Tolerability Assessment of Formulation pH in New Zealand White Rabbits Following Intravitreal Administration.

Development of intravitreal drugs presents several challenges due to the delicate ocular environment and volume constraints of what can be safely administered in the eye. Formulation development of intravitreally administered drugs may necessitate the use of nonphysiological pH in order to accommodate manufacturing processes or achieve favorable drug properties. Clinical and nonclinical data show that intravitreal drugs formulated in the pH 5.5 to 7.4 range are well tolerated. The aim of this study was to provide ocular toxicity data for formulations in the pH 4.0 to 5.5 range following intravitreal administration in New Zealand White rabbits. This range was evaluated as part of formulation development for an intravitreal drug that necessitated the use of pH outside the available tolerability data for formulations. Toxicity was assessed by ophthalmic examinations, intraocular pressure (IOP) measurement, clinical observations, body weights, and microscopic analysis of ocular tissue. Histidine chloride pH 5.0 to 5.5 and acetate chloride pH 4.0 to 5.0 solutions were well tolerated, and no test article-related ocular inflammation, IOP changes, or gross or microscopic findings were observed in any eye. The data presented here add to the knowledge of pH ranges that can be explored for intravitreal drug formulation development.

The safety evaluation of long-acting ocular delivery systems.

The safety evaluation of ocular long-acting delivery (LAD) technologies is a nascent field. Here, we detail the challenges in assessing the safety of novel LAD technologies, and well as the most common types of toxicity encountered during early toxicity testing. A detailed understanding of the route of administration, pharmacology, and functionality and/or pharmacokinetics (PK) of the LAD, along with all of its component parts, including the active pharmaceutical ingredient and excipients, is crucial for the successful development of next-generation long-acting ocular therapeutics.

Evaluation of the Toxicity of Intravitreally Injected PLGA Microspheres and Rods in Monkeys and Rabbits: Effects of Depot Size on Inflammatory Response.

Purpose: Poly(lactic-co-glycolic) acid (PLGA) inserts have been successfully developed for the treatment of posterior eye disease as a means of reducing injection frequency of intravitreally administered therapeutics. PLGA microspheres are also of interest for the delivery of intravitreal drugs, since they offer the advantage of being easily injected without surgical procedures or large injectors. Methods: In the current study, the toxicity of PLGA microspheres and rods was investigated in nonhuman primates (NHPs) and rabbits. An in vitro assessment of cytokine responses to PLGA in peripheral blood mononuclear cells (PBMCs) and macrophages was also performed. Results: Intravitreal administration of 3, 10, or 12.5 mg/eye of PLGA microspheres in NHPs resulted in a severe immune response characterized by a foreign body response. Follow-up studies in the rabbit confirmed this finding for PLGA microspheres ranging in size from 20 to 100 µm. In contrast, administration of PLGA rod implants with a similar PLGA mass did not elicit a significant immune response. In vitro assays in PBMCs and macrophages confirmed proinflammatory cytokine release upon treatment with PLGA microspheres but not PLGA rods. Conclusions: These data demonstrate a lack of tolerability of PLGA microspheres upon intravitreal injection, and suggest that the size, shape, and/or surface area of PLGA depots are critical attributes in determining ocular toxicity.

Lens Capsule Perforation Without Inflammation in 4 Rabbits From Intravitreal Injection Studies.

Historically, it was thought that lens protein was sequestered, and injury to the lens capsule causing release of lens material into the eye would always result in ocular inflammation. Currently, it is believed that lens antigens are recognized as self, subject to normal T-cell tolerance. Three different single-dose intravitreal injection/implantation studies of 4 different test materials, ranging from 4 to 6 weeks in length, were performed in New Zealand White rabbits. The test materials included polymer microspheres, polymer rods, a solvent, and a hydrogel. Intravitreal injection/implantation procedures were performed on day 1, and indirect ophthalmoscopy and slit-lamp biomicroscopy examinations were performed by board-certified veterinary ophthalmologists periodically throughout the course of each study. None of the affected animals received corticosteroids or other immunomodulatory agents during the course of the studies. Four rabbits had perforation of the posterior lens capsule during the injection/implantation procedure on day 1, visible on clinical ophthalmic examination as lens capsule alterations described as "lens hits" and/or incipient posterior cataracts. Findings on slit-lamp biomicroscopy examination were limited to vitreous cells in 2 of the animals, although not centered on the area of lens capsule disturbance. Histologically, there was no evidence of inflammation in association with extruded lens protein material in any of the affected eyes. These results indicate that iatrogenic damage to the lens capsule during aseptically performed intravitreal injections/implantations does not appear to induce inflammation in rabbits.

Characterization of the pH and Temperature in the Rabbit, Pig, and Monkey Eye: Key Parameters for the Development of Long-Acting Delivery Ocular Strategies.

Many long-acting delivery strategies for ocular indications rely on pH- and/or temperature-driven release of the therapeutic agent and degradation of the drug carrier. Yet, these physiological parameters are poorly characterized in ocular animal models. These strategies aim at reducing the frequency of dosing, which is of particular interest for the treatment of chronic disorders affecting the posterior segment of the eye, such as macular degeneration that warrants monthly or every other month intravitreal injections. We used anesthetized white New Zealand rabbits, Yucatan mini pigs, and cynomolgus monkeys to characterize pH and temperature in several vitreous locations and the central aqueous location. We also established post mortem pH changes in the vitreous. Our data showed regional and species differences, which need to be factored into strategies for developing biodegradable long-acting delivery systems.

Neutral endopeptidase-resistant C-type natriuretic peptide variant represents a new therapeutic approach for treatment of fibroblast growth factor receptor 3-related dwarfism.

Achondroplasia (ACH), the most common form of human dwarfism, is caused by an activating autosomal dominant mutation in the fibroblast growth factor receptor-3 gene. Genetic overexpression of C-type natriuretic peptide (CNP), a positive regulator of endochondral bone growth, prevents dwarfism in mouse models of ACH. However, administration of exogenous CNP is compromised by its rapid clearance in vivo through receptor-mediated and proteolytic pathways. Using in vitro approaches, we developed modified variants of human CNP, resistant to proteolytic degradation by neutral endopeptidase, that retain the ability to stimulate signaling downstream of the CNP receptor, natriuretic peptide receptor B. The variants tested in vivo demonstrated significantly longer serum half-lives than native CNP. Subcutaneous administration of one of these CNP variants (BMN 111) resulted in correction of the dwarfism phenotype in a mouse model of ACH and overgrowth of the axial and appendicular skeletons in wild-type mice without observable changes in trabecular and cortical bone architecture. Moreover, significant growth plate widening that translated into accelerated bone growth, at hemodynamically tolerable doses, was observed in juvenile cynomolgus monkeys that had received daily subcutaneous administrations of BMN 111. BMN 111 was well tolerated and represents a promising new approach for treatment of patients with ACH.

Evaluation of the therapeutic potential of a CNP analog in a Fgfr3 mouse model recapitulating achondroplasia.

Achondroplasia (ACH), the most common form of dwarfism, is an inherited autosomal-dominant chondrodysplasia caused by a gain-of-function mutation in fibroblast-growth-factor-receptor 3 (FGFR3). C-type natriuretic peptide (CNP) antagonizes FGFR3 downstream signaling by inhibiting the pathway of mitogen-activated protein kinase (MAPK). Here, we report the pharmacological activity of a 39 amino acid CNP analog (BMN 111) with an extended plasma half-life due to its resistance to neutral-endopeptidase (NEP) digestion. In ACH human growth-plate chondrocytes, we demonstrated a decrease in the phosphorylation of extracellular-signal-regulated kinases 1 and 2, confirming that this CNP analog inhibits fibroblast-growth-factor-mediated MAPK activation. Concomitantly, we analyzed the phenotype of Fgfr3(Y367C/+) mice and showed the presence of ACH-related clinical features in this mouse model. We found that in Fgfr3(Y367C/+) mice, treatment with this CNP analog led to a significant recovery of bone growth. We observed an increase in the axial and appendicular skeleton lengths, and improvements in dwarfism-related clinical features included flattening of the skull, reduced crossbite, straightening of the tibias and femurs, and correction of the growth-plate defect. Thus, our results provide the proof of concept that BMN 111, a NEP-resistant CNP analog, might benefit individuals with ACH and hypochondroplasia.

Differential gene expression between juvenile and adult dura mater: a window into what genes play a role in the regeneration of membranous bone.

BACKGROUND: Although reossification of large calvarial defects is possible in children, adults lack this tissue engineering capacity. In this study, the authors compared the differences in gene expression between juvenile and adult dura mater using a mouse cDNA microarray with 42,000 unique elements. METHODS: Non-suture-associated parietal bone was harvested from 6-day-old and 60-day-old mice. The dura mater was carefully dissected from the calvarial disk and snap-frozen. RNA was extracted from pooled dura mater for microarray analysis. The 25 most differentially expressed genes were listed, as were selected bone-related genes. In addition, quantitative real-time reverse-transcriptase polymerase chain reaction confirmation of selected genes-BMP-2, BMP-4, and BMP-7; and osteopontin (OP), osteocalcin (OC), and FGFR-1-was performed. RESULTS: Juvenile dura mater expressed significantly greater amounts of BMP-2 and OP. Minimal difference in OC expression was observed between juvenile and adult dura mater. Extracellular matrix proteins (Col3a1, 5a1, 6a1, and fibronectin 1), osteoblast differentiation markers (Runx2/Cbfa1, Itm2a, and FGFR-1), and the growth factor Ptn were among other genes with greater expression in juvenile dura mater. Markers of osteoclasts (Acp5, MMP9, Ctsk) and the multiple candidate gene Ntrk2 were also expressed at higher levels in the juvenile dura mater. CONCLUSIONS: These findings suggest a more differentiated osteoprogenitor population to exist along with a greater presence of osteoclasts in the juvenile dura mater relative to adults. In addition to establishing a baseline difference in gene expression between juvenile and adult dura mater, new genes potentially critical to the regenerative potential of juvenile calvaria were identified.

Osteoprotegerin in human milk: a potential role in the regulation of bone metabolism and immune development.

Osteoprotegerin (OPG) is a member of the tumor necrosis factor superfamily. It is a soluble "decoy" receptor for tumor necrosis factor-related apoptosis-inducing ligand and ligand of the receptor activator of NF-kappaB. As such, OPG inhibits osteoclast activity and regulates the immune system. Human milk is a complex biologic fluid that supplies nutritional and protective factors to the breast-fed infant. In the present study, human milk samples at various times postpartum were assessed for the presence of OPG. Using biochemical as well as immunologic and biologic techniques we showed that human milk contains OPG at a level that is 1000-fold higher than that found in normal human serum. We observed that human breast milk cells and the human mammary epithelial cell line MCF-7 express OPG, indicating that both cell types are possible sources of milk OPG in vivo. In vitro studies demonstrated that milk OPG is biologically active and suggested that it may contribute to the antiresorptive activity of milk on bone, as well as tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of T cell proliferation. OPG-like activity was also observed in bovine colostrum and milk. Furthermore, we were able to detect human OPG in the sera of rats gavaged with human milk. We discuss the relevance of our findings for the breast-fed infant and for the prevention of immune and bone disorders.

Flavonoid quercetin decreases osteoclastic differentiation induced by RANKL via a mechanism involving NF kappa B and AP-1.

Flavonoids are micronutrients widely present in food of plant origin. They have been attributed pharmacological properties such as anticancer and prevention of age-related pathologies. It has been recently hypothesized that flavonoids increase bone mass and prevent osteoporosis. However, little is known about the in vitro effects of flavonoids on osteoclast activities. We investigated the effects of quercetin, one of the most commonly occurring flavonoids, on osteoclast differentiation which is a critical determinant step of in vivo bone resorption. Two in vitro models of osteoclast differentiation were used in this study: a murine one, involving the culture of RAW 264.7 cells in presence of receptor activator of NF kappa B ligand (RANKL), and a human model consisting of differentiating peripheral blood monocytic cells (PBMC) isolated from peripheral blood in presence of RANKL and macrophage-colony stimulating factor (M-CSF). Osteoclastogenesis was assessed by osteoclast-like number, tartrate resistant acid phosphatase (TRAP) activity, and bone resorbing activity. We showed that quercetin (0.1-10 microM) decreased osteoclastogenesis in a dose dependent manner in both models with significant effects observed at low concentrations, from 1 to 5 microM. The IC(50) value was about 1 microM. Analysis of protein-DNA interaction by electrophoretic mobility shift assay (EMSA) performed on RAW cells showed that a pre-treatment with quercetin inhibited RANKL-induced nuclear factor kB (NF kappa B) and activator protein 1 (AP-1) activation. NF kappa B and AP-1 are transcription factors highly involved in osteoclastic differentiation and their inhibition could play an important role in the decrease of osteoclastogenesis observed in the presence of quercetin. In conclusion, the present results demonstrate for the first time that quercetin, a flavonoid characterized by antioxidant activities, is a potent inhibitor of in vitro osteoclastic differentiation, via a mechanism involving NF kappa B and AP-1.

Potent inhibitory effect of naturally occurring flavonoids quercetin and kaempferol on in vitro osteoclastic bone resorption.

Several recent studies have suggested that flavonols, a class of phytochemicals with many biological activities, might exert a protective effect against post-menopausal bone loss. In the present study, we investigated the effects of quercetin and kaempferol, two of the major naturally occurring flavonols on the in vitro bone resorbing activity of osteoclasts. Our results indicate that both compounds, at concentrations ranging from 0.1 to 100 microM reduce bone resorption in a time and dose-dependent manner. Significant inhibitory effects were observed at concentrations as low as 0.1 microM especially with kaempferol. The IC(50)s, or concentration inhibitory of 50% of basal resorption, calculated for quercetin and kaempferol were 1.6 and 5.3 microM, respectively. Using highly purified rabbit osteoclasts, we showed that both flavonols directly induce apoptosis of mature osteoclasts in the same dose-range effective for inhibiting bone resorption. When osteoclasts were treated with 50 microM of quercetin and kaempferol, intracellular reactive oxygen species levels decreased significantly by 75 and 25%, respectively, indicating these molecules keep their antioxidant properties at this concentration. However, at concentrations below 50 microM, neither quercetin nor kaempferol exerted antiradical action, suggesting that antioxidant properties cannot fully explain the inhibitory effect on bone resorption. Finally, we report that kaempferol-, but not the quercetin-induced inhibition of bone resorption was partially abolished by the presence of the pure anti-estrogen ICI 182780 suggesting that kaempferol's estrogenic effect could be involved in the inhibition of bone resorption. The present study demonstrates that flavonols widely distributed in human diet such as quercetin and kaempferol, exert a potent inhibitory effect on in vitro bone resorption.

Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity.

Lactoferrin (LF) is a key modulator of inflammatory response. Since bone and immune systems are genetically and functionally linked, we were interested to know if LF could influence bone remodeling. Bovine LF (bLF) inhibited in vitro bone resorbing activity (IC50, 200 microg/ml) in a rabbit mixed bone cell culture, consisting of authentic osteoclasts in an environment of osteoblast and stromal cells. Using human CD14 selected cells committed toward osteoclasts, bLF (10 microg/ml) stimulated cell proliferation, however, led to an inhibition of calcitonin receptor mRNA expression, a main marker of osteoclast phenotype, and decreased the global resorbing activity. No modulation of RANK mRNA expression was observed and mRNA for RANKL and OPG were not detected in this culture system, suggesting that bLF inhibits osteoclastogenesis and reduces bone resorption through a mechanism independent of OPG/RANKL/RANK. In conclusion, bLF appears to modulate bone remodeling. Its mechanism of action remains to be elucidated.