Exploration of α1-antitrypsin treatment protocol for islet transplantation: dosing plan and route of administration

Abstract

Life-long weekly infusions of human α1-antitrypsin (hAAT) are currently administered as augmentation therapy for patients with genetic AAT deficiency (AATD). Several recent clinical trials attempt to extend hAAT therapy to conditions outside AATD, including type 1 diabetes. Since the endpoint for AATD is primarily the reduction of risk for pulmonary emphysema, the present study explores hAAT dose protocols and routes of administration in attempt to optimize hAAT therapy for islet-related injury. Islet-grafted mice were treated with hAAT (Glassia™; i.p. or s.c.) under an array of clinically relevant dosing plans. Serum hAAT and immunocyte cell membrane association were examined, as well as parameters of islet survival. Results indicate that dividing the commonly prescribed 60 mg/kg i.p. dose to three 20 mg/kg injections is superior in affording islet graft survival; in addition, a short dynamic descending dose protocol (240→120→60→60 mg/kg i.p.) is comparable in outcomes to indefinite 60 mg/kg injections. While hAAT pharmacokinetics after i.p. administration in mice resembles exogenous hAAT treatment in humans, s.c. administration better imitated the physiological progressive rise of hAAT during acute phase responses; nonetheless, only the 60 mg/kg dose depicted an advantage using the s.c. route. Taken together, this study provides a platform for extrapolating an islet-relevant clinical protocol from animal models that use hAAT to protect islets. In addition, the study places emphasis on outcome-oriented analyses of drug efficacy, particularly important when considering that hAAT is presently at an era of drug-repurposing towards an extended list of clinical indications outside genetic AATD.

Keywords: diabetes; inflammation.

The American Society for Pharmacology and Experimental Therapeutics.

Figures

Fig. 1.

The effect of Glassia on pancreatic islet responses and dendritic cell maturation during inflammatory conditions. (A and B) Primary mouse islets (35 per well in triplicate) were cultured for 48 hours in the absence (CT) or presence of IL-1β and IFNγ (5 ng/ml each), with or without overnight pretreatment with Glassia (0.5 mg/ml). (A) Islet viability and insulin release. (B) Supernatant levels of nitric oxide, IL-6, MCP-1, and IL-10. (C) BMDCs (3 × 105 cells per well in triplicates) were stimulated with IL-1β and IFNγ (5 ng/ml each) overnight in the presence or absence of Glassia (0.5 mg/ml). Cells were analyzed by flow cytometry. Representative results of three independent experiments. Mean ± S.E.M., *P < 0.05, **P < 0.01.

Fig. 2.

Protection of pancreatic islet allografts by Glassia. Mice were rendered hyperglycemic by single-dose STZ (225 mg/kg) and then subjected to islet allograft transplantation. Experimental groups included: 15 mg/kg (n = 3), 30 mg/kg (n = 13), 60 mg/kg (n = 10), 120 mg/kg (n = 5), and 240 mg/kg (n = 5). Control group animals received PBS vehicle (n = 9). (A) Graft survival curve. (B) Graft site histology, K signifies kidney tissue and G signifies graft site. Representative images of rejected untreated mouse graft (top), 60 mg/kg-treated rejected graft (bottom left), and accepted graft (bottom right).

Fig. 3.

The effect of intraperitoneal Glassia dose distribution treatment on hAAT serum concentration and islet graft survival. (A) Mice (n = 3–5) were treated with 60 mg/kg i.p Glassia as indicated (arrow). Serum levels of human AAT were determined with specific ELISA. Results of 3 independent experiments, mean ± S.E.M. (B) Mice were stimulated with ThG for 72 hours after treatment with Glassia (240 mg/kg i.p.). Peritoneal lavage was performed 16 hours later, and membrane-associated hAAT levels were measured by flow cytometry (top panel). Groups included nonstimulated, untreated mice (CT, red); ThG-stimulated untreated mice (ThG, yellow); nonstimulated Glassia-treated mice (CT/Glassia, blue), and ThG-stimulated Glassia-treated mice (ThG/Glassia, green), n = 3. Results are representative of 3 independent repeats. For cell-associated analysis (bottom panel), peritoneal macrophages (1×106, in triplicate) were pulsed with Glassia (0.5 mg/ml). hAAT binding was evaluated by Western blotting. (C) Mice were treated with 30 mg/kg (left) or 20 mg/kg (right) Glassia intraperitoneally at indicated time points (arrow; n = 3 or 4 in each group). Serum levels of human AAT were determined. Results of 3 independent experiments, mean ± S.E.M. (D) Islet graft survival curve. Groups include: ×3 of 20 mg/kg i.p (n = 3, yellow) and ×2 of 30 mg/kg i.p (n = 6, red). CT and 60 mg/kg treatment groups are duplicates of Fig. 2A.

Fig. 4.

The effect of subcutaneous Glassia treatment on hAAT serum concentration and islet graft survival. (A) Mice were treated with 60 and 20 mg/kg Glassia subcutaneously at indicated time points (arrow; n = 3 or 4 in each group). Serum hAAT levels were measured. Results are representative of three independent experiments, mean ± S.E.M. (B) Islet grafted mice were treated with Glassia either intraperitoneally (dashed) or subcutaneously (solid). Groups include untreated mice (CT; gray. n = 9); ×1 of 60 mg/kg i.p (n = 10); ×1 of 60 mg/kg s.c (n = 6); ×2 of 30 mg/kg i.p. (n = 6); ×2 of 30 mg/kg s.c. (n = 5); ×1 of 30 mg/kg i.p (n = 13); ×1 of 30 mg/kg s.c. (n = 2); ×1 of 15 mg/kg i.p (n = 3); ×1 of 15 mg/kg s.c. (n = 1). Islet graft survival curve.

Fig. 5.

The effect of Glassia dynamic dose treatment on graft survival. Islet-grafted mice were treated with Glassia using dynamic dose regimen. (A) Illustration representing the treatment protocol. (B) Islet graft survival curve under Glassia dynamic dose treatment (blue area). Comparison of intraperitoneal (red; n = 6) and subcutaneous (purple; n= 3) dynamic dose treatment.