Clinical proof of concept for anti-FGF2 therapy in exudative age-related macular degeneration (nAMD): phase 2 trials in treatment-naïve and anti-VEGF pretreated patients.

BACKGROUND/OBJECTIVE: Intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents are the first-line treatment for exudative age-related macular degeneration (nAMD). Due to the limitations of these standard therapies, targeting alternative mechanisms of action may be helpful for treatment of this very common disease. Here, we investigated an anti-fibroblast growth factor-2 (FGF2) aptamer, umedaptanib pegol, a next generation therapeutic for the treatment of nAMD. METHODS: Three phase 2 studies were designed. First, a multicentre, randomized, double-masked TOFU study assessed the efficacy of intravitreal injections of umedaptanib pegol monotherapy or in combination with aflibercept, compared to aflibercept monotherapy in 86 subjects with anti-VEGF pretreated nAMD. Second, 22 subjects who had exited the TOFU study received 4 monthly intravitreal injections of umedaptanib pegol (extension, RAMEN study). Third, as an investigator-sponsored trial (TEMPURA study), a single-center, open-label, 4-month study was designed to evaluate the safety and treatment efficacy of umedaptanib pegol in five naïve nAMD patients who had not received any prior anti-VEGF treatment. RESULTS: The TOFU study demonstrated that umedaptanib pegol alone or in combination with aflibercept did not improve best-corrected visual acuity (BCVA) and central subfield thickness (CST) over aflibercept alone. However, the change in BCVA and CST at primary endpoint was marginal in all the three treatment groups, suggesting that umedaptanib pegol is effective to prevent the disease progression. The RAMEN study confirmed the cessation of disease progression. In the TEMPURA study, naïve nAMD patients showed improvement and no further macular degeneration, with striking improvement of visual acuity and central subfield thickness in some of the patients. CONCLUSIONS: These results demonstrate, for the first time, clinical proof of concept for aptamer based anti-FGF2 therapy of nAMD.

Safety and tolerability of intravitreal umedaptanib pegol (anti-FGF2) for neovascular age-related macular degeneration (nAMD): a phase 1, open-label study.

OBJECTIVE: To evaluate the efficacy and safety of a single-dose intravitreal umedaptanib pegol (anti-FGF2, investigational new drug) for the treatment of neovascular age-related macular degeneration (nAMD). METHODS: Nine participants who had a diagnosis of refractory nAMD were enrolled and received a single intravitreal injection of umedaptanib pegol at increasing doses of 0.2, 1.0 or 2.0 mg in the study eye. RESULTS: All three doses of umedaptanib pegol evaluated in the study were safe and well tolerated. No severe adverse event (AE) was observed in the study. There was an improvement in retinal fluid measured by central subfield thickness (CST) in most subjects. Remarkably, all three subjects who received 2.0 mg/eye showed improvement of more than 150 µm. CONCLUSIONS: Intravitreal umedaptanib pegol was safe, well tolerated, and demonstrated an indication of bioactivity in participants that have persistent subretinal fluid refractory to the treatment with anti-VEGFs.

Nucleic acid ligands act as a PAM and agonist depending on the intrinsic ligand binding state of P2RY2.

G protein-coupled receptors (GPCRs) play diverse roles in physiological processes, and hence the ligands to modulate GPCRs have served as important molecules in biological and pharmacological approaches. However, the exploration of novel ligands for GPCR still remains an arduous challenge. In this study, we report a method for the discovery of nucleic acid ligands against GPCRs by an advanced RNA aptamer screening technology that employs a virus-like particle (VLP), exposing the GPCR of interest. An array of biochemical analyses coupled with a cell-based assay revealed that one of the aptamers raised against purinergic receptor P2Y2 (P2RY2), a GPCR, exhibits an activation potency to unliganded receptor and prohibits a further receptor activation by endogenous ligand, behaving like a partial agonist. However, the aptamer enhances the activity of intrinsic ligand-binding P2RY2, thereby acting as a positive allosteric modulator (PAM) to liganded receptor. Our findings demonstrate that the nucleic acid aptamer conditionally exerts PAM and agonist effects on GPCRs, depending on their intrinsic ligand binding state. These results indicate the validity of our VLP-based aptamer screening targeting GPCR and reemphasize the great potential of nucleic acid ligands for exploring the GPCR activation mechanism and therapeutic applications.

Anti-Angiogenic and Anti-Scarring Dual Action of an Anti-Fibroblast Growth Factor 2 Aptamer in Animal Models of Retinal Disease.

Currently approved therapies for age-related macular degeneration (AMD) are inhibitors against vascular endothelial growth factor (VEGF), which is a major contributor to the pathogenesis of neovascular AMD (nAMD). Intravitreal injections of anti-VEGF drugs have shown dramatic visual benefits for AMD patients. However, a significant portion of AMD patients exhibit an incomplete response to therapy and, over the extended management course, can lose vision, with the formation of submacular fibrosis as one risk factor. We investigated a novel target for AMD treatments, fibroblast growth factor 2 (FGF2), which has been implicated in the pathophysiology of both angiogenesis and fibrosis in a variety of tissue and organ systems. The anti-FGF2 aptamer, RBM-007, was examined for treatment of nAMD in animal models. In in vivo studies conducted in mice and rats, RBM-007 was able to inhibit FGF2-induced angiogenesis, laser-induced choroidal neovascularization (CNV), and CNV with fibrosis. Pharmacokinetic studies of RBM-007 in the rabbit vitreous revealed high and relatively long-lasting profiles that are superior to other approved anti-VEGF drugs. The anti-angiogenic and anti-scarring dual action of RBM-007 holds promise as an additive or alternative therapy to anti-VEGF treatments for nAMD.

Alkaline-tolerant RNA aptamers useful to purify acid-sensitive antibodies in neutral conditions.

Recently, several oligonucleotides have been launched for clinical use and a number of therapeutic oligonucleotides are under clinical trials. Aptamer is one of the oligonucleotide therapeutics and has received a lot of attention as a new technology and an efficacious pharmaceutical compound comparable to antibody. Aptamer could be used for various purposes, not only therapeutics but also diagnostics, and applicable to affinity chromatography as a carrier molecule to purify proteins of interest. Here we demonstrate the usage and advantages of RNA aptamer to Fc region of human IgG (i.e., IgG aptamer) for purification of human antibodies. IgG aptamer requires divalent cations for binding to IgG and bound IgG dissociates easily upon treatment with chelating reagent, such as EDTA, under neutral conditions. This elution step is very mild and advantageous for maintaining active conformations of therapeutic antibodies compared to the widely used affinity purification with Protein A/G, which requires acidic elution that often damages the active conformation of antibodies. In fact, of several monoclonal antibodies tested, three antibodies were prone to aggregate on acidic elution from the Protein A/G resin, while remained fully active upon neutral elution from the IgG aptamer resin. The IgG aptamer was fully manipulated to alkaline resistant by ribose 2'-modifications, and thereby reusable numerous times with 1 N NaOH washing. The capacity of the aptamer resin to bind IgG was equivalent to that of the Protein A/G resin. Therefore, the IgG aptamer will provide us with a unique tool to uncover and purify human monoclonal antibodies, which hold therapeutic potential but lose the activity upon acidic elution from Protein A/G-based affinity resin.

A novel selective androgen receptor modulator, NEP28, is efficacious in muscle and brain without serious side effects on prostate.

Age-related androgen depletion is known to be a risk factor for various diseases, such as osteoporosis and sarcopenia. Furthermore, recent studies have demonstrated that age-related androgen depletion results in accumulation of ß-amyloid protein and thereby acts as a risk factor for the development of Alzheimer's disease. Supplemental androgen therapy has been shown to be efficacious in treating osteoporosis and sarcopenia. In addition, studies in animals have demonstrated that androgens can play a protective role against Alzheimer's disease. However, androgen therapy is not used routinely for these indications, because of side effects. Selective androgen receptor modulators (SARMs) are a new class of compounds. SARMs maintain the beneficial effects of androgens on bone and muscle while reducing unwanted side effects. NEP28 is a new SARM exhibiting high selectivity for androgen receptor. To investigate the pharmacological effects of NEP28, we compared the effects on muscle, prostate, and brain with mice that were androgen depleted by orchidectomy and then treated with either placebo, NEP28, dihydrotestosterone, or methyltestosterone. We demonstrated that NEP28 showed tissue-selective effect equivalent to or higher than existing SARMs. In addition, the administration of NEP28 increased the activity of neprilysin, a known Aß-degrading enzyme. These results indicate that SARM is efficacious for the treatment of not only osteoporosis and sarcopenia, but also Alzheimer's disease.

XLGαolf regulates expression of p27Kip1 in a CSN5 and CDK2 dependent manner.

XLGα(olf) is an extra large transcriptional variant of the heterotrimeric G protein, Gα(olf), which we previously reported to be localized in the Golgi apparatus and interacted with Rab3A and Rab8A through its N-terminal region. However, many physiological functions of XLGα(olf) remain to be elucidated. In this study, performance of yeast two-hybrid screening with XLGα(olf) allowed isolation of COP9 signalosome subunit 5 (CSN5), known to regulate the p27(Kip1) protein level through a proteasome dependent pathway. Co-immunoprecipitation experiments followed by Western blotting also showed association of CSN5 with XLGα(olf) linked to down-regulation of p27(Kip1). Gene silencing of endogenous CSN5 by siRNA attenuated the XLGα(olf)-mediated down-regulation, which was also demonstrated to require CDK2. Both knock down of CDK2 and the treatment with a CDK2 inhibitor reversed the reduction of p27(Kip1) due to XLGα(olf). Our findings provide important clues to understanding physiological functions of XLGα(olf).

Subcellular localization of a novel G protein XLGalpha(olf).

XLGalpha(olf) was identified as a transcriptional variant of the heterotrimeric G protein, Galpha(olf). Previous work showed that XLGalpha(olf) couples with adenosine A2a receptor and dopamine D1 receptor in vitro. However, physiological functions of XLGalpha(olf) remain to be elucidated. In this study, we performed indirect immunofluorescence confocal analyses to examine the subcellular localization of XLGalpha(olf). With overexpression, surprisingly, many large endosomes resulted. We also observed that XLGalpha(olf) localizes at the Golgi apparatus. The N-terminal region of XLGalpha(olf) appears necessary for both endosome formation and the Golgi localization. The results indicate that XLGalpha(olf) and Galpha(olf) play distinctly separate roles. Moreover, XLGalpha(olf) colocalized with Rab3A and Rab8A, as well as partially with Rab11A, but not with other endocytotic endosomes. We could confirm the interaction between XLGalpha(olf) and Rab3A/Rab8A by co-immunoprecipitation experiments. Our study provides important clues toward understanding physiological functions of XLGalpha(olf).

In vitro stabilization and minimum active component of polygalacturonic acid synthase involved in pectin biosynthesis.

Polygalacturonic acid (PGA) synthase successively transfers galacturonic acid to oligogalacturonic acid by an alpha1,4-linkage to synthesize PGA, the backbone of plant pectic homogalacturonan. PGA synthase has not been purified to date due to its instability in vitro. In this study, we found stable conditions in vitro and separated a minimum active component of the enzymes from pea and azuki bean epicotyls. The PGA synthase lost its activity in 500 mM of sodium chloride or potassium chloride, while it was relatively stable at low salt concentrations. Under low salt concentrations, three peaks bearing PGA synthase activity were separated, by gel filtration and sucrose density gradient centrifugation. The molecular masses of these enzymes solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid were estimated to be 21,000, 5,000, and 590 kDa. The two higher molecular mass PGA synthases converted to smaller PGA synthase proteins when treated with high salt concentrations, while retaining their activity, indicating that PGA synthase has a minimum active component for its activity.

p21WAF1 modulates NF-kappaB signaling and induces anti-apoptotic protein Bcl-2 in Tax-expressing rat fibroblast.

Of the cell cycle-associated genes regulated by human T-cell leukemia virus type-1 (HTLV-1) Tax, cyclin-dependent kinase (CDK) inhibitor p21WAF1 is upregulated in HTLV-1-infected cells. Previously, we reported that p21WAF1 stimulated Tax-dependent NF-kappaB activation which influences a variety of cellular processes, including proliferation, differentiation, and apoptosis. In HTLV-1-infected cells, Tax is primarily involved in the constitutive activation of NF-kappaB signaling. Here, we demonstrate that p21WAF1 affects Tax-dependent NF-kappaB signaling by inducing p100/52, an NF-kappaB-related protein. W4, a Tax-transformed rat fibroblast cell line, exhibits the constitutive activation of NF-kappaB signaling, potentially mediated by overexpression of RelB. Ectopic expression of p21WAF1 in W4 cells, which lack endogenous expression due to methylation of the p21WAF1 promoter, induces the expression of p100/52. Bcl-2 expression was also upregulated by ectopic p21WAF1 in this cell line, suggesting that p21WAF1 plays an important role in the regulation of apoptosis by modulating NF-kappaB signaling in Tax-expressing rat fibroblasts. We also address the expression of NF-kappaB-related proteins in HTLV-1-infected cells.

Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-alpha-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates.

Polygalacturonate 4-alpha-galacturonosyltransferase (pectin synthase) was solubilized from pollen tubes of Petunia axillaris and characterized. To accomplish this, an assay method using fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as acceptor substrates was developed. When the pollen tube enzyme was solubilized with 0.5% (v/v) Triton X-100 and was incubated with PA-OGA and UDP-galacturonic acid (UDP-GalUA), successive transfer activity of more than 10 GalUAs from UDP-GalUA to the nonreducing end of PA-OGA was observed by diethylaminoethyl high-performance liquid chromatography. This activity was time- and enzyme concentration-dependent. The optimum enzyme activity was observed at pH 7.0 and 30 degrees C. Among the PA-OGAs investigated, those with a degree of polymerization of more than 10 were preferred as substrates. The crude pollen tube enzyme had an apparent K(m) value of 13 microM for the PA-OGA with a degree of polymerization 11 and 170 microM for UDP-GalUA. The characteristics of the P. axillaris pollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed.