Sustained peripheral depletion of amyloid-β with a novel form of neprilysin does not affect central levels of amyloid-β

Abstract

Alzheimer's disease is characterized by the accumulation of amyloid deposits in the brain and the progressive loss of cognitive functions. Although the precise role of amyloid-β in disease progression remains somewhat controversial, many efforts to halt or reverse disease progression have focussed on reducing its synthesis or enhancing its removal. It is believed that brain and peripheral soluble amyloid-β are in equilibrium and it has previously been hypothesized that a reduction in peripheral amyloid-β can lower brain amyloid-β, thereby reducing formation of plaques predominantly composed of insoluble amyloid-β; the so-called peripheral sink hypothesis. Here we describe the use of an amyloid-β degrading enzyme, the endogenous metallopeptidase neprilysin, which is fused to albumin to extend plasma half-life and has been engineered to confer increased amyloid-β degradation activity. We used this molecule to investigate the effect of degradation of peripheral amyloid-β on amyloid-β levels in the brain and cerebrospinal fluid after repeated intravenous dosing for up to 4 months in Tg2576 transgenic mice, and 1 month in rats and monkeys. This molecule proved highly effective at degradation of amyloid-β in the periphery but did not alter brain or cerebrospinal fluid amyloid-β levels, suggesting that the peripheral sink hypothesis is not valid and is the first time that this has been demonstrated in non-human primates.

Keywords: Alzheimer’s disease; amyloid-β; neprilysin; peripheral sink hypothesis.

Figure 1

Amyloid-β_1–40 (Aβ1–40) and/or amyloid-β_1–42 (Aβ1–42) concentrations in plasma, CSF and brain of female Tg2576 mice after twice-weekly intravenous administration of MSA-mNEPv (0 and 25 mg/kg) for 4 months. (A) Mean (± SEM) amyloid-β_1–40 concentrations in plasma (expressed as per cent of mean vehicle concentration at the equivalent time) after 5 weeks (10th dose), 9 weeks (18th dose), 12 weeks (24th dose) or 18 weeks (34th and 35th dose) of treatment (open circle = vehicle, filled circle = MSA-NEPv 25 mg/kg). Amyloid-β_1–40 concentrations in vehicle-treated animals ranged from 520 to 9400 pg/ml. Samples were taken from 10–12 of 34–36 mice per treatment group at each time point in a rolling scheme such that each mouse was bled only once a month except at termination (24 h after the 35th dose) where all animals were sampled. ***P < 0.001; **P < 0.01. (B) Mean (± SEM) amyloid-β_1–40 (left) and amyloid-β_1–42 (right) concentrations in CSF. Samples were taken 24 h after the final dose. Data only presented for samples without blood contamination (n = 14–19 per group). ns = not statistically significant. (C) Mean (± SEM) soluble amyloid-β_1–40 (left) and amyloid-β_1–42 (right) concentrations in brain at 24 h after the final dose (n = 33–34 per group). (D) Mean (± SEM) insoluble amyloid-β_1–40 (left) and amyloid-β_1–42 (right) concentrations in brain at 24 h after the final dose (n = 33–34 per group).

Figure 2

Soluble amyloid-β_1–40 (Aβ1–40) and/or amyloid-β_1–42 (Aβ1–42) concentrations in plasma, CSF and brain of male and female rats after twice-weekly intravenous administration of HSA-hNEPv (0, 5, 50 and 143 mg/kg) for 1 month. All plots include animals that were positive for anti-drug antibodies at the end of the study. ns = not statistically significant. (A) Mean (± SEM) amyloid-β_1–40 concentrations in plasma (expressed as per cent of pre-dose). Arrows denote times of dosing prior to collection of samples for analysis. Amyloid-β levels on Days 21 and 28 are shown pre- and post-dose to illustrate the degree of suppression during the study. Pre-dose plasma amyloid-β_1–40 concentrations ranged from 30–100 pg/ml [n = 6–10, 10–12, 10–11 and 10–11 animals/time point for 0 (open circle), 5 (filled square), 50 (filled triangle) and 143 (open triangle) mg/kg, respectively]. ***P < 0.001, **P < 0.01, *P < 0.05. (B) Amyloid-β _1–40 concentrations in plasma of individual animals (expressed as pg/ml) at termination (Day 32; n = 10 per group, bar represents mean). **P < 0.01. (C) Amyloid-β_1–40 concentrations in CSF of individual animals at termination (Day 32). Filled squares refer to animals that tested negative for anti-drug antibody and open squares refer to animals that were positive for anti-drug antibody (n = 15, 20, 18 and 18 for 0, 5, 50 and 143 mg/kg, respectively; bar represents mean). (D) Soluble amyloid-β_1–40 (left) and soluble amyloid-β_1–42 (right) concentrations in brain of individual main study animals at termination (Day 32). Filled squares refer to animals that tested negative for anti-drug antibody and open squares refer to animals that were positive for anti-drug antibody (n = 10 per group, bar represents mean). BLQ = below the limit of accurate quantification.

Figure 3

Amyloid-β_1–40 (Aβ1–40) and/or amyloid-β_1–42 (Aβ1–42) concentrations in plasma, CSF and brain of male cynomolgus monkeys after twice-weekly intravenous administration of HSA-hNEPv (0, 5, 50 and 143 mg/kg) for 1 month. Only animals that maintained exposure to HSA-hNEPv to the end of the study (i.e. that did not develop clearing antibodies) are included (n = 3 animals/time point at 0 mg/kg and n = 2 at 5, 50 and 143 mg/kg). (A) Mean (± SEM) amyloid-β_1–42 concentrations in plasma (expressed as per cent of pre-dose) at 0 (open circle), 5 (filled square), 50 (filled inverted triangle), and 143 (filled triangle) mg/kg. Arrows denote times of dosing. Amyloid-β levels on Day 11 are shown pre- and post-dose to illustrate the degree of suppression during the study. Pre-dose plasma amyloid-β_1–42 concentrations ranged from 20–100 pg/ml. (B) Amyloid-β _1–40 (left) and amyloid-β_1–42 (right) concentrations in CSF of individual animals at termination (Day 31, bar represents mean). (C) Soluble Aβ_1–40 (left) and soluble amyloid-β_1–42 (right) concentrations in brain of individual animals at termination (Day 31, bar represents mean). BLQ = below the limit of accurate quantification.

Figure 4

Amyloid-β_1–40 (Aβ1–40) and/or amyloid-β_1–42 (Aβ1–42) concentrations in plasma and CSF of male cynomolgus monkeys with implanted CSF catheters after twice-weekly intravenous administration of HSA-hNEPv (0, 5 and 50 mg/kg) for 1 month. Arrows denote times of dosing. Amyloid-β levels on Days 7, 18 and 25 are shown pre- and post-dose to illustrate the degree of suppression during the study. All plots include animals where exposure was reduced presumably as a consequence of development of anti-drug antibodies. (A) Mean (± SEM) amyloid-β_1–42 concentrations (expressed as per cent of pre-dose) in plasma after dosing of HSA-NEPv at 0 (filled circle), 5 (filled square) and 50 (open inverted triangle) mg/kg. Pre-dose plasma amyloid-β_1–42 concentrations ranged from 20–50 pg/ml. (B) Mean (± SEM) amyloid-β_1–42 concentrations in CSF after dosing HSA-NEPv at 0 (filled circle), 5 (filled square) and 50 (open inverted triangle) mg/kg. Pre-dose CSF Aβ_1–42 concentrations ranged from 300–1000 pg/ml. (C) Mean (± SEM) amyloid-β_1–40 concentrations in CSF after dosing at 0 (filled circle), 5 (filled square), and 50 (open inverted triangle) mg/kg. Pre-dose CSF amyloid-β_1–40 concentrations ranged from 400–3500 pg/ml.

Figure 5

Expression of APP in male cynomolgus monkeys after twice-weekly intravenous administration of HSA-hNEPv (0, 5, 50 and 143 mg/kg) for 1 month. (A) Protein extract from frontal cortex samples taken 3 days after the last dose (Day 31) analysed by western blot probed with anti-APP and anti-actin. Dose level (mg/kg) is indicated, asterisk indicates animals that lost exposure to the drug because of immunogenicity. (B) APP expression levels normalized to the signal from anti-actin. Signal intensity for APP and actin in the western blot were quantified and the APP signal divided by the actin signal. Only animals which maintained exposure to HSA-hNEPv to the end of the study (i.e. which did not develop clearing antibodies) are included (n = 3 at 0 mg/kg and n = 2 at 5, 50 and 143 mg/kg). Bar represents mean.