N-Acetylcysteine for Hereditary Cystatin C Amyloid Angiopathy: A Nonrandomized Clinical Trial

Abstract

Importance: Hereditary cystatin C amyloid angiopathy (HCCAA) is a lethal, dominantly inherited disease primarily affecting Icelandic young adults that leads to severe cerebral amyloid angiopathy, with no effective therapy.

Objective: To investigate safety, tolerance, and therapeutic potential of N-acetylcysteine (NAC) in lowering disease-associated biomarkers in sequence variation carriers.

Design, setting, and participants: This phase 2a open-label clinical trial was conducted from March 2019 to December 2021 at a single study center at Landspitali University Hospital in Reykjavik, Iceland, and included 17 confirmed carriers of the L68Q-CST3 sequence variation.

Intervention: High-dose NAC treatment was administered at 2400 mg daily for 9 months. Participants underwent regular monitoring for hemorrhages and disease progression, including blood and skin biopsy samples obtained every 3 months for biomarker testing.

Main outcomes and measures: The primary outcomes were drug tolerability and safety, cognitive status, and reduction in disease-associated biomarkers in skin biopsies. Secondary outcomes included changes in blood and plasma biomarker levels.

Results: Of 17 carriers treated, 6 were male and 11 were female, and mean (SD) participant age was 40.0 (4.2) years. Analysis of the primary outcomes showed that NAC was safe and well tolerated. Five cerebral bleeds occurred during the treatment period without permanent neurological sequela; no death occurred. There was significant reduction in median (IQR) disease-specific biomarker levels in skin after treatment, including collagen IV (baseline: 3.69% [2.48%-5.16%]; after treatment: 2.60% [1.99%-2.97%]; P < .001), fibronectin (baseline: 3.17% [2.09%-5.05%]; after treatment: 2.37% [1.87%-3.42%]; P = .01), vimentin (baseline: 1.60% [1.24%-2.37%]; after treatment: 1.31% [0.97%-1.68%]; P < .001), and SMAD (baseline: 2.25% [0.55%-4.36%]; after treatment: 1.56% [0.20%-2.54%]; P < .001) via Wilcoxon matched-pairs signed rank test. Secondary outcomes included a significant increase in reduced glutathione levels and decreased high-molecular-weight cystatin C aggregate levels in plasma after NAC treatment.

Conclusions and relevance: In this single-center nonrandomized clinical trial, NAC was safe and well tolerated and decreased disease-associated biomarker and amyloid deposition, suggesting NAC may offer a preventive strategy against HCCAA.

Trial registration: ClinicalTrialsRegister.eu Identifier: 2017-004776-56.

Figure 1.. Study Flow Diagram and Patient Characteristics

A, Study flow diagram showing enrollment of patients with hereditary cystatin C amyloid angiopathy (HCCAA) and analyses. B, Characteristics of patients with HCCAA at baseline. CT indicates computed tomography; NAC, N-acetylcysteine.

Figure 2.. Skin Biopsies at Baseline (V1) and Following 9 Months of Treatment (V4)

Statistical analysis was done with the Wilcoxon matched-pairs signed rank test (P < .05) for comparing percentage immunoreactivity per region of interest (ROI) in biopsy V1 to biopsy V4 in 15 patients. All plots show change in median (IQR) percentage biomarker staining per ROI from V1 to V4 in carrier biopsies and controls. A, Skin biopsy from control (healthy family member) showing no L68Q–human cystatin C (hCC) amyloid complex deposition. In carriers, moderate hCC amyloid complex deposition is seen in the basement membrane (BM), between epidermis and dermis, and in upper dermis in V1 and less intensive deposition in V4. For hCC, V1: 0.89% (0.51%-4.75%); V4: 0.65% (0.19%-4.10%); P = .27; controls: 0.003% (0.0015%-0.01%). B, Skin biopsy from control with collagen IV staining in BM between epidermis and dermis and BMs in dermis. In carriers, more intense staining is seen in V1, especially in the BM between epidermis/dermis and in fibroblasts in upper dermis (arrowheads), and less extensive staining in seen in V4, mainly in fibroblasts. For collagen IV, V1: 3.69% (2.48%-5.16%); V4: 2.60% (1.99%-2.97%); P < .001; control: 1.97% (1.29%-2.44%). C, Skin biopsy from control with fibronectin staining in BMs, more intense staining in V1 from carrier, especially in fibroblasts in upper dermis (arrowhead) and less intense staining in fibroblasts in V4. For fibronectin, V1: 3.17% (2.09%-5.05%); V4: 2.37% (1.87%-3.42%); P = .01; control: 2.95% (2.06%-4.29%). One control biopsy had intense fibronectin immunoreactivity. D, Skin biopsy from control with vimentin staining, showing normal fibroblasts in upper dermis, biopsy V1 from carriers showing an elevated number of fibroblasts in the upper dermis with activated appearance (arrowheads) and less staining and more normal appearance in fibroblasts in V4. For vimentin, V1: 1.60% (1.24%-2.37%); V4: 1.31% (0.97%-1.68%); P < .001; control: 0.88% (0.68%-1.08%). E, Skin biopsy from control participant showing normal fibroblasts with SMAD 2/3 immunoreactivity in upper dermis, biopsy V1 from carrier showing an increased number of fibroblasts with SMAD 2/3 immunoreactivity in the upper dermis between V1 and V4. For SMAD, V1: 2.25% (0.55%-4.36%); V4: 1.56% (0.20%-2.54%); P < .001; control: 1.35% (1.16%-1.56%). All figures were taken with 20 × objective. Scale bar represents 100 μm on all figures. C indicates controls.

Figure 3.. Free Glutathione (GSH) and GSH:Glutathione Disulfide (GSSG) Ratio Analysis in Patients With Hereditary Cystatin C Amyloid Angiopathy (HCCAA)

The plots show the results from analysis for free GSH and GSH:GSSG ratio at baseline (V1) and following 9 months of treatment (V4) for all 15 patients with HCCAA and for 6 N-acetylcysteine (NAC)–naive (NAC-) patients. Statistical analysis was done with the Wilcoxon test (P < .05) and plots were conducted in GraphPad and represent median with interquartile range. A, There was not a significant change in median (IQR) free GSH between V1 and V4 in patients with HCCAA (V1: 1.68% [0.90%-10.85%]; V4: 1.56% [0.76%-18.72%]; P = .93). B, There was not a significant change in median (IQR) GSH:GSSG ratio in patients with HCCAA (V1: 1.64% [0.96%-13.52%]; V4: 2.01% [0.06%-80.53%]; P = .12). C, There was a significant change in median (IQR) free GSH between V1 and V4 in NAC- patients (V1: 6.64% [1.18%-18.02%]; V4: 18.64% [16.57%-18.78%]; P = .03). D, There was a measurable change, albeit not significant, in median (IQR) GSH:GSSG ratio between V1 and V4 in NAC- patients (V1: 18.26% [1.67%-22.15%]; V4: 25.31% [12.62%-35.40%]; P = .12).

Figure 4.. N-Acetylcysteine (NAC) Treatment Reduced High-Molecular-Weight (HMW) Human Cystatin C (hCC) Levels in Plasma Samples of Patients With Hereditary Cystatin C Amyloid Angiopathy (HCCAA)

High-molecular-weight (HMW) human cystatin C (hCC) was detected by Western blot (WB) in plasma samples of L68Q-hCC carriers. A, Representative figure of HMW hCC levels in WB analysis shows reduction in HMW hCC levels following NAC therapy. B, HMW hCC levels were determined by WB and quantitated by optical density. Bars represent means, error bars represent standard deviation, and individual data points are depicted as dots. Data were normalized to the V1 time point of each patient for accurate comparison (n = 15 patients for time points V1, V2, and V3; n = 11 for time point V4). Normal distribution was tested, and comparisons between the V1 and V4 sample population were made using 2-tailed t tests. There was significant reduction in mean (SD) HMW hCC levels between baseline sample (V1) (100% [0]) and treatment at 9 months (V4) (73.6% [27.9%]) with 2-tailed Student t test (P = .001). C, hCC ratio in L68Q-hCC carriers analyzed by mass spectrometry who were not previously treated with NAC prior to clinical trial enrollment (left group) and patients who were receiving a low- to medium-dose NAC (middle group) vs high-dose NAC (right group) upon study enrollment. Bars represent mean and error bars represent standard deviation of hCC ratio between V4 and V1 and show reduction in HMW levels at V4. Individual data points are depicted as dots.