Detection of TTR Amyloid in the Conjunctiva Using a Novel Fluorescent Ocular Tracer

Detection of TTR Amyloid in the Conjunctiva Using a Novel Fluorescent Ocular Tracer

February 18, 2024 – Background: Transthyretin amyloidosis (ATTR) is a significant cause of cardiomyopathy and other morbidities in the elderly and Black Americans. ATTR can be treated with new disease-modifying therapies, but large shortfalls exist in its diagnosis. The objective of this study was to test whether TTR amyloid can be detected and imaged in the conjunctiva using a novel small-molecule fluorescent ocular tracer, with the implication that ATTR might be diagnosable by a simple eye examination.

Methods: Three approaches were used in this study. First, AMDX-9101 was incubated with in vitro aggregated TTR protein, and changes in its excitation and emission spectra were quantified. Second, a cadaver eye from a patient with familial amyloid polyneuropathy type II TTR mutation and a vitrectomy sample from an hATTR patient were incubated with AMDX-9101 and counterstained with Congo Red and antibodies to TTR to determine whether AMDX-9101 labels disease-related TTR amyloid deposits in human conjunctiva and eye. Last, imaging of in vitro aggregated TTR amyloid labeled with AMDX-9101 was tested in a porcine ex vivo model, using a widely available clinical ophthalmic imaging device.

Results: AMDX-9101 hyper-fluoresced in the presence of TTR amyloid in vitro, labeled TTR amyloid deposits in postmortem human conjunctiva and other ocular tissues and could be detected under the conjunctiva of a porcine eye using commercially available ophthalmic imaging equipment.

Conclusions: AMDX-9101 enabled detection of TTR amyloid in the conjunctiva, and the fluorescent binding signal can be visualized using commercially available ophthalmic imaging equipment.

Translational relevance: AMDX-9101 detection of TTR amyloid may provide a potential new and noninvasive test for ATTR that could lead to earlier ATTR diagnosis, as well as facilitate development of new therapeutics.

Conflict of interest statement

Disclosure: J. Pilotte, Amydis (E); A.S. Huang, Amydis (F); S. Khoury, Amydis (E); X. Zhang, None; A. Tafreshi, Amydis (C); P. Vanderklish, Amydis (E); S.T. Sarraf, Amydis (O); J.S. Pulido, Amydis (F); T. Milman, None

Figures:

Figure 1. Spectroscopic and binding properties of AMDX-9101 combined with aggregated wild type TTR. (A) Fluorescent emission of AMDX-9101 before (blue) and after (purple) mixing with TTR aggregates. (B) Plot of the fluorescence intensity (λmax = 545 nm) as a function of the concentration of AMDX-9101 in the presence of aggregated TTR peptides (5 µM) in solution. Fitting this data to the equation: y = Bx/(Kd + x) revealed a Kd of 1.476 ± 0.3452 µM for association of AMDX-9101 to aggregated TTR peptides. (C) Spectroscopic properties of AMDX-9101 and wt TTR. Fold increase determined from the emission with 4 µM AMDX and 5 µM aggregated TTR at the maximum emission of AMDX + TTR.

Figure 2. Ex vivo co-staining of AMDX-9101 and TTR in human ATTR amyloidosis eye. FFPE ocular sections from a human ATTR amyloidosis eye were either (1) stained with Congo red and examined in bright and polarized light using a Leica microscope equipped with a polarizer and analyzer. The characteristic green birefringent polarization color was taken as proof of the presence of amyloid in the tissue (left column) or (2) FFPE sections were co-stained with AMDX-9101 (green) and TTR antibody (red). Overlay of all channels shows colocalization in yellow. Scale bar = 100 µm. Images taken from 87-year-old man with FAP type II TTR mutation (serine 84) TTR amyloidosis: (A) Sclera adjacent to emissarial canals (ES, episclera; S, sclera; CB, ciliary body; arrows, amyloid); (B) Optic nerve sheath (arrows, amyloid); (C) Retina and vitreous, amyloid deposits around retinal vessels (arrows) and in the vitreous (arrowhead); (D) Posterior ciliary arteries and nerves (arrows); (E) Conjunctiva, epithelium (arrows), and vessels in the stroma (arrowheads). (F) Vitreous amyloid from an hATTR patient (TTR c.148G>A; p.Val50Met heterozygous missense, autosomal dominant).

Figure 3. Ex vivo ocular images in human controls. FFPE ocular sections from human control eyes were either stained with Congo red and examined in bright and polarized light using a Leica microscope equipped with a polarizer and analyzer (the characteristic green birefringent polarization color was taken as a proof of the presence of amyloid in the tissue, left column) or co-stained with AMDX-9101 (green) and the TTR antibody (red). Overlay of all channels shows absence of colocalization in yellow. Scale bar = 100 µm. (A–C) From a 62-year-old patient with uveal melanoma: (A) optic nerve sheath (Sh), sclera (Sc), and posterior ciliary arteries/nerves (arrowheads). Note intrinsic yellow-white birefringence of the collagen, (B) sclera (S), and extraocular muscles (M), and (C) posterior ciliary arteries (arrow) and nerves (arrowhead). (D) Conjunctiva from three-year-old patient, epithelium (arrow), vessels in the stroma (arrowheads). (E) Conjunctiva from healthy adult, epithelium (arrow), vessel (arrowheads).

Figure 4. Quantification of AMDX-9101 signal intensity. Regions were selected from four separate fields. All images were analyzed by MATLAB R2022b. The fluorescent signal-to-noise ratio was calculated as the average of the foreground pixels over the average of the background pixels. Results were plotted as box plots, indicating the twenty-fifth percentile (bottom boundary), median (middle line), seventy-fifth percentile (top boundary), and nearest observations within 1.5 times the interquartile range (whiskers).

Figure 5. Visualization of TTR with AMDX-9101 in an ex vivo porcine eye model. (A) Nitrocellulose membrane discs (NMDs) were cut and incubated with TTR protein and AMDX-9101 and stained with Ponceau-S (top panel). Fluorescence images were taken using a Spectralis angiographic camera, demonstrating fluorescence with AMDX-9101. (B)

Julie Pilotte 1, Alex S Huang 2, Sami Khoury 1, Xiaowei Zhang 2, Ali Tafreshi 1, Peter Vanderklish 1, Stella T Sarraf 1, Jose S Pulido 3, Tatyana Milman 3, 4

1 Amydis, Inc., La Jolla, CA, USA.
2 Hamilton Glaucoma Center, Shiley Eye Institute, Viterbi Family Department of Ophthalmology, University of California San Diego, La Jolla, CA, USA.
3 Vickie and Jack Farber Vision Research Center and MidAtlantic Retina Service, Wills Eye Hospital, Philadelphia, PA, USA.
4 Pathology Department, Wills Eye Hospital, Philadelphia, PA, USA.

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