Systemic Dendrimer-Peptide Therapies for Wet Age-Related Macular Degeneration

Abstract

Wet age-related macular degeneration (AMD) is an end-stage event in a complex pathogenesis of macular degeneration, involving the abnormal growth of blood vessels at the retinal pigment epithelium driven by vascular endothelial growth factor (VEGF). Current therapies seek to interrupt VEGF signaling to halt the progress of neovascularization, but a significant patient population is not responsive. New treatment modalities such as integrin-binding peptides (risuteganib/Luminate/ALG-1001) are being explored to address this clinical need but these treatments necessitate the use of intravitreal injections (IVT), which carries risks of complications and restricts its availability in less-developed countries. Successful systemic delivery of peptide-based therapeutics must overcome obstacles such as degradation by proteinases in circulation and off-target binding. In this work, we present a novel dendrimer-integrin-binding peptide (D-ALG) synthesized with a noncleavable, "clickable" linker. In vitro, D-ALG protected the peptide payload from enzymatic degradation for up to 1.5 h (~90% of the compound remained intact) in a high concentration of proteinase (2 mg/mL) whereas ~90% of free ALG-1001 was degraded in the same period. Further, dendrimer conjugation preserved the antiangiogenic activity of ALG-1001 in vitro with significant reductions in endothelial vessel network formation compared to untreated controls. In vivo, direct intravitreal injections of ALG-1001 and D-ALG produced reductions in the CNV lesion area but in systemically dosed animals, only D-ALG produced significant reductions of CNV lesion area at 14 days. Imaging data suggested that the difference in efficacy may be due to more D-ALG remaining in the target area than ALG-1001 after administration. The results presented here offer a clinically relevant route for peptide therapeutics by addressing the major obstacles that these therapies face in delivery.

Keywords: AMD; hydroxyl PAMAM dendrimer; integrin; neovascularization; peptide conjugates; targeted delivery.

Figure 1

Synthesis and characterization of D-ALG1001. (A) Synthesis scheme of D-ALG1001: surface of generation-6 hydroxyl-terminated dendrimer was functionalized with alkyne-terminated linkers. ALG-1001 peptide was then attached using a copper-catalyzed click reaction to yield D-ALG conjugates. (B) The D-ALG conjugate was characterized by HPLC (left) with a retention time of 16.6 min compared to precursor materials ALG-1001 (12.3 min) and D-hexyne (19.58 min) with the chromatogram trace color corresponding to the text color. ¹H NMR characterization of the precursor and final conjugate (right) shows the appearance of characteristic signals.

Figure 2

In vitro enzymatic degradation in the presence of proteinase K. HPLC chromatographs of D-ALG and ALG-1001 after coincubation with proteinase K show a decrease in the AUC of the peak associated with the free ALG-1001 peptide (top). The trace of D-ALG shows a negligible decrease in AUC (middle). The peaks corresponding to ALG-1001 and D-ALG are outlined in red. The curve plotting the amount degraded is shown by normalizing peaks of analyte taken at set time points to the starting peak obtained at 0 min (bottom). About 90% of ALG-1001 was degraded by 90 min whereas only 10% of D-ALG was degraded.

Figure 3

In vitro inhibition of vessel formation. (A) Image analysis of HUVECs seeded onto a Matrigel matrix using the Angiogenesis Analyzer plug-in for ImageJ. The relevant metrics extracted for the integrity and expanse of vessel formation were the number (#) of times vessels intersected one another (junctions), the number (#) of spaces enclosed by vessels (meshes), the number (#) of connected vessels (segments), and the number (#) of isolated vessels (isolated segments). Significant disruptions in vessel formation resulted in reduced junctions, segments, and meshes while the number of isolated segments increased. Cells treated with 1 mM ALG-1001 and D-ALG significantly reduced vessel formation compared to untreated cells. * p < 0.05, ** p < 0.01, and **** p < 0.0001 (B) Representative images of vessel formation from untreated cells (control), and cells treated with D-ALG and ALG-1001 at a 1 mM concentration.

Figure 4

Activation of ERK and FAK pathway in response to VEGF stimulation. Relative protein expression was calculated by analyzing protein bands associated with ERK (42 and 44 kDa) and FAK (110 kDa) and normalizing the content to an internal control (cyclophilin B) (top). Representative images of blots are shown (bottom).

Figure 5

Expression of proinflammatory cytokine of RAW264.7 cells in response to LPS stimulation. Compared to untreated controls, RAW cells stimulated with LPS produced robust expressions of IL1β and TNFα. Pretreatment with ALG-1001 produced a slight effect at attenuating TNFα expression at 1 mM concentration; on the other hand, treating with D-ALG brought the level of TNFα to a level not statistically different than untreated controls. p-values denoted here compare the level of IL1β and TNFα expression to untreated controls. * p < 0.05, ** p < 0.01.

Figure 6

Biodistribution of intraperitoneally injected Cy5-D-ALG-Cy3 and ALG-1001-Cy3 at areas of choroidal neovascularization. CNVs were stained with isolectin (green), ALG-1001 was labeled with Cy3 (yellow), and the dendrimer was labeled with Cy5 (red). Cy5-D-ALG-Cy3 conjugates stayed in the target area over a period of 4 days with the signal staying around the CNV area. A majority of ALG-1001-Cy3 stayed in the target area over 2 days, and most were cleared by day 3.

Figure 7

Quantification of ERK and FAK pathway activation and expression of proinflammatory/angiogenic cytokines. (A) Quantification of FAK, phosphor-FAK (Y397), p44/42 ERK, phospho-p44/42 ERK proteins using ELISA kits. Trends indicate a reduced phosphorylation of p44/42 ERK over both 7 and 14 days compared to controls in both treatment groups. In D-ALG-treated animals, there is also a trend in reduced phosphor-FAK protein expression across both time points. ** p < 0.01 (B) Quantification of VEGF-A, TNFα, and IL1β through qPCR. The trend shows a reduction in VEGF-A and TNFα at day 7 for animals treated with D-ALG and a reduction in IL1β production on day 14. ALG-1001-treated animals had a trend of lower TNFα at both time points and lower VEGF-A and IL1β on day 14.

Figure 8

In vivo inhibition of CNV formation in a laser CNV mouse model, following intraperitonial administration of free ALG-1001 and D-ALG-1001. (A) Area of CNV as quantified using ImageJ. The trend suggests D-ALG- and ALG-1001-treated animals had smaller CNV areas compared to untreated animals at day 7. The degree of CNV reduction is retained in D-ALG-treated animals on day 14 and the difference reaches statistical significance. (B) Representative images of CNV on day 14. * p < 0.05.