Mihir Shah

A rapamycin-binding protein polymer nanoparticle shows potent therapeutic activity in suppressing autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome

Sjögrens syndrome (SjS) is a chronic autoimmune disease characterized initially by lymphocytic infiltration and destruction of exocrine glands, followed by systemic organ damage and B-cell lymphoma. Conventional treatment is based on management of symptoms and there is a shortage of therapies that address the underlying causes of inflammation at source exocrine tissue. The aim of this study was to test a novel protein polymer-based platform consisting of diblock copolymers composed from Elastin-like Polypeptides (ELPs) fused with FKBP12, to deliver a potent immunosuppressant with dose-limiting toxicity, rapamycin (Rapa) also known as Sirolimus, and evaluate its effects on the inflamed lacrimal gland (LG) of non-obese diabetic mouse (NOD), a classic mouse model of SjS. Both soluble and diblock copolymer ELPs were fused to FKBP12 and characterized with respect to purity, hydrodynamic radii, drug entrapment and release. Both formulations showed successful association with Rapa; however, the nanoparticle formulation, FSI, released drug with nearly a 5 fold longer terminal half-life of 62.5h. The strong interaction of FSI nanoparticles with Rapa was confirmed in vivo by a shift in the monoexponential pharmacokinetic profile for free drug to a biexponential profile for the nanoparticle formulation. When acutely administered by injection into NOD mice via the tail vein, this FSI formulation significantly suppressed lymphocytic infiltration in the LG relative to the control group while reducing toxicity. There was also a significant effect on inflammatory and mammalian target of Rapamycin (mTOR) pathway genes in the LG and surprisingly, our nanoparticle formulation was significantly better at decreasing a proposed tear biomarker of SjS, cathepsin S (CATS) compared to free drug. These findings suggest that FSI is a promising tool for delivering Rapa for treatment of SjS in a murine model and may be further explored to meet the unmet medical challenge of SjS.

Elastin-based protein polymer nanoparticles carrying drug at both corona and core suppress tumor growth in vivo.

Numerous nanocarriers of small molecules depend on either non-specific physical encapsulation or direct covalent linkage. In contrast, this manuscript explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for nanoparticulate drug delivery. To explore this approach, genetically engineered diblock copolymers were constructed from elastin-like polypeptides (ELPs) that assemble small (<100nm) nanoparticles. ELPs are protein polymers of the sequence (Val-Pro-Gly-Xaa-Gly)n, where the identity of Xaa and n determine their assembly properties. Initially, a screening assay for model drug encapsulation in ELP nanoparticles was developed, which showed that Rose Bengal and Rapa have high non-specific encapsulation in the core of ELP nanoparticles with a sequence where Xaa=Ile or Phe. While excellent at entrapping these drugs, their release was relatively fast (2.2h half-life) compared to their intended mean residence time in the human body. Having determined that Rapa can be non-specifically entrapped in the core of ELP nanoparticles, FK506 binding protein 12 (FKBP), which is the cognate protein target of Rapa, was genetically fused to the surface of these nanoparticles (FSI) to enhance their avidity towards Rapa. The fusion of FKBP to these nanoparticles slowed the terminal half-life of drug release to 57.8h. To determine if this class of drug carriers has potential applications in vivo, FSI/Rapa was administered to mice carrying a human breast cancer model (MDA-MB-468). Compared to free drug, FSI encapsulation significantly decreased gross toxicity and enhanced the anti-cancer activity. In conclusion, protein polymer nanoparticles decorated with the cognate receptor of a high potency, low solubility drug (Rapa) efficiently improved drug loading capacity and its release. This approach has applications to the delivery of Rapa and its analogs; furthermore, this strategy has broader applications in the encapsulation, targeting, and release of other potent small molecules.

Biodegradation of elastin-like polypeptide nanoparticles

Protein polymers are repetitive polypeptides produced by ribosomal biosynthetic pathways; furthermore, they offer emerging opportunities in drug and biopharmaceutical delivery. As for any polymer, biodegradation is one of the most important determinants affecting how a protein polymer can be utilized in the body. This study was designed to characterize the proteolytic biodegradation for a library of protein polymers derived from the human tropoelastin, the Elastin‐like polypeptides (ELPs). ELPs are of particular interest for controlled drug delivery because they reversibly transition from soluble to insoluble above an inverse phase transition temperature (Tt). More recently, ELP block copolymers have been developed that can assemble into micelles; however, it remains unclear if proteases can act on these ELP nanoparticles. For the first time, we demonstrate that ELP nanoparticles can be degraded by two model proteases and that comparable proteolysis occurs after cell uptake into a transformed culture of murine hepatocytes. Both elastase and collagenase endopeptidases can proteolytically degrade soluble ELPs. To our surprise, the ELP phase transition was protective against collagenase but not to elastase activity. These findings enhance our ability to predict how ELPs will biodegrade in different physiological microenvironments and are essential to develop protein polymers into biopharmaceuticals.

Tear cathepsin S as a candidate biomarker for Sjögren's syndrome.

The diagnosis of Sjögrens syndrome (SS) in routine practice is largely a clinical one and requires a high index of suspicion by the treating physician. This great dependence on clinical judgment frequently leads to delayed diagnosis or misdiagnosis. Tear protein profiles have been proposed as simple and reliable biomarkers for the diagnosis of SS. Given that cathepsin S activity is increased in the lacrimal glands and tears of NOD mice (a murine model of SS), the aim of this study was to explore the clinical utility of using tear cathepsin S (CTSS) activity as a biomarker for SS.

Rapamycin Eye Drops Suppress Lacrimal Gland Inflammation In a Murine Model of Sjögren's Syndrome.

Purpose To evaluate the efficacy of topical rapamycin in treating autoimmune dacryoadenitis in a mouse model of Sjögrens syndrome. Methods We developed rapamycin in a poly(ethylene glycol)-distearoyl phosphatidylethanolamine (PEG-DSPE) micelle formulation to maintain solubility. Rapamycin or PEG-DSPE eye drops (vehicle) were administered in a well-established Sjögrens syndrome disease model, the male nonobese diabetic (NOD) mice, twice daily for 12 weeks starting at 8 weeks of age. Mouse tear fluid was collected and tear Cathepsin S, a putative tear biomarker for Sjögrens syndrome, was measured. Lacrimal glands were retrieved for histological evaluation, and quantitative real-time PCR of genes associated with Sjögrens syndrome pathogenesis. Tear secretion was measured using phenol red threads, and corneal fluorescein staining was used to assess corneal integrity. Results Lymphocytic infiltration of lacrimal glands from rapamycin-treated mice was significantly (P = 0.0001) reduced by 3.8-fold relative to vehicle-treated mice after 12 weeks of treatment. Rapamycin, but not vehicle, treatment increased tear secretion and decreased corneal fluorescein staining after 12 weeks. In rapamycin-treated mice, Cathepsin S activity was significantly reduced by 3.75-fold in tears (P < 0.0001) and 1.68-fold in lacrimal gland lysates (P = 0.003) relative to vehicle-treated mice. Rapamycin significantly altered the expression of several genes linked to Sjögrens syndrome pathogenesis, including major histocompatibility complex II, TNF-α, IFN-γ, and IL-12a, as well as Akt3, an effector of autophagy. Conclusions Our findings suggest that topical rapamycin reduces autoimmune-mediated lacrimal gland inflammation while improving ocular surface integrity and tear secretion, and thus has potential for treating Sjögrens syndrome–associated dry eye.

Myoepithelial cell-driven acini contraction in response to oxytocin receptor stimulation is impaired in lacrimal glands of Sjögren’s syndrome animal models

The purpose of the present studies was to investigate the impact of chronic inflammation of the lacrimal gland, as occurs in Sjögren’s syndrome, on the morphology and function of myoepithelial cells (MECs). In spite of the importance of MECs for lacrimal gland function, the effect of inflammation on MECs has not been well defined. We studied changes in MEC structure and function in two animal models of aqueous deficient dry eye, NOD and MRL/lpr mice. We found a statistically significant reduction in the size of MECs in diseased compared to control lacrimal glands. We also found that oxytocin receptor was highly expressed in MECs of mouse and human lacrimal glands and that its expression was strongly reduced in diseased glands. Furthermore, we found a significant decrease in the amount of two MEC contractile proteins, α-smooth muscle actin (SMA) and calponin. Finally, oxytocin-mediated contraction was impaired in lacrimal gland acini from diseased glands. We conclude that chronic inflammation of the lacrimal gland leads to a substantial thinning of MECs, down-regulation of contractile proteins and oxytocin receptor expression, and therefore impaired acini contraction. This is the first study highlighting the role of oxytocin mediated MEC contraction on lacrimal gland function.

A novel elastin-like polypeptide drug carrier for cyclosporine A improves tear flow in a mouse model of Sjögren's syndrome

&NA; As a potent macrolide immunosuppressant, cyclosporine A (CsA) is used to treat multiple autoimmune diseases, including non‐autoimmune and autoimmune‐mediated dry eye disease, rheumatoid arthritis and psoriasis. Despite its potency, CsA has poor solubility, poor bioavailability, and can cause serious adverse reactions such as nephrotoxicity and neurotoxicity. To overcome these limitations, we invented a new strategy to carry CsA by fusing its cognate human receptor, cyclophilin A (CypA), to a 73 kDa elastin‐like polypeptide (ELP) termed A192 using recombinant protein expression. Derived from human tropoelastin, ELPs are characterized by the ability to phase separate above a temperature that is a function of variables including concentration, molecular weight, and hydrophobicity. The resultant fusion protein, termed CA192, which assembles into a dimeric species in solution, effectively binds and solubilizes CsA with a Kd of 189 nM, comparable to that of endogenous CypA with a Kd of 35.5 nM. The release profile of CsA from CA192 follows a one phase decay model with a half‐life of 957.3 h without a burst release stage. Moreover, CA192‐CsA inhibited IL‐2 expression induced in Jurkat cells through the calcineurin‐NFAT signaling pathway with an IC50 of 1.2 nM, comparable to that of free CsA with an IC50 of 0.5 nM. The intravenous pharmacokinetics of CA192 followed a two‐compartment model with a mean residence time of 7.3 h. Subcutaneous administration revealed a bioavailability of 30% and a mean residence time of 15.9 h. When given subcutaneously for 2 weeks starting at 14 weeks in male non‐obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis used to study Sjögrens syndrome (SS), CA192‐CsA (2.5 mg/kg, every other day) significantly (p = 0.014) increased tear production relative to CA192 alone. Moreover, CA192 delivery reduced indications of CsA nephrotoxicity relative to free CsA. CA192 represents a viable new approach to deliver this effective but nephrotoxic agent in a modality that preserves therapeutic efficacy but suppresses drug toxicity.