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Biomarkers in Alzheimer’s disease drug developmentKaj Blennow

Biomarkers may be of great value in Alzheimer’s disease drug development to select the most optimal drug candidates for large and expensive phase 3 clinical trials. Biomarkers will also be important to provide evidence that a drug affects the underlying pathophysiology of the disease, which, together with a beneficial effect on the clinical course, will be essential for labeling the drug as having a disease-modifying effect.

Twenty-five years ago, our knowledge of the molecular pathogenesis of Alzheimer’s disease was very limited. In the mid-1980s, research-ers succeeded in identifying amyloid-β (Aβ) and tau as the main components of plaques and tangles, which are the hallmarks of the dis-ease. Together, these important achievements marked the start of modern Alzheimer’s dis-ease research, and subsequent research efforts have led to a detailed knowledge of amyloid precursor protein (APP) metabolism, Aβ gen-eration and the pathogenic events involved in plaque development and of tau homeostasis and tangle formation1. The prevailing hypoth-esis for Alzheimer’s disease pathogenesis is the amyloid cascade hypothesis, which states that Aβ aggregation with plaque formation is the central event, ultimately leading to neuronal degeneration and clinical symptoms2.

These research advances have led to a large number of drug candidates that are now in

clinical trials and may have disease-modifying effects. The vast majority of these new treat-ments aim to inhibit Aβ toxicity and include secretase inhibitors inhibiting the production of Aβ from APP, immunotherapies aimed at increasing the clearance of Aβ from the brain and inhibitors of Aβ aggregation1. If this type of anti-Aβ treatment proves able to reduce plaque pathology and have a beneficial effect on cognition in people with Alzheimer’s dis-ease, then the recent years of Alzheimer’s dis-ease research will be a success story.

Unfortunately, reports of clinical trials showing negative results have begun to accu-mulate. In recent years, there have been sev-eral clinical trials where drug candidates with an Aβ-targeting mode of action have failed to show any substantial effects on primary cog-nitive outcome measures, such as tramipro-sate (that binds soluble Aβ, thus maintaining it in a nonfibrillar form) and tarenflurbil (a γ-secretase modulator)3. These disappoint-ing results are causing concern that the amy-loid cascade hypothesis will be proved wrong and that the potential success will turn into a failure. However, there are several other possible explanations for this. These include that the trials have been done in subjects with Alzheimer’s disease with dementia (that is, at a far too advanced stage of the disease), that clinical diagnostic procedures have been too unspecific to allow identification of a homo-genous group of patients with Alzheimer’s dis-ease pathology and that the drug candidates tested have been ineffective despite promis-ing, but misleading, data from preclinical development.

Are we too far down the road in clinical trials?It is logical to assume that disease-modifying drugs targeting Aβ will have only minor effects on the clinical course of the disease

in Alzheimer’s disease cases with advanced neurodegeneration. A potential example of this is the follow-up study of cases in the Elan AN1792 phase 1 Aβ immunotherapy trial. In subjects who underwent post-mortem assess-ment, there was evidence of marked plaque removal, despite their clinical deterioration to severe dementia before death4. Thus, arrest-ing Aβ aggregation or even plaque removal may have limited effect in Alzheimer’s disease cases with dementia, owing to severe neuronal and synaptic loss and heavy tangle load at this stage of disease.

It is likely that we need to design trials on the basis of diagnostic algorithms that rec-ognize the predementia stage (prodromal Alzheimer’s disease) or even the asymptomatic phase of the disease (preclinical Alzheimer’s disease) to give promising drug candidates a chance of showing a disease-modifying effect. This proposal is supported by studies in trans-genic mouse models of Alzheimer’s disease, which suggest that drugs would be most effi-cacious early on in the disease process, before severe plaque pathology is present5.

Mild cognitive impairment (MCI) may be regarded as a transitional state between nor-mal aging and Alzheimer’s disease. However, MCI is an etiologically heterogeneous entity; only ~50% of people with MCI have pro-dromal Alzheimer’s disease, whereas others have a benign form of MCI as part of the normal aging process and some have other disorders6. Given that individuals with MCI have only mild disturbances in episodic memory and other characteristic symptoms of Alzheimer’s disease are absent or vague, it is a great challenge for the clinician to identify which MCI cases have prodromal Alzheimer’s disease.

Testing Alzheimer’s disease drugs in clini-cal trials in which unspecified MCI cases are included will thus mean that around half of

Kaj Blennow is at the Clinical Neurochemistry

Laboratory, Institute of Neuroscience and

Physiology, Department of Psychiatry and

Neurochemistry, The Sahlgrenska Academy at

University of Gothenburg, Mölndal, Sweden.

e-mail: kaj.blennow@neuro.gu.se

Published online 21 September 2010;

doi: 10.1038/nm.2221

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the cases do not have prodromal Alzheimer’s disease, the disorder for which the drug is intended, which may seriously affect the pos-sibility of identifying clinical effects. One pos-sible example so far is the large trial on the cholinesterase inhibitor donepezil, in which more than 700 subjects with unspecified MCI were treated for 3 years without any signifi-cant effect of donepezil treatment7.

The addition of positive biomarkers as an inclusion criterion in such MCI trials would enrich the proportion of subjects with under-lying Alzheimer’s disease pathology and, thereby, increase the possibility of identify-ing a positive effect of a drug. Several studies have consistently shown that the combina-tion of high cerebrospinal fluid (CSF) levels of total tau (T-tau) and phosphorylated tau (P-tau) and low levels of the 42–amino-acid isoform of Aβ (Aβ42) has a high predictive value for identifying cases of prodromal Alzheimer’s disease in people with MCI, with one study reporting a sensitivity of 95%8. A high predictive value has been verified in large multicenter studies, including the Alzheimer’s Disease Neuroimaging Initiative study9, the Descripa study10 and the Swedish Brain Power project11. Numerous studies have also shown that magnetic resonance imaging (MRI) measurement of hippocampal atrophy and positron emission tomography (PET) imaging with the 2-[18F]fluoro-2-deoxy-d-glucose (FDG) ligand and β-amyloid–binding tracers such as Pittsburgh Compound-B (PiB) have high predictive values of approximately 85–90% for prodromal Alzheimer’s disease in the MCI stage of the disease12,13. Thus, bio-markers will be key tools for enrichment of clinical trials with true prodromal Alzheimer’s disease cases.

Is Alzheimer’s disease too heterogeneous?Trials involving clinically diagnosed sub-jects with Alzheimer’s disease may enroll patients with different pathologies, owing to misdiagnoses. Apart from the fact that the relative intensity of plaques and tangles may differ markedly among individuals with Alzheimer’s disease14, there is also a sizable overlap in pathology between Alzheimer’s disease and other dementias, such as Lewy body dementia and vascular dementia15,16. Current clinical criteria for Alzheimer’s dis-ease may also be too nonspecific to identify cases of pure Alzheimer’s disease with high certainty. For example, recent data show that approximately 20% of clinically diagnosed Alzheimer’s disease cases enrolled in clini-cal trials may have negative PiB-PET scans17. This diagnostic uncertainty introduces a high

degree of noise into trials, which may mark-edly reduce the possibility of identifying any effect of a drug.

It is likely that the effectiveness of disease-modifying drugs targeting Aβ will vary between cases depending on the severity of the Aβ plaque pathology. In other words, clinically diagnosed Alzheimer’s disease cases with biomarker evidence of a disturbance in Aβ metabolism—for example, low CSF Aβ42 or high PiB binding on PET—may be more responsive to anti-Aβ drugs than individuals not showing such a disturbance. Biomarkers reflecting the central pathogenic processes in Alzheimer’s disease may thus be valuable for post hoc data analyses of clinical trials to stratify patient cohorts, enabling identifica-tion of subgroups with more homogeneous neuropathology.

Are we taking poor drug candidates into phase 3 clinical trials?The effect of disease-modifying, Aβ-targeting drug candidates on plaque pathology is com-monly evaluated in APP and presenilin trans-genic mice. There are numerous drugs that cause a substantial reduction in ‘Aβ burden’

(the number or extent of Aβ plaques in the brains of Alzheimer’s disease–transgenic mice) but have no beneficial effect in people with Alzheimer’s disease, indicating that these mouse models have a very low predictive power of treatment success in patients with sporadic Alzheimer’s disease1. Alzheimer’s disease–transgenic mice have a huge over-expression of Aβ and develop plaques much faster (months instead of decades) than people who develop Alzheimer’s disease and are thus probably much more responsive to anti-Aβ treatment than humans with sporadic Alzheimer’s disease. Indeed, several anti-Aβ drug candidates that were shown to reduce Aβ burden in Alzheimer’s disease–transgenic mice, such as tramiprosate (an Aβ antiaggre-gation compound), tarenflurbil (a γ-secretase modulator), rosiglitazone (a type 2 diabetes drug that also acts as a β-secretase inhibitor), statins, nicotine, phenserine (a cholinest-erase inhibitor that also reduces Aβ amounts by decreasing APP mRNA translation) and estrogens, have now failed in clinical trials of patients with Alzheimer’s disease1,3. It is likely that the promising data from the Alzheimer’s disease–transgenic mice models may have

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Figure 1 Flow chart for hypothetical use of theragnostic biomarkers in Aβ-targeting clinical trials. Theragnostic biomarkers can be divided into primary biomarkers (orange), used to identify and monitor the specific biochemical effect, or mode of action, of the drug, and downstream biomarkers (blue), used to identify and monitor effects on pathogenic processes downstream of the drug target. At right, validated biomarkers that may be valuable to monitor specific pathogenic processes are indicated with green boxes, whereas promising biomarkers that need further development and validation of methods are indicated with yellow boxes. sAPP, soluble APP.

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had a large impact on decisions to proceed with these drugs to phase 3 clinical trials.

One example of this might be tarenflurbil, which is the R-enantiomer derivative of flur-biprofen, a nonsteroidal anti-inflammatory drug (NSAID). Data from experiments in cell culture and in Alzheimer’s disease–transgenic mice suggest that this drug selectively lowers Aβ42 amounts and acts as a γ-secretase mod-ulator18. The authors of this study cautiously concluded that “rigorous clinical testing of R-flurbiprofen will be necessary to determine if it has the ability to lower Aβ42 in humans and therapeutic efficacy in Alzheimer’s dis-ease.”18. However, despite data showing that the drug, even at high doses, does not affect CSF Aβ42 concentrations or cause a shift toward shorter Aβ isoforms in CSF in humans19, and a phase 2 trial showing no overall effect on primary outcome measures in individuals with Alzheimer’s disease20, the drug was taken to a large phase 3 clinical trial. This 18-month trial on more than 1,600 subjects with Alzheimer’s disease showed no significant effects on cognitive or functional primary outcomes21.

Biomarkers used to detect and monitor the biochemical effects of therapeutics may be called ‘theragnostic’ biomarkers22. These types of biomarkers may be valuable in drug development to bridge the gap between animal studies and large clinical trials. Biomarker evi-dence from small-scale trials that a drug has a true effect on the Alzheimer’s disease process directly in humans would help in selecting the most promising drug candidates, thereby improving the success rate of large clinical phase 2 and 3 trials. Such trials could be short-term proof-of-principle studies on a limited number of healthy volunteers in phase 1 (ref. 23), as well as proof-of-concept studies on individuals with Alzheimer’s disease in phase 2 (ref. 24). This type of early clinical biomarker study would aid decision making regarding whether to embark on large and expensive phase 3 trials.

What constitutes a proficient theragnostic biomarker?A theragnostic biomarker should reflect the central pathogenic processes of the dis-ease. In contrast to drug development for other neurodegenerative disorders, such as Parkinson’s disease, Alzheimer’s disease drug development has the benefit of several such biomarkers readily available. CSF concentra-tions of T-tau reflect cortical axonal degenera-tion, whereas P-tau reflects tangle pathology and Aβ42 reflects brain amyloid pathology22. Further, MRI imaging of hippocampal atro-phy gauges progression of neurodegenera-

tion in a manner that correlates well with both neuropathological measures of tangle load and cognitive symptoms12. Last, PET imaging with the FDG ligand allows for the assessment of the glucose metabolism rate in specific brain regions, whereas fibrillar Aβ deposits and plaques in the brain can be visu-alized with β-amyloid–binding tracers such as PiB13. These biomarkers may serve as valuable theragnostic tools in Alzheimer’s disease drug development.

Treatment with symptomatic drugs (such as cholinesterase inhibitors) is usually allowed as background medication in Alzheimer’s dis-ease clinical trials. Therefore, it is important that theragnostic biomarkers used to monitor the effect of an anti-Aβ drug are not affected by this type of treatment. Several studies have shown that the CSF biomarkers Aβ42, T-tau and P-tau do not change during cholinesterase inhibitor treatment and also have a very low intraindividual variability, both in short-term and long-term trials25,26. These results indi-cate that even minor changes in biomarker amounts can be monitored with CSF biomark-ers. It is noteworthy that studies have shown that treatment with donepezil may retard both the decline in brain glucose metabolism evaluated by FDG-PET27,28 and the progres-sion rate of hippocampal atrophy measured by MRI29,30. Thus, cholinesterase treatment might be a potential confounder when evalu-ating the effect of disease-modifying therapies with these types of biomarkers.

Unresolved issues for Alzheimer’s disease biomarkersBoth imaging and CSF biomarkers show promise as diagnostic and theragnostic tools in clinical trials, but they have in common a need for standardization. Although biological and within-laboratory variability for CSF bio-markers is low, there is a variation in biomarker levels between laboratories. Furthermore, there are no available calibrators (certified reference standards) for these biomarkers and no consen-sus on what assays should be used. This com-plicates multicenter trials and also precludes the introduction of generally applicable cutoff levels. In response to this problem, a global quality-control program for CSF biomarkers, supported by the Alzheimer’s Association, has recently been launched22. The aim of this program is to serve as the basis for efforts to minimize variation caused by differences in preanalytical and laboratory procedures. A substantial part of the variability is probably also caused by assay-related factors, espe-cially batch-to-batch variation of assays. This involves a need for assay vendors to implement improved standards for quality control in assay

production. Further information on the qual-ity control program is available at http://www.neurochem.gu.se/TheAlzAssQCprogram/.

For structural MRI imaging of medial tem-poral lobe atrophy, there is no consensus on which structure is the region of interest (hip-pocampus, amygdala, entorhinal cortex or parahippocampal gyrus), whether to use visual rating or computerized algorithms for evalu-ation of atrophy and whether age-dependent cutoffs should be applied for hippocampal atrophy to correct for age-related changes12. The utility of structural MRI as a biomarker in clinical trials will thus be increased by stan-dardization of acquisition and analysis meth-ods12. A task force to develop a harmonized procedure for evaluation of hippocampal atrophy is ongoing under the auspices of the Alzheimer’s Association.

Similar standardization issues also apply to PET imaging. For amyloid PET, there is no consensus on the optimal brain region to quantify amyloid retention and very limited data comparing the performance of the vari-ous radioligands (PiB, FDDNP, AV-45 and AZD2184)13. For FDG-PET, further data is needed on which brain regions to evaluate in each stage of the disease, that is, hypometabo-lism in the parietotemporal cortex to diagnose Alzheimer’s disease, in the medial temporal lobe to predict MCI and in the posterior cin-gulate cortex to predict progression from MCI to Alzheimer’s disease13.

Implementing theragnostic biomarkers in clinical trialsConceptually, it may be useful to divide bio-markers into primary (specific) and second-ary (downstream) biomarkers (Fig. 1). A primary biomarker can be used to identify and monitor the specific biochemical effect, or mode of action, of a drug. In trials with Aβ-targeting compounds such as secretase inhibitors or Aβ immunotherapy, examples of primary biomarkers include CSF Aβ42 and Aβ40, β-secretase (BACE1) activity and sol-uble APP-β and amyloid-PET. Downstream biomarkers are used to identify and monitor effects on pathogenic processes downstream of the drug target, for example, CSF T-tau and MRI measurements of brain atrophy to identify and monitor an effect on the rate of neuronal degeneration in an anti-Aβ trial.

A key question will also be which of the biomarkers to include in a trial. For example, several studies have shown a strong correla-tion between low CSF Aβ42 concentration and high binding of the PiB ligand on PET, suggesting that these biomarkers will give similar information on the amount of Aβ plaque pathology31,32. For a clinical trial,

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factors such as the willingness among clini-cians to perform lumbar puncture on the one hand, and the availability of cyclotrons and PET scanners and financial consideration on the other, will form a basis for a decision on which of these biomarkers to choose.

Can we find new biomarkers?A surrogate biomarker can be used as a sub-stitute for a clinical end point in a clinical trial33. In the case of Alzheimer’s disease, such a biomarker should correlate with cognitive or functional clinical outcomes. Synapses are the primary functional unit for neuronal communication, and the degree of synaptic degeneration has also been found to show the best correlation with severity of dementia in Alzheimer’s disease34. Synaptic proteins might thus serve as valuable surrogate CSF biomark-ers, as the abundance of these molecules is likely to correlate with cognitive function and disease progression. Although synaptic pro-teins, such as synaptotagmin and rab3a, have been shown to be present in human CSF35, assay development for this class of proteins has been difficult, owing to their low concen-trations in CSF.

Probably for the same reason, assay development has been difficult for Aβ oli-gomers in CSF. Aβ oligomers would be a valuable biomarker in Alzheimer’s disease drug development for several reasons. First, recent experimental data suggest that soluble Aβ oligomers inhibit long-term potentia-tion and have synaptotoxic properties, and thereby have a central role in Alzheimer’s disease pathogenesis36. Second, it is difficult to predict in which direction CSF Aβ42 may change in an Aβ immunotherapy trial. Thus, CSF Aβ oligomers might be an important biomarker for Alzheimer’s disease. Some pre-liminary studies on Aβ oligomers in CSF have been published; a recent study with a previ-ously undescribed ELISA assay found a clear increase in CSF Aβ oligomers in Alzheimer’s disease37.

Targeted proteomics studies have shown that, in addition to Aβ1–42 and Aβ1–40, there are several shorter Aβ isoforms truncated at the carboxy terminus. By a technique based on immunoprecipitation with an Aβ-specific monoclonal antibody combined with matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry, all Aβ isoforms can be quantified simultane-ously38. Cell culture studies have shown that the longer isoforms (Aβ1–17 up to Aβ1–42) are produced by the amyloidogenic pathway, by the concerted action of β-secretase and γ-secretase, whereas the short Aβ isoforms (Aβ1–14, Aβ1–15 and Aβ1–16) are pro-

duced by a previously unknown pathway of APP processing38. A series of studies in dogs, monkeys and Alzheimer’s disease–transgenic mice have shown that γ-secretase inhibitor treatment results in a marked increase in Aβ1–14, Aβ1–15 and Aβ1–16 abundance, probably as a result of increased substrate availability of the C99 APP stub induced by γ-secretase inhibition39–41. A recent clinical trial on the γ-secretase inhibitor LY450139 in Alzheimer’s disease found no effect on the CSF concentrations of Aβ1–40 or Aβ1–42, as measured by ELISA42. In another study in the same trial, a marked dose-dependent increase in Aβ1–14, Aβ1–15 and Aβ1–16 was found by the immunoprecipitation–MALDI-TOF technique24. Thus, these short Aβ isoforms may be valuable and sensitive biomarkers to monitor the biochemical effect of γ-secretase inhibitor treatment in clinical trials.

Use of biomarkers to label a drug ‘disease modifying’Ideally, a change in the slope of cognitive decline may identify differences between symptomatic and disease-modifying drugs. Clinical trials designed with a delayed start (placing patients on drug or placebo for a period at the start of the trial and later ini-tiating treatment also in the placebo group) or staggered withdrawal (placing patients on active drug at the trial start and later ran-domizing them to drug or placebo) have been suggested to allow this differentiation. This approach may be theoretically attractive, but it has not been successful, probably owing to the very large variability in the rate of cognitive change between cases, both in the dementia and the MCI stages of Alzheimer’s disease.

Academic institutions, the pharmaceuti-cal industry and regulatory organizations agree that biomarkers have a crucial role in the drug development process and that evi-dence obtained from biomarker studies show-ing that a drug candidate affects the central disease processes in Alzheimer’s disease will, together with a beneficial effect on cognition, be essential for a drug to be labeled disease modifying33.

How far have we come today?Apart from animal studies, there is only pre-liminary evidence that biomarkers may be useful as theragnostic markers in humans. For CSF biomarkers, a phase 2 study of the Aβ clearance-enhancing compound PBT2 showed a dose-dependent reduction in the amount of the primary biomarker Aβ42 in CSF43. Furthermore, in the AN1792 immunother-apy trial, there was a decrease toward normal amounts of the downstream biomarker T-tau

in CSF, suggesting that the treatment may have reduced the rate of neuronal degeneration44. For MRI measurements of atrophy, a decrease in atrophy rate would be the logical expec-tation for a drug with a disease-modifying effect. However, the AN1792 trial showed an unexpected increase in the brain atrophy rate in antibody responders, possibly as a result of removal of Aβ plaques45. A recent report on the bapineuzumab immunotherapy trial showed a reduced cortical PiB retention during treatment compared with both baseline and placebo17. These results bring hope that amy-loid PET will be valuable to assess the effect of Aβ-targeting disease-modifying therapies on cortical fibrillar Aβ load in clinical trials.

Thus, there are only limited data from Alzheimer’s disease trials suggesting that bio-markers have a place as theragnostic markers in phase 1–2 studies or surrogate end points in phase 3 studies. A challenge in this field of research is the lack of drugs with an estab-lished disease-modifying effect that can be used to validate a biomarker and, at the same time, that biomarker evidence that a drug candidate affects the central disease processes will be required to label a drug as disease modifying33. This means that Alzheimer’s disease drug development and biomarker research need to go forward hand in hand. The increasing number of clinical trials on disease-modifying drug candidates that include biomarkers for enrichment of pure Alzheimer’s disease cases or as end points will hopefully keep providing accumulative evidence for the usefulness of biomarkers in Alzheimer’s disease clinical trials and serve as the basis for approval by the regulatory authorities of disease-modifying drugs for this devastating disease.

COMPETING FINANCIAL INTERESTSThe author declares competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/.

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