Songqi Gao

Biodistribution and pharmacokinetic studies of bone-targeting N-(2-hydroxypropyl)methacrylamide copolymer-alendronate conjugates

The biodistribution and pharmacokinetics of bone-targeting N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-alendronate conjugates were evaluated following intravenous administration of radioiodinated conjugates to young healthy BALB/c mice. The synthesis of a polymerizable and cathepsin K cleavable alendronate derivative, N-methacryloylglycylglycylprolylnorleucylalendronate, enabled the preparation of HPMA copolymer-alendronate conjugates with varying composition. Using the RAFT (reversible addition-fragmentation chain transfer) polymerization technique, four conjugates with different molecular weight and alendronate content and two control HPMA copolymers (without alendronate) with different molecular weight were prepared. The results of biodistribution studies in mice demonstrated a strong binding capacity of alendronate-targeted HPMA copolymer conjugates to bone. Conjugates with low (1.5 mol%) alendronate content exhibited a similar bone deposition capacity as conjugates containing 8.5 mol % of alendronate. The molecular weight was an important factor in the biodistribution of the HPMA copolymer conjugates. More conjugate structures need to be evaluated, but the data suggest that medium molecular weights (50-100 kDa) might be effective drug carriers for bone delivery.

QLT091001, a 9-cis-Retinal Analog, Is Well-Tolerated by Retinas of Mice with Impaired Visual Cycles

PURPOSE Investigate whether retinas of mice with impaired retinal cycles exposed to light or kept in the dark tolerate prolonged high-dose administration of QLT091001, which contains as an active ingredient, the 9-cis-retinal precursor, 9-cis-retinyl acetate. METHODS Four- to six-week-old Lrat(-/-) and Rpe65(-/-) mice (n = 126) as well as crossbred Gnat1(-/-) mice lacking rod phototransduction (n = 110) were gavaged weekly for 6 months with 50 mg/kg QLT091001, either after being kept in the dark or after light bleaching for 30 min/wk followed by maintenance in a 12-hour light ≤ 10 lux)/12-hour dark cycle. Retinal health was monitored by spectral-domain optical coherent tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) every other month and histological, biochemical, and visual functional analyses were performed at the end of the experiment. Two-photon microscopy (TPM) was used to observe retinoid-containing retinosome structures in the RPE. RESULTS Retinal thickness and morphology examined by SD-OCT were well maintained in all strains treated with QLT091001. No significant increases of fundus autofluorescence were detected by SLO imaging of any strain. Accumulation of all-trans-retinyl esters varied with genetic background, types of administered compounds and lighting conditions but retinal health was not compromised. TPM imaging clearly revealed maintenance of retinosomes in the RPE of all mouse strains tested. CONCLUSIONS Retinas of Lrat(-/-), Rpe65(-/-), and crossbred Gnat1(-/-) mice tolerated prolonged high-dose QLT091001 treatment well.

Synergistically acting agonists and antagonists of G protein–coupled receptors prevent photoreceptor cell degeneration

Systems pharmacology reveals a combination of GPCR-targeted drugs that prevent retinal degeneration. Preventing blindness Loss of photoreceptors in the retina results in visual impairment and eventually blindness. Light can damage the retina through processes that involve G protein–coupled receptors (GPCRs). Chen et al. took a systems pharmacology approach to identify combinations of drugs that activate or inhibit specific GPCRs to prevent light-induced retinal damage in a mouse model of progressive retinal degeneration. This approach identified a photoreceptor-protecting combination of FDA-approved drugs that activated the Gi/o-coupled dopamine receptors D2R and D4R, inhibited the Gs-coupled dopamine receptor D1R, and inhibited the Gq-coupled α1A-adrenergic receptor. This study not only provides a potential therapeutic strategy for preventing blindness due to retinal degeneration but also suggests that systems pharmacology approaches could lead to the discovery of new combinations of available drugs to promote therapeutic changes in signaling networks. Photoreceptor cell degeneration leads to visual impairment and blindness in several types of retinal disease. However, the discovery of safe and effective therapeutic strategies conferring photoreceptor cell protection remains challenging. Targeting distinct cellular pathways with low doses of different drugs that produce a functionally synergistic effect could provide a strategy for preventing or treating retinal dystrophies. We took a systems pharmacology approach to identify potential combination therapies using a mouse model of light-induced retinal degeneration. We showed that a combination of U.S. Food and Drug Administration–approved drugs that act on different G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) exhibited synergistic activity that protected retinas from light-induced degeneration even when each drug was administered at a low dose. In functional assays, the combined effects of these drugs were stimulation of Gi/o signaling by activating the dopamine receptors D2R and D4R, as well as inhibition of Gs and Gq signaling by antagonizing D1R and the α1A-adrenergic receptor ADRA1A, respectively. Moreover, transcriptome analyses demonstrated that such combined GPCR-targeted treatments preserved patterns of retinal gene expression that were more similar to those of the normal retina than did higher-dose monotherapy. Our study thus supports a systems pharmacology approach to identify treatments for retinopathies, an approach that could extend to other complex disorders.

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Significance Vertebrate rhodopsin (Rh) has been a model system for many G protein-coupled receptors for over a decade. However, due to its thus-far limited repertoire of active ligands, its use in assisting the development of new therapeutic modalities and drugs has been limited. This study elucidates a photocyclic G protein activation by Rh bound with a six-carbon ring retinal (Rh6mr), and thus broadens the diversity of such Rh signaling modulators. Rh6mr does not release its chromophore after light activation, but instead the resulting photoproduct is thermally reisomerized back to its inactive state, abrogating the necessity for a complex retinoid cycle to renew its chromophore. This photocyclic behavior of Rh6mr opens up several avenues for using optogenetic tools based on vertebrate Rhs. Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged Gt activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates Gt in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure–function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis–to–11,13-dicis isomerization.

Manganese-Enhanced MRI for Preclinical Evaluation of Retinal Degeneration Treatments

PURPOSE Apply manganese-enhanced magnetic resonance imaging (MEMRI) to assess ion channel activity and structure of retinas from mice subject to light-induced retinal degeneration treated with prophylactic agents. METHODS Abca4(-/-)Rdh8(-/-) double knockout mice with and without prophylactic retinylamine (Ret-NH2) treatment were illuminated with strong light. Manganese-enhanced MRI was used to image the retina 2 hours after intravitreous injection of MnCl2 into one eye. Contrast-enhanced MRIs of the retina and vitreous humor in each experimental group were assessed and correlated with the treatment. Findings were compared with standard structural and functional assessments of the retina by optical coherence tomography (OCT), histology, and electroretinography (ERG). RESULTS Manganese-enhanced MRI contrast in the retina was high in nonilluminated and illuminated Ret-NH2-treated mice, whereas no enhancement was evident in the retina of the light-illuminated mice without Ret-NH2 treatment (P < 0.0005). A relatively high signal enhancement was also observed in the vitreous humor of mice treated with Ret-NH2. Strong MEMRI signal enhancement in the retinas of mice treated with retinylamine was correlated with their structural integrity and function evidenced by OCT, histology, and a strong ERG light response. CONCLUSIONS Manganese-enhanced MRI has the potential to assess the response of the retina to prophylactic treatment based on the measurement of ion channel activity. This approach could be used as a complementary tool in preclinical development of new prophylactic therapies for retinopathies.

MicroRNA-processing Enzymes Are Essential for Survival and Function of Mature Retinal Pigmented Epithelial Cells in Mice.

Age-related macular degeneration (AMD) is a major cause of irreversible vision loss. The neovascular or “wet” form of AMD can be treated to varying degrees with anti-angiogenic drugs, but geographic atrophy (GA) is an advanced stage of the more prevalent “dry” form of AMD for which there is no effective treatment. Development of GA has been linked to loss of the microRNA (miRNA)-processing enzyme DICER1 in the mature retinal pigmented epithelium (RPE). This loss results in the accumulation of toxic transcripts of Alu transposable elements, which activate the NLRP3 inflammasome and additional downstream pathways that compromise the integrity and function of the RPE. However, it remains unclear whether the loss of miRNA processing and subsequent gene regulation in the RPE due to DICER1 deficiency also contributes to RPE cell death. To clarify the role of miRNAs in RPE cells, we used two different mature RPE cell-specific Cre recombinase drivers to inactivate either Dicer1 or DiGeorge syndrome critical region 8 (Dgcr8), thus removing RPE miRNA regulatory activity in mice by disrupting two independent and essential steps of miRNA biogenesis. In contrast with prior studies, we found that the loss of each factor independently led to strikingly similar defects in the survival and function of the RPE and retina. These results suggest that the loss of miRNAs also contributes to RPE cell death and loss of visual function and could affect the pathology of dry AMD.

A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4−/−Rdh8−/− mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential.Mutations that lead to misfolding of rhodopsin can cause retinitis pigmentosa. Here, the authors carry out a high throughput screen to identify a small molecule chaperone of rod opsin, and show that it protects mouse models of retinitis pigmentosa from retinal degeneration.

Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-viral Gene Therapy for Leber’s Congenital Amaurosis

Development of a gene delivery system with high efficiency and a good safety profile is essential for successful gene therapy. Here we developed a targeted non-viral delivery system using a multifunctional lipid ECO for treating Leber’s congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65−/− mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2.

A combination of G protein-coupled receptor modulators protects photoreceptors from degeneration

Degeneration of retinal photoreceptor cells can arise from environmental and/or genetic causes. Since photoreceptor cells, the retinal pigment epithelium (RPE), neurons, and glial cells of the retina are intimately associated, all cell types eventually are affected by retinal degenerative diseases. Such diseases often originate either in rod and/or cone photoreceptor cells or the RPE. Of these, cone cells located in the central retina are especially important for daily human activity. Here we describe the protection of cone cells by a combination therapy consisting of the G protein–coupled receptor modulators metoprolol, tamsulosin, and bromocriptine. These drugs were tested in Abca4−/−Rdh8−/− mice, a preclinical model for retinal degeneration. The specificity of these drugs was determined with an essentially complete panel of human G protein–coupled receptors. Significantly, the combination of metoprolol, tamsulosin, and bromocriptine had no deleterious effects on electroretinographic responses of wild-type mice. Moreover, putative G protein–coupled receptor targets of these drugs were shown to be expressed in human and mouse eyes by RNA sequencing and quantitative polymerase chain reaction. Liquid chromatography together with mass spectrometry using validated internal standards confirmed that metoprolol, tamsulosin, and bromocriptine individually or together penetrate the eye after either intraperitoneal delivery or oral gavage. Collectively, these findings support human trials with combined therapy composed of lower doses of metoprolol, tamsulosin, and bromocriptine designed to safely impede retinal degeneration associated with certain genetic diseases (e.g., Stargardt disease). The same low-dose combination also could protect the retina against diseases with complex or unknown etiologies such as age-related macular degeneration.

New GABA modulators protect photoreceptor cells from light-induced degeneration in mouse models

No clinically approved therapies are currently available that prevent the onset of photoreceptor death in retinal degeneration. Signaling between retinal neurons is regulatedby the release and uptake of neurotransmitters, wherein GABA is the main inhibitory neurotransmitter. In this work, novel 3‐chloropropiophenone derivatives and the clinical anticonvulsants tiagabine and vigabatrin were tested to modulate GABA signaling and protect against light‐induced retinal degeneration. Abca4−/− Rdh8−/− mice, an accelerated model of retinal degeneration, were exposed to intense light after prophylactic injections of one of these compounds. Imaging and functional assessments of the retina indicated that these compounds successfully protected photoreceptor cells from degeneration to maintain a full‐visual‐field response. Furthermore, these compounds demonstrated a strong safety profile in wild‐type mice and did not compromise visual function or damage the retina, despite repeated administration. These results indicate that modulating inhibitory GABA signaling can offer prophylactic protection against light‐induced retinal degeneration.—Schur, R. M., Gao, S., Yu, G., Chen, Y., Maeda, A., Palczewski, K., Lu, Z.‐R. New GABA modulators protect photoreceptor cells from light‐induced degeneration in mouse models. FASEB J. 32, 3289–3300 (2018). www.fasebj.org