CardionomicsTM Human Heart Failure Detection Technology
Microarrays provide a versatile platform for utilizing information from the Human Genome Project to benefit human health. However, to date this technique has been shown to have low specificity, sensitivity, and reproducibility when using human samples for identification of genes involved in the pathogenesis of heat failure. This is evident in conflicting published data identifying diverse gene expression patterns by means of array analysis using human heart failure tissue. The extent to which this deficiency is linked to individual differences such as sex, race, diverse etiology of the disease, drug therapy vs. tissue collection and handling has not been fully addressed until now.
Our unique approach to develop this technology resides first in having clearly identified and categorized tissue samples through access to full medical records and demographics. We have true non-failing control samples unlike other investigators. Our medical team of heart failure clinicians carefully categorizes all of our samples. Tissue harvesting and handling have been carefully developed to insure the highest quality of samples and RNA isolation.
Our technology is based on a subtractive hybridization screening analysis (SSH). We have used this analysis approach as a screening tool to develop a focused heart failure microarray containing 1,143 differentially expressed genes. This array has been used to quantify further the expression of gene products in female and male non-failing and failing hearts subsequent to definitive diagnosis of idiopathic dilated cardiomyopathy (IDCM) as well as identify a unique fingerprint for other etiologies of heart failure.
Each individual heart failure (HF) sample has been normalized to gender-specific and age matched pooled normal heart samples to standardize for normal differences in expression values between the two genders. In this regard, we have identified a gender-specific gene expression pattern in female patients that presented with idiopathic dilated cardiomyopathy despite having similar clinical characteristics as male counterparts. We have also identified a unique fingerprint profile for patients with alcohol induced heart failure.
Application: Drug discovery and clinical diagnostics
Large pharmaceutical companies with drug discovery programs can use our heart failure microarray to identify novel targets to be used in drug development for the treatment of heart failure. Our technology can be used for chemical library screening and identification of molecular medicine approaches, e.g., gene transfer as well as small molecule approaches. Furthermore, our technology can be applied to pharmaco-genomics (correlations between therapeutic responses to drugs and the genetic profiles of patients) and toxico-genomics (correlations between toxic responses to toxicants and changes in the genetic profiles of patients) prior to enrollment in clinical trails as well as during clinical trials. Our technology would enable the rapid identification of responders and non-responders.
Device companies can apply our technology to their hardware creating a clinical monitoring device for disease diagnosis and evaluating therapeutic interventions and outcomes. Device companies would therefore be able to use our microarray on human clinical specimens obtained by physicians.
Presently, blood markers (e.g. creatine kinase, troponin T, troponin I, brain natriuretic peptide) are used to document poor heart function. It is known that the time for detection of some of these markers is less than ideal particularly with regard to preventative approaches and impacting clinical outcomes. Our product would be better in that it would say which specific pathogenetic pathway is malfunctioning as opposed to a simple read out of "the heart is in trouble". Importantly, our technology would quickly diagnose idiopathic dilated cardiomyopathy as opposed to being a diagnosis when others have been ruled out. This alone would save time, money, and possibly lives. Furthermore, our technology can easily be used and interfaced in the daily medical management of patients with heart failure.
Our goal is to create a new and more comprehensive gold standard for clinical diagnosis and clinical outcomes analysis. Our goal is to license the human heart failure array as a product to be combined with an easy to use and affordable platform offered by a device company manufacturing diagnostic devices.
While the market that we have targeted (i.e. idiopathic dilated cardiomyopathy) is substantial, there exist other large markets, such as drug or toxin induced heart failure, where our technology would be equally applicable. For example Doxorubicin (DOX), an anticancer drug, has a wide array of therapeutic uses in adults and children (breast and bladder cancers, Hodgkin's lymphomas, leukemia and others). Despite its frequent clinical use, the utility of DOX as well as other drugs in this class is severely compromised by dose-limiting cardiac toxicities. A recent study demonstrated that while many patients exhibit cumulative dose dependent toxicities, others experience acute life-threatening toxicity with their first dose. Other patients receiving the anti-cancer drug have delayed reactions and develop heart failure months to years following cessation of therapy. The use of our technology could predict the likelihood for the development of delayed heart failure and might even prevent the occurrence of cardiac toxicity through detection of early changes in gene profiles warning of possible future drug induced heart failure and the early initiation of therapeutic intervention. We can combine our technology with any heart toxic agent.
Application: Target identification through to drug discovery
Drug discovery is highly time and resource sensitive with a high level of risk during the drug approval process. Drugs require, prior to commercialization, regulatory clearance by the FDA and by comparable agencies in most foreign countries. The nature and extent of regulation differs with respect to different products. In order to test, produce and market certain therapeutic products in the United States, mandatory procedures and safety standards, approval processes, and manufacturing and marketing practices established by the FDA must be satisfied.
An Investigational New Drug application, referred to as an IND, is required before human clinical use in the United States of a new drug, compound, or biological product can commence. The IND includes results of pre-clinical animal studies evaluating the safety and efficacy of the drug and a detailed description of the clinical investigations to be undertaken.
Clinical trials are normally done in three Phases, although the Phases may overlap. Phase I trials are concerned primarily with the safety of the product. Phase II trials are designed primarily to demonstrate effectiveness and safety in treating the disease or condition for which the product is indicated. These trials typically explore various doses and regimens. Phase III trials are expanded clinical trials intended to gather additional information on safety and effectiveness needed to clarify the product's benefit-risk relationship, discover less common side effects and adverse reactions, and generate information for proper labeling of the drug, among other things. The FDA receives reports on the progress of each phase of clinical testing and may require the modification, suspension or termination of clinical trials if an unwarranted risk is presented to patients. When data is required from long-term use of a drug following its approval and initial marketing, the FDA can require Phase IV, or post-marketing, studies to be conducted.
After clinical testing is successfully completed, a new drug application for approval is submitted. After initial FDA or foreign health authority approval has been obtained, further studies, including Phase IV post-marketing studies, may be required to provide additional data on safety and might be required to gain approval for the use of a product as a treatment for clinical indications other than those for which the product was initially tested. In addition, the FDA or foreign regulatory authority will require post-marketing reporting to monitor the side effects of the drug. Results of post-marketing programs may limit or expand the further marketing of the products. Further, if there are any modifications to the drug, including any change in indication, manufacturing process, labeling or manufacturing facility, an application seeking approval of such changes may be required to be submitted to the FDA or foreign regulatory authority.
The commercialization process of a new drug is likely to take ten or greater years and requires the expenditure of substantial resources. Gwathmey's business strategy is to enter into a strategic alliance with a large pharmaceutical company and to license our technology. The technology can be used to identify targets for drug development. Twenty percent of the genes are of unknown function. Our human heart failure array in combination with our large animal model that shows a high level of congruence with the human condition with associated heart failure array could significantly reduce the time frame of pre-clinical testing. This alone would result in significant cost savings and faster time to market (i.e. synergistic and expionential cost savings). Our technology in summary not only spans the pre-clinical testing and screening process in drug discovery, but transitions to the clinical testing and clinical trial enrollment. Using our technology would result in greater confidence in successful clinical trial outcomes through enrollment of only likely responders. Our business model would result in an early revenue stream for Gwathmey, Inc. Our long-term plan is to sign with a strategic partner such as a larger biotechnology or pharmaceutical company to use our array for drug discovery with Gwathmey, Inc. providing pre-clinical testing of small molecule (NCEs) or molecular medicine candidates. Our ultimate goal is to make the array available on an exclusive basis to a drug discovery company or through non-exclusive licensing agreements.
In summary, our proposed technology is a technically complex system that requires significant investment in order to achieve the required high throughput use needed to maximize on financial returns. We foresee follow on products as well. Custom arrays can be created that are race specific, heart chamber specific (arrhythmia detection) and stage-specific (e.g. early heart dysfunction). We can also make customized arrays based solely on gender or develop a database on fingerprint profiles for other causes of heart failure.
Whole genome DNA microarrays are useful for a broad range of biological problems. This approach, however, typically results in large data sets that contain measurements on thousands of genes associated with what we "believe" is a specific single condition. When managing such large data sets it is critical to summarize the data to be able to extract important information. This can be a daunting task with investigator bias influencing data handling and processing.
Our approach reduces the number of genes that must be analyzed and filtered thereby allowing full utilization of the information contained on our array. This then allows investigators to focus on an enriched pool of heart specific genes thereby uncovering genes that may have been masked as subtle changes or that may have been lost in the wealth of information acquired by a whole genome microarray approach.
There is no system other than clinical phenotype and improvement of symptoms used in the clinical diagnosis or projected prognosis for heart failure. These tools though useful are not very exact with regard to clinical outcomes and survival. The goal for the use of our technology, simply stated, is the accurate definitive diagnosis of idiopathic dilated cardiomyopathy as well as support for better medical management of patients with the disease and better clinical outcomes. This will reduce the clinical diagnosis time in half. We have had successful manufacturing of our technology (low technical risk), believe it is safe, specific, sensitive, reliable, and robust (manageable medical risk), and satisfactory profit potential (acceptable commercial risk) in our product development program. We must now successfully transition to the commercialization stage.
The gold standard for heart failure diagnosis is heart size determined by imaging modalities. Idiopathic dilated cardiomyopathy is a diagnosis by default and is based on exclusion of ischemic heart failure, viral infection or other toxin induced causes of heart failure. There are no clear clinical markers for idiopathic dilated cardiomyopathy unlike markers used for ischemic heart failure e.g. creatine kinase and troponin T and I.
The benefit to patients of healthcare providers using our array is a definitive diagnosis of their disease as opposed to simply based on exclusion of other causes. Furthermore, the responsiveness to treatment or the lack thereof can save precious time in identifying proper medical management paradigms for patients as individuals resulting in an increased likelihood of favorable clinical outcomes and survival. In addition, the saving of life as well as resources (financial and otherwise) would be significant. Financial savings would eliminate the use of other less specific clinical markers e.g. troponin T $131 or creatine kinase $159). However, our array is an imperfect approach in that we have not narrowly defined which markers could be exclusively placed on a custom clinical diagnostic and medical management array. Furthermore, we have not generated a database of genomic fingerprints for patients with heart failure of differing etiologies.
The new "kill early and often" paradigm used by large pharmaceutical companies will almost certainly increase the likelihood of false negatives. Our technology might save drugs that might be aborted that are late blooming blockbuster drugs or kill drugs early and save lives as well as millions of dollars invested in clinical trials. Aborting a clinical trial early can save tens of millions of dollars. A late blooming blockbuster drug, e.g. Lipitor, resulted in a $1 billion dollar drug. We are, therefore confident that this technology is poised for commercialization and use by a larger strategic partner with the resources and needs to proceed at an accelerated pace.
Clinical cardiologists have long been interested in the pathogenesis, development, progression and response to treatment for heart failure. Currently, the leading manufacturer of microarrays "Affymetrix" has collaborated with scientists from Duke University to seek a better understanding of heart failure. It is important to note that they are using a mouse animal model in combination with a mouse whole genome microarray. They hope to extrapolate and compare the mouse model to the human condition. We have taken a similar approach in combination with our human heart failure microarray. We have created and validated a large animal model, i.e. avian model of heart failure that is highly congruent with the human condition. The model has been shown to be highly predictive of human clinical trial outcomes with therapeutic agents targeted to treat heart failure. A heart failure microarray has been created for this model as a way to screen drug candidates and identify clinical markers for use in a clinical trial.
Our microarray is unique first because it was generated from human heart samples. Furthermore, our array was generated from an enriched pool of IDCM heart specific genes (disease vs. normal human). Our human microarray is particularly unique because it takes into consideration race and gender profiles of IDCM, which has never before been performed. We were able to develop this array because we maintain a large human heart tissue bank and through significant financial support from the Heart, Lung, and Blood Institute at the National Institutes of Health. Our company has the needed research and development (R&D) infrastructure as well as the combined clinical and microarray fabrication knowledge that was needed for the successful creation of such a tool.
Importantly, we have developed and validated both a turkey heart failure microarray as well as our human heart failure microarray. Our avian heart failure model can be used as a large animal model to evaluate and understand mechanisms of heart failure in a longitudinal manner. The turkey model has been shown to be highly congruent with the human condition at the functional, biochemical and now genomic level. The model, therefore, can be used to screen drugs targeted to treat heart failure. Because the animal model allows for well "controlled" longitudinal studies, the natural history of heart failure can be studied as well as the pathogenesis. The impact of therapeutic interventions being tested in the model on gene expression levels might be important for selecting drug targets for small molecules, end-points in clinical trials, as well as clinical markers for evaluation during a clinical trial.
Approach: SSS/HTDS screening for differentially expressed genes
Traditional techniques e.g. Northern Blots, RT-PCR, differential display, representational difference analysis (RDA), enzymatic degradation subtraction or linker capture subtraction have all been used to isolate differentially expressed sequences. Some of these have been used to identify new genes that might be involved in heart failure. However, all of these methods suffer from serious drawbacks and are very labor intensive. Furthermore, all of these techniques strongly favor the isolation of abundant transcripts because the disproportion of rare versus abundant transcripts is maintained throughout the isolation procedure. Furthermore, the subtraction efficiency (the removal of sequences common to both tissues) is often low. A drawback of conventional differential display methods is that they restrict the analysis of differentially expressed genes to those that are expressed relatively abundantly. Therefore, we have applied a novel differential screening technique to create our microarray that combines subtractive hybridization (SH), suppressive PCR, and suppression subtraction screening (SSS) with a high throughput differential screen (HTDS). This experimental strategy enables the efficient and rapid cloning of hundreds of differentially expressed, abundant and rare transcripts in a single hybridization experiment and reduces the number of false positive clones obtained. In contrast to the usual 10- to 20-fold enrichment of differentially expressed sequences, SSS/HTDS yields up to 100-fold enrichment in a single experiment and the efficiency of subtraction can be monitored.
We have already generated a first and second generation of our human heart failure cDNA array using subtracted cDNA libraries and the SSS/HTDS approach.
The major advantage of our array technology is that the expression of all 1143 genes can be examined in a single hybridization experiment, and since only sequence-verified DNA sequences are arrayed, their identity is known within minutes after the hybridization experiment is complete. The use of our invaluable microarray will enable users to identify quickly critical tissue-specific, differentially expressed genes during differing stages of heart failure.
Cost and marketing promotion could also influence the clinical use of our technology worldwide. Approximately 46 million Americans have no health insurance (~15.7 percent of the population). With healthcare being affordable by 84.3 percent of the American population, health care providers as well as insurance providers must broadly accept our technology. For use in clinical trials, it must be accepted by regulatory agencies. Despite the proven questionable utility of creatine kinase ($159) and troponin T or I ($131) in a diagnostic mode, they are still highly used in clinical settings. Our customized array will be heart specific and include CK and the troponins. An estimated cost is approximately $200 per hybridization.
It is known that creatine kinase is a marker of myocardial damage and does not correlate with early heart failure. The same is true for troponin T and I, which are again primarily used in the diagnosis of acute ischemic heart failure. None of these tools is specific for the diagnosis of idiopathic dilated cardiomyopathy (IDCM).
Our objective was to create a sensitive, highly specific, robust human heart failure microarray. We have been successful. This technology now must be commercialized with a low financial risk strategic partner through licensing agreements. Our technology can be made available to multiple types of users e.g. device manufacturers, clinical research centers, pharmaceutical companies etc). This new technology promises to lower the cost of clinical trials by better focusing on patient inclusion criteria, clinical monitoring during clinical trials and routine patient medical management.
We have developed a unique human heart-specific oligonucleotide array (race and gender specific). Our validation by RT-PCR as well as at the protein level gives us a high level of confidence in the quality, reproducibility, and reliability of clinical read outs using our microarray.
These arrays may be used for the following applications:
Intellectual Property Status
A patent application to the microarray technology has been filed in the U.S. Patent and Trademark Office. Twelve months from this filing date, Gwathmey, Inc. plans to file a Patent Cooperation Treaty (PCT) application. Cost effective protection of intellectual property will be discussed with our patent lawyers to file national stage applications in countries where patent protection is desired.
A new registered trademark CardionomicsTM has also been generated during the process of research and development of the human heart failure microarray. The registered trademark used to brand the IDCM detection technology will be available as part of the licensing package.
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