Scientists worldwide use zebrafish to study the development of vertebrates, the group to which humans also belong. Zebrafish are only a few centimeters long and their embryos are transparent, which is why researchers can observe every change under the microscope. In the early development of zebrafish, the heart is like a tube surrounded by a pump of epithelial cells.

The upper and lower sides of an epithelial cell are clearly different. The upper side, which forms a boundary between organs and either tissue fluid, hollow spaces, or the environment, has a different function than the lower side facing the connective tissue. Both poles of an epithelial cell are vital for the functioning of the heart and other organs.

Different proteins steer cell polarity

Until now, the scientists had assumed that the development of the cell poles was steered by a protein complex which scientists call Crumbs/Nagie oko complex. “What is surprising is that, depending on the tissue, the protein complex has a different composition while being fully functional,” Dr. Abdelilah-Seyfried explained. Thus, some proteins in the heart are not needed, but for the development of epithelial cells of the retina they are fundamental. “The program as understood previously may apply for individual cells in the laboratory,” Dr. Abdelilah-Seyfried said, “but in a complex organism like the zebrafish, there are many deviations.” In the opinion of the researchers, these deviations indicate that the epithelial cells have adapted to their tasks in the respective tissue. Until now, these processes have not been fully elucidated. Next, the MDC researchers want to find out whether still more proteins are involved in epithelial cell polarization.

*Divergent polarization mechanisms during vertebrate epithelial development mediated by the Crumbs complex protein Nagie oko

Nana Bit-Avragim1,2,*, Nicole Hellwig1,*, Franziska Rudolph1, Chantilly Munson3, Didier Y.S. Stainier3 and Salim Abdelilah-Seyfried1,‡

1. Max Delbrück Center (MDC) for Molecular Medicine, Berlin, Germany
2. Department of Cardiology, The Charité University Medical School of Berlin, Campus Buch, Campus Virchow Clinics, Berlin, Germany
3. Department of Biochemistry and Biophysics and Programs in Developmental Biology, Genetics, and Human Genetics, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-2711, USA

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Disorders of fat and carbohydrate metabolism make a major contribution to common diseases such as diabetes, obesity, hyperlipidemia, and premature arthrosclerosis. In recent years, science has turned its attention to how endogenous substances circulating in the blood can affect metabolism, not least in relation to lifestyle, for example dietary, changes.

Fibroblast growth factor 21 (FGF21) is an endogenous substance that has recently been shown to reduce increased glucose levels in the blood in both rodents and monkeys. In mice, FGF21 seems to be closely associated with the metabolism of fatty acids and has been shown to increase dramatically after the ingestion of a high-fat diet and after a period of fasting, which is seen as a possible explanation for the positive effects of such dietary changes. FGF21 is therefore a very interesting candidate for the treatment of Type II (age-related) diabetes.

However, there has been no data to indicate whether FGF21 has any physiological importance in humans. Swedish scientists at Karolinska Institutet have now studied levels of circulating FGF21 in humans and how they can be affected by the ingestion of food, fasting and during therapy with lipid-reducing drugs.

“We’ve found evidence that FGF21 levels in the blood can be regulated in humans,” says Professor Mats Rudling, who has led the study together with Professor Bo Angelin. “However, it seems as much more extreme circumstances are required, such as long-term fasting on the borderline to starvation.”

The study, which was conducted at the Clinic for Endocrinology, Metabolism and Diabetes at Karolinska University Hospital in Huddinge, shows that FGF21 levels in blood are not affected by fasting for up to 48 hours or by treatment with a fat-rich ‘ketogenic’ diet (an extreme form of the Atkins Diet). The changes recorded are much more modest in scope than those observed in rodents. According to the researchers, this contradicts the idea that FGF21 is the hormone responsible for the positive effects ascribed to the Atkins Diet.

“The importance of making comparative studies between different species on metabolism and its regulation is obvious, and we now have to learn more about what treatment with this interesting hormone can lead to in humans,” says Professor Rudling.

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No reliable responses were seen in the 900mg and 1500mg CX717 groups, but procedural problems were detected by the Data Safety Monitoring Board (DSMB) for this study, which was authorized by the protocol to monitor safety and responses on an interim basis. Corrective procedural changes were instituted before the initiation of the last group of subjects in the 2100mg segment of the study.

“While we initiated this study using oral doses of CX717 and had only eight subjects per treatment group, we were pleased to obtain statistical significance using such small study groups,” said Dr. Roger Stoll the CEO of Cortex. The primary objective was to simply verify that the mechanism, which was seen functioning in animal studies, would also be operative in humans. Substantial investments were required to develop an intravenous dosage form of CX717, including formulation development and stability studies for such a dosage form as well as two species toxicology trials. The Company now feels that it can proceed with such studies. Cortex recently received verification of three months of accelerated stability for the experimental intravenous formulation of CX717 and plans to initiate toxicology trials in the fourth quarter 2008.

A second respiratory depression study has been performed by a group in Frankfurt, Germany. This study uses a single dose of 1500mg of CX717 and focuses on both the respiratory depression and the analgesic effects associated with alfentanil. The analysis of the data has been initiated and related results should be reported within a few weeks. Studies of CX717 in animal models by Dr. John Greer at the University of Alberta have shown that the AMPAKINE drugs do not interfere with the analgesic effects of opiates.

Cortex continues to advance other AMPAKINE compounds, particularly those newer compounds that have potential patent lives to 2028. It will also be reported at the BMO Capital Markets conference that Cortex has initiated human phase one safety and kinetic trials with CX1739 in normal volunteers. Assuming successful Phase I human trials with this compound, the Company plans to rapidly pursue the Attention Deficit-Hyperactivity disorder indication for CX1739 in the second half of 2009. The Company will also report on an initial animal trial with CX1942, a unique pro-drug analog of another low impact AMPAKINE drug that is highly water soluble, ideally suited for an intravenous dosage form. This compound rapidly hydrolyzes to the parent AMPAKINE drug in vivo. CX1942 has shown exceptionally rapid results in reversing respiratory depression due to intravenously administered fentanyl in rats. The Company plans to initiate toxicology studies with this unique analog during the last quarter of 2008.

High impact AMPAKINE compounds from recent patent applications are also being advanced with a focus on neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, as well as orphan drug indications like Huntington’s and Fragile X disease.

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