Anavex Life Sciences Corp (AVXL)

[Investor2014]

Here is a summary of how I read the Goguadze et al paper divided into Physiological (normal) and Pathological (disease) conditions.

Physiological conditions:


Data are expressed as percentage of the no-drug value.

When the paper says

The most effective compound was PRE-084 with a ROS increase of + 24%at 1 µM (Fig. 1a). DHEA showed a + 26% increase at 10 µMdonepezil, + 12%at 3 µM AN1-41, + 11% at 1 µM and AN3-71, and + 12% at 0.3–1 µM. Among agonists, only AN2-73 was ineffective (Fig. 1b).

It is a good thing that only AN2-73 was ineffective, and here is why:

How mitochondria produce reactive oxygen species

The production of ROS (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus

In other words you want a drug to be ineffective at producing ROS under normal conditions.

Since previous work suggested a modulation of NOX activity (Natsvlishvili et al. 2015), we examined the impact of s1R drugs on NOX activity, in mitochondrial preparations, and on all NOX in whole homogenate preparations (Fig. 2). NOX activity in mitochondria was significantly inhibited by SOD or the NOX inhibitor VAS3947 but not by s1R agonists or antagonists at 1 or 10 µM (Fig. 2a). In homogenates, s1R antagonists at the highest concentration decreased NOX activity but this effect was only significant for PROG (Fig. 2b).

PRE-084 was without effect, but the ANAVEX compounds AN1-41 and AN2-73 tended to increase or significantly increased NOX activity at the 1 µMconcentration (Fig. 2b). The effects remained however limited in a ± 10% range. The contribution of NOX-induced ROS production on the s1R effect on ROS remained marginal.

Fig. 2 The s1R ligands failed to markedly affect NOX activities (a, b) and SOD activity (c, d) in mouse forebrain mitochondria (a, c) and homogenates (b, d) under physiological conditions.

Above describes the undesirable effect of modulating NOX activity, which all the compounds failed to markedly affect including the ANAVEX compounds.

In order to explain the s1R-mediated generation of ROS, we specifically examined the electron transfer chain (ETC) complex activities. Complex I activity was analyzed spectrophotometrically by measuring NADH oxidation (Fig. 4a). PRE-084 and NE-100 were analyzed in the 0.3–10 µM concentration range and other compounds at 1 and 10 µM. We observed that PRE-084 significantly increased by + 40% the complex I activity in physiological conditions. The s1R antagonists had no effect, but the ANAVEX compounds also increased markedly complex I activity (Fig. 4b).

It is my reading that above is a good thing since:

Mitochondrial Respiratory Complex I: Structure, Function and Implication in Human Diseases

Complex I is the first enzyme of the respiratory chain. It oxidizes NADH, which is generated through the Krebs cycle in the mitochondrial matrix, and uses the two electrons to reduce ubiquinone to ubiquinol. Ubiquinol is reoxidized by the cytochrome bc1 complex and transfers electrons to reduce molecular oxygen to water at complex IV. The redox energy released during this process is used to transfer protons from the mitochondrial matrix to the periplasmic space that generates proton-motive force across the inner mitochondrial membrane at complex I, III, and IV. Complex V uses this proton-motive force to produce ATP from ADP and inorganic phosphate. This entire process constitutes OXPHOS. Because complex I is the major entry point for electrons to the respiratory chain and is suggested as the rate-limiting step in overall respiration, it plays a central role in energy metabolism.

We are good so far, all check!

Pathological conditions:

Direct application ofAß1–42, between 0 and 4 µM, induced a trend to decreased state 3 and a concentration-dependent decrease in RCR, significant at concentration higher than 2 µM (Fig. 7a). The s1R drugs were therefore tested against
the Aß1–42 (4 µM)-induced alteration of respiration. PRE-084 significantly prevented the Aß1–42-induced RCR decrease at the concentration of 1 µM (Fig. 7b). The antagonists NE-100 and PROG failed to affect it or even worsened it with NE-100
at 10 µM. AN1-41 and AN2-73, but not AN3-71, attenuated the Aß1–42-induced RCR decrease, since the data with both concentrations of compounds were not significantly different from the control level (Fig. 7c).

Attenuated means reduced and my reading is this is a good thing as we want to keep Aß1–42-induced RCR decrease in check.

Futhermore:

Aß1–42 affects directly complex I and complex IV activities. We confirmed this data, by observing that the peptide concentration-dependently decreased both complex I (Fig. 8a) and complex IV (Fig. 8c) activities. At 4 µM, Aß1–42 induced a - 20% decrease in complex I and a - 30% decrease in complex IV. Among the drug tested, PRE-084 significantly prevented the Aß1–42-induced decrease in complex I activity (Fig. 8b). Other drugs were not effective. PRE-084 significantly prevented, while AN1-41, AN2-73, and AN3-71 attenuated the Aß1–42-induced decrease in complex IV activity (Fig. 8d). NE-100 and PROG were not effective.

Again good thing in favour of A2-73, check!

And finally in the conclusions:

The s1R agonists (PRE-084,AN1-41, and to a lesser extent AN3-71) decreased Aß1–42-induced increase in mitochondrial ROS. s1R antagonists did not affect Aß1–42-induced increase in mitochondrial ROS. This observation confirmed that, in pathological conditions, s1R agonists have direct antioxidant potential in mitochondria. In order to determine whether this effect was due to a restoration of ETC, we analyzed the oxidative respiration and observed that PRE-084, AN1-41, and AN2-73 attenuated or significantly prevented the Aß1–42-induced decrease in RCR, indicating that the ETC worked more efficiently. Indeed, Aß1–42 altered in the same concentration range, both complex I and complex IV activities. These effects were significantly prevented by PRE-084 for complex I and IV activities and the ANAVEX compounds for complex IV activity. The s1R antagonists remained without effect. These data confirmed the direct mitochondria protective effect of s1R agonists. The mechanism of action may combine a general effect on TCA cycle efficacy and up-regulation of cellular anti-oxidant pathways. First, amyloid toxicity decrease TCA cycle enzymes, including isocitrate dehydrogenase and a-ketoglutarate dehydrogenase (Mastrogiacomo et al. 1993, 1996; Ko et al. 2001). Therefore, by chaperoning Ca2+ flow, thus balancing Ca2+-dependent TCA cycle enzymes, s1R agonists may be able to maintain
NADH pool inside the matrix. Direct measures of s1R ligands effects on TCA enzymes must now be performed to confirm this hypothesis. Second, s1R agonists have been shown to modulate cellular anti-oxidants, including for instance the
NF-?B pathway (Meunier and Hayashi 2010). For instance in a neurodegenerative condition, Hyrskyluoto et al. (2013) reported that PRE084 is able to decrease the levels of ROS and oxidative stress in neuronal PC6.3 cells expressing mutant huntingtin proteins via the up-regulation of NF-?B. The present study therefore confirmed that s1R activity is closely related to mitochondrial physiopathology. In physiological conditions, s1R agonists are able to generate a moderate oxidative stress, through ROS production involving complex I activity. In pathological conditions, such as Aß-induced stress, s1R activity may help to contribute to a rapid restoration of mitochondrial physiology. Detailed analyses on other mitochondrial functional responses and morphological changes by s1R ligands must now be performed.
Acknowledgements

I am not trained in biology or medicine. Nevertheless papers like this needs to be read carefully, which I believe I have and come to the opposite conclusions to those drawn by OFP. That really ought to not happen in science, so one of us are probably wrong.