Estrogens and Novel Analogs

Why Estrogens?

A key observation, published in 1996 by Tang et al in the British medical journal the Lancet (and subsequently confirmed by others), was that post-menopausal women who had taken estrogens as hormone replacement therapy (HRT) showed substantial reductions in the incidence and age of onset of Alzheimer’s disease. For example, by the time they were 85 years old, 25% of the women in the study who had never received HRT were showing symptoms of AD, versus less than 5% of those who had been on HRT for at least 1 year. The divergence becomes even more striking as the women aged, with over 50% of the women who had not received HRT showing AD symptoms by the time they were 90, versus less than 10% incidence for the women who had received HRT (Lancet, 438:429-432, 1996).

Many scientists who studied these data have attributed such profound responses to the hormonal effects of estrogen and its well-documented capacity to affect brain physiology. However, it has become clear from the research from many labs that estrogens are able to protect neurons, and a host of other cells, from a wide variety of noxious stimuli, including the tissue ischemia that occurs during a stroke or myocardial infarction.

Three key observations made by scientists at Apollo BioPharmaceutics, a subsidiary of MitoKor, have altered the direction of research in this field. First, they found that estrogen analogs lacking the hormonal effects (e.g., 17-a estradiol) were equally as protective as authentic estrogen (17-b estradiol). This opened an entirely new arena for development of novel compounds that conferred protection, but that were non-feminizing. Second, they found that the potency of estrogen and its non-feminizing analogs, was greatly amplified when the glutathione status of cells was experimentally augmented. Glutathione is a small molecule composed of 3 amino acids that operates in cells with several enzymes, such as glutathione peroxidase and glutathione reductase, to scavenge reactive free radicals that can destabilize cell function. Third, Apollo scientists determined that the key structural element required for cell protection is the presence of a phenolic ring in the A position (polycyclic phenolic compounds or “PPCs”).

The synergy with glutathione, and the requirement that the ring structure contain a phenol in the A position, has helped to illuminate how estrogen and its non-feminizing analogs are likely be working in the cell. The available data indicate that these PPCs intercalate into the cellular and mitochondrial membranes where they interrupt lipid peroxidation, a free radical-induced chain reaction that undermines membrane integrity. In doing so, however, the PPC molecule itself becomes oxidized, which renders it ineffective for further protective reactions. It is at this point that glutathione outside the membrane restores the PPC to its original conformation, allowing it to again interrupt membrane oxidation. By cycling catalytically in this way, small amounts of PPCs in the membrane provide access to the large reservoir of antioxidant capacity stored in the intracellular glutathione pools. It is worth noting in this context that mitochondria maintain very high glutathione concentrations (in the millimolar range). Thus, these PPCs protect cells and mitochondria by tapping into ongoing cellular metabolism to augment the cell’s antioxidant defenses. This process is directly analogous to the well-described antioxidant coupling between vitamins E and C, where the former, having stopped oxidation within the membrane, is recycled by the latter.

The evidence is now compelling that estrogen and its non-feminizing analogs are protective via several mechanisms, one of which is the stabilization of mitochondrial function. For example, the feminizing form 17b estradiol:

ULB Bordet

Reduces the severity and volume of injured brain tissue in animal models of stroke.
Protects PC12 cells expressing mutant presenilin from apoptosis induced by amyloid or serum withdrawal, and in so doing prevents loss of mitochondrial membrane potential (DYm), reduces free radical production and preserves cellular ATP pools.
Protects cortical neurons from hydroxyl radical and amyloid toxicity by moderating intraneuronal free Ca2+ and preserving the function of membrane proteins such as Na-K-ATPase and glucose transporters.
Protects neuroblastoma cells from the mitochondrial toxin 3-nitroproprionic acid by preserving DYm, thereby moderating both ATP depletion and ROS production.
Protects spinal cord cultures from mice expressing the human mutant SOD associated with familial amyotrophic lateral sclerosis (Lou Gehrig’s disease) against excitotoxicity by preserving mitochondrial function and reducing radical production.
Similarly, the non-feminizing 17a estradiol also:

Protects hippocampal neurons from apoptosis induced by H2O2, amyloid, glutamate, and glutathione depletion.
Protects domaminergic neurons in vivo and in culture against the mitochondrial toxin MPTP, excitotoxicity, and free radical exposure.
Reduces stroke volume and mortality in animal models of both ischemic stroke and subarachnoid hemorrhage.

Clinical Developments

MitoKor and Apollo have entered into a strategic alliance with Wyeth Corporation related to the development of estrogens and estrogen-like compounds for treatment of Alzheimer’s disease. Wyeth is funding a Phase III clinical trial evaluating the use of estrogens in the prevention and treatment of Alzheimer’s disease in post-menopausal women. This trial is part of the Women’s Health Initiative Study, and has enrolled over 7,500 women.

In addition, an oral non-feminizing estrogen analog, ABP-150, has recently completed Phase I trials. The compound will be evaluated for use in chronic neurodegenerative diseases such as Parkinson’s disease.

The data indicate that estradiols exert their protective effects by preserving mitochondrial function, at least in part by stabilizing membrane structure during exposure to free radicals. This mechanism is particularly germane to mitochondria where glutathione concentrations are maintained at extremely high concentrations. Because the vital parameters of cellular energetics and oxidative status are predominantly determined by mitochondrial function, MitoKor and Apollo have shown that estrogens and its analogs yield beneficial effects in a wide variety of models. This has supported establishment of a strong intellectual property position in a wide variety of both acute and chronic pathologies.

Several proprietary formulations will be entering the clinic for acute and long-term neuronal loss resulting from stroke.

Our elucidation of the molecular mechanisms responsible for cytoprotection by polycyclic phenols continues to yield novel compounds, several of which are in advanced pre-clinical development. These PPCs, and other novel compounds being generated in house and at the Mitophenomesubsidiary Mimotpoes in Australiia, are being evaluated in a comprehensive suite of assays for evaluating mitochondrial function, MitoMetrics™ Profile. Mitophenome has also developed technology for rapid discovery of novel molecular targets. Mitophenome IVD discovery pipeline for cytoprotection will continue to yield novel compounds of potential utility to our other on-going programs, such as stabilization of pancreatic beta function in diabetes or preservation of chondrocyte integrity in osteoarthritis.