Ischemia-Reperfusion Injury

Overview
Mitochondria play crucial roles controlling cell viability after transient loss of blood flow in a variety of tissues. Recent findings have identified mitochondrial failure as the pivotal event controlling cell death, particularly the activation of programmed cell death, or apoptosis. This systematic deconstruction of the cell is responsible for loss of neurons following neuronal ischemia/reperfusion injury that accompanies stroke, death of heart cells following myocardial infarction, and non-viability of cells after ischemia arising from surgery or transplantation (among others). MitoKor has incorporated these findings into its MitoMetrics™ drug discovery program by developing new strategies for the discovery of novel compounds that will treat mitochondrial dysfunction and hence ameliorate tissue injury after an ischemic attack.

The Role of Mitochondria in Stroke

Stroke is the third leading cause of death in the United States. Each year, an estimated 400,000 Americans suffer a stroke and approximately 150,000 die from its consequences. A stroke occurs when a region of the brain loses blood flow, usually due to an obstruction in the vessel, but sometimes due to a vessel bursting, and neurons die as a result of this sudden ischemic event. Under these conditions, mitochondrial function fails and ATP concentration in the neurons drops within minutes after cessation of blood supply. At the center of the infarct this causes osmotic cell lysis and necrotic death. Radiating outward from this necrotic core is a region where perfusion from collateral vessels is sufficient to supply varying amounts of oxygen and hence support mitochondrial ATP production. However, even here mitochondria gradually fail over the ensuing days resulting in secondary cell death via apoptosis. Regardless of whether neuronal death is acute or secondary, disruption of energy production, increased free radical production, intracellular calcium dyshomeostasis, and induction of the apoptotic cascade are all results of mitochondrial failure.

The Role of Mitochondria in Myocardial Infarction

Almost exactly the same vascular events and deleterious consequences occur in the heart muscle during the transient ischemia associated with a heart attack. Obstruction of blood flow precipitates mitochondrial collapse in myocytes at the center of the infarct, with secondary death ensuing in the surrounding penumbra. Of course, the timing and relative importance of specific events is different between CNS stroke and myocardial infarct. For example, myocardial mitochondria are more tolerant of elevated Ca2+ levels than those in CNS, and so recover better and generate fewer free radicals after comparable Ca2+ exposure. Indeed, differences in mitochondrial stability also highlight opportunities for development of therapeutic agents with tissue-specific functional profiles.

  IVD Target Discovery Strategy

MitoKor has developed strategies to curtail cell death by impeding mitochondrial dysfunction after an infarct. The Company’s integrated drug and target discovery program exploits the functional involvement of mitochondria in cell death and presents a strategic opportunity for drug discovery. A principal feature of the MitoKor drug discovery strategy involves a novel drug screening and drug target identification system that employs simultaneous assessment of multiple drug targets.

Whole Cell-Based High-Throughput Screening

The front end of this system is a proprietary, whole cell-based, high throughput, screening assay that allows real-time measurement of changes in mitochondrial function. Primary cell cultures and proprietary cell lines containing a selection of pre-existing mitochondrial defects are routinely used for this assay. Compounds are rapidly screened in this assay for their ability to stabilize mitochondrial function under conditions that mimic transient ischemia.

Drug Target Discovery Program

In parallel with drug screening, Mitophenome  has established a target discovery program that is based on the analysis of differential gene and protein expression. Identification and validation of new targets can be performed using Mitophenome’s cellular models of mitochondrial dysfunction. This approach can also be used to uncover protein targets that mediate the cytoprotective effects of active compounds that are identified in the high throughput screening assay. The gene expression profiles generated in response to a test compound can then be compared to those of reference cytoprotective agents. Validated genes can be used to generate valuable animal models using transgene expression or gene knockout technologies. The gene/protein expression profiles can also be used as endpoints for drug evaluation in model systems and humans.
Clinical Development
The feminizing hormone estrogen (17-b-estradiol) exerts cytoprotective effects in a variety of cell and animal models. For example, hormone replacement therapy postpones the age of onset for Alzheimer’s disease (Tang et al., Lancet, 438:429-432, 1996) and estrogen significantly reduces the volume of injured brain tissue in animal models of stroke.

In a crucial series of experiments, scientists affiliated with MitoKor have shown that non-feminizing estrogen analogs (e.g., 17-a -estradiol) also show comparable cytoprotective effects. That have also shown that such protection can be substantially augmented by increasing the cellular glutathione pools, a small peptide antioxidant found in all cells and mitochondria.

Cytoprotection by estrogen and several of its analogs has now been documented in numerous models from a host of different laboratories, thus encouraging the clinical development of these polycyclic phenols for use in stroke and other ischemic pathologies. To that end, MitoKor is now completing the formulation studies for Neurostat™ and Neurextend™ , two proprietary sub-cutaneous injectible forms of estrogen and its non-feminizing analogs for acute and chronic treatment of stroke, respectively. Both have ideal pharmacokinetics, and are also being considered for potential utility in myocardial infarction.

Strategic Opportunity

In addition to new chemical entities emerging from SAR studies of polycyclic phenols, MitoKor’s MitoMetrics™ drug and target discovery strategy offers significant advantages over the “one molecule – one target” method of library screening in that potential targets for a given molecule can be identified while defining the molecular pathway for the action of each compound class. This program, therefore, has the potential to accelerate the development of effective new therapeutic agents for stroke/ischemia by:

Enabling rapid screening of compound libraries using proprietary functional assays that assess mitochondrial function.
Obviating the initial need for a known site of action, thus facilitating the discovery of novel lead structures.
Providing the technology for rapid identification and validation of protein targets that mediate the neuroprotective activities of the novel chemical entities.

Lieven Gevaert, Bio-ir