18 Jul 2008

PBT2, a zinc/copper ligand that may act like an ionophore, shows dramatic effects on cognition in transgenic mouse models of AD, according to a paper in the July 10 Neuron. Researchers led by Ashley Bush, University of Melbourne, Victoria, Australia, report that the compound significantly reduces brain Aβ within a day of administration, and restores cognitive abilities to rival, or even surpass those of normal mice. “The bottom line is that this orally bioavailable compound is having probably the most potent and fastest effect on transgenic animal models for Alzheimer’s disease that have been yet reported,” said Bush in an interview with ARF. Whether that will translate into similar success in AD patients is still under active investigation. According to Bush, Phase 2 clinical trial data on PBT2 will be released this month at the International Conference on Alzheimer’s Disease.

Exactly how PBT2 works is not clear, but in the Neuron paper, Bush and colleagues demonstrate several actions of the compound that may contribute to its ability to improve cognition in transgenic mice. Joint first authors Paul Adlard, Robert Cherny, and colleagues show that PBT2 can prevent deficits in long-term potentiation in hippocampal slices treated with synthetic Aβ. How this is achieved is not known, but Bush said that Aβ binds best to cell membranes in the presence of copper. This suggests that PBT2 might prevent the peptide from attaching to cellular targets. The researchers also found that PBT2 reduces the generation of hydrogen peroxide by Aβ, which has also been linked to toxicity of the peptide (see Tabner et al., 2003).

In vivo, PBT2 had an impressive effect on learning as judged by performance in the Morris water maze. Given PBT2 (30 mg/Kg), both male and female mice, from two different transgenic strains (APP/PS1 and Tg2576), learned to find the hidden platform much faster than transgenic littermates given a placebo. PBT2-treated transgenics even outperformed wild-type animals. By day six of training, they were finding the platform nearly 20 seconds faster than wild-type animals or transgenics administered clioquinol. Probe trials conducted 24 hours after training showed that PBT2 also had statistically significant effects on retention.

It is not clear how PBT2 works in vivo, either, but the parameter that best parallels the rapid improvement in cognition is reduction in brain interstitial Aβ (AβISF), as measured by microdialysis. In APP/PS1 mice there was also a reduction in insoluble Aβ40 (30 percent), Aβ42 (41 percent), and Aβ43 (35 percent) after 11 days of treatment. However, in identically treated Tg2576 mice, the picture was different. The researchers measured a reduction in insoluble Aβ37 (73 percent), Aβ39 (79 percent), and Aβ2-46 (92 percent). They did observe reductions in Aβ40 and Aβ42, but these were not statistically significant because of one outlier. Also, while reactivity to the A11 antibody that recognizes oligomeric Aβ was significantly lower in APP/PS1 mice, the researchers did not detect any decrease in A11 reactivity in Tg2576 animals. “From our current findings, the toxic mediator of the acute cognitive deficits was most likely AβISF, which was conspicuously decreased by treatment in both strains of Tg mice and in approximate register with the short time frame of cognitive improvement,” write the authors. Interestingly, data overwhelmingly support the idea that a decrease in CSF Aβ is a marker for disease progression. How this relates to a drop in interstitial Aβ elicited by PBT2 remains to be determined.

The authors offer several possibilities for the actions of PBT2 based on its ability to mobilize copper. Capturing copper may reduce redox chemistry associated with Aβ, reduce oligomerization, and restore cellular copper resulting in activation of matrix metalloproteases that degrade Aβ and kinase cascades that regulate tau phosphorylation. They did find reduced insoluble tau in PBT2-treated Tg2576 mice, and reduced phosphorylated tau in PBT2-treated APP/PS1 animals.

PBT2 is a second-generation Zn/Cu chelator based on clioquinol, an 8-hydroxy quinoline derivative originally used as an antibiotic. Prana Biotechnology Ltd., Parkville, Victoria, Australia, of which Bush is a co-founding partner, developed the chelator as a potential treatment for AD based on the premise that reducing copper might attenuate plaque formation and Aβ toxicity, which have been linked to copper-Aβ interactions (see ARF related news story). The story, however, may be more complex than that. In this latest paper Bush and colleagues promote the idea that PBT2 is acting as an ionophore that mobilizes extracellular copper, contributing to post-synaptic uptake of the metal. “That is compatible with many observations,” suggested Thomas Bayer, University of Goettingen, Germany. Bayer, together with Gerd Multhaup, Free University, Berlin, Germany, demonstrated that this may be the mode of action of clioquinol (see Treiber et al., 2004), though Bayer also noted that in their hands clioquinol acts as a broad-based chelator reducing copper in the blood of transgenic animals. “I do not know if PBT2 works in the same fashion or not, but it is based on the clioquinol scaffold, so it needs to be clarified whether it is a broad-based chelator,” Bayer told ARF. The answer may be important for future development since Bayer and colleagues have reported a premature death phenotype in transgenic AD mouse models treated with clioquinol, which may be related to its chelating properties. Bush and colleagues report that PBT2 mobilizes copper and zinc from extracellular medium into M17 neuroblastoma cells and that it is much more potent than clioquinol in this regard. In addition, it has no effect on metal levels (copper, iron, manganese, and zinc) in brain, liver, kidney, or plasma of treated animals. The in vitro and in vivo data support the idea that PBT2 acts more like an ionophore than a chelator.

However PBT2 works, the success of the treatment in animal models really underscores the value of the metals hypothesis of Alzheimer's disease, suggested Bush. “We think that targeting abnormal metal-bound Aβ is a more disease-specific therapeutic strategy than approaches that target all forms of Aβ, and so usefully differentiates our approach from secretase inhibitors and immunotherapy,” he told ARF. Whether the approach will be as successful in humans needs to be evaluated. As Bayer pointed out, there have been many therapies that work very well in animal models but have stumbled when it came to efficacy in humans (see Blennow et al., 2006). He and Multhaup are proponents of the theory that lack of intracellular copper contributes to AD pathology and that administering daily copper supplements may actually help slow or prevent the disease. Clinical trial results just published by that group show that copper given in this manner has very little benefit, however (see Kessler et al., 2008). Bush believes that while copper alone may help transgenic mice, which are copper deficient, in humans merely giving copper will have no effect unless accompanied by some compound that helps it distribute into the brain. The Phase 2 clinical trial data for PBT2 may help decide if that is true.—Tom Fagan

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References

News Citations

1. Two Ways to Attack Amyloid: Metal Chelator and Antibody 22 Nov 2002

Paper Citations

1. Tabner BJ, Turnbill S, El-Agnaf OM, Allsop. Direct Production of Reactive Oxygen Species from Aggregating Proteins and Peptides Implicated in the Pathogenesis of Neurodegenerative Diseases. Curr Med Chem Immun Endoc & Metab Agents. 2003 Dec;3(4):299-308.
2. Treiber C, Simons A, Strauss M, Hafner M, Cappai R, Bayer TA, Multhaup G. Clioquinol mediates copper uptake and counteracts copper efflux activities of the amyloid precursor protein of Alzheimer's disease. J Biol Chem. 2004 Dec 10;279(50):51958-64. PubMed.
3. Blennow K, de Leon MJ, Zetterberg H. Alzheimer's disease. Lancet. 2006 Jul 29;368(9533):387-403. PubMed.
4. Kessler H, Bayer TA, Bach D, Schneider-Axmann T, Supprian T, Herrmann W, Haber M, Multhaup G, Falkai P, Pajonk FG. Intake of copper has no effect on cognition in patients with mild Alzheimer's disease: a pilot phase 2 clinical trial. J Neural Transm. 2008 Aug;115(8):1181-7. PubMed.

External Citations

1. International Conference on Alzheimer’s Disease

Further Reading

News

1. Two Ways to Attack Amyloid: Metal Chelator and Antibody 22 Nov 2002
2. Anti-Amyloid Drug Clears Phase 2a Hurdle 19 Mar 2008
3. Side-Reactions Sideswipe Clioquinol—Prevent Clinical Trial 11 Apr 2005