Molecular Targeting of Immunosuppressants Using a Bi-functional Elastin-Like Polypeptide
Bioconjugate chemistry
Yaping Ju, Hao Guo, Frances Yarber, Maria C Edman, Santosh Peddi, Srikanth R Janga, John Andrew MacKay, Sarah F Hamm-Alvarez
2019
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.
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Berunda Polypeptides: Biheaded Rapamycin Carriers for Subcutaneous Treatment of Autoimmune Dry Eye Disease
Molecular pharmaceutics
Changrim Lee, Hao Guo, Wannita Klinngam, Srikanth R Janga, Frances Yarber, Santosh Peddi, Maria C Edman, Nishant Tiwari, Siyu Liu, Stan G Louie, Sarah F Hamm-Alvarez, J Andrew MacKay
2019
The USFDA-approved immunosuppressive drug rapamycin (Rapa), despite its potency, is limited by poor bioavailability and a narrow therapeutic index. In this study, we sought to improve bioavailability of Rapa with subcutaneous (SC) administration and to test its therapeutic feasibility and practicality in a murine model of Sjögren’s syndrome (SS), a systemic autoimmune disease with no approved therapies. To improve its therapeutic index, we formulated Rapa with a carrier termed FAF, a fusion of the human cytosolic FK506-binding protein 12 (FKBP12) and an elastin-like polypeptide (ELP). The resulting 97 kDa FAF (i) has minimal burst release, (ii) is “humanized”, (iii) is biodegradable, (iv) solubilizes two Rapa per FAF, and (v) avoids organic solvents or amphiphilic carriers. Demonstrating high stability, FAF remained soluble and monodisperse with a hydrodynamic radius of 8 nm at physiological temperature. A complete pharmacokinetic (PK) analysis of FAF revealed that the bioavailability of SC FAF was 60%, with significantly higher blood concentration during the elimination phase compared to IV FAF. The plasma concentration of Rapa delivered by FAF was 8-fold higher with a significantly increased plasma-to-whole blood ratio relative to free Rapa, 24 h after injection. To evaluate therapeutic effects, FAF–Rapa was administered SC every other day for 2 weeks to male non-obese diabetic (NOD) mice, which develop an SS-like autoimmune-mediated lacrimal gland (LG) inflammation and other characteristic features of SS. Both FAF–Rapa and free Rapa exhibited immunomodulatory effects by significantly suppressing lymphocytic infiltration, gene expression of IFN-γ, MHC II, type I collagen and IL-12a, and cathepsin S (CTSS) activity in LG compared to controls. Serum chemistry and histopathological analyses in major organs revealed no apparent toxicity of FAF–Rapa. Given its improved PK and equipotent therapeutic efficacy compared to free Rapa, FAF–Rapa is of further interest for systemic treatments for autoimmune diseases like SS.
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Identifying Biomarkers for Parkinson’s Disease with Reflex Tears (S10. 001)
Neurology
Mark Lew, Danielle Feigenbaum, Dan Freire, Srikanth Janga, Wendy Mack, Maria Edman, Curtis Okamoto, Sarah Hamm-Alvarez
2019
To evaluate whether the protein composition of reflex tears differs in individuals with Parkinson’s disease (PD) versus Healthy Controls (HC).
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PP2A: A Novel Target to Prevent Cathepsin S-Mediated Damage in Smoking-Induced COPD
American journal of respiratory and critical care medicine
Srikanth R Janga, Sarah F Hamm-Alvarez
2019
Chronic Obstructive Pulmonary Disease (COPD) is a recognized global health crisis, with smoking the most important and well-studied risk factor for disease development and progression (1). The World Health Organization (WHO) estimates that 80 million individuals live with moderate to severe COPD, and this disease will become the third leading cause of death worldwide by 2030 (https://www. who. int/respiratory/copd/en/). COPD is characterized by chronic inflammation and obstructed airflow usually originating with long term exposure to particulates, with the most egregious offender being cigarette smoke. Various signaling pathways are implicated in the induction of lung inflammation associated with COPD pathogenesis.
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Longitudinal analysis of tear cathepsin S activity levels in male non-obese diabetic mice suggests its potential as an early stage biomarker of Sjögren’s Syndrome
Biomarkers
Srikanth R Janga, Mihir Shah, Yaping Ju, Zhen Meng, Maria C Edman, Sarah F Hamm-Alvarez
2019
CTSS activity was measured in tears and lacrimal glands (LG) from male 1–6 month (M) NOD and 1 and 6 M BALB/c mice. Lymphocytic infiltration was quantified by histopathology, while disease-related proteins (Rab3D, CTSS, collagen 1) were quantified using q-PCR and immunofluorescence.
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