Intralacrimal Sustained Delivery of Rapamycin Shows Therapeutic Effects without Systemic Toxicity in a Mouse Model of Autoimmune Dacryoadenitis Characteristic of Sjögren's Syndrome.

Sjögren's syndrome (SS) is an autoimmune disease associated with severe exocrinopathy, which is characterized by profound lymphocytic infiltration (dacryoadenitis) and loss of function of the tear-producing lacrimal glands (LGs). Systemic administration of Rapamycin (Rapa) significantly reduces LG inflammation in the male Nonobese Diabetic (NOD) model of SS-associated autoimmune dacryoadenitis. However, the systemic toxicity of this potent immunosuppressant limits its application. As an alternative, this paper reports an intra-LG delivery method using a depot formulation comprised of a thermoresponsive elastin-like polypeptide (ELP) and FKBP, the cognate receptor for Rapa (5FV). Depot formation was confirmed in excised whole LG using cleared tissue and observation by both laser-scanning confocal and lightsheet microscopy. The LG depot was evaluated for safety, efficacy, and intra-LG pharmacokinetics in the NOD mouse disease model. Intra-LG injection with the depot formulation (5FV) retained Rapa in the LG for a mean residence time (MRT) of 75.6 h compared to Rapa delivery complexed with a soluble carrier control (5FA), which had a MRT of 11.7 h in the LG. Compared to systemic delivery of Rapa every other day for 2 weeks (seven doses), a single intra-LG depot of Rapa representing 16-fold less total drug was sufficient to inhibit LG inflammation and improve tear production. This treatment modality further reduced markers of hyperglycemia and hyperlipidemia while showing no evidence of necrosis or fibrosis in the LG. This approach represents a potential new therapy for SS-related autoimmune dacryoadenitis, which may be adapted for local delivery at other sites of inflammation; furthermore, these findings reveal the utility of optical imaging for monitoring the disposition of locally administered therapeutics.

Molecular Targeting of Immunosuppressants Using a Bifunctional Elastin-Like Polypeptide.

Elastin-Like Polypeptides (ELP) are environmentally responsive protein polymers which are easy to engineer and biocompatible, making them ideal candidates as drug carriers. Our team has recently utilized ELPs fused to FKBP12 to carry Rapamycin (Rapa), a potent immunosuppressant. Through high affinity binding to Rapa, FKBP carriers can yield beneficial therapeutic effects and reduce the off-site toxicity of Rapa. Since ICAM-1 is significantly elevated at sites of inflammation in diverse diseases, we hypothesized that a molecularly targeted ELP carrier capable of binding ICAM-1 might have advantageous properties. Here we report on the design, characterization, pharmacokinetics, and biodistribution of a new ICAM-1-targeted ELP Rapa carrier (IBPAF) and its preliminary characterization in a murine model exhibiting elevated ICAM-1. Lacrimal glands (LG) of male NOD mice, a disease model recapitulating the autoimmune dacryoadenitis seen in Sjögren's Syndrome patients, were analyzed to confirm that ICAM-1 was significantly elevated in the LG relative to control male BALB/c mice (3.5-fold, p < 0.05, n = 6). In vitro studies showed that IBPAF had significantly higher binding to TNF-α-stimulated bEnd.3 cells which overexpress surface ICAM-1, relative to nontargeted control ELP (AF)(4.0-fold, p < 0.05). A pharmacokinetics study in male NOD mice showed no significant differences between AF and IBPAF for plasma half-life, clearance, and volume of distribution. However, both constructs maintained a higher level of Rapa in systemic circulation compared to free Rapa. Interestingly, in the male NOD mouse, the accumulation of IBPAF was significantly higher in homogenized LG extracts compared to AF at 2 h (8.6 ± 6.6% versus 1.3 ± 1.3%, respectively, n = 5, p < 0.05). This accumulation was transient with no differences detected at 8 or 24 h. This study describes the first ICAM-1 targeted protein-polymer carrier for Rapa that specifically binds to ICAM-1 in vitro and accumulates in ICAM-1 overexpressing tissue in vivo, which may be useful for molecular targeting in diverse inflammatory diseases where ICAM-1 is elevated.

NOD and NOR mice exhibit comparable development of lacrimal gland secretory dysfunction but NOD mice have more severe autoimmune dacryoadenitis.

The male Non-Obese Diabetic (NOD) mouse is an established model of autoimmune dacryoadenitis characteristic of Sjögren's Syndrome (SS), but development of diabetes may complicate studies. The Non-Obese Diabetes Resistant (NOR) mouse is a MHC-II matched diabetes-resistant alternative, but development of autoimmune dacryoadenitis is not well-characterized. We compare features of SS in male NOD and NOR mice at 12 and 20 weeks. Stimulated tear secretion was decreased in 12 week NOD relative to BALB/c mice (p < 0.05), while by 20 weeks both NOD and NOR showed decreased stimulated tear secretion relative to BALB/c mice (p < 0.001). Tear CTSS activity was elevated in NOD and NOR relative to BALB/c mice (p < 0.05) at 12 and 20 weeks. While NOD and NOR lacrimal glands (LG) showed increased LG lymphocytic infiltration at 12 and 20 weeks relative to BALB/c mouse LG (p < 0.05), the percentage in NOD was higher relative to NOR at each age (p < 0.05). Gene expression of CTSS, MHC II and IFN-γ in LG were significantly increased in NOD but not NOR relative to BALB/c at 12 and 20 weeks. Redistribution of the secretory effector, Rab3D in acinar cells was observed at both time points in NOD and NOR, but thinning of myoepithelial cells at 12 weeks in NOD and NOR mice was restored by 20 weeks in NOR mice. NOD and NOR mice share features of SS-like autoimmune dacryoadenitis, suggesting common disease etiology. Other findings suggest more pronounced lymphocytic infiltration in NOD mouse LG including increased pro-inflammatory factors that may be unique to this model.

Increased expression of cathepsins and obesity-induced proinflammatory cytokines in lacrimal glands of male NOD mouse.

PURPOSE: Lacrimal glands (LGs) of male NOD mice, a model of Sjögren's syndrome (SjS), exhibit immune cell infiltration and lipid deposition. The mechanism of SjS was further investigated by characterizing gene expression profiles of NOD mouse LGs in comparison with those of healthy control mice. Differentially expressed genes were further investigated at the protein level to correlate changes in location and abundance with development of disease. METHODS: Microarray followed by real-time RT-PCR was conducted to compare the gene expression in 12-week-old male NOD mouse LG relative to that in matched BALB/c mouse LG. Immunofluorescence and Western blot analyses were used to localize and quantify proteins of interest. Enzymatic assays measured catalytic activity of cathepsins. RESULTS: Cathepsin H (Ctsh), S (Ctss), and Z (Ctsz) and proinflammatory factors, including tumor necrosis factor (Tnf), interleukin 6 (Il6), and interleukin 1 beta (Il1b), were upregulated at the mRNA level. Increased cathepsin S immunofluorescence was detected in lysosomes and secretory vesicle-like organelles in LG acinar cells and CD68-positive infiltrating macrophages in NOD mouse LG. Cathepsin S (CATS) and cathepsin H (CATH) activities were significantly higher in NOD mouse LG lysate than in control lysates, and CATS was also significantly elevated in NOD mouse tears. CONCLUSIONS: Expression of CATS and CATH increases in parallel with proinflammatory cytokines during the development of autoimmune inflammatory disease in the NOD mouse disease model. Tear CATS may represent a biomarker for diagnosis of dacryoadenitis in SjS.