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Evaluate the safety, tolerability and amyloid beta (Aβ) response to a γ-secretase inhibitor (LY450139) in Alzheimer's disease.
Multi-center, randomized, double-blind, dose-escalation, placebo-controlled trial.
Community based clinical research centers.
51 participants with mild to moderate AD were randomized (placebo=15, 100mg=22, 140mg=14), with 43 completing the treatment phase.
Subjects randomized to LY450139 received 60mg daily for 2 weeks followed by 100mg for 6 weeks, then re-randomized to 100mg or 140mg for 6 additional weeks.
Primary outcome measures consisted of adverse events, plasma and cerebrospinal fluid Aβ levels, vital signs, electrocardiogram data, and laboratory safety tests. Secondary outcome measures included the ADAS-cognitive subscale and the ADCS-Activities of Daily Living scale.
Group differences were seen in “skin and subcutaneous tissue” complaints (p=0.052). These included 3 possible drug rashes and 3 reports of hair color change in the treatment groups. There were 3 adverse-event-related discontinuations, including one report of transient bowel obstruction. Plasma Aβ40 was reduced by 58.2% for the 100mg group and 64.6% for the 140mg group (P<0.001). No significant reduction was seen in CSF Aβ. No group differences were seen in cognitive or functional measures.
LY450139 was generally well tolerated at doses of up to 140mg taken daily for 14 weeks with several findings indicating the need for close clinical monitoring in future studies. Decreases in plasma Aβ concentrations were consistent with inhibition of γ-secretase.
LY450139 is a functional γ-secretase inhibitor that is currently under development as a disease-modifying therapy for Alzheimer's disease (AD). As reported previously, LY450139 rapidly reduces Aβ concentrations in the brain, cerebrospinal fluid (CSF) and plasma of transgenic APPV717F mice (“PDAPP” mice)1, 2 and in plasma of humans.3 Additionally, administration of LY450139 at doses of 30mg/kg once daily for 5 months to PDAPP mice results in reduced accumulation of Aβ in hippocampus and cortex as measured by ELISA.4 Previous clinical studies using LY450139 with either normal volunteers5 or patients with AD6 have shown acute reductions in plasma Aβ40 up to approximately 40% using single daily doses up to 50mg. A single-dose escalation study using 60mg, 100mg or 140mg in normal volunteers demonstrated a dose-proportional increase of drug levels in plasma and CSF, as well as a dose-dependent reduction in plasma Aβ.3
Tolerability of LY450139 in clinical studies of up to 50mg for 2 weeks5 or 40mg for 6 weeks6 duration was generally good. However, there is concern for cumulative toxicity that may not be revealed in short low dose trials or higher single dose biomarker studies. A potential cause of clinical toxicity for γ-secretase inhibitors is the inhibition of Notch cleavage by these compounds.7 Notch cleavage is integral to cell differentiation pathways in many organ systems including the gastrointestinal tract and lymphoid cell lines. The effect of multiple dose administration of 100mg and 140mg of LY450139 in subjects with AD has not been reported previously.
In this study we aimed to demonstrate the safety and tolerability of LY450139 over 14-weeks using single daily doses of 100mg and 140mg. We wished to determine if extended exposure to these higher doses of LY450139 would be tolerated and result in expected changes in plasma and CSF Aβ levels.
A total of 51 participants were enrolled at 6 academic research centers between October 2005 and December 2006. The protocol was reviewed and approved by the institutional review board for each participating site. All research participants and caregivers gave written informed consent. An independent data and safety monitoring board provided oversight on an ongoing basis.
Participants were ≥50 years old with a diagnosis of probable AD as defined by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer's Disease and Related Disorders Association (ADRDA) criteria.8 Participants on stable doses of cholinesterase inhibitor drugs or memantine were included. Participants were excluded if they had a history of irritable bowel syndrome, chronic diarrhea, peptic ulcer, or gastroesophageal reflux disease, a history of cardiac disease, significant electrocardiogram abnormalities, hematologic disorders, hepatic or renal disease, active malignancy within five years, or clinically important depression, neuropsychiatric, cerebrovascular or respiratory disease.
The study was a multi-center, randomized, double-blind, placebo-controlled, dose-escalation trial (Figure 1). Participants were randomized to LY450139 or placebo using a 2:1 randomization scheme via a telephone based interactive voice response system. Subjects randomized to LY450139 received 60mg once daily for 2 weeks, then 100mg once daily for the next 6 weeks. At eight weeks the treatment arm was re-randomized to receive 6 additional weeks of treatment at either 100mg or 140mg per day. Dose reductions were allowed for dose-limiting adverse events during the treatment phase.
All adverse events occurring during the study were characterized. Safety data such as vital signs, electrocardiogram (ECG) data and laboratory test results were collected throughout the trial. Vital signs were recorded at each study visit, with ECGs performed at baseline and weeks 2, 4, 8, 10, 14 and 16.
Routine hematology and clinical chemistry laboratory tests were obtained at screening and weeks 2, 4, 8, 10, and 14 as well as at a 26-week follow-up visit. Measurements of CD4, CD8, CD19, IgG, IgA, and IgM cells were obtained at baseline, treatment end, and the 26 week follow-up visit. T-cell function was assessed by flow-cytometry3. Urinalysis was tested at screening, weeks 8 and 14. Stool samples were tested for occult blood at each study visit after baseline. Optional DNA testing for apolipoprotein E subtype was performed.
Plasma samples for biomarkers and drug levels were collected at baseline, weeks 14 and 16. At week 14 multiple samples were collected over 6 hours for pharmacodynamic modeling (30 minutes pre-drug dose, and post dose hours 0.5, 1, 2, 4 and 6). CSF samples were collected into polypropylene tubes by means of a lumbar puncture at baseline and approximately 6 hours after the last dose of LY450139 at week 14.3 Plasma and CSF concentrations of Aβ were measured from all samples3, and concentrations of LY450139 from all samples after baseline.5
Adverse event data are reported using intention-to-treat (ITT) analyses (n=51) over 26 weeks, with a Completers analyses for all comparisons of laboratory and clinical measures through week 14 (n=43). To maximizing sensitivity of safety measures, no adjustments were made for multiple comparisons. All statistical tests were two-sided. A p value <0.05 was considered statistically significant for biomarker and clinical measures, while a p value <0.10 was used for safety variables to indicate potential clinical significance. All analyses were performed using the R statistical package, Version 2.3.111
Data from all randomized participants over 26 weeks were included in the safety analyses. Fisher's Exact Test was performed to assess group differences in proportions of participants reporting specific types of adverse events (AEs) and serious adverse events (SAEs).
Analysis of covariance (ANCOVA) was used to assess 14 week change scores in all efficacy and biomarker outcomes of interest with treatment group as a factor. Significance was based on the overall F statistic for interactions between all three treatment arms. Baseline values of outcome measures were entered as covariates into each model to account for any baseline group differences. In addition, any variable found to have significant between-group differences at baseline and correlate with the outcome measure being tested, was entered as a confounder into a post-hoc ANCOVA model. Outcome variables included vital signs, laboratory measures, Fridericia-corrected QT intervals (QTcF), log mean CSF Aβ40 and Aβ42 measures, ADAS-COG11 and ADCS-ADL. A two-way ANCOVA was used to investigate whether the treatment effect on the change in ADAS-Cog11 was modified by use of donepezil. Post-hoc Bayesian estimates were used to determine pharmacokinetic parameters.5 At week 14 mean change in LY450139 and absolute mean plasma Aβ levels were compared for each time point over six hours using Kruskal-Wallis non-parametric tests of means.
Of the 71 participants who were screened for the trial, 51 were eligible and randomized. 43 subjects completed the treatment phase of the trial (Figure 2). There were no differences in drop-out rates (84%) or medication compliance (96-99%) between the three groups.
The three groups were balanced at baseline with regard to all demographics, clinical measures (except MMSE), APOE ε4 distribution, and acetylcholine esterase inhibitor and memantine use (Table 1). Differences in baseline laboratory values are shown in Table 1.
Adverse events that occurred in two or more participants are presented in Table 2. Only the category of “Skin and Subcutaneous tissue” complaints reached statistical significance for differences between groups (p=0.052). In this category, there were 3 possible “drug-related” rashes and 3 reports of hair color change (lightening) in the treatment groups, with none in placebo. Although not reaching statistical significance, the number of subjects reporting nausea, vomiting or diarrhea was 26.6% in the treatment groups and only 13.3% for placebo (p=0.41). In addition, when combining reports of somnolence, fatigue, lethargy and asthenia from different organ classes we found 39.6% of treatment subjects complained of one or more of these symptoms while only 13.3% in the placebo group had these symptoms (p=0.18). There were four SAEs (Table 3). A small bowel obstruction was considered to be possibly related to drug; no medical intervention was required, the study drug was discontinued, and the obstruction resolved spontaneously.
Eight participants dropped out of the trial during the treatment phase. Reasons included adverse events (n=3), personal conflict (n=3), physician decision due to difficulty managing increasing agitation and paranoia (n=1), and a protocol violation consisting of a subject reporting a history of gastroesophageal reflux disease after randomization (n=1). The adverse events included: 1) diarrhea, 2) heme-positive stool (required drop-out per protocol), and 3) the previously described bowel obstruction. There were three site-initiated dose reductions for rash, abdominal discomfort, and nausea.
Between group differences were seen in some safety laboratory value changes during the therapy phase (Table 4). None of these changes were considered clinically important based on the degree of change and lack of associated adverse events.
There were no statistically significant group differences in mean change in ECG QTcF interval over 14 weeks. The 140mg group showed the greatest numeric change with a prolongation of 19.3ms (4.8%) compared to an increase of 2.8 ms in the placebo group (p=0.18).
At week 14, a rapid increase in plasma drug levels was seen after dosing (Figure 3). Peak plasma levels occurred at 1.26 hours (t1/2=2.23hrs) for the 100mg group and 1.68 hours (t1/2=2.59hrs) for 140mg group.
A CSF sample could not be obtained for one participants in the 140mg group. Six hours post dosing at week 14, the 100mg and 140mg groups had mean CSF drug concentrations of 77.4±34.3ng/mL and 102.1±30.3ng/mL respectively (Wilcoxon Rank Sum p=0.017).
At week fourteen, just prior to dosing, absolute mean plasma Aβ40 levels were increased by 32.0%±34.6 in the 100mg group and 35.2%±60.6 in the 140mg group compared to study baseline (Kruskal Wallis p=0.009) (Figure 4). No significant elevations were seen in Aβ42.
After drug administration at week 14, mean plasma Aβ40 declined rapidly in both treatment groups (Figure 4). Concentrations dropped significantly below baseline by hour 2 for both treatment groups (p=0.017), and continued to decline up to 6 hours post-dose (p<0.001). The maximum reduction in Aβ40 concentration was 58.2% for the 100mg group and 64.6% for the 140mg group (Figure 4). There was no significant difference in Aβ40 reduction between the 100mg and 140mg groups. Over this six hour period, fourteen out of thirty-six treated subjects had one or more plasma Aβ42 level drop below the lower limit of quantification (LLOQ) measured by ELISA (28.0pg/mL), making these data statistically non-interpretable.
No significant reductions were seen in mean log CSF Aβ40 (F statistic=2.025; p=0.15) or Aβ42 (F statistic=0.73; p=0.49) in the full ANCOVA model. Percent change from baseline of absolute means are presented in Figure 5. Spearman correlations showed trend association between csf drug levels and percent change in csf Aβ in the 140mg group (Aβ40 r=-0.51, p=0.094; Aβ42 r=-0.51, p=0.094) but not in the 100mg group (Aβ40 r=-0.35, p=0.17; Aβ42 r=-0.2, p=0.44). Change in absolute csf Aβ levels showed trend associations with drug levels for both 140mg group (Aβ40 r=-0.43, p=0.17; Aβ42 r=-0.55, p=0.067) and the 100mg group (Aβ40 r=-0.47, p=0.062; Aβ42 r=-0.21, p=0.42)
No significant differences were seen in ADAS-Cog11 (p=0.36) or ADCS-ADL (p=0.63) after 14 weeks of treatment between any of the three groups (Table 5). This lack of treatment effect was not modified by inclusion of donepezil use in the ANCOVA model.
MMSE was the only variable found to be a confounding measure. The three treatment groups differed at baseline on MMSE scores, with a possible relationship found between baseline MMSE scores and change in CSF Aβ40 (Spearman rho=0.30, p=0.058). An ANCOVA model including MMSE as a covariate revealed a trend towards significant reductions in CSF Aβ40 at week fourteen compared to study baseline in the treatment groups compared to placebo(F statistic=2.90; p=0.068)(Figure 5).
This longitudinal study in elderly subjects with AD was necessary for assessing safety and possible Notch-related toxicities, which would not likely be revealed by single dose studies.12 Notch toxicity encompasses much of the drug related tolerability concerns for gamma secretase inhibitors.13 Notch is a transmembrane protein that plays a role in nuclear signaling, and, like the amyloid precursor protein, it appears to be cleaved by a presenilin-dependent γ-secretase complex.7 It plays an important role in programmed cellular death. Organ systems with rapid cellular turnover have therefore been the primary concern for Notch-related toxicity. Both gastrointestinal and immune cell functions have been altered in preclinical studies with LY450139 (data on file, Eli Lilly and Company). No previous human study with LY450139 has demonstrated clinically significant toxic effects on the immune system.3, 5, 6 Diffuse macular rash, on the extremities and torso, as well as hair color changes in some participants were likely the result of treatment with LY450139. However, there was no evidence of other toxicities associated with these. Both the rash and the hypopigmentation were reversible. Gastrointestinal symptoms, somnolence/asthenia and ECG changes were not found to be statistically different between groups, yet they should still be considered potentially important drug related adverse events given the known mechanism of action of this drug and the limited statistical power of this study.
As shown in Figure 4, the plasma level of Aβ40 is above study baseline prior to dosing at visit fourteen. In previous single dose studies a biphasic response of plasma Aβ to LY450139 is observed at 60 mg, 100mg or 140mg, with an initial reduction in plasma Aβ followed by an elevation above baseline levels eight to ten hours after the dose.3 Doses of 100mg or greater prolonged plasma reductions and reduced plasma elevations over 24 hours compared to 60mg.3 Similar patterns of peripheral changes in Aβ have been seen in pre-clinical studies of guinea pigs14 with LY450139 and in studies of other γ-secretase inhibitors15. But, similar increases in CSF or brain Aβ levels have not been demonstrated in previous pre-clinical studies of LY450139 at multiple post-dosing intervals up to 24 hours (data on file). In addition, studies using very low doses of LY450139 showed an increase in plasma Aβ without a period of reduction.5, 14 Thus, one possible explanation for a transient increase in plasma Aβ is that, in peripheral but not in central tissue(s), γ-secretase inhibitors have a stimulatory effect on the enzyme at low concentrations that is overcome by the inhibitory effects at higher concentrations. Although steady state Aβ reductions would be desirable, twice daily dosing is not possible due to observed Notch-related toxicity in multiple organ systems in pre-clinical studies of Fischer 344 rats and dogs (data on file). The clinical implications of this are unclear.
Clear changes in CSF Aβ levels were not demonstrated in this study as expected despite robust reductions in plasma Aβ40 and dose related LY450139 concentrations in the CSF. Correlation analyses between csf drug levels and Aβ levels suggest a pharmicodynamic response, but perhaps lack in statistical power. In preclinical studies CSF Aβ-lowering effects have been seen in both PDAPP mice1 and dogs (data on file). Lack of CSF changes in the setting of clear serum changes may reflect rapid transport of Aβ from CSF into plasma and a lengthened time period for Aβ to reach equilibrium in CSF. In a trial of a similar γ-secretase inhibitor, changes in CSF Aβ lagged behind changes in plasma Aβ, with significant decreases seen at 12 hours.15 One study demonstrated that 13C labeled Aβ did not reach equilibrium in lumbar CSF until approximately 13 hours after the beginning of the infusion.16 In our current study, Aβ levels were measured at approximately six hours after morning drug dosing. It may require longer periods of time to identify CSF changes. Furthermore, transgenic PDAPP mice1, wildtype mice1, and guinea pigs14 show a clear association between the reduction of plasma Aβ and brain Aβ following administration of LY450139. Whether or not CSF changes in Aβ can be detected in people with AD after oral dosing of LY450139 requires additional studies.
The long term efficacy of this drug is not known. Given the slow rate of clinical progression in AD, we did not expect to see drug effects on measures of cognition or ADLs in this fourteen week trial. Without this, a full risk to benefit assessment cannot be made. This current trial sufficiently demonstrated that LY450139 could be tolerated, though not without risk. Given the potential for disease modifying effects of this Aβ lowering agent, and the arguably acceptable tolerance and safety profile of LY450139 demonstrated in this study, further large scale efficacy trials are justified. Based in part on the results from this phase 2 study, Eli Lilly and company is launching a multinational phase 3 trial in the second quarter of 2008, enrolling 1500 AD subjects.
All data was collected and analyzed by the Alzheimer's Disease Cooperative Study in a collaborative effort with Eli Lilly and co., with all funding provided by Eli Lilly and co. The ADCS had access to all data with full, unrestricted, publication rights. Study design was developed by Eli Lilly in consultation with the ADCS. This study was conducted by the ADCS, with data collection, management, analysis, and interpretation of the data by the ADCS. Manuscript preparation was completed by the ADCS in consultation with Eli Lilly and company. The ADCS infrastructure is supports by a National Institute on Aging cooperative grant UO1AG10483.
Disclosure: Dr. Siemers and Dr. Dean are employees of Eli Lilly, the manufacturer of the study drug. Eli Lilly and company provided all funding for this study. All study sites were subcontracted through the Alzheimer's Disease Cooperative Study at the University of California, San Diego, which held the primary contract with Eli Lilly. Dr. Farlow has received research grant funding support from Eli Lilly which was independent and unrelated to this trial. For all other authors there are no disclosures to be made of any personal financial support from, or equity positions in, manufacturers of drugs or products mentioned in the manuscript. All authors have agreed to conditions noted on the Author Disclosure Form. All of the authors have been instrumental in the interpretation of the data and its presentation in this paper. This manuscript has not been simultaneously submitted for publication to any other journal. There is no patient identifiable data presented in this manuscript. Written permission for publication has been obtained from all authors.