Modification of translationally controlled tumor protein-derived protein transduction domain for improved intranasal delivery of insulin

Abstract

Carrier peptides, termed protein transduction domains (PTDs), serve as provide promising vehicles for intranasal delivery of macromolecular drugs. A mutant PTD derived from human translationally controlled tumor protein (TCTP-PTD 13, MIIFRALISHKK) was reported to provide enhanced intranasal delivery of insulin. In this study, we tested whether its efficiency could be further improved by replacing amino acids in TCTP-PTD 13 or changing the amino acids in the carrier peptides from the l- to the d-form. We assessed the pharmacokinetics of PTD-mediated transmucosal delivery of insulin in normal rats and the activity of insulin in alloxan-induced diabetic rats. The safety/toxicity profile of the carrier peptides was evaluated based on the release of lactate dehydrogenase (LDH) in nasal wash fluid, body weight changes, and several biochemical parameters. Pharmacokinetic and pharmacodynamic studies showed that the l-form of a double substitution A6L, I8A (MIIFRLLASHKK), designated as l-TCTP-PTD 13M2 was the most effective carrier for intranasal insulin delivery. The relative bioavailability of insulin co-administered intranasally with l-TCTP-PTD 13M2 was 37.1% of the value obtained by the subcutaneous route, which was 1.68-fold higher than for insulin co-administered with l-TCTP-PTD 13. Moreover, co-administration of insulin plus l-TCTP-PTD 13M2 reduced blood glucose levels compared to levels in diabetic rats treated with insulin plus l-TCTP-PTD 13. There was no evidence of toxicity. These results suggest that the newly designed PTD is a useful carrier peptide for the intranasal delivery of drugs or biomolecules.

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

Figure 1.

(a) Amino acid sequence of l-TCTP-PTD 13 and its analogs. Amino acid modifications in l-TCTP-PTD 13 are underlined. (b) Cellular uptake of FITC-labeled peptides in BEAS-2B cells analyzed by a flow cytometer. (c) Histograms of l-TCTP-PTD 13, l-TCTP-PTD 13M1, and l-TCTP-PTD 13M2. Each bar represents the standard deviation of three independent replicates. The mean fluorescence intensity of FITC-labeled l-TCTP-PTD 13 in BEAS-2B cells was set to 100%. *p < .05 versus FITC-labeled l-TCTP-PTD 13.

Figure 2.

(a and b) Plasma insulin concentration in normal rats following intranasal administration of insulin in the presence of 0.1 mM (a) or 0.25 mM (b) TCTP-PTD analogs containing l-amino acids. (c) Plasma insulin concentration following intranasal administration of insulin with 0.25 mM d-TCTP-PTD analogs. Insulin doses were 5 and 1 IU/kg for insulin alone and insulin plus PTD, respectively. Vertical bars indicate means ± SEM (n = 5–7). (d) LDH leakage in nasal fluid of normal rats following intranasal administration of insulin (1 IU/kg) with different PTDs. Sodium taurodeoxycholate (5% w/v) served as a positive control. Each bar represents mean ± SEM (n = 6). *p < .01 versus insulin alone.

Figure 3.

Changes in blood glucose levels in rats with alloxan-induced diabetes following intranasal administration of insulin plus l-TCTP-PTD analogs. Insulin doses were 2 and 1 IU/kg for administration by the nasal route and s.c. injection, respectively. Vertical bars indicate means ± SEM (n = 6–8).

Figure 4.

(a) Body weight of normal mice (n = 6) after daily intraperitoneal injection of l-TCTPPTD 13M2 for 10 days. (b) Biochemical analysis of AST, ALT, BUN, and CRE levels.