Epigenetics: The sins of the father

In the recent paper in Nature (2014, vol 507, p. 22-24), Virginia Hughes reports the experiments carried out by Dr Dias and Dr Ressler from the University of Atlanta in recent years. They have studied the involvement in inheritance imprints in mice as a result of a fear-based reaction associated to acetophenone. As a result, they found a larger than normal expression of M71 glomeruli receptors in their offspring’s noses. These receptors are encoded by a single gene, known as Olfr151.

This elegant, but still inconclusive cause-effect mechanism approach, brings a possible explanation to a controversial observation back to the 19th century when French biologist Jean-Baptiste Lamark pointed out the pass of acquired traits to future generations. Since then, scientists have observed this phenomenon in plants, animals and even humans.

Although some scientists are still sceptical about the transmitance method, nobody denies the phenomenon. Finding an explanation to this complex problem would involve a deeper study on reproductive biology and to study both mother and father lines over few generations.

The strong suggestion that this heriditary transmission of environmental factors is due to epigenetics, a concept introduced in the 2000’s, where there are some changes in the way that DNA is packed and expressed without altering its sequence, is one of the strong lines of thought, where chemical tags (methylation) on DNA can turn genes on and off.

But even if epigenetics is directly involved in the inheritance, through marks on the material contained in the sperm, the first question to be addressed would be to understand how the effects of environmental/ health legacy get embedded into the animal’s germ cells.

Epigenetics is still unable to explain how this observed phenomenon gets passed down through multiple generations, surviving several rounds of genetic re-programing. Other suggested agents might involve histones (proteins which has been observed that they can be passed down through generations) or short RNA molecules which role would be to latch on DNA and affect further into gene expression.

Scientists are optimistic about finding a cause-effect relationship in the years to come for a phenomenon which has proved elusive for researchers in the past hundred years.

Predicting cancer targets modulated by Ayurvedic medicines

The recent availability of databases that provide both phenotypic descriptions and the chemical structures of the constituent compounds in traditional Chinese and Indian medicines, have enabled Bender et al  (J. Chem. Inf. Model. 2013 (53) 661 – 673, DOI: 10.1021/ci3005513 , http://www.andreasbender.de/) to develop a cool algorithm to predict the mode of action (MOA) of these compounds and to predict novel protein targets for cancer therapies.

Traditional medicine has been utilised by human for thousands of years and normally viewed as complementary or alternative to mainstream therapies.  However, both Chinese and traditional Indian medicine (Ayervedic) have provided us with important drugs for instance Artemisinin an antimalarial drug and reserpine an antihypertensive drug.

From 1981 to 2007, 67% of the pharmaceuticals or new molecular entities (NMEs) introduced into the market were natural product based or a derivative there of.  These natural products often have desirable properties which make them good drugs; they are soluble despite breaking Lipinski’s Rule of Five, they embody privileged structures that are more frequently found to bind a variety of proteins in different organisms, and they are safe and well-tolerated, often having been commonly used for centuries.

However, there are major challenges that need to be resolved that enable the development of a new drug from a traditional medicine.  These include the isolation of the active constituents, the synthesis of the active constituents, the elucidation of the mode of action and finally the development of the compound as a “drug”.

The recent availability of databases that provide chemical structures and their corresponding phenotypes have enabled Bender et al to predict the MOA of compounds found in TCM and Ayurveda addressing one of these major challenges.  First they developed a classifier using bioactive compounds from the ChEMBL database, ChEMBL biological targets, ECFP_4 fingerprints for each compound and a Naïve Bayes classifier.


Figure 1: The compounds were represented using the Extended Connectivity Fingerprints, with a diameter of 4 bonds ECFP_4.  The ECPF is derived from the Morgan algorithm and was implemented in Sitegic’s Pipeline Pilot (Accelrys Inc).  Each atom identifier contains topological information on the atom that includes the number of immediate heavy atoms, the atom’s mass, its charge, the number of hydrogens attached, the valance minus the number of hydrogens and whether it is part of a ring.

This was used to predict which compound (fingerprint) would inhibit each protein target.  Then by creating fingerprints for each traditional medicine compound they could predict which protein targets they would hit.  For example they predicted the protein targets for some of the active ingredients of Panaz ginseng

FP5Figure 2:  Predicted targets for some of the active ingredients in Panax Ginseng

Next they correlated different proteins targets with different phenotypes. Predicting which molecular targets were modulated by the compounds in each different phenotype.  This enabled them to identify the protein targets most frequently modulated by Ayurvedic medicines, with possible anti-tumour effects.  The 10 most enriched protein targets are shown in the table below. The progesterone receptor currently has over 10 inhibitors with FDA approval.  Other proteins identified by this methods include regulators of other well-known cancer targets.


Figure 3:  Top 10 cancer targets in predicted to be inhibited by Ayurvedic medicines.

Targeting leukaemias with a therapeutic human antibody

Currently marketed anticancer monoclonal antibodies (mAbs) recognise extracellular proteins or those expressed on the cell surface. Generally these are not tumour-specific, as oncogenic proteins tend to be nuclear or cytoplasmic. These intracellular proteins can however, be presented on the cell surface as T cell epitopes by the major histocompatibility complex (MHC). These epitopes are recognised by T cell receptors (TCRs). Therefore, the generation of ‘TCR-like’ mAbs that can recognise cell surface epitopes that are derived from tumours are an exciting potential cancer therapy.

Dao et al., recently published a paper in Science Translational Medicine, in which they have exploited phage display technology to produce a specific mAb (ESK1) against Wilms tumor 1 (WT1), an oncoprotein that is overexpressed in both leukaemia and a range of solid tumours such as ovarian cancer and mesothelioma, but is rare in normal tissues. It was also recently ranked as a top cancer target for immunotherapy by the NIH. ESK1 targets a 9mer WT1 derived peptide (RMF) that is processed and presented by HLA-A0201. RMF induces cytotoxic T cells that are able to kill WT1+ tumour cells in vitro.

ESK1 was shown to bind to acute myeloid leukaemia (AML) CD34+/CD33+ cells expressing HLA-A02 and WT1 but not to normal peripheral blood mononuclear cells. The specificity of the binding was confirmed with other cell types with no general cross-reactivity to healthy or leukemic cells that do not express WT1. Radioimmunoassay experiments then demonstrated that there was adequate RMF expressed on the surface of many cancer and leukaemia cells, whilst the levels of epitope on WT1 negative healthy cells are low.

mAbs can cause cytotoxicity in four ways: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), antibody-dependent cellular phagocytosis (ADCP) and inducing apoptosis. ESK1 was shown to be active in ADCC assays against the following cell types: JMN mesothelioma, BV173 leukaemia, ovarian carcinoma, colon carcinoma cell lines and AML cells. No other form of mAb-mediated cytotoxicity was observed.

The efficacy of ESK1 was then tested in vivo in mice that had been xenografted with BV173 acute lymphoblastic leukaemia (ALL) cells or BA25 acute lymphocytic leukaemia. Two intravenous doses of 100ug of ESK1, when administered in conjunction with human effector cells, suppressed the growth of  leukemic cells in both animal models. Prolonged or, in some cases, leukaemia-free survival were observed. Two control studies confirmed that the RMF/A2 epitope was required for the therapeutic effect observed. Further, no evidence of toxicity was observed in transgenic mice when given therapeutic doses of ESK1.


Survival of NSG mice with BV173 leukaemia. **P<0.01 for all treatment groups compared to untreated control animals or animals treated with isotype control hIgG (log-rank Mantel-Cox test).

The ESK1 antibody could therefore be promising as a new cancer drug, with a large clinical impact for those patients with WT1+ tumours or leukaemia with HLA-A02 expression.


Aggregation false positives in cell based assays?

An article dealing with the common problem of compounds that are false positives in screening assays was published recently (http://www.ncbi.nlm.nih.gov/pubmed/23437772).  One cause of compounds acting as false positives in screening assays is that they can self-aggregate, forming colloidal particles. This aggregation effectively sequesters the protein from its target and prevents activity. This has been a common problem in drug screening assays, particularly with soluble protein methods. In this publication the Shoichet lab at University of California, and others, have been investigating a further scope of the problem by examining  GPCR assays using a cell based format.

They took four compounds that were known to form aggregates and measured the activity against a variety of receptors using the Beta- Arrestin assay. The results show that these compounds were acting as antagonists against the receptors when they were stimulated with their agonist ligand, and this activity could be reversed with the addition of detergent or the use of centrifugation.

They also observed inverse agonism when the compounds were tested against the receptor in the absence of the activating ligand of the receptor, maybe via membrane perturbation.

It all highlights a type of assay artefact, which was thought to be more prevalent in soluble protein assays, can also have a bearing in cell based formats.The steps show by the authors (centrifugation and detergent usage) should be included to reduce the chance of false positives even if you are using a cell based method.

gw1Figure extracted from: Sassano, M. F., Doak, A. K., Roth, B. L., & Shoichet, B. K. (2013). Colloidal aggregation causes inhibition of g protein-coupled receptors. Journal of medicinal chemistry, 56(6), 2406–14. doi:10.1021/jm301749y


How do molecules get into cells…?…the continuing debate

Prompted by a number of recent comments in meetings and blogs, although the paper came out last year it is worth highlighting again the strongly held and opposing opinions on this topic.

If you pick up Drug Discovery Today from 2011, and read the Kell and Dobson article, you would be forgiven for thinking that you had missed a significant development in the understanding of diffusion into cells, and immediately start disregarding all the passive permeability data you’re generating.  Essentially, the argument in this paper, forcefully made and apparently with no clear challenge or comment during review & revision, is that passive diffusion plays no significant contribution to the entry of molecules into cells.  Instead, all drugs are actively transported by a diverse range of transporters – some know and some yet to be identified as illustrated by figure from paper below:


This paper is at pains to refute an article in Drug Discovery Reviews the previous year discussing the co-existence of passive and active transport mechanisms.  The argument is based on the observation that when comparing the rates of drug transport in natural versus artificial membranes, there are discrepancies of over 100x, with the natural membranes demonstrating higher permeability.  The paper very appropriately points out the pitfalls of blindly using Caco-2 and/or MDCK systems and highlights numerous examples of drugs which don’t fit to the widely held views.

All these points were strongly (and convincingly) made, and some have read this paper and associated references, and taken all the points strongly to heart.

However, the co-existers rose up and fought back in the same journal, publishing an article the following year which is essentially a rebuttal of all the Kell and Dobson claims and a reassertion of the 2010 DDR paper.  In particular, it makes a strong re-assertion that passive diffusion is the major mechanism for blood-brain barrier permeation of lipophilic small molecules (Figure below from article; ref 31 = Tsinman, Pharm Res, 2011, 337).


These arguments look reasoned and balanced, and importantly are based on considerable data. The door is still open to the presence of many other active transport processes, as well as to the approximate nature of current permeability screening assays, however a complete disregard of passive diffusion appears premature at best.

These papers have been already well covered by the excellent critique by Derek Lowe, however, the lack of awareness across some of our community of the existence of this debate and the uncompromising nature of the language in both papers circulating today (and the fun of seeing groups slogging it out over the pages of Drug Discovery Today) warranted a second airing…

Promising New Frontiers for RNAi Therapy

In 2001 Elbashir and Tuschl (1) published they had managed to silence gene expression in mammalian cells using small interfering RNA (siRNA). This was the catalyst for an explosion of research using siRNA to demonstrate the effect of knocking out targets without the need to identify compounds capable of doing this job. Potentially highly specific, the opportunity to use these as therapeutics was rapidly explored. Ten years later the first clinical trials are coming through; impressively fast.

Probably the largest obstacle to therapeutic siRNA has been delivery, which often is either toxic, or not very effective – as any of us who have tried to transfect siRNA into primary cells will be able to appreciate. Advances in materials science are seemingly solving this problem encapsulating siRNA in nanoparticles, resulting in safe and effective delivery. CALAA-01 (Calando Pharmaceuticals) lead the way  being the first to deliver siRNA therapeutically demonstrating phase I efficacy and safety, as well as localisation to melanoma metastases.

One particular advantage of siRNA is that once delivery has been optimised, it is possible for several different siRNAs for different targets to be contained within on package. This could enable simultaneous delivery of different targets simultaneously effecting different aspects of the same disease, i.e. metastasis and well as tumour growth. By also hitting a known resistance pathway this duel delivery could enable the chemotherapy to be more effective.

A recently published article (2) has done just this, delivering two siRNAs in lipid nanoparticles (known as ALN-VSP) for both VEGF-A (vascular endothelial growth factor-A) and kinesin spindle protein (KSP) for the treatment of advanced solid tumours with liver metastases.  KSP is involved in cancer proliferation and VEGF-A in the growth of new blood vessels.

The study was phase I, dose escalation, on patients who had already been heavily pre-treated with chemotherapy and/or anti VEGF/VEGFR agents with the ALN-VSP administered as 15 minute IV every 2 weeks for 1 month.

The safety profile was very encouraging with ALN-VSP being generally well-tolerated with mainly low-grade fatigue, nausea and fever noted in 15-24% of patients. The lipid nanoparticle of ALN-VSP distributes primarily to the liver and spleen and the delivery was also excellent. Liver biopsies were performed on 12 patients before the first dose and then at 2 and 7 days post dose. qPCR identified VEGF siRNA present in all 12 patients and KSP siRNA present in 11 of the 12.

Of the patients treated, 7 had no disease progression (measured by computerized tomography [CT] scan) after the treatment cycles and continued onto an extension study. One patient in particular with endometrial cancer achieved a complete response after 20 months of treatment

This is clearly fantastic progress for ALN-VSP, and specifically for the handful of patients who were positively affected from participation in the trial. The results from this study also demonstrate the ability for safe delivery of multiple siRNA to specific sites tumour and this extends the promising start for these methods of siRNA delivery which may open up previously un-druggable targets.


1.           Elbashir SM, Harborth J, Lendeckel W, Yalcin a, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–8, 2001.

2.           Tabernero J, Shapiro GI, Lorusso PM, Cervantes A, Schwartz GK, Weiss GJ, Paz-Ares L, Cho DC, Infante JR, Alsina M, Gounder MM, Falzone R, Harrop J, Seila White AC, Toudjarska I, Bumcrot D, Meyers RE, Hinkle G, Svrzikapa N, Hutabarat RM, Clausen V a, Cehelsky J, Nochur S V, Gamba-Vitalo C, Vaishnaw AK, Sah DWY, Gollob J a, Burris H a. First-in-Man Trial of an RNA Interference Therapeutic Targeting VEGF and KSP in Cancer Patients with Liver Involvement. Cancer discovery (January 28, 2013). doi: 10.1158/2159-8290.CD-12-0429.




A Staple Diet for Peptide Mimetics

Among other things, the PPI-net conference at the Royal Society in London earlier this year gave some interesting insight into different techniques that researchers are using to synthesize stable proteomimetics. One such technique makes use of hydrocarbon ‘staples’ between amino acids along one side of an α-helical polypeptide in order to fix its conformation as well as provide some protection against proteases when in vivo.

Intramolecular bridging of α-helical peptides has been attempted in many different ways, such as lactams or disulfide bridges. In 1998, Grubbs developed the use of ring closing metathesis to form peptide ‘staples’, which were both stable and relatively easy to access synthetically. Since then, this technique has been refined so that hydrocarbon staples can be incorporated into known α-helices, allowing for relatively stable competitors for therapeutically targeted PPI’s.

TM01Scheme 1: RCM by Grubbs catalyst, to form an i, i+4 stapled peptide

 This technique was pioneered by Korsmeyer, who in 2004 published a paper detailing the use of stapled peptides to target the BH3 domain on BCL-2 family proteins. Korsmeyer was able to create peptides with nanomolar binding constants (KD = 38.8 nM), which proved to be both protease-resistant as well as exhibiting good bioavailability when tested in vivo. Other PPI’s have since been targeted by this technique, such as another ‘holy grail’ of cancer therapeutics, the p53-HDM2/HDMX interaction.


Figure 1: Binding affinity of stapled peptides to Bcl-x

More recently, some creative use of this chemistry has allowed the synthesis of stapled helices with some interesting physical properties. One such example is the work done by Rudolf Allemann at the University of Cardiff. Allemann takes advantage of the physical properties of azobenzenes, whereby they are able to undergo light induced trans-cis isomerization. By incorporating the azobenzene functionality into an intramolecular peptide bridge, this property can be transferred onto the helix. By attaching the staple in an i, i+7 conformation (or i, i+4 for shorter sequences), the helix will be stabilized when in the cis conformation, and therefore activated light energy. Alternatively, by using an i, i+11 it is the trans conformation has increased stability, and the protein is therefore deactivated by light.

TM03Scheme 2: Trans-cis isomerization of azobenzene peptide bridges

Experimentally, this technique has been shown to work using in vitro protein assays on a range of proteins. The most notable of which is probably our old friend the BH3 domain, this time using Bcl-x as the target protein. Three different photocontrollable peptides were synthesized by modification of a known Bcl-x inhibitor, using an azobenzene bridge in the i, i+7, i, i+11, and i, i+4 positions. Each three of these were shown to have activity dependent on being in either the trans or cis conformation. The i, i+7 and i, i+4 bridged peptides both showed around 20-fold increase in activity after irradiation with light, with the i, i+4 shifting from a KD [nM] of 1275 ± 139 to 55 ± 4 after isomerization.

TM04Table 1: Binding affinities of photocontrollable Bcl-x inhibitors

Although it is not immediately obvious on face value how this work could be transferred into the therapeutic environment, it provides an interesting example of how peptide staples can provide more than just stabilization of the parent α-helix.

Safe azide or oxymoron?

The Huisgen cycloaddition or ‘click chemistry’ could certainly take part of the blame for the resurgence of one of the bad boys of organic chemistry: azide. Coming from a large – Risk averse – pharma, I have always tried to avoid such fragments, possibly due to the implications and paperwork if (or more likely when) it goes wrong. So when an article with ‘Azide’, ‘Facile’ and ‘Safe’ is published it’s certainly worth reading.

The article from Wang’s group is looking at the diazotransfer reaction converting a primary amine to the corresponding azide.

The pre-Goddard-Borger and Stick era was using triflate azides as diazotransfer reagents which proved to be prone to explosion (Figure 1).

azide1Figure 1

The Wang article here is looking at a safe protocol to the imidazole-1-sulfonyl azide (compound 4, Figure 2) reagents developed and optimised over the last 6 years.

azide2Figure 2

Key requirements to the described ‘safe’ route were to avoid the presence of NaN3 with strong acids, minimise the excess of NaN3 and avoid the formation of explosive intermediates.  Previously reported procedures to prepare diazotransfer reagents such as those depicted in Figure 2 all seem to engage sulfonyl chloride, leading to the generation of (N3)2SO2 as a highly explosive byproduct. Wang starts from sulfuryl diimidazole which after mono methylation is treated with NaN3 to give the  imidazole sulfuryl azide reagent (Figure 3).


Figure 3

Worth noting that dimethylation of the sulfuryl diimidazole is not observed, so no highly explosive (N3)2SO2 species were observed and that sulfuryl diimidazole itself proved unreactive with NaN3 (Figure 4).

azide4Figure 4

Furthermore, Wang report that the aqueous conditions the reaction is performed in prevent the formation of the explosive (N3)2SO2 intermediate from the diazotransfer reagent itself (Figure 5).


Figure 5

Does that make the whole process safe? What about stability and storage? Wang et al. prepared the  imidazole-1-sulfonyl azide (compound 4, Figure 2) in over 100g scale but seems to have used it in-situ….

Imidazole-1-sulfonyl azide (the preferred diazotransfer reagent from the Figure 2 bunch) is usually prepared as a HCl salt. The ‘safety update’ from Goddard-Borger and Stick, published in 2011 as a follow up to their original 2007 ‘shelf-stable’ imidazole-1-sulfonyl azide, reports that ‘imidazole-1-sulfonyl azide hydrochloride is hygroscopic and reacts slowly with water to produce hydrazoic acid. Concentration of the mother liquors from which imidazole-1-sulfonyl azide hydrochloride crystallised has resulted in an explosion. This solution may contain sulfonyl diazides and/or hydrazoic acid byproducts which are both extremely sensitive, explosive substances’.


The Sejer group seems to work regularly with such diazotransfer reagents and reports that ‘rigorous drying of the HCl salt of imidazole-1-sulfonyl azide followed by storage at -20⁰C makes it stable for >1 year’ and that tetrafluoroborate and hydrogensulfate salts of imidazole-1-sulfonyl azide were found to be much better with respect to shelf life.


Back to Wang et al.’s conclusion that the protocol can be applied to large scale preparation in both academia and industry…. I will ensure it’s on my day off!

Novel method for assessing cardiotoxicity

One of the most common reasons for failure of compounds in drug discovery programmes is cardiac toxicity. Therefore assessing this in the early stages of any drug discovery programme is key. Currently the earliest indicator for cadio-tox is interaction between compounds and a voltage gated potassium channel hERG (the human Ether-à-go-go-Related Gene) channels. Generally electrophysiological techniques are used in hERG, although fluorescent-based kits are now available.  Although hERG is the most common interaction for cardiotoxicity, this does not measure other potential interactions that could result in cardiotox. Sirenko et al. have taken advantage of the recent emergence of inducible pluripotent stem cells (iPSC) to explore the effects of compounds on cardiomyocytes in more detail.

Culture of iPSC is not as straightforward as immortalised cells, however the cells were obtained from a commercial source (Cellular Dynamics) saving the differentiation process and these cells are able to be reproducibly differentiated into a large quantity and cryopreserved until use. The use of iPSC derived cardiomyocytes for toxicity testing has the clear advantage that these cells express full cardiomyocyte functionality and even have the beating characteristic expected of primary cardiomyocytes. Since this beating is analogous to the beating of a heart, and ion channel block can present as drug-induced arrhythmias, it is not hard to see how this can be used to identify cardiotoxic compounds. At this point most groups would have struggled to have the technology available to analyse these beatings, but since the authors were from Molecular Devices, they conveniently had a FLIPR tetra and image xpress high content screening (HCS) platform along with the technical knowledge to enable these beatings to be quantified.

Sirenko et al. were able to quantify these beatings on both systems. On the HCS, they used time-lapse images and quantified the beating using their own differential algorithm that measured the number of beats per minute. On the FLIPR, they used the Calcium 5 Assay Kit which enabled the beating to be measured using Ca2+ influx in beats/min. Since the HCS could not acquire images as quickly as the FLIPR, the HCS approach was less sensitive than using the FLIPR and less work was performed using this method, however, they were still able to obtain IC50’s for known agonists adrenaline and isoproterenol.

The increased sensitivity and throughput possible using the FLIPR, enabled more studies to be performed using it. As such compound-induced dose-dependent atypical beating patterns induced by known cardio-toxic compounds such as hERG, Ca2+ and Na+ channel blockers were measured. They were also able to validate an automated analysis algorithm that was used to calculate the effect of a variety of compounds in high-throughput format, with positive or negative chronographic effects such as digoxin and propranolol. Furthermore, due to the increased sensitivity of the FLIPR it was also possible to quantify components of each individual Ca2+ peak (i.e. amplitude, peak width, decay time etc.) rather than just the number of beats. This meant they were able to measure more compound specific effects that have more physiological deviations such as QT prolongation, i.e. cisapride that increased both peak spacing and peak width.

A large number of drug withdrawals from the market as well as late stage clinical trials are from cardiac toxicity. These withdrawals are clearly very expensive, but more importantly potentially very damaging to health. The use of hERG testing clearly identifies many cardiotoxic compounds, however, the use of iPSC cardiomyocytes and measuring Ca2+ influx and beating enables many more facets of cardiac toxicity to be measured. The study by Sirenko demonstrates the use of this technique to identify compounds already known to effect different facets of cardiac toxicity. The real test, however, would be to put compounds that were withdrawn from the market, or clinical trials due to cardiac toxicity that were missed by other tox screens. If the technique set out by Sirenko et al., were to have picked up this toxicity it would demonstrate a step forward in early determination.

Amyloid in Alzheimer’s Disease – The End of the Beginning or the Beginning of the End?

In terms of drug discovery, there are four general ways of identifying new drugs: 1) there is serendipity, where a chance preclinical or clinical observation is translated into a novel therapeutic (with the initially cardiovascular Viagra being an example, the clinical utility of which you could quite literally hang your hat on); 2) Iteration, in which a new drug is an improvement upon an existing drug (e.g. reduced side effects or better pharmacokinetics); 3) repositioning, whereby a drug approved or initially evaluated for Indication A proves efficacious in Indication B and finally; 4) hypothesis-driven drug discovery, in which drugs are targeted towards a pathway or protein specifically implicated (e.g. genetically or pathologically) in a disease process. This latter process is the most rational and intellectually satisfying and forms the basis of the multiple amyloid-related approaches to treating Alzheimer’s disease since the amyloid core at the centre of the hallmark senile plaques  as well as the genetics of familial cases of AD all point the scientific finger of guilt towards the amyloid pathway.

As regards the amyloid hypothesis of Alzheimer’s Disease, it is now a few months ago that we discussed the big summer of data that lay ahead with Bapineuzumab and Solanezumab. Well, the data has now been chewed over and digested and as the year draws to a close, it is a good time to be reflective and assess where the field stands. So, in alphabetical, chronological and clarity of what-happens-next? order let us first consider Bapineuzumab. The data for the first Phase III study, Study 301, was disappointing but not surprising since the AD patients were the ApoE4-carrier subpopulation that the Phase II study suggested were less susceptible to the potential benefits. Hopes were therefore pinned on data from the ApoE4-noncarrier Study 302. However, these data were unambiguously negative (follow the links for data of Studies 301 and 302 presented at the September meeting of the European Federation of Neurological Societies) with the complete lack of ambiguity resulting in the termination of the two additional ex-US and incomplete studies (Studies 3000 and 3001). So, all-in-all, quite an emphatic end of story for i.v. Bapineuzumab.

The story for Eli Lilly’s Solaneuzumab is, however, not quite so clear cut. To recap, the EXPEDITION1  and EXPEDITION2 were pivotal Phase III studies. The EXPEDITION1 study missed its primary end-point but showed a significant effect on a secondary end-point, namely cognition in mild AD. This secondary end-point was then used as the primary end-point in the EXPEDITION2 study, but there were no significant effects of Solanezumab. However, combining data from these 2 studies – one of which showed efficacy, the other one not – showed a significant effect in mild but not more advanced moderate AD patients. This is entirely consistent with the way the field has been moving, namely that amyloid-related treatments need to be as early in the disease process as possible. There was a brief flirtation with the prospect that because of the large unmet need regulators may find a way to approving Solanezumab based on the existing data. However, subsequent to discussions with the FDA, Eli Lilly accept that approval would require a new Phase III study in mild AD patients, although their press release does note that “It is possible that different courses of action could be taken in different jurisdictions.”. Given their recent run of bad luck with neuroscience Phase III compounds (the γ-secretase inhibitor Semagacestat in 2010 and then this year the mGlu2/3 agonist pomaglumetad methionil and Solanezumab), one can only admire the depth of the company’s neuroscience financial trouser pocket and their obvious commitment to the area.

If the Solaneuzumab data tells us that treating earlier is the way to go, then the ultimate extension of this approach is a prevention trial. Such a trial, which commences in the spring of 2013 and is being organised by the Banner Alzheimer’s Institute in Phoenix, is being conducted as part of the 5-year, $100 million Alzheimer’s Prevention Initiative and will focus on a family in Columbia with genetic mutation associated with on onset of Alzheimer’s disease in their late 40s. Subjects with the mutation will receive Crenezumab, an anti-amyloid antibody developed by Roche/Genentech and licensed from the Swiss company AC Immune. An additional “branch study” will also take place in the US and will include an additional 150 US mutation carriers. A second prevention trial is also due to start in early 2013 and will be conducted by the Dominantly Inherited Alzheimer Network Trials Unit (DIAN TU) and will evaluate the effects of three different drugs on subjects (160 carriers and 80 non-carriers) with AD-causing mutations.  These three drugs were selected from the more than a dozen drugs proposed by the 10 pharmaceutical companies that comprise the DIAN Pharma Consortium and include the anti-amyloid antibodies Solanezumab and Gantenerumab, a Roche antibody currently in a Phase III trial for very early, presymptomatic (prodromal) AD known exotically as SCarlet RoAD, with a third drug, the Lilly BACE inhibitor currently in Phase II, also being selected for potential inclusion.

So, there remains life in the amyloid hypothesis. But what about other approaches? Well, as we mentioned at the top of this article, drug repositioning (or drug repurposing) is an attractive potential alternative since it is a route accessible to research councils and academic centres (i.e., it lacks the huge development costs of novel therapies). Recently, a number of drugs currently in clinical use for other indications have been shown to have an effect on amyloid metabolism or the associated neuroinflammatory response in animal models, including, for instance, the anticancer (cutaneous T-cell lymphoma) drug Bexarotene, the antiepileptic drug Levetiracetam and the blood pressure drug Prazosin. Nevertheless, the extrapolation between effects in animal models and human is a large and tenuous one with, for example, Rosiglitazone producing marked effects in transgenic mice but there were no signs of efficacy in two  Phase III studies. So, despite claims that “Drug giants give up on Alzheimer’s cure” (The Independent, 19th September, 2012) it would appear that there still remains a major commitment to the development of new therapeutics for Alzheimer’s Disease and that recent developments in the field represent the end of the beginning rather than the beginning of the end.