Cancer stem cells- a new target to inhibit tumour growth


One of the keys for drug discovery is to be able to target the diseased pathways/cells without affecting the healthy cell/pathways. This is particularly evident in cancer, where the challenge is to target the cancerous cells without affecting healthy cells. Three recent publications have raised the bar of this for drug discovery. These studies have identified stem cells at the heart of the tumour which appear to be the drivers for certain types of cancer that are resistant to current chemotherapy. Simultaneously providing potentially revolutionary novel targets for cancer, whilst also increasing the challenge to hit these cells without targeting the healthy cells.

To put these findings in context, for some time there has been debate as to whether stem cells sit at the heart of cancers and the role that these cells may have. Up until this point, the evidence for stem cells has mainly been from immunohistochemical staining/FACs sorting and assaying them in vitro. The problem with using these methods is whether the in vivo phenotype is being altered by in vitro culturing. However, by use of lineage tracing, three separate groups (Luis Parada at the South Western Medical School, University of Texas; Cedric Blanpain of the Free University of Brussels and Hans Clevers at the Hubrecht Institute in Utrecht) in different tumours, in the brain, gut and skin, have demonstrated in each, a subpopulation of stem cells that may propagate and spread the cancer.

Glioblastoma multiforme (GMB) is an aggressive tumour that initially responds to chemotherapy however, the cancer nearly always returns. As such GBM is considered incurable and has a median survival of 15 months. In the first of these studies Chen et al., (1) used mice bred to develop GBM, in which they labelled healthy adult neural stem cells, but not their descendants, with a genetic marker. They found all the tumours again contained at least a few labelled cells, along with the unlabelled cells. Chemotherapy with temozolomide killed the unlabelled cells, but the tumours returned. When the animals were tested again, the tumours contained unlabelled cells that came from the labelled stem cells. When they used the chemotherapeutic treatment alongside a technique to supress the labelled stem cells they found the tumours shrank back to “residual vestiges” that bore no resemblance to GBM. Hence, they identified a chemotherapeutically resistant tumour cell that behaved more like stem cells. These cells themselves do not rapidly divide; however, they give rise to rapidly dividing progeny that are susceptible to current chemotherapy.

In a similar way Schepers et al., (3) used genetically engineered mice to label healthy gut cells and stem cells in benign intestinal tumours, a precursors of cancer. These labels carried a drug-inducible marker, that, when activated, fluoresce one of four colours. They found that even though the tumours consist of many different cell types, each tumour fluoresced the same colour, suggesting they arose from one single stem cell. To double check this, the researchers added a lower dose of the drug that caused the stem cells to fluoresce a different colour. In doing so, they demonstrated the stem cells were consistently producing progeny of different cell types.

In the final study Driessens et al., (2) also used linage tracing and identified distinct proliferative cell compartments within a benign papilloma. The majority of the cells had only limited proliferative potential, however a fraction had the capacity to persist long term. The former population gave rise to a terminally differentiated cell population; however, the more persistent population had stem cell-like characteristics and produced many progeny. In addition, as the tumours became more aggressive they were also more likely to produce new stem cells and less likely to produce terminally differentiated cells.

These studies present clear evidence of the existence of cancer stem cells, explaining the re-occurrence of some cancers after successful chemotherapy, but also critically providing new lines of research for drug targeting. Clearly research will now be focussed on killing these cells to eradicate the cancer, however, targeting the stem cell’s proliferative capacity, or encouraging them to differentiate into non-dividing cells may also be as effective.  Since the numbers of stem cells within the tumours appear to be so small and readily able to differentiate, isolating them to study in vitro is unlikely to be fruitful. However, the labelling and tracking the stem cells and their progeny in vivo demonstrated by these groups enable these cells to be studied in vivo and will be vital for drug discovery programmes. This will enable micro-dissection; genomic sequencing and micro-array analysis to be more easily performed on a pure population of cancer stem cells to be used for target identification/validation. In addition by enabling the tracking of these cells and their progeny in vivo the efficacy of any compounds targeted to either kill the stem cells, or prevent the production of progeny could be assed relatively easily.

The challenge of targeting a small cell population that relatively little is known about, without damaging healthy tissue-resident stem cells will be great. However, the research provides great tools to aid this process together with a new way in to provide novel therapeutic agents for cancer therapy.

1.           Chen J, Li Y, Yu T-S, McKay RM, Burns DK, Kernie SG, Parada LF. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature (August 1, 2012). doi: 10.1038/nature11287.

2.           Driessens G, Beck B, Caauwe A, Simons BD, Blanpain C. Defining the mode of tumour growth by clonal analysis. Nature (August 1, 2012). doi: 10.1038/nature11344.

3.           Schepers a. G, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M, Clevers H. Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas. Science 730, 2012.

One-Pot Synthesis of Substituted Ureas Directly from Primary Alcohols


Publications that describe novel and unusual transformations or interesting one-pot procedures always grab my attention and the latest paper by Chi Zang et al. in Synthesis eFirst is a prime example of this.

Zang set out to develop a phosgene and transition metal free, easily handled, and mild method to synthesise substituted ureas. In this paper Zang describes a one-pot transformation of primary alcohols and amines into unsymmetrical ureas (scheme 1). This work builds on and optimises some previous work from Zang’s group where they use iodobenzenedichloride and sodium azide in acetonitrile to convert primary alcohols into carbamoyl azides, albeit in low yields.

After screening a selection of solvents, hypervalent iodine reagents and the number of equivalents of reagents required Zang’s best conditions used 5 equivalents of iodobenzenedichloride and 10 equivalents of sodium azide in ethyl acetate.

To test the generality of these conditions a variety of primary alcohols were converted to their respective carbamoyl azides (table 1). It was found that, regardless of the electronic or steric properties of the alcohols, all of the reactions proceeded smoothly. It is also noteworthy that no racemisation was observed with chiral alkyl alcohol 1h (table 1, entry 8).

With the synthesis of the first stage of the reaction optimised Zang proceeded to test his hypothesis that both symmetrical and unsymmetrical ureas could be synthesised from alcohols. Zang chose 4-methylbenzyl alcohol (1b) and aniline as the model substrates and as expected, 1-phenyl-3-p-tolylurea (3a) was isolated in high yields (table 2). It was also shown that by increasing the number of equivalents of aniline a small improvement in the yield could be achieved as well as a reduction in the reaction time.

Utilising these reaction conditions Zang explored the range of anilines to gage the generality for this new one-pot procedure (table 3). Satisfyingly all of the anilines examined, which included electron poor, electron rich and sterically hindered molecules, produced the desired products in good to excellent yields. Alkyl amines (table 3, entries 11-13) also gave the desired ureas in good yields. Zang acknowledges that 8 equivalents of the amine coupling partner is not ideal but he does explain that a majority of this excess can be recovered by a simple workup and gave the example that 4-bromo-aniline was recovered in a 78% yield (table 3, entry 4).

On the basis of the above experimental results and his previous related work, a stagewise reaction mechanism for the present one-pot transformation of primary alcohols to substituted ureas is proposed: i) oxidation of the primary alcohols to the corresponding aldehydes, ii) conversion of the aldehydes into acyl azides, iii) formation of carbamoyl azides from the acyl azides via Curtius rearrangement and subsequent addition of hydrazoic acid, and iv) transformation of the carbamoyl azides with amines to the corresponding ureas (scheme 2).

 

With this proposed mechanism Zang wanted to understand why the reaction gave higher yields when run in ethyl acetate compared to acetonitrile. He used benzyl alcohol to investigate the kinetic formation of benzaldehyde and benzoyl azide. The studies showed that the benzyl alcohol was completely oxidised to benzaldehyde within 30 minutes at 0 °C in both acetonitrile and ethyl acetate with only trace amounts of benzoyl azide observed. After stirring for a further 4 hours all of the benzaldehyde in ethyl acetate was converted to benzoyl azide but even after stirring for 5 hours only a small proportion of the benzaldehyde in acetonitrile was converted to benzoyl azide. [Bis(azido)iodo]benzene, the active intermediate generated when sodium azide is added iodobenzenedichloride, has been shown to be unstable at 0 °C and to decompose to iodobenzene and nitrogen gas. It was seen that in contrast to the reaction in ethyl acetate when the reaction was run in acetonitrile a large amount of gas was generated from the reaction mixture. A ligand-exchange experiment was undertaken to attempt to understand these differences. These results showed that the speed of the ligand-exchange reaction between iodobenzenedichloride and sodium azide is faster in acetonitrile than that in ethyl acetate; consequently, the concentration of the reactive intermediate [bis(azido)iodo]benzene would be higher in acetonitrile and thus kinetically result in the faster decomposition of [bis(azido)iodo]benzene.

 

In conclusion this paper demonstrates a phosgene and transition metal free, easily handled, and mild one-pot method to synthesise substituted ureas. It also includes a good explanation for the differences in yields that were observed when the reaction was run in different solvents. I believe that this methodology could be modified to synthesise carbamates in one-pot by the addition of an excess of an alcohol (instead of the amine) to the carbamoyl azide intermediate.

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.