Recruitment, Retainment, and Biomarkers of Response; A Pilot Trial of Lithium in Humans With Mild Cognitive Impairment

Ashleigh Duthie, Lidy van Aalten, Cara MacDonald, Alison McNeilly, Jennifer Gallagher, John Geddes, Simon Lovestone, Calum Sutherland, Ashleigh Duthie, Lidy van Aalten, Cara MacDonald, Alison McNeilly, Jennifer Gallagher, John Geddes, Simon Lovestone, Calum Sutherland

Abstract

Lithium has been used for decades to treat Bipolar Disorder. Some of its therapeutic benefits may be through inhibition of Glycogen Synthase Kinase (GSK)-3. Enhanced GSK3 activity associates with development of Alzheimer's disease (AD), therefore lithium is a currently used therapeutic with potential to be repurposed for prevention of Dementia. An important step toward a clinical trial for AD prevention using lithium is to establish the dose of lithium that blocks GSK3 in Mild Cognitive Impairment (MCI), a high-risk condition for progression to AD. We investigated volunteer recruitment, retention, and tolerance in this population, and assessed biomarkers of GSK3 in MCI compared to control and after lithium treatment. Recruitment was close to target, with higher than anticipated interest. Drop out was not related to lithium blood concentration. Indeed, 33% of the withdrawals were in the first week of very low dose lithium. Most made it through to the highest dose of lithium with no adverse events. We analyzed 18 potential biomarkers of GSK3 biology in rat PBMCs, but only four of these gave a robust reproducible baseline signal. The only biomarker that was modified by acute lithium injection in the rat was the inhibitory phosphorylation of Ser9 of GSK3beta (enhanced in PBMCs) and this associated with reduced activity of GSK3beta. In contrast to the rat PBMC preparations the protein quality of the human PBMC preparations was extremely variable. There was no difference between GSK3 biomarkers in MCI and control PBMC preparations and no significant effect of chronic lithium on the robust GSK3 biomarkers, indicating that the dose reached may not be sufficient to modify these markers. In summary, the high interest from the MCI population, and the lack of any adverse events, suggest that it would be relatively straightforward and safe to recruit to a larger clinical trial within this dosing regimen. However, it is clear that we will need an improved PBMC isolation process along with more robust, sensitive, and validated biomarkers of GSK3 function, in order to use GSK3 pathway regulation in human PBMC preparations as a biomarker of GSK3 inhibitor efficacy, within a clinical trial setting.

Keywords: GSK3; biomarker; clinical trial; lithium; safety.

Figures

FIGURE 1
FIGURE 1
Lithium intervention Study Design (Arm 2). Eleven volunteers were given low dose (100 mg daily) for 3 weeks, then medium dose (200 mg daily) for 3 weeks, high dose (400 mg daily) for 3 weeks, prior to a 3 week washout period. Blood samples were taken for safety measures, PBMC preparation and Lithium measure at baseline (visit 1), 1 and 3 weeks after starting each dose (visit 2–7), and after washout (visit 8).
FIGURE 2
FIGURE 2
Consort diagram detailing the recruitment process for the clinical studies.
FIGURE 3
FIGURE 3
Biomarkers of GSK3 in rat tissues following lithium injection. Rats were given an i.p. injection of vehicle or lithium chloride as detailed in section “Materials and Methods,” and tissues isolated 3 h later. Representative examples of Western blots of rat protein lysates isolated from (A) rat PBMCs, (B) Hippocampus, or (C) Cortex, are provided using the antibodies stated. In (A), we also provide a comparison of vehicle treated rat PBMC lysate with rat brain cortex lysate with every antibody as evidence for the utility of each antibody (antibody control). The ratio of expression of GSK3 phosphorylation at the N-terminal inhibitory site to the overall isoform expression was quantified from the images as detailed in section “Materials and Methods,” and is provided for each tissue (±SD) in the table inserted into A. The red box highlights the major biomarker changes in response to lithium, although only the P-GSK3β-total GSK3β ratio was significantly increased by lithium (p < 0.05, student t test).
FIGURE 4
FIGURE 4
Comparison of Biomarkers of GSK3 in human PBMCs isolated from volunteers with MCI and controls (Study Arm 1). Protein lysates were prepared from PBMCs isolated from two groups of human volunteers, one with a diagnosis of MCI, the other age matched but with no MCI. In each case the volunteer provided two blood samples for PBMC preparation, taken 12 weeks apart (Visit 1 and 2). (A) Representative Coomassie protein staining of PBMC lysates from 8 of the 17 volunteers in Study Arm 1, where equal total protein was loaded on the gel. (B) Representative immunoblots of biomarkers within the PBMCs of four of the volunteers in Study arm 1. (C) Immunoprecipitation and assay of GSK3 isoforms from 50 μg of protein lysate in each of the PBMC samples. The specific activity was quantified for GSK3alpha and GSK3beta from all PBMC preparations and the average activity (±SE) for the MCI and control groups at each visit, as well as the two visits combined, is given.
FIGURE 5
FIGURE 5
Comparison of Biomarkers of GSK3 in human PBMCs isolated from volunteers with MCI treated with a stepwise increasing dose of Lithium (Study Arm 2). Protein lysates were prepared from PBMCs isolated from volunteers with MCI who underwent lithium treatment (see Figure 1). (A) Protein profiles (Coomassie staining) from the PBMC lysates of the five volunteers that completed eight visits. Equal amounts of protein were loaded in each lane (by Bradford assay). (B) Immunoblot of all of the lysates from the same five volunteers using the phosphoser9/21 GSK3 antibody. The box around visit 6 and 7 highlights the samples with the peak plasma lithium levels (Table 3). (C) GSK3β isoform specific activity in PBMCs isolated across the eight visits for four of the five volunteers that completed the study. The average GSK3β activity for each visit and the average GSK3β activity relative to the baseline activity for each volunteer are given under the graph. The box highlights samples (6 and 7) with the peak plasma lithium levels (Table 3).

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