Optimization of formulation for enhanced intranasal delivery of insulin with translationally controlled tumor protein-derived protein transduction domain

Abstract

Intranasal delivery of insulin is an alternative approach to treat diabetes, as it enables higher patient compliance than conventional therapy with subcutaneously injected insulin. However, the use of intranasal delivery of insulin is limited for insulin's hydrophilicity and vulnerability to enzymatic degradation. This limitation makes optimization of formulation intranasal insulin for commercial purpose indispensable. This study evaluated bioavailability (BA) of various formulations of insulin intranasally delivered with protein transduction domain (PTD) derived from translationally controlled tumor protein. The therapeutic efficacy of newly formulated intranasal insulin + PTD was compared in vivo studies with normal and alloxan-induced diabetic rats, to those of free insulin and subcutaneously injected insulin. BA of insulin in two new formulations was, respectively, 60.71% and 45.81% of subcutaneously injected insulin, while the BA of free insulin was only 3.34%. Histological analysis of tissues, lactate dehydrogenase activity in nasal fluid, and biochemical analysis of sera revealed no detectable topical or systemic toxicity in rats and mice. Furthermore, stability analysis of newly formulated insulin + PTD to determine the optimal conditions for storage revealed that when stored at 4 °C, the delivery capacity of insulin was maintained up to 7 d. These results suggest that the new formulations of intranasal insulin are suitable for use in diabetes therapy and are easier to administer.

Keywords: Drug formulation; insulin; intranasal delivery; protein transduction domain; translationally controlled tumor protein.

Figure 1.

Selection of effective formulations for intranasal insulin + PTD with low toxicity. (a–c) Overnight-fasted normal rats were intranasally administered with each formulations the blood glucose levels were determined. The insulin dose was 5 IU/kg and 0.25 IU/kg for nasal route and s.c. injection, respectively (n = 4–7). (a) Blood glucose in normal rats after intranasal administration. Data from insulin s.c. of both graphs originated from the same experiment. (b) Relative blood glucose level relative to 0 min was calculated as shown as graph. As in (a), data from insulin s.c. of both graphs originated from the same experiment. (c) OD_490nm means LDH activity from nasal wash solution. 5% NaTDC was used as positive control known to be toxic. ***p < .001 by Student’s t-test compared to untreated group. Data are presented mean ± s.d.

Figure 2.

Pharmacokinetic and pharmacodynamic analysis of intranasal insulin + PTD formulations. (a) Normal rats were administered as indicated, followed by plasma insulin measurement. Insulin dose was 5 IU/kg for nasal route, 0.25 IU/kg for s.c. injection, and 1 IU/kg for administration with PTD (n = 5–8). (b) Blood glucose in alloxan-induced diabetic rats following each administration. Insulin dose was 2 IU/kg for nasal route, 1 IU/kg for s.c. injection, and 2 IU/kg for administration with PTD (n = 5–7). Data are presented mean ± s.d.

Figure 3.

In vivo toxicity analysis of intranasal insulin + PTD formulations. (a–d) Normal mice were treated as indicated once a day for 10 d (n = 5). (a and b) Representative images of H&E staining of nasal cavity (a) and main organs (b). (c) OD_490nm means LDH activity from nasal wash solution. 5% NaTDC was used as positive control known to be toxic. ***p < .001 by Student’s t-test compared to untreated group. (d) Serum level of biochemical variables following nasal administration once a day for 10 d. Data are presented as mean ± s.d.

Figure 4.

Biological evaluation of 3-3 formulation’s stability at two temperatures. (a, b) After incubation of indicated time at room temperature or 4 °C, insulin + PTD in 3–3 formulations was intranasally administered into normal rats. Dose of insulin administered was 1 IU/kg. (a) Blood was collected after various period time of administration, then plasma insulin level was measured by ELISA. (b) Areas under the curves were calculated for each group of rats. (c) Soluble insulin + PTD in 3–3 formulations was analyzed by Tricine-SDS PAGE under non-reducing conditions. Solid line arrow, insulin; Dashed line arrow, PTD. Data are presented as mean ± s.d.