ENCODE: A new tool for drug discovery?


Only a small proportion (<2%) of the total genome codes for proteins and the remainder had up to now been termed non-coding or ‘junk DNA’. The aim of the ENCODE (Encyclopaedia of DNA elements) project was to attempt to characterize these undefined regions.  The consortium has recently published 30 papers detailing, amongst much data, regions of transcription and regulatory areas that were previously unreported.

One of these papers by Maurano et al., used a technique to map sites of regulatory elements within the DNA and compare these with noncoding variant polymorphisms associated with common diseases that have been identified through genome-wide association studies (GWAS).

The group examined many different cell types including primary cells, immortalized, malignancy derived or pluripotent cell lines, hematopoietic cells, progenitor cells as well as some fetal tissue samples. They used Deoxyribonuclease 1 (DNase1) hypersensitive sites (DHSs) of increased chromatin accessibility as a marker for binding sites of regulatory elements such as transcription factors and thus mapped the regulatory regions in this material. In total, they identified DHS positions spanning 42.2% of the genome, a higher density of regulatory regions than previously appreciated. They then examined the position of single nucleotide polymorphisms (SNPs) identified by GWAS and found a 40% enrichment of these SNPs in DHSs. This analysis shows that the common genetic variants associated with disease are often located at recognition sequences of transcription factors. The authors also demonstrated that these regulatory regions may control the expression of genes that are distant (>250kb) rather than solely the expression of the nearest gene.

Further interesting data from the consortium was obtained through the study of cancer lines. Over 40 cancer lines of different origin were examined and data obtained showing that cancer lines possess regulatory DNA regions that are not present in normal cells (Stamatoyannopoulous, J. A., 2012).

The new information provided by ENCODE is not yet readily applicable to drug discovery, however, this data could provide a map of transcriptional and regulatory regions that could help to identify novel therapeutic targets. In a recent article in Nature Drug Discovery, Michael Snyder one of the principal investigators of the ENCODE consortium explains that changes in gene expression through a change in regulatory sequence could enable identification of proteins that could make useful drug targets.

Applications that could be useful in drug discovery settings include the use of knockdown technologies to screen for biological effects, or zinc finger nuclease technology that can introduce mutations to regulatory elements to determine if changes in these regulatory regions are causal of disease.

Systematic localization of common disease-associated variation in regulatory DNA.

Maurano MT, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H, Reynolds AP, Sandstrom R, Qu H, Brody J, Shafer A, Neri F, Lee K, Kutyavin T, Stehling-Sun S, Johnson AK, Canfield TK, Giste E, Diegel M, Bates D, Hansen RS, Neph S, Sabo PJ, Heimfeld S, Raubitschek A, Ziegler S, Cotsapas C, Sotoodehnia N, Glass I, Sunyaev SR, Kaul R, Stamatoyannopoulos JA.

Science. 2012 Sep 7;337(6099):1190-5. doi: 10.1126/science.1222794. Epub 2012 Sep 5.

What does our genome encode?, Stamatoyannopoulous, J. A. 2012, Genome Research 22: 1602-1611

An audience with Michael Snyder, Nature reviews Drug Discovery Oct 2012. 11: 744

The ENCODE papers are available online at go.nature.com/iN6Ezx.

 

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Faster metabolism


However well new compounds in development perform in vitro, the real confirmation is if they have desired effect in the body, and without major side effects. A key parameter in this understanding is the effect of the body’s metabolism on the compounds. It is highlighted and discussed in this article , where the authors have developed an early method to determine the functional effect of the metabolites formed on the drug target.

The Authors took human H4 receptor ligands which had been well characterised as active inverse agonists in a 384 well functional cell based assay using H4 receptor linked  to a reporter gene (β-Galactosidase ) and incubated them with liver microsomes (containing the cytochrome enzymes). The cytochrome enzymes converted the compounds into their respective metabolites (as would occur in the liver). The metabolites were then separated and identified using a LC/MS (electrospray ionization in positive ion mode). The individual metabolites were then collected and reformatted into separate wells in a microtitre plates. A freeze drying process was employed to remove organic solvents such as acetonitrile and formic acid which were required by the liquid chromatography, and the metabolites were re-solubilised in DMSO. One concern the authors did address is obtaining a full solubilisation of the freeze dried metabolite in the DMSO solution, however the metabolites that they were using had a number of protonated nitrogen atoms and were relatively polar so poor solubility was not an issue in this case.  The authors however suggested if dealing with very non-polar compounds, 10% DMSO could be added before the freeze drying step, which would dry into a DMSO film which would aid with re-solubilisation step. Another suggestion would be to use further analytical techniques such as ELSD (evaporative light scattering detection), to determine the true concentration of the metabolite preparation and therefore correct any activity measurements determine the true concentration curve for the metabolite

Once the metabolites were re-solubilised in DMSO, the author’s re- tested them in the 384 well cell based reporter gene assay.  This allowed determination of the functional response of the metabolites in comparison to parent compounds. With the optimisation of the fraction collection procure, two individual compounds, with a full profiling run from the LS/MS can be screened on a 384 plate. This allows key compounds from structure activity profiled in a timely manner to be profiled.

The results from this work were quite interesting, the first finding was that there was a contaminant in all four of the preps of the compounds, and this contaminant was a histamine receptor antagonist underlining the importance of QC on compounds that you are testing in any drug discovery programme, otherwise structure activity relationships could be mislead. When the individual metabolites were tested, one was shown to be a competitive antagonist compared to its parent compound being an inverse agonist. Again this is important to determine to drive further optimisation of your lead compound.  Other metabolites appeared to be inactive or still have the same functional response as their parent compound.

The key from this study is the process development which allows a fast turnaround of key series in the same assay format used for SAR studies and can be integrated into a screening cascade. That can only help in assisting the drug design process.

Central Nervous System Drug Discovery For Dummies


The Pfizer neuroscience group have published several papers over the recent years which have tried to simplify the complexities involved with successful design of CNS-penetrating drugs.  The CNS MPO papers – ‘Moving beyond Rules: The Development of a Central Nervous System Multiparameter Optimization (CNS MPO) Approach To Enable Alignment of Druglike Properties’ and ‘Defining Desirable Central Nervous System Drug Space through the Alignment of Molecular Properties, in Vitro ADME, and Safety Attributes’ whilst being additions to the battery of rules/guidelines with which to beat medicinal chemists have also provided some practical tools for assessing the ‘CNS drug-likeness’ across a range of potential chemical series and structures in early project phases.

A recent perspective in J Med Chem ‘Demystifying Brain Penetration in Central Nervous System Drug Discovery’ continues this theme, but unlike the papers above, does not provide any significant analysis of data, but instead is a slightly strange article to find in this journal, as it essentially provides a glossary of terms for CNS PK and a reiteration of the basic concepts of CNS drug discovery.  However, that being said, this compilation of terms covering compartments, transporters, assays and general principles should provide a useful recap for anyone working in the field.

The concepts described cover unbound drug concentrations, unbound & total brain-to-plasma ratios, fraction unbound, BBB passive permeability and efflux ratios:

And then explores these parameters across a retrospective analysis of 32 Pfizer CNS clinical drug candidates according to the flow scheme in Figure 2 within the paper.  These compounds partitioned into 14 each in Groups I and II and 4 in Group III.  The flow analysis and subsequent conclusions that Group I are best to progress are not exactly surprising, although it would be interesting to know what confidence building measures enabled the progression of the molecules in Group III…?  The motivation for highlighting this paper, however, lies in the section ‘Clarification of Misconceptions about the BBB’ which is essentially the debunking of 8 CNS drug discovery urban myths which are claimed to be regularly encountered.  This does provide helpful material to combat the continuing obsession with brain/plasma ratios, and reiterates the need to focus instead on the ratio of unbound brain/plasma concentrations as the meaningful parameter against which to optimise.

Additionally, the clarification of the use of CSF is helpful, for which the authors state that the CSF drug concentration can sometimes be a surrogate for unbound drug concentrations in the brain, but these data can be misleading, particularly for drugs which are actively transported (P-gp at blood-CSF barrier pumps into CSF in contrast to P-gp in blood-brain barrier).  Finally, the data from across the Pfizer compound set was used to make the valuable observation that CNS PK in higher species did not increase the confidence of achieving good CNS penetration in man and that the rodent PK alone was sufficient for pre-clinical evaluation.

Cancer Drug Targets: The March of the Lemmings


Whilst at a joint meeting with the ICR Computational Biology and Chemogenomics Team, and the Blundell Bioinformatics Group at Cambridge, this article in Forbes by Bruce Booth was brought to my attention.

This article discussed the current oncology portfolios being developed by major Pharma.  Their analysis illustrates that over 20% of current clinical oncology projects are focused on just 8 targets, each of which has at least 24 projects in clinical development, and further projects in pre-clinical development.

Bruce points out several advantages to this approach, for instance that by developing smarter follow-on molecules, some of the liabilities of the earlier molecules may be diminished possibly improving patient outcomes.  Also drugs based on different chemotypes may lead to differential responses and exhibit different toxicity profiles.  However, he concludes that this focus on a limited range of targets is a waste of resources. Although pursuing established targets may reduce the biological and chemical risk during the early stages of drug development,  “ the differentiation risks skyrocket” – at later stages of development. In particular he highlights that the downstream drugs may fail at a later, more costly stage.

Whilst agreeing that theses are all excellent targets, he suggests that resource should be more focused on the preclinical stages of drug discovery, identifying and validating new cancer targets, rather than chasing incremental improvements in drugs against existing ones.

 

Frances

Eph in ALS


A recent publication from Van Hoecke and colleagues (Van Hoecke et al., 2012) suggests a novel therapeutic approach to the treatment of amyotrophic lateral sclerosis (ALS; also called Lou Gehrig’s disease). ALS is a progressive degenerative disorder that affects 1-2/100,000 people per year and results in death, normally by respiratory failure, 3-5 years after onset. It is caused by a loss of the motor neurons that control muscle movement. There is a hereditary component in about 10% of all ALS cases and in these familial ALS subjects, a variety of genes have been implicated (Al Chalabi et al., 2012), including SOD1, TARDBP and FUS and which encode superoxide dismutase 1 (SOD-1; which was the initial gene reported to be associated with familial ALS in 1993) TAR DNA binding protein (TDP-43) and the Fused in Sarcoma protein, respectively. Recently, a hexanucleotide repeat expansions that occurs in the chromosome 9 open-reading frame 72 gene (C9ORF72) has been described to be associated with ALS with frontotemporal dementia (DeJesus-Hernandez et al, 2011; Renton et al, 2011).

The huge advances in our understanding of the genetics underlying the familial form of ALS have yet to result in breakthrough therapies for this disorder and Riluzole remains the only FDA-approved treatment for ALS. It was approved in 1995 on the basis of clinical studies that demonstrated that it increased survival times in patients, yet the effects are relatively modest and there is a clear need for new and improved treatments for ALS. Since Riluzole was approved, there have been over 30 clinical trials of new treatments but for a variety of reasons (including poor clinical trial design and drug delivery or dose selection issues) none have reached the market, although dexpramipexole, which enhances mitochondrial function, is currently undergoing Phase III trials sponsored by Knapp (Cudcowicz et al, 2011).

A key challenge to the development of new drugs based upon the genetic information derived from familial ALS, as well as genes associated with sporadic ALS, is to understand how mutations in the various genes produce a similar clinical and pathological phenotype. In other words, what is the final common pathway by which these genetic mutations produce ALS? Generic explanations such as mitochondrial dysfunction or alterations in protein degradation pathways have been suggested but how these processes are affected by genetic influences remain vague. However, it is not necessary to understand the mechanism if one can develop a screen that rescues the phenotype produced by different mutations, and this is what Van Hoecke and colleagues did. Hence, they screened for different morpholinos (antisense oligos in which ribose or deoxyribose is replaced by a morpholine ring) that rescued a SOD-1 induced axonopathy in zebra fish. The most protective morpolino targeted the zebra fish Rtk2 gene, which has 67% identity to the human EPHA4 gene that encodes for the Epha4 receptor tyrosine kinase that can bind both type A and type B ephrins. Knock down of the Rtk2 gene rescued the phenotype in zebra fish with various SOD1 mutants (A4V, G37R and G93A) and SOD-1-induced axonopathy could also be rescued pharmacologically by inhibition of Epha4 using 2,5-dimethylpyrrolyl benzoic acid.  Importantly, knockdown of Rtk1, which is a paralog with 83% identity to human Epha4, was able to rescue the axonopathy induced by either mutant SOD-1, TDP-43 or knockdown of Smn1 in zebra fish, indicating that inhibition of EphA4 is protective against motor neuron degeneration irrespective of the genetic determinant of vulnerability. Having identified Epha4 as a potential modifier of SOD1-mediated pathology, the authors also studied the effects of a deletion of the Epha4 gene in mice overexpressing the G93A mutant SOD1 and were able to show that in heterozygotes, a 50% reduction in Epha4 was able to prolong survival.

As regards ALS itself, EphA4 mRNA expression in total blood was inversely collected to the age of onset such that patients with lower levels of EphA4 expression had an age of onset older than those with higher levels of expression. Suggesting that reduced EphA4 expression is associated with a reduced disease severity. Collectively, these data shed light onto an intriguing pathway in which rescue of the axonopathy is achieved irrespective of the genetic cause. A further understanding of the mechanism by which Epha4 exerts these effects could provide the basis for novel therapeutic approaches to treating ALS.

References:

Al-Chalabi, A., Jones, A., Troakes, C., King, A., Al-Sarraj, S. and van den Berg, L.H. (2012) The genetics and neuropathology of amyotrophic lateral sclerosis. Acta Neuropathol., 124:339-352.

Cudkowicz, M., Bozik, M.E., Ingersoll, E.W., Miller, R., Mitsumoto, H., Shefner, J., Moore, D.H., Schoenfeld, D., Mather, J.L., Archibald, D., Sullivan, M., Amburgey, C., Moritz, J. and Gribkoff, V.K. (2011) The effects of dexpramipexole (KNS-760704) in individuals with amyotrophic lateral sclerosis. Nat. Med., 17:1652-1656.

DeJesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., et al. (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron, 72:245-256.

Renton, A.E., Majounie, E., Waite, A., et al., A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron, 72:257-268.

Van Hoecke, A., Schoonaert, L., Lemmens, R., Timmers, M., Staats, K.A., Laird, A.S., Peeters, E., Philips, T., Goris, A., Dubois, B., Andersen, P.M., Al-Chalabi, A., Thijs, V., Turnley, A.M., van Vught, P.W., Veldink, J.H., Hardiman, O., Van Den Bosch, L., Gonzalez-Perez, P., Van Damme, P., Brown, R.H. Jr., van den Berg, L.H. and Robberecht, W. (2012) EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans. Nat. Med., Aug 26. doi: 10.1038/nm.2901. [Epub ahead of print]

Bapineuzumab in Alzheimer’s Disease – Keep Calm and Carry On


The news from Pfizer released on the 23rd July stating that Bapineuzumab failed the first of four Phase III studies in Alzheimer’s Disease (AD) might have induced a wailing and gnashing of teeth and Henny Penny behaviour in certain quarters but now is not the time to panic, the sky is not falling (yet). The press release stated that in a Phase III study of 18-month duration in around 1,100 mild-to-moderate Alzheimer’s Disease (AD) patients, Bapineuzumab failed to meet either of its primary endpoints which were a significant improvement relative to placebo in cognitive performance measured using the Alzheimer’s Disease Assessment Scale-Cognitive subscale (ADAS-Cog) and functional outcomes assessed using the Disability Assessment for Dementia (DAD) scale. Nevertheless, there were signs that Bapineuzumab was having some effect, albeit with respect to side effects rather than efficacy, in that the most commonly observed, treatment-related serious adverse events were the so-called amyloid-related imaging abnormalities-edema (or ARIA-E) observed by MRI.

Bapineuzumab is a humanized version of the mouse monoclonal antibody 3D6 which recognizes the N-terminus of amyloid-β and is administered by intravenous infusion. It is being co-developed by Pfizer and Janssen Alzheimer Immunotherapy (the latter of which is a division of the healthcare giant Johnson and Johnson that acquired rights from Elan – a pharmaceutical company headquartered in Dublin – to co-develop bapineuzumab in September 2009). The rationale is that the small amount of antibody that crosses the blood-brain barrier will bind to amyloid-β within the brain and facilitate its removal by microglial-mediated phagocytosis. It is therefore a passive amyloid-β immunotherapy in which the antibody is administered directly to the patient. This distinguishes Bapineuzumab from a previous active immunization approach with Elan’s AN1792 in which the synthetic, pre-aggregated 42-amino acid amyloid-β peptide was administered along with a immunogenic adjuvant (QS-21) to stimulate an immune response in patients who then themselves produced antibodies against amyloid. Unfortunately, however, 6% of patients developed meningoencephalitis (Orgogozo et al., 2003, Neurology 61:46-54), possibly as a consequence of a pro-inflammatory T-cell response.

Before discussing the implications of the recently-released data on Bapineuzumab, it is worth considering the current state of AD therapeutics. Treatment of the symptoms associated with Alzheimer’s Disease is dominated by the cholinesterase inhibitors donepezil (tradename Aricept), galantamine (Reminyl or Razadyne) and rivastigmine (Exelon) plus the N-methyl-D-aspartate (NMDA) receptor antagonist memantine (Ebixa or Namenda). Although approved for the symptomatic treatment of Alzheimer’s Disease, these cognition-enhancing therapies leave a lot to be desired both in terms of efficacy and tolerability. Consequently, the Holy Grail for the treatment of Alzheimer’s Disease is the prevention or reversal of this debilitating disease of the elderly but unfortunately this remains a distant objective. However, a more achievable goal, a Grail of Significant (if not quite religious) Importance if you will, is that of slowing disease progression. In order to modify the disease process, it is necessary to  understand the underlying pathological processes. In this regard, we are fortunate (although that term always seems inappropriate for such a dreadful disease) to have clues from the pathological hallmarks of the disease which Alois Alzheimer first described over a hundred years ago, namely the extracellular deposits of amyloid that comprise the senile plaque and the intracellular accumulations of hyperphosphorylated tau (a protein that ordinarily plays an important part in maintaining the microtubules that comprise the scaffolding of the neuron).

There is much discussion over the relative importance of the role of amyloid-β peptide versus tau in the disease process, resulting in the moniker of BAPtists (βamyloid peptide-ists) for those believing that the abnormal production of the amyloid-β peptide from the amyloid precursor protein (APP) is the primary pathological process whereas those espousing the importance of the tau protein abnormalities comprise the tauist camp. Although the BAPtist and tauist labels makes for a linguistically convenient division in research activities it is clearly a gross oversimplification since the two pathologies must clearly be linked albeit in a manner that is currently unclear. Nevertheless, it would be fair to say that over the last 20 years or so, the BAPtists have taken the lion’s share of the spotlight based on the convincing genetic evidence that familial AD is associated with mutations in either APP or one of the subunits (either presenilin 1 or presenilin 2) that constitute the γ-secretase enzyme which, along with a second enzyme, the β-site APP cleaving enzyme type 1 (BACE1), plays a role in cleaving APP to produce β-amyloid.

Anyway, back to Bapineuzumab and the pressing question of what the recently-announced failure of the Phase III study with Bapineuzumab actually means for the amyloid hypothesis. Well, not a lot actually. First of all, the data relate to one of four Phase III currently underway and although Study 302 is the first for which data has been publically announced, it is not the key clinical trial. Hence, the AD patients in Study 302 had an ApoE4 genotype yet the Phase II data for Bapineuzumab (Salloway et al., 2009, Neurology, 73:2061-2070) showed that in this patient population, there was no effect; Phase II efficacy was only observed in those patients that did not possess the ApoE4 genotype and it is therefore data from the two Phase III studies with these non-carrier patients (Study 301 primarily based within the US and Study 3000 based primarily outside the US) that are of greatest interest. Data from Studies 301 and 302 will be presented at the Stockholm meeting of the European Federation of Neurological Sciences to be held from 8-11th September so within the next few weeks the future of Bapineuzumab should become clearer.

It is currently an important time for the amyloid hypothesis since Phase III data for the Eli Lilly antibody Solanezumab (also known as LY2062430) is also expected in the very near future. Amyloid antibodies are not all the same and whereas Bapineuzumab targets the N-terminal domain of the amyloid peptide, Sol recognizes the amino acids in the central region of the peptide (Aβ13-28). Moreover, Bapineuzumab binds more strongly to amyloid in plaques rather than soluble amyloid whereas Solanezumab preferentially binds to soluble amyloid-β. This distinction between antibodies can be detected clinically in so far as the fact that although Bapineuzumab produced ARIA-E, no such abnormalities were observed with Solanezumab (Farlow et al., 2012, Alz. Dementia, 8:261-271). Nevertheless, the expectations for Bapineuzumab and Solanezumab are not high. Hence, in June, the news agency Reuters reported that a survey of around 150 investors gave Bapineuzumab odds of about 5:1 for hitting its primary endpoints in the ApoE4 non-carrier Phase III studies whereas Solanezumab got the longer odds of 7:1. Although they may lack a deep scientific understanding of the underlying science, these investors nevertheless give a good indication of the expectations of the Wall Street community. Moreover, this expectation reflects the perception of Bapineuzumab and Solanezumab as being high risk, high reward assets. In other words, both antibodies have a low probability of success but if they do work, then they will justify their huge development costs not only in terms of market size but also, and more importantly, from the patient perspective.

The low expectations for Bapineuzumab and Solanezumab are related in part to the evidence emerging most notably from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) that suggests amyloid deposition occurs very early in the disease process and well before clinical signs appear (Jack et al., 2010, Lancet Neurol., 9:119-128) and that consequently amyloid-related therapeutics need to be targeted much earlier in the disease process (Karran et al., 2011, Nat. Rev. Drug Discov., 10:698-712).  In addition, the probably of success is further tempered by the difficulties inherent in the development of disease-modifying treatments for AD as highlighted by the recent Phase III failures of the Eli Lilly γ-secretase inhibitor Semagacestat (LY450139; Eli Lilly), which actually made cognition worse rather than better (plus it increased the risk of skin cancer), the Russian antihistamine latrepirdine (Dimebon or Dimebolin; Medivation/Pfizer), R-flurbiprofen (Tarenflurbil or Flurizan; Myriad Genetics/Lundbeck), for which Lundbeck signed a $350 million deal barely a month before the clinical data were released and finally homotaurine (Alzhemed or tramiprosate; Neurochem).

Irrespective of the outcome of the Bapineuzumab and Solanezumab trials, the US National Institutes of Health (NIH) announced in May that it will help sponsor the Alzheimer’s Prevention Initiative, which constitutes probably the most rigorous test of the amyloid cascade hypothesis in that it aims to prevent the development of AD in an at-risk population that show no signs of dementia.  This clinical trial, which is scheduled to commence in 2013, is being led by the Banner Alzheimer’s Institute in Phoenix, Arizona and plans to use the Genentech antibody Crenezumab (MABT; licensed from the Swiss company AC Immune) to treat pre-symptomatic members of an extended Colombian family living in and around Medellin. Within this family, there is a high incidence of early-onset AD which is caused by a mutation, E280A, in the presenilin 1 gene that is a part of the γ-secretase complex (Acosta-Baena et al., 2011, Lancet Neurol., 10:213-220). Family members with the mutation start to show cognitive impairment at around age 45 with full dementia developing by about age 51 (New York Times, 15 May, 2012). Crenezumab was chosen in part because it is an IgG4 antibody (Bapineuzumab and Solanezumab are IgG1) that activates microglia enough to help clear β-amyloid but not enough to produce the inflammatory signal that is thought to underlie some of the edema and microhemorrhages seen with other antibodies in clinical development (Adolfsson et al., 2012, J. Neuroscience, 32:9677–9689). The approximately $100 million trial will be funded by a mixture of philanthropic (Banner Institute), public (NIH) and private (Genentech) funding in a roughly $15:$16:$65 million split. This ground-breaking trial will be carried out on 300 hundred members of the 5000-strong Colombian family, with 100 carriers of the mutation receiving drug whereas a further 100 will receive a placebo and an additional 100 non-carriers will receive a placebo, with this latter arm being included since many family members do not want to know if they carrier the genetic mutation for the disease which is called locally La Bobera – the foolishness.

While the Alzheimer’s disease community awaits the outcome of the amyloid antibody Phase III studies with Bapineuzumab and Solanezumab, and despite the challenges in developing disease-modifying drugs for AD, a recent publication describes what could essentially be viewed as clinical proof-of-concept that amyloid lowering agents could be beneficial in the treatment of AD. Thus, Jonsson and colleagues (Jonsson et al., 2012, Nature, in press doi: 10.1038/nature11283) described a mutation in APP that protects against AD in the Icelandic population. Moreover, this mutation was adjacent to the BACE1 cleavage site of APP and in a cellular model, introduction of the mutation into APP resulted in an approximate reduction of 40% in the production of β-amyloid peptide relative to non-mutated APP. These data therefore support the strategy of BACE1 inhibitors as potential therapies for treating AD and imply that a BACE1 inhibition in the region of 40% may be sufficient. This publication is especially timely given the recent description at the Alzheimer’s Association International Conference held between 14-19th July in Vancouver, Canada, at which Eli Lilly, Merck and Eisai all described Phase I clinical data with BACE1 inhibitors (designated LY2886721, MK-8931 and E2609, respectively) (http://www.alzforum.org/new/detail.asp?id=3222). Hence, after more than a decade of struggle, during which time it was considered that it might not be possible to inhibit BACE1 with small molecules that were also brain penetrant and not substrates for P-glycoprotein (which pumps drug out of the brain), it would appear that significant progress is being made.

So, in summary, the recent announcement of the failure of Bapineuzumab to demonstrate any benefit in a Phase III study in ApoE4-positive AD is consistent with the Phase II data and is therefore not unexpected. The critical data in ApoE4 non-carriers should be available in early September. These data plus the additional Bapineuzumab Phase III studies as well as the soon-to-be-announced Phase III data with Solanezumab represent a key fork in the road of AD therapeutics. If the data are positive, then it is full steam ahead down the road to a regulatory filing and the eagerly awaiting AD patient population. If, however, these collective data are negative, the discussion will turn to whether the amyloid hypothesis has actually been tested early enough in the disease process or, alternatively, have side effects limited the doses of Bapineuzumab and Solanezumab to an extent that the clinical failures can be ascribed to shortcomings in  the antibodies themselves. If it is the latter, and given that all therapeutic antibodies are not equal, then this will encourage the further development of additional antibodies such as Gantenerumab (Roche), Ponezumab (Pfizer) and Crenezumab (Genentech) as well as encourage the further development of small molecular inhibitors of BACE1. If, on the other hand, the biomarkers included in the Bapineuzumab and Solanezumab clinical trials (amyloid imaging and CSF amyloid peptide measurements) give confidence that there are significant levels of target engagement, and that modulation of amyloid is not sufficient to produce clinical benefit in AD patients with established symptoms, then there will be a pause at the fork in the road. The AD research community will then have to ponder the signpost pointing down the difficult road towards earlier diagnosis or the equally difficult and poorly lit road towards alternative, non-amyloid (e.g. tau- or ApoE4-related) disease modifying approaches. But such decisions clearly need to be data-driven and so until all the Phase III results for Bapineuzumab and Solanezumab are available, it is prudent at this stage to just keep calm and carry on.