of cases. Oral cancer begins with a long phase of hyperplasia, which then transforms into dysplasia and subsequently becomes carcinoma. This disease progresses substantially before the patients search for cure and is a key contributing factor to the disease severity.
Oral cancer is primarily a growth-related disorder, and cyclins are the prime regulators of cell division. Their periodic synthesis and degradation is linked with different phases of cell division. Aberrantly expressed cell cycle-related cyclins are highly associated with various tumour types. These important molecules are regulated in many ways to achieve a gain in function and are involved in promoting neoplastic growth. Cyclins are associated with the pathogenesis of oral cancer and are considered valuable biomarkers for diagnosis and prognosis. Substantial experimental evidences support a link between oncogenic signaling pathways and the deregulation of cyclins in oral cancer. There is also substantial evidence to indicate that tumor growth is inhibited when cyclins are targeted. The causes of most cyclin over-expression are different as mentioned in Figure 1.
Success of therapeutic intervention strategies to control the uncontrolled cyclins in oral cancer is much dependent on understanding the mode of its deregulation. Among the cyclin molecules, cyclin D is frequently deregulated. Cyclin D is controlled by many signaling pathways in oral cancer and may be considered a potential target. Cyclin regulation by hormone stimulation, proteolytic processing, viruses, other pathways (e.g., EGFR, Wnt, MAPK), TFs and miRNAs represents a new avenue for designing novel therapies. Similarly, disrupting protein-cyclin interactions with a blocking peptide or restricting the nuclear-cytoplasmic translocation of cyclins may be another approach for therapeutic development. It is known that cyclin targeting sensitizes cancer cells to cancer drugs and inhibits cell growth. Similar strategies may be adopted as adjuvant therapies with traditional chemotherapeutic drugs to combat OSCC.
Although these cyclins have been reported for the last 2 decades, their value is just beginning to be understood. Additional studies on cyclins may lead to their identification as important cancer diagnostic and prognostic indicators as well as their use as possible therapeutic tools for oral cancer intervention in the future. So far, much attention has been given to targeting higher-order master molecules and membrane receptors/tyrosine kinases for therapeutic purposes without having a significant advantage. Thus, targeting cyclins may be a novel approach to treat oral neoplasms, that would block the accelerated cell cycle and thus rapid cell division. The application of advanced molecular biological and biochemical methodologies to elucidate cyclins as biomarkers may aid in early detection; however, much more work must be done for this information to be effectively applied in the clinical setting.
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