. Imatinib treatment and Aβ42 in humans. Alzheimers Dement. 2013 Dec 10; PubMed.

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  1. We welcome a discussion on the problem with irreproducibility of published research that we recently encountered, as outlined in the Alzforum news story. In this context, the recent editorial guidelines from Nature (1) might represent one step to reduce this problem. A checklist for methodological and statistical information, together with unlimited space for methods, statistical description, and access to raw data, are commendable. In our mind, however, at least two problems remain. One is related to the difference in the requirement of independent replication between clinical and preclinical research, another to the technical requirements on the methods used for quantifying biochemical changes. Due to the issue with poor replication of results, Nature Genetics stipulated that novel genetic associations must be verified in at least two independent cohorts (2). Correspondingly, replication in independent cohorts is a core feature of any high-quality publication in clinical biomarker research (3). In contrast, in high-impact basic research journals, publication is common with very small numbers of inbred animals or cell experiments, without replication in independent sets. One example is the imatinib/GSAP paper (4), in which many critical conclusions were based on n=3 experiments, a number that actually is too low to do statistical calculations. We appreciate that the variability in animal models may be lower than in clinical samples, but at the same time these models may be highly skewed and not representative of the clinical disease they are supposed to reflect (5). To reduce the problem with irreproducibility, any finding should be replicated in two independent disease models, or at least two independent experiments with a number of cell cultures or animals that allows proper statistical testing. Another bias is related to the methodology used for quantification. Clinical studies often include validated methods and provide data on assay performance, e.g., between-run variability. In contrast, it is common in basic research to use non-validated semi-quantitative methods, e.g., Western blot, without presenting any information on method precision, variability, or specificity. For this, again, the imatinib/GSAP paper can serve as an example (4). In our view, such low-quality quantitative data may underlie irreproducibility, yet high-impact journals are full of them. We believe that addressing these two problems, in addition to the actions presented in the recent editorial, will solve a large part of the problem with irreproducible data from preclinical research.

    View all comments by Kaj Blennow
  2. I have not tried to replicate He et al. 2010 myself, so I can only comment based on my readings about this topic. I am particularly interested in the regulation of γ-secretase, and carefully followed the research about GSAP. Two papers report a lack of interaction between GSAP and APP-CTFβ, also called C99 (Hussain et al., 2013, and Deatherage et al., 2012). Moreover, Hussain et al. report that GSAP has no effect on γ-secretase mediated ε cleavages of both APP and Notch (Fig. 5 and 6). Finally, Hussain et al. report that imatinib does not lower Aβ in rats in vivo. However, while we were particularly interested in the findings by He et al., we mentioned in a recent review (Barthet et al., 2012) some discrepancies found in the paper. Most notably, the authors reported that GSAP stimulates production of Aβ while it reduces AICD, the product of γ-secretase cleavage of APP at the ε site, a phenomenon that is difficult to conceive. Finally, the paper by Olsson et al. in Alzheimer’s & Dementia reports that imatinib does not reduce Aβ in humans. All together, three independent groups report evidence that raises doubts about the effects of imatinib and GSAP on γ-secretase.

    View all comments by Gael Barthet
  3. I did not try to reproduce He et al. 2010 myself but agree with the shortcomings of this data as reported in this story. If patient data suggest there will be no efficacy, I question whether it is worth resolving the remaining data discrepancies at this point.

    View all comments by Raphael Kopan
  4. Unfortunately, many studies in the AD field have failed to be replicated and it is then difficult to get these data published. Journals tend to be reluctant to publish out of concern that failed replication studies might tarnish their image. I have been moderately successful in publishing failed replications, but it takes an enormous effort. Without such publication, however, the original paper continues to get cited, often with a reference to the effect of “but these findings are somewhat controversial." We should also acknowledge a real fear of retaliation in our community. Given very competitive NIH funding and promotions on the line, not everyone wants to antagonize a colleague who might be a future reviewer, even if it is the right thing to do.

    View all comments by Sangram Sisodia
  5. The irreproducibility of the literature data also impacts the speed and cost of discovering new therapies. The commitment to start an industry drug-discovery program is an expensive proposition, and risky if based only on the scientific literature because of the knowledge gaps created by the bias against publishing negative and contradictory findings. The biology is rarely as straightforward as presented in the first few publications on a new target. Thus, companies have to conduct internal research to fill in the knowledge gaps, and for competitive reasons, that knowledge is not made public immediately. The shrinking of industry drug-discovery groups results in fewer scientists working on target identification and a movement toward well-documented targets, which in turn results in homogenization of industry pipelines. Innovation and the discovery of game-changing therapies slows. Journals could help to address this problem by acknowledging the value of work that both replicates the data of others as well as work that may bring into question conclusions of previous papers. With the societal costs and human suffering that results from Alzheimer's disease, this field in particular needs to acknowledge the issue of publication biases.

    View all comments by Barbara Tate
  6. Robust enough for drug discovery?

    From an industry perspective, we follow the emergence of new biology with the hope that the new findings point to a potential new target or pathway for us to work toward producing candidate drugs. It is generally recognized, however, that a new finding regularly needs to be investigated further before we are ready to put the machinery in motion to design new drugs to a new target.

    In deciding whether a new target is ready, industry highly values replication of the original finding. But absolute replication can be challenging, and Madolyn Bowman Rogers’ article outlines just a few of the reasons why replication may not give the same results. I support the growing culture of sharing detailed experimental protocols, however, even the most detailed and accurate protocol may not work the first time, or initially give you the same results in a new setting. In industry, transferring new techniques or protocols to the labs of colleagues, or collaborators having researchers discuss the methods, and visit each other including doing the work together, is commonplace.

    In deciding if a target is robust enough, we also look to generate additional experiments that scope around the original finding and generate broader, hopefully supportive knowledge. In this stage we may find the original finding does not translate well. For example, some of us have been unlucky enough to have worked on targets that were subsequently found not to be expressed in the relevant tissues in humans, or have a markedly different pharmacological profile across species. We spend time on these and other such questions, and it is a platform of evidence that suggests whether a target is robust enough. If you look at new therapies that have come to market, you will find that they often have decades of biology research behind them—the hedgehog molecular signaling pathway and Genentech’s new skin cancer drug is one recent such story (see Bazurto article).

    Ultimately whether a new target does in disease what we think it does comes with human clinical trials. But here on the first clinical test we need to be aware that there are two questions remaining—does this target operate in humans the way it has in preclinical experiments, and does the molecule that we are using achieve the target engagement needed in order to see the expected effect? In the discussion on GSAP, the field has had the boon of a clinical-ready compound, enabling us to skip the process of designing a molecule so we can interrogate the human question directly. But clinical experiments, just like preclinical experiments, are designed to ask a question and the conditions of the experiment need to support asking that question.

    When thinking of the Olsson et al. imatinib study, my first question is whether there was enough target engagement: In cellular experiments with imatinib the potency has been reported around the 5 uM range, while the human studies had mean plasma levels of 1.46 uM. Even if translation for this mechanism is linear across systems, the clinical exposure is a total concentration and lower. Translation of potency across species and systems is rarely linear, and so a deep understanding of the pharmacology and pharmacokinetics is needed to underpin whether a drug is in sufficient exposure to have an effect, or to decide if the mechanism doesn’t translate. Indeed, in a review of 44 programs to reach Phase 2, Pfizer scientists determined that demonstrating target engagement (showing that the drug occupies the desired target sufficiently to elicit its pharmacological effect) is crucial for success (Morgan et al., Drug Discovery Today 2011). Time and dose response studies in vivo that measure effects in the same (plasma) compartment are needed.

    As a field searching for potential therapies for Alzheimer’s disease, we need the biology discoveries to continue. We need to expand our knowledge around any new target. We need to openly discuss and share findings. Comments to this article and in wider groups highlight that the current culture of publication is perhaps not best supporting these aims—aims which I am certain each of these labs share.

    Many years back AlzForum pioneered the Alzgene database, which addressed issues with replication or strength of linkage for the genetics of Alzheimer’s disease. Perhaps a similar representation of targets and the associated biology findings would help display the biology associated to potential targets.

    View all comments by Samantha Budd
  7. Co-authored by William Netzer and Paul Greengard, The Rockefeller University; Yueming Li and Darren Veach, Memorial Sloan-Kettering Cancer Center; Wenjie Luo, Weill Cornell Medical College; Fred Gorelick, Yale University School of Medicine; and Dongming Cai and Sam Gandy, Icahn School of Medicine at Mount Sinai, New York.

    Madolyn Rogers’ news article, and some of the comments in response to that article, draw attention to interlaboratory variability in the replication of two studies published by our research group.  In our original paper on this topic (Netzer et al., 2003), we reported that Gleevec (imatinib) reduced Aβ generation in cell culture and in vivo in guinea pig brain.  In the second paper (He et al., 2010), we described the identification of γ–secretase-activating protein (GSAP), an endogenous regulator of Aβ formation.  Here we focus primarily on studies published subsequent to Netzer et al. that have evaluated the Aβ-lowering effects of imatinib.

    Studies of imatinib in intact cell systems

    Netzer et al. reported that imatinib lowered Aβ40 levels in rat primary neurons and lowered Aβ40 and 42 in N2a (mouse neuroblastoma) cells stably transfected with APP 695 or PS1ΔE9/APP Swe.  Subsequent to that publication, we tested imatinib in Chinese hamster ovary cells transfected with wild-type human APP695 (CHO-hAPP), in 3T3 fibroblasts transfected with wild-type human APP695, and in mouse embryonic fibroblasts (MEFs) transfected with Swedish mutant human APP695.  In each of these three cell lines, imatinib reduced total Aβ by 40 to 80 percent (Netzer, unpublished results).  The observation that imatinib reduces Aβ generation in five different cell types—namely rat primary neurons, N2a, CHO, 3T3, and MEF cells—does not exclude the possibility that some cell types or cell lines might be resistant to imatinib-induced reduction in Aβ generation (see below).

    Independent confirmation of the imatinib effect in the cell types used by Netzer et al. (2003) 

    Hussain et al. confirmed in N2a cells our published observations (in that same cell line) that imatinib causes a 50 to 60 percent reduction in Aβ40 and 42 secretion.

    Arslanova et al. tested 100 μM imatinib in CHO-hAPP cells in culture and demonstrated that secretion of Aβ40 and 42 was reduced by 30 percent and 80 percent, respectively.  The published results of Arslanova et al.  were confirmed in the unpublished studies of Netzer mentioned above.

    Extension of the studies of Netzer et al. (2003) to three additional cell types

    Eisele et al. extended the list of imatinib-responsive cells to include H4-APPwt cells in their demonstration of an effect of imatinib on Aβ generation over a drug concentration range of 3.7 to 20μM.  A 50 percent reduction in secretion of Aβ40 and 42 was observed in the presence of 10μM imatinib. 

    Hussain et al. tested imatinib in SHSY5Y-SPA4CT cells and found that 10μM imatinib reduced secretion of Αβ40 and 42 by about 40 percent, adding yet another cell line to the roster of those that display imatinib-responsive Αβ generation.

    Mertens et al. reported that 10μΜ imatinib lowered Aβ40 and 42 secretion by 70 percent and 75 percent, respectively, in human differentiated neurons in culture, consistent with our observations using rat primary neuronal cultures. 

    Failure to replicate the imatinib effect in three previously untested cell types

    Olsson et al. reported the only published failure to reproduce the imatinib effect in intact cells.  They reported that imatinib does not lower Aβ42 secretion from either: (i) HEK293 cells (stably transfected with human APP Swe); (ii) human Down’s syndrome embryonic stem cell-derived cortical projection neurons; or (iii) mouse primary cortical neurons.  The protocol published by Olsson et al. provided no obvious explanation for why their results differed from those derived from multiple other cell types in the hands of multiple other investigators.  As of this writing, we are unaware of any other laboratory that has tested any of the cell types used by Olsson et al.  At present, we cannot determine whether the Olsson results are technical in nature or whether all the cell types they studied are imatinib-resistant.

    In summary, four of the five studies published since Netzer et al. have confirmed the Aβ-lowering effect of imatinib in cell culture.  We have been unable to identify any obvious explanation for this disparity in careful scrutiny of the published protocols from those failing to replicate the imatinib effect.  In an effort to resolve the issue as efficiently as possible, we would welcome visits to our laboratory by any scientists seeking a detailed demonstration of our methods of measuring the effect of imatinib on Aβ secretion by cultured cells.

    Demonstration of the imatinib effect in a cell-free system

    In addition to the results obtained in intact cell systems, a study by Fraering et al. reported that 75μM imatinib reduced Aβ40 and 42 formation by 50 percent in a cell-free γ–secretase assay.

    Studies of imatinib in intact rodents and in humans

    Netzer et al. reported that continuous intrathecal infusion of imatinib by osmotic pump reduced Aβ levels in the intact guinea pig brain. Intrathecal infusion was chosen as the route of administration because imatinib is rapidly pumped out of the brain by the p-glycoprotein pump (PGP) at the blood-brain-barrier (see Dai et al.).  As a result of robust PGP action, imatinib that is peripherally injected or ingested will appear in brain only to a negligible extent (Leis et al.).  For this reason, Netzer et al. explicitly stated that imatinib administered peripherally would not be useful as a therapeutic for Alzheimer’s disease (AD).  Nevertheless, both Hussain et al. (i.p. imatinib in rats) and Olsson et al. (oral imatinib in humans) studied the effects of peripherally injected or orally ingested imatinib.  Since most plasma Αβ is derived from the CNS (DeMattos et al.; Ghersi-Egea et al.; Shibata et al.), the failures of Hussain et al. and Olsson et al. to observe Αβ–reducing effects of peripherally injected or orally ingested imatinib on brain or plasma Aβ levels were the predicted results.  Therefore, the data of Hussain et al. and Olsson et al. are irrelevant to any assessment of the potential for therapeutic success using a CNS imatinib-like compound or a GSAP-modulating compound.

    Since we remain interested in developing an AD therapeutic based on imatinib, the Olsson et al. study in humans merits particular attention.  These investigators measured plasma Aβ42 levels in chronic myelogenous leukemia patients (n = 51) before and during treatment with imatinib.  They estimated that plasma concentrations of imatinib in these patients was 1.46 μM (the concentration of imatinib in the brain would have been much lower).  We have only observed reductions in Aβ secretion by imatinib for imatinib concentrations of 5μΜ or greater (Netzer, unpublished results).  Thus, the dose employed by Olsson et al. would not be expected to have an impact on peripheral generation of Aβ (if any such Aβ generation exists).   

    Studies of GSAP

    With respect to GSAP, four members of our laboratory and one publication from another laboratory (Hussain et al.) have confirmed the observation reported in He et al. that GSAP knockdown reduces Aβ secretion by cells in culture.  There have been no contradictory publications reporting a failure of GSAP knockdown to lower Aβ secretion in intact cells.  Two additional groups reported higher expression levels of GSAP in human tissues (including AD brain) that correlated with increased expression of γ-secretase components and increased Aβ deposition (Satoh et al.; Nogalska et al.).  We continue to actively investigate the molecular mechanism(s) by which GSAP modulates Aβ accumulation.  

    Two research groups (Hussain et al. and Deatherage et al.) have reported failure to reproduce some of the results reported by He et al. We are investigating the basis for these discrepancies.

    View all comments by William Netzer
  8. We read with interest the response by Netzer, Greengard, and colleagues. It is clear that we have different views not only on the biological effect of GSAP but also on Αβ (patho)physiology in general.

    There are some strong claims in the response, e.g., "that most plasma Αβ is derived from the CNS." Netzer and Greengard cite some papers, which they claim support this statement. One of these (Ghersi-Egea et al., 1996) showed that a large portion of Αβ40 injected into lateral ventricle CSF of normal rats ends up in blood, while another (Shibata et al., 2000) showed that Αβ40 injected into brains of mice is cleared from it by the LRP-1 receptor. Although interesting in themselves, these articles do not tell us whether or not most plasma Αβ is derived from the CNS.

    There are many additional papers, not cited by Netzer and Greengard, that are highly relevant for the question of whether plasma Αβ is CNS-derived. Walker and co-workers showed that intravenous administration of a fusion protein of the Aβ-degrading enzyme neprilysin (NEP), which resembles the orientation of the native NEP in membranes, in mice resulted in a fast and very marked dose-dependent reduction in plasma Aβ without affecting either soluble or formic acid-extractable brain Aβ levels, or CSF Aβ levels (Walker et al., 2013). These results also have been confirmed using NEP engineered to have an extended plasma half-life and an increased Aβ degradation activity. In long-term studies on mice, rats, and monkeys, a marked decrease of Aβ was found in plasma without any change in brain or CSF Aβ levels (Henderson et al., 2014). This paper was also commented on at the AlzForum website.

    Further, pharmacodynamic studies in man on BACE1 inhibitors also show that the decrease in plasma Aβ is much faster, and also more pronounced, than the decrease in CSF Aβ (May et al., 2011). Similar results were found in a Phase 2 trial on a gamma-secretase inhibitor (Fleisher et al., 2008). 

    Taken together, neither the NEP studies in animals nor the clinical trials in man support the existence of any robust peripheral Aβ efflux sink, and thus do not support "that most plasma Αβ is derived from the CNS." 

    Last, it well known that Aβ is generated in considerable amounts in many organs and cell types outside the CNS, such as platelets, the neuromuscular junctions of skeletal muscle, and arterial walls (Li et al., 1998; Kuo et al., 2000; Roher et al., 2009). Undoubtedly, these peripheral organs contribute to the pool of circulating Aβ in plasma.

    In summary, the literature suggests that there is a (large) pool of Αβ in the plasma that is not derived from the CNS, and that compounds that suppress amyloidogenic processing of APP can shrink that pool. We found no such effect in patients treated with imatinib. We are happy to see continued discussion on the important topic of validating data from cell and animal experiments by translational studies in humans. However, the statement by Netzer and Greengard that "the data of Hussain et al. and Olsson et al. are irrelevant to any assessment of the potential for therapeutic success using a CNS imatinib-like compound or a GSAP-modulating compound" seems to be based on a misconception due to selective referencing of available data. Instead, an unbiased literature review tells us that the forecast of imatinib-like or GSAP-modifying compounds as Aβ-lowering therapies in AD does not look bright.

    View all comments by Kaj Blennow

This paper appears in the following:

News

  1. GSAP Revisited: Does It Really Play a Role in Processing Aβ?