FO9: Potential Neurotrophic Effects of a Multiuse Drug

Luke Nunnelly, Jesus Campanga, Karen Poksay , Dale E. BredesenAlzheimer’s Drug Discovery Network (ADDN), Buck Institute for Research on Aging, Novato, CA, 94945 USA

Introduction

CONCLUSIONS

REFERENCES

Methods

Coull, JT., Nobre AC., Frith CD (2001, January). The noradrenergic alpha2 agonist clonidine modulates behavioural and neuroanatomical correlates of human attentional orienting and alerting. Cereb Cortex. 11(1):73-84.

Mohr E., Schlegel J., Fabbrini G., Williams J., Mouradian MM., Mann UM., Claus JJ., Fedio P., Chase TN (1989, April). Clonidine treatment of Alzheimer’s disease. Arch Nuerol. 46(4):376-8.

Riekkinen M., Laakso MP., JakalaP (1999). Clonidine impairs sustained attention and memory in Alzheimer’s disease. Neuroscience. 92(3):975-82. 

FO9, an α2 adrenergic agonist, is a multifunctional drug used currently as an anti-hypertensive drug as well as a treatment to subdue ADHD symptoms. Its interaction with the adrenergic system and its anti-hypertensive properties indicate that it works mainly with the vascular system. However, some recent data would indicate that FO9 has a profound effect on key biomarkers associated with the treatment of AD symptoms. Therefore FO9 became a candidate for further study for the ADDN. The goal of my study of FO9 was to better understand the key receptors involved in its ability to reduce toxic biomarkers in In Vitro studies as well as to discern what vehicle works best for the delivery of FO9 in In Vitro studies.

APP Cleavages

D664

CTFβ

APPneo

Drug Testing Flow Scheme

Clinical Compound Library

CHO-7W and SH-SY5Y cell lines = primary screen

J20 (PDAPP) mice primary neurons = secondary screen

J20 mice drug level (PK) and biomarkers (PD) = tertiary screen

AlphaLISA Assay

Multiplexed Assay

Supernatant/media Cell lysate

sAPP, sAPP A42 APPneo, Tau

Β-cleavage

sAPP, sAPP or A42 antibody

γ-cleavage

APPneo antibody

ACKNOWLEDGEMENTS

I would like to thank the entire Bredesen Lab as well as the REU program for this opportunity and their support.

sAPPα/Aβ 1-42 Ratio:Improvement for Doxazosin mesylate and FO9

sAPPα and Aβ1-42 Results[sAPPα]=AU/[Aβ1-42]=pg/mL

CHO 7W Cells treated with 1uM FO9Experiments performed by Jesus Campagna

Past Results

First Test

Second Test

Third Test

Abstract

AlphaLISA kits from PerkinElmer were used to quantify human sAPPα (AL231C), Aβ 1-42 (AL276C) and APPneo (custom) from diluted media samples added to an AlphaPlate-384 (6005350). This technique is a no-wash procedure that is high-throughput and sensitive. Five ul of acceptor bead antibody was added to 2 ul of diluted sample and allowed to incubate for 1 hr at room temp. Next, 5 ul of donor bead antibody was added and allowed to incubate in the dark for 30 min at room temp. Fluorescence was measured on an EnsPire 384-well plate reader (PerkinElmer).

β

α

695

D664

γ

The progression towards Alzheimer’s Disease is a complex system, with a diverse number of contributing factors that ultimately lead to AD symptoms in patients. In mainstream medicine, AD treatment involves attempts to slow a patient’s decline into the symptoms typical of AD pathology. In contrast, the Bredesen Lab attempts to find treatments and drugs capable of curing the disease and returning patients to normal brain function. The key to accomplishing this is the Bredesen Lab’s focus on the cleavages of Amyloid Precursor Protein (APP). APP cleaves in two distinct ways. Trophic cleavages include sAPPα and αCTF and promote the maintenance of synapse and lead to neurite outgrowth. Toxic cleavages include Aβ 1-42, sAPPβ, and Jcasp and lead to neuronal retraction and programmed cell death of neurons. In healthy brains, there is a balance between toxic and trophic cleavages of APP. However, in AD brains, the toxic cleavages of APP become expressed more than the trophic cleavages. The Alzheimer’s Drug Detection Network (ADDN) headed by the Bredesen Lab focuses on searching for drugs capable of switching the cleavage back from the toxic to the trophic. FO9 is one such drug that has been found to be extremely promising. In Vitro tests using SH-SY 5Y neural blastomal cells illustrate this promise, although they are largely inconclusive.

Initially, my In Vitro experiments with FO9 were done using CHO-7W (Chinese Hamster Ovarian) cells that are stably transfected with the expression of human APP. However, these data proved to be un-reliable because of the complex receptor interactions of FO9 and its analogs. FO9 and its family of drugs have interactions with the α1 and α2 adrenergic receptors, the dopamine D2 receptor, the I1 and I2 receptors, and the 5-HT3 receptor. The reason that CHO cells would not work is because, since they are not neuronal cells, it is unclear which receptors are truly present in these cells. Therefore, in order to achieve the most useful and reliable data, I decided to use SH-SY 5Y neural blastomal cells because, although they are cancerous cells, they are neuronal and therefore would more likely have the target receptors desired.I plated the SH-SY 5Y cells in a 48 well plate in a high confluency and then treated overnight with 1uM of drug/control solution. The drugs and controls used as well as the target receptors of each were FO9 in water vehicle (α2), FO9 in DMSO vehicle (α2), old FO9 in DMSO vehicle (α2), Phenoxybenzamine hydrochloride (α1), Doxazosin mesylate (α1), Bromocriptine mesylate (D2), Guanabenz acetate (α2/I2), Moxonidine hydrochloride (I1), B-HT 933 dihydrochloride (α2), B-HT 920 (D2/5-HT3), and two control groups. After an overnight incubation, I burst the cells using 1X αLISA buffer and a freeze/thaw cycle. Using undiluted sample, I ran an ELISA immunoassay to measure Aβ 1-42 levels. Usually, the Aβ levels in SH cells is too low to measure using this technique, but because of the bursting of the cells, we could measure the Aβ not only in the media, but also intracellular Aβ in the ELISA. After diluting the remaining sample, the sAPPα levels were measured using the αLISA immunoassay. Then a ratio of sAPPα/Aβ was formed to determine which one performed best relative to the controls.

In earlier experiments, I determined that there was no difference between the old and the newer stock FO9 solutions. Therefore, for this test, consider the old FO9 another test of the DMSO vehicle.In one of the controls, the Doxazosin mesylate, Phenoxybenzamine hydrochloride, and Bromocriptine mesylate sections of the culture plate, I could not achieve the same confluency as I could on the rest of the plate for the rest of the samples. This lower confluency resulted in a lower concentration of both biomarkers measured in this test. That being said, the ratios were still valid and comparable to the rest of the samples, meaning these data are reliable and useful.

FO9 had a profound positive effect on the sAPPα/Aβ1-42 ratio in this first experiment. Initially, this test

seemed to confirm the potential of FO9. However, the massive

difference does present issues about the reliability of these data.

In this follow up test, FO9 Did not perform well relative to the control

group. In fact, the FO9 treated cells show a decrease in the the

sAPPα/Aβ1-42 ratio, results that are generally considered discouraging.

Once again, FO9 could not recreate the results of the previous

experiment. In this test, the sAPPα/Aβ1-42 ratio increased, once

again demonstrating potential of FO9.

In order to determine the operative receptor, I included several analogs of FO9 in this experiment to act as potential positive controls. Doxazosin mesylate, the α1 adrenergic agonist and the older FO9 in the DMSO vehicle were the only treatments to improve relative to the controls. It can be concluded that the α adrenergic family of receptors is likely the genesis of the improvement of biomarkers, but which one specifically cannot be discerned from these data. However, this test does demonstrate FO9’s ability to improve key biomarkers associated with AD treatment. However, the inconsistency with which FO9 affects the biomarkers. Because of this inconsistency, a clean conclusion cannot be drawn from this experiment. Much like earlier tests (Figure 4), FO9 shows great potential for use as an AD treatment. However, FO9 does not perform consistently enough to be considered an effective treatment just yet.

FUTURE DIRECTIONS

Further In Vitro tests as well as tests on primary hippocampal neurons and In Vivo tests are needed to substantiate FO9’s potential as an AD treatment.