Subconjunctival delivery of p75NTR antagonists reduces the inflammatory, vascular, and neurodegenerative pathologies of diabetic retinopathy

The p75NTR is a novel therapeutic target validated in a streptozotocin mouse model of diabetic retinopathy. Intravitreal (IVT) injection of small molecule p75NTR antagonist THX-B was therapeutic and resolved the inflammatory, vascular, and neurodegenerative phases of the retinal pathology. To simplify clinical translation, we sought a superior drug delivery method that circumvents risks associated with IVT injections. METHODS. We compared the pharmacokinetics of a single 40 lg subconjunctival (SCJ) depot to the reported effective 5 lg IVT injections of THX-B. We quantified therapeutic efficacy, with endpoints of inflammation, edema, and neuronal death. RESULTS. The subconjunctival depot affords retinal exposure equal to IVT injection, without resulting in detectable drug in circulation. At week 2 of diabetic retinopathy, the SCJ depot provided therapeutic efficacy similar to IVT injections, with reduced inflammation, reduced edema, reduced neuronal death, and a long-lasting protection of the retinal structure. CONCLUSIONS. Subconjunctival injections are a safe and effective route for retinal delivery of p75NTR antagonists. The subconjunctival route offers an advantageous, less-invasive, more compliant, and nonsystemic method to deliver p75NTR antagonists for the treatment of retinal diseases.Fil: Galan, Alba. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Barcelona, Pablo Federico. Mc Gill University. Lady Davis Research Intitute; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Nedev, Hinyu. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Sarunic, Marinko V.. University Fraser Simon; CanadáFil: Jian, Yifan. University Fraser Simon; CanadáFil: Saragovi, H. Uri. Mc Gill University. Lady Davis Research Intitute; Canad

Small-molecule agonists of the RET receptor tyrosine kinase activate biased trophic signals that are influenced by the presence of GFRa1 co-receptors

Glial cell line?derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor ?1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET?GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.Peer reviewe

Raft-Dependent Endocytosis of Autocrine Motility Factor/Phosphoglucose Isomerase: A Potential Drug Delivery Route for Tumor Cells

Autocrine motility factor/phosphoglucose isomerase (AMF/PGI) is the extracellular ligand for the gp78/AMFR receptor overexpressed in a variety of human cancers. We showed previously that raft-dependent internalization of AMF/PGI is elevated in metastatic MDA-435 cells, but not metastatic, caveolin-1-expressing MDA-231 cells, relative to non-metastatic MCF7 and dysplastic MCF10A cells suggesting that it might represent a tumor cell-specific endocytic pathway.Similarly, using flow cytometry, we demonstrate that raft-dependent endocytosis of AMF/PGI is increased in metastatic HT29 cancer cells expressing low levels of caveolin-1 relative to metastatic, caveolin-1-expressing, HCT116 colon cells and non-metastatic Caco-2 cells. Therefore, we exploited the raft-dependent internalization of AMF/PGI as a potential tumor-cell specific targeting mechanism. We synthesized an AMF/PGI-paclitaxel conjugate and found it to be as efficient as free paclitaxel in inducing cytotoxicity and apoptosis in tumor cells that readily internalize AMF/PGI compared to tumor cells that poorly internalize AMF/PGI. Murine K1735-M1 and B16-F1 melanoma cells internalize FITC-conjugated AMF/PGI and are acutely sensitive to AMF/PGI-paclitaxel mediated cytotoxicity in vitro. Moreover, following in vivo intratumoral injection, FITC-conjugated AMF/PGI is internalized in K1735-M1 tumors. Intratumoral injection of AMF/PGI-paclitaxel induced significantly higher tumor regression compared to free paclitaxel, even in B16-F1 cells, known to be resistant to taxol treatment. Treatment with AMF/PGI-paclitaxel significantly prolonged the median survival time of tumor bearing mice. Free AMF/PGI exhibited a pro-survival role, reducing the cytotoxic effect of both AMF/PGI-paclitaxel and free paclitaxel suggesting that AMF/PGI-paclitaxel targets a pathway associated with resistance to chemotherapeutic agents. AMF/PGI-FITC uptake by normal murine spleen and thymus cells was negligible both in vitro and following intravenous injection in vivo where AMF/PGI-FITC was selectively internalized by subcutaneous B16-F1 tumor cells.The raft-dependent endocytosis of AMF/PGI may therefore represent a tumor cell specific endocytic pathway of potential value for drug delivery to tumor cells

Small-Molecule Ligands that Bind the RET Receptor Activate Neuroprotective Signals Independent of but Modulated by Coreceptor GFR alpha 1

Glial cell line-derived neurotrophic factor (GDNF) binds the GFR alpha 1 receptor, and the GDNF-GFR alpha 1 complex binds to and activates the transmembrane RET tyrosine kinase to signal through intracellular Akt/Erk pathways. To dissect the GDNF-GFR alpha 1-RET signaling complex, agents that bind and activate RET directly and independently of GFR alpha 1 expression are valuable tools. In a focused naphthalenesulfonic acid library from the National Cancer Institute database, we identified small molecules that are genuine ligands binding to the RET extracellular domain. These ligands activate RET tyrosine kinase and afford trophic signals irrespective of GFR alpha 1 coexpression. However, RET activation by these ligands is constrained by GFR alpha 1, likely via an allosteric mechanism that can be overcome by increasing RET ligand concentration. In a mouse model of retinitis pigmentosa, monotherapy with a small-molecule RET agonist activates survival signals and reduces neuronal death significantly better than GDNF, suggesting therapeutic potential. SIGNIFICANCE STATEMENT A genuine ligand of RET receptor ectodomain was identified, which acts as an agonist. Binding and agonism are independent of a coreceptor glial cell line-derived neurotrophic factor family receptor a, which is required by the natural growth factor glial cell line-derived neurotrophic factor, and are selective for cells expressing RET. The lead agent protects neurons from death in vivo. This work validates RET receptor as a druggable therapeutic target and provides for potential leads to evaluate in neurodegenerative states. We also report problems that arise when screening chemical libraries.Peer reviewe

The route of administration influences the therapeutic index of an anti-proNGF neutralizing mAb for experimental treatment of Diabetic Retinopathy

<div><p>Many neurodegenerative retinal diseases are treated with monoclonal antibodies (mAb) delivered by invasive intravitreal injection (IVT). In Diabetic Retinopathy there is a scarcity of effective agents that can be delivered using non-invasive methods, and there are significant challenges in the validation of novel therapeutic targets. ProNGF represents a potential novel target, and IVT administration of a function-blocking anti-proNGF mAb is therapeutic in a mouse model of DR. We therefore compared invasive IVT to less invasive systemic intravenous (IV) and local subconjunctival (SCJ) administration, for therapy of Diabetic Retinopathy. The IV and SCJ routes are safe, afford sustained pharmacokinetics and tissue penetration of anti-proNGF mAb, and result in long–term therapeutic efficacy that blocks retinal inflammation, edema, and neuronal death. SCJ may be a more convenient and less-invasive approach for ophthalmic use and may enable reduced frequency of intervention for the treatment of retinal pathologies.</p></div

The increase of cytotoxic factors in diabetes is prevented by systemic and subconjunctival administration of anti-proNGF mAb.

<p>Protein expression in whole retina analyzed by Western blot of 6-week-diabetic mice ± treatment with injection of anti-proNGF at week 2.5 of diabetes; compared to age-matched control healthy mice. <b>(A)</b> α₂M and proNGF expression after IV injection of anti-proNGF mAb. The IV treatment prevented the increase of α₂M and inhibited the increase of proNGF. <b>(C)</b> α₂M, TNFα and proNGF expression after SCJ injection of anti-proNGF at week 2.5 of diabetes. The SCJ treatment prevented the increase of TNFα, α₂M, and proNGF. <b>(B, D)</b> Quantification of Western blot data, * p< 0.05, ** p < 0.01, versus healthy animals (n = 6 animals per group). <b>(E)</b> Confocal microscopy images of retina sections of 6-week diabetic mice ± treatment with SCJ injection of anti-proNGF or vehicle at week 2.5 of diabetes, compared with age-matched naive control animals. Localization of α₂M (green) is in the GCL, whereas TNFα (green) is around the end-feet of CRALBP-positive (red) Muller cells on the GCL. Nuclei were counterstained with DAPI. The expression of both TNFα and α₂M was reduced on diabetic mice treated with anti-proNGF. Data are normalized to TNFα and α₂M area values (± SEM) relative to healthy control. A total of 10 images at 20X magnification were taken from n = 3 retinas per group, *p < 0.05; ***p<0.001. One-way ANOVA with significance established at α<0.05, followed by Bonferroni’s correction for multi-comparisons was used. Scale bar, 25 μm. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL; inner plexiform layer; INL, inner nuclear layer; PhR, photoreceptor layer; RPE, retinal pigment epithelium.</p

Systemic and subconjunctival administration of anti-proNGF mAb in diabetes reduces pathological vascular permeability in diabetes.

<p>Confocal images of retinal sections from 8-week-diabetic mice ± treatment with anti-proNGF mAb at week 2.5 of diabetes. <b>(A)</b> IV injection (n = 2 mice) <b>(B)</b> SCJ injection (n = 3 mice); compared to age-matched healthy controls or diabetic non-treated mice (n = 3). Red signal depicts leakage of Evans blue into the retina. Nuclei are counterstained with DAPI. Scale bar 25 μm. <b>(C, D)</b> Data are showed as normalized Evans Blue area (± SEM) relative to control healthy mice. Treatment by either the IV or the SCJ route reduces plasma extravasation). One-way ANOVA with significance established at α<0.05, followed by Bonferroni’s correction for multi-comparisons, *** p< 0.001, * p< 0.05. A total of 10 images were taken from each retina. Images were taken at 20X.</p

Detection and pharmacokinetics of anti-proNGF antibody in vitreous humor and retina after local administration.

<p>Analysis of the pharmacokinetics of biotinylated anti-proNGF mAb by Western blot in <b>(A)</b> vitreous and <b>(B)</b> retina, at the time points indicated after IVT or SCJ injection of 2 μg or 20 μg of anti-proNGF•biotin mAb. As standard controls, 5 ng of anti-proNGF•biotin mAb are shown. For retina 1/15 of the whole retina sample was loaded per lane; and for vitreous 1/3 of the total sample was loaded per lane. <b>(C-D)</b> Quantification of the anti-proNGF mAb in vitreous and retina at 30 and 60 min after IVT and SCJ injections (one-way ANOVA, followed by Bonferroni post-hoc analysis, * p< 0.05, in IVT <i>versus</i> SCJ retina samples at 30 min post-injection; n = 3 mice per group).</p

Systemic and subconjunctival delivery of anti-proNGF mAb protects retinal structure.

<p><b>(A)</b> Representative sections of B-scans OCT images from diabetic mice (at 5, 6 and 8 weeks of diabetes) ± treatment, one-time injection of anti-proNGF mAb IV (100 μg bolus dose) at week 2.5 of diabetes; compared to age-matched control healthy animals. Vertical red lines delimit the thickness of the retinal layers. Scale bar 100 μm. NGI: the neuronal structure comprising Nerve fiber layer-Ganglion cell layer-Inner plexiform layer; INL: inner nuclear layer; ONL: outer nuclear layer; IS/ES: internal/external segments. <b>(B)</b> Histogram of time-dependent changes in NGI thickness ± SEM (n = 3 independent experiments with total n = 9 mice per group). Anti-proNGF IV significantly protected the NGI structure from week 6 to at least week 8 (p<0.001). <b>(C)</b> Percent of NGI damage relative to vehicle-treated diabetic retinas (100% NGI damage) at weeks 6 and 8 after onset of diabetes. Systemic treatment significantly reduced NGI damage compared to diabetic vehicle-treated retinas (*** p< 0.001, n = 3 independent experiments, total of 9 mice per group up to week 6; n = 2 mice per group at week 8). <b>(D)</b> Time-dependent changes in NGI thickness ± SEM in mice with 4, 5, 6, 8 and 10 weeks of diabetes ± treatment, one time injection of anti-proNGF mAb SCJ (20 μg) at week 2.5 of diabetes; compared to age-matched control healthy animals (2 independent experiments, n = 8 mice per group). SCJ treatment significantly protected the NGI structure at week 10 (p<0.05). All statistical analysis (panels A-D) were one-way ANOVA with significance established at α<0.05, followed by Bonferroni’s correction for multi-comparisons. <b>(E)</b> Percent of NGI damage relative to diabetic vehicle-treated retinas (100% NGI damage) at weeks 5, 6, 8 and 10 after the onset of diabetes. SCJ treatment significantly reduced NGI damage compared to diabetic vehicle-treated retinas at both time points (t-test, *** p< 0.001, * p<0.05, 2 independent experiments, n = 6 mice per group up to week 8; n = 3 mice per group at week 10).</p