Targeting the translationally controlled tumor protein by a monoclonal antibody improves allergic airway inflammation in mice.

Secretion of translationally controlled tumor protein (TCTP) was found in body fluids during the late phase of allergic reactions, implicating TCTP in allergic diseases. Furthermore, blocking TCTP has been shown to be helpful in treating asthma and allergies in animal models. The objectives of this study were to produce anti-TCTP monoclonal antibodies (mAbs), test their ability to inhibit the cytokine-like function of dimeric TCTP (dTCTP) in vitro and to assess their therapeutic effects in a murine model of ovalbumin (OVA)-induced airway inflammation. We first verified the inhibitory effects of 4 anti-TCTP mAbs on dTCTP-induced secretion of IL-8 in BEAS-2B cells. To investigate the anti-inflammatory effect of anti-TCTP mAbs on allergic airway inflammation, we treated OVA-sensitized mice with anti-TCTP mAbs before OVA challenge. The changes in bronchoalveolar lavage fluid (BALF) cells, IL-4, IL-5, and IL-13 levels in both BALF and lung homogenates, plasma levels of OVA-specific IgE, and lung tissues were analyzed. We found that JEW-M449 anti-TCTP mAb bound to the flexible loop of TCTP and significantly inhibited dTCTP-induced IL-8 release, making it the most effective inhibitor in our study. We also found that treatment with JEW-M449 significantly reduced the infiltration of inflammatory cells and suppressed the OVA-induced upregulation of type 2 cytokines in both BALF and lung homogenates in a dose-dependent manner. In addition, JEW-M449 significantly attenuated the degree of goblet cell hyperplasia and mucus secretion. Our results demonstrate that specific targeting of the flexible loop of TCTP is a potent strategy for treating airway inflammatory diseases.

Meclizine, a piperazine-derivative antihistamine, binds to dimerized translationally controlled tumor protein and attenuates allergic reactions in a mouse model.

Translationally controlled tumor protein (TCTP), a highly conserved protein present in most eukaryotes, is involved in numerous biological processes. Only the dimeric form of TCTP (dTCTP) formed during inflammatory conditions exhibits cytokine-like activity. Therefore, dTCTP is considered as a therapeutic target for allergic diseases. Because monomeric TCTP (mTCTP) and dTCTP share a high topological similarity, we hypothesized that small molecules interacting with mTCTP would also bind to dTCTP and interfere with dTCTP-based cellular processes. In this study, nine compounds listed in the literature as interacting with mTCTP were investigated for their ability to suppress the activity of extracellular dTCTP in bronchial epithelial cells. It was found that one of the nine, meclizine, a piperazine-derivative antihistamine, significantly reduced IL-8 release and suppressed the NF-κB pathway. The direct interaction of meclizine with dTCTP was confirmed by surface plasmon resonance (SPR). Also, we found that meclizine can attenuate ovalbumin (OVA)-induced airway inflammation in mice. Therefore, meclizine might be a potential anti-allergic drug as an inhibitor for dTCTP.

PEGylation improves the therapeutic potential of dimerized translationally controlled tumor protein blocking peptide in ovalbumin-induced mouse model of airway inflammation.

Dimerized translationally controlled tumor protein (dTCTP) initiates a variety of allergic responses in mouse models and that dTCTP-binding peptide 2 (dTBP2) attenuates the allergic inflammation by targeting dTCTP. However, the usefulness of peptide-based drugs is often limited due to their short half-lives, rapid degradation, and high levels of clearance after systemic administration. In this study, we chemically conjugated dTBP2 with 10 kDa polyethylene glycol (PEG) to improve its therapeutic potential. N-terminal mono-PEGylated dTBP2 (PEG-dTBP2) was characterized by in vitro bioactivity assay, pharmacokinetics study, and in vivo efficacy. When compared to the unmodified dTBP2, PEG-dTBP2 reduced proinflammatory cytokine IL-8 secretion in human bronchial cells by 10 to 15% and increased plasma half-life by approximately 2.5-fold in mice. This study specifically demonstrated that PEG-dTBP2 shows higher inhibitory action against ovalbumin (OVA)-induced airway inflammation in mice compared to dTBP2. Importantly, PEG-dTBP2, when administered once at 1 mg/kg, significantly reduced the migration of inflammatory cells and the levels of cytokines in the bronchoalveolar lavage fluids as well as OVA-specific IgE levels in serum. In addition, the degree of goblet cell hyperplasia and mucus secretion were significantly attenuated in the PEG-dTBP2 group compared with the control group. These results suggest that PEG-dTBP2 can be considered a potential candidate drug for regulating allergic inflammation.

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

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.

Enhanced intranasal insulin delivery by formulations and tumor protein-derived protein transduction domain as an absorption enhancer.

One of the key factors for successful development of an intranasal insulin formulation is an absorption enhancer that would deliver insulin efficiently across nasal membranes without causing damage to mucosa or inducing protein aggregation under physiological conditions. In the present study, a protein transduction domain (PTD1) and its L-form with the double substitution A6L and I8A (PTD4), derived from human translationally controlled tumor protein, were used as absorption enhancers. PTD4 exhibited higher compatibility with insulin in terms of biophysical properties analyzed using µDSC, DLS, and CD. In addition, thermodynamic properties indicated stable complex formation but higher propensity of protein aggregation. Arginine hydrochloride (ArgHCl) was used to suppress protein aggregation and carbohydrates (i.e., mannitol, sucrose, and glycerin) were used as osmolytes in the formulation. The relative bioavailability of insulin co-administered intranasally using PTD4, 16 mg/mL glycerin and 100 mM ArgHCl was 58% and that using PTD4, 1 w/v% sucrose, and 25 mM ArgHCl was 53% of the bioavailability obtained via the subcutaneous route. These values represented a remarkable increase in bioavailability of intranasal insulin, causing a significant decrease in blood glucose levels within one hour. The pharmacokinetic properties of intranasal absorption were dependent on the concentration of carbohydrates used. These results suggest that the newly designed formulations with PTD represent a useful platform for intranasal delivery of insulin and other biomolecules.

Modified translationally controlled tumor protein-derived protein transduction domain enhances nasal delivery of exendin-4 as shown with insulin.

Protein transduction domains (PTDs) have been shown to promote the delivery of therapeutic proteins or peptides into the living cells. In a previous study, we showed that the double mutant of TCTP-PTD 13, TCTP-PTD 13M2, was more effective in the delivery of insulin than the wild-type TCTP-PTD 13. In this study, we applied this approach to the nasal delivery of a different peptide, exendin-4, using as carriers, several modified TCTP-PTDs, such as TCTP-PTD 13M1, 13M2, and 13M3. Nasal co-administration of TCTP-PTD 13M2 with exendin-4 showed the highest exendin-4 uptake among the three analogs in normal rats, and also decreased blood glucose levels by 43.3% compared with that of exendin-4 alone and by 18.6% compared with that of exendin-4 plus TCTP-PTD 13 in diabetic mice. We also designed an additional covalently linked conjugate of TCTP-PTD 13M2 and exendin-4 and evaluated its hypoglycemic effect after subcutaneous or intranasal delivery. Subcutaneous administration of exendin-4 that its C-terminus is covalently linked to TCTP-PTD 13M2 showed hypoglycemic effect of 42.2% compared to that in untreated group, whereas intranasal delivery was not successful in diabetic mice. We conclude that a simple mixing TCTP-PTD 13M2 with peptide/protein drugs can be potentially a generally applicable approach for intranasal delivery into animals.

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

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.

Potential of Translationally Controlled Tumor Protein-Derived Protein Transduction Domains as Antigen Carriers for Nasal Vaccine Delivery.

Nasal vaccination offers a promising alternative to intramuscular (i.m.) vaccination because it can induce both mucosal and systemic immunity. However, its major drawback is poor absorption of large antigens in the nasal epithelium. Protein transduction domains (PTDs), also called cell-penetrating peptides, have been proposed as vehicles for nasal delivery of therapeutic peptides and proteins. Here, we evaluated the potential of a mutant PTD derived from translationally controlled tumor protein (designated TCTP-PTD 13) as an antigen carrier for nasal vaccines. We first compared the l- and d-forms of TCTP-PTD 13 isomers (l- or d-TCTP-PTD 13) as antigen carriers. Studies in mice demonstrated that nasally administered mixtures of the model antigen ovalbumin (OVA) and d-TCTP-PTD 13 induced higher plasma IgG titers and secretory IgA levels in nasal washes than nasally administered OVA alone, OVA/l-TCTP-PTD 13, or i.m.-injected OVA. Plasma IgG subclass responses (IgG1 and IgG2a) of mice nasally administered OVA/d-TCTP-PTD 13 showed that the predominant IgG subclass was IgG1, indicating a Th2-biased immune response. We also used synthetic CpG oligonucleotides (CpG) as a Th1 immune response-inducing adjuvant. Nasally administered CpG plus OVA/d-TCTP-PTD 13 was superior in eliciting systemic and mucosal immune responses compared to those induced by nasally administered OVA/d-TCTP-PTD 13. Furthermore, the OVA/CpG/d-TCTP-PTD 13 combination skewed IgG1 and IgG2a profiles of humoral immune responses toward a Th1 profile. These findings suggest that TCTP-derived PTD is a suitable vehicle to efficiently carry antigens and to induce more powerful antigen-specific immune responses and a more balanced Th1/Th2 response when combined with a DNA adjuvant.

On employing a translationally controlled tumor protein-derived protein transduction domain analog for transmucosal delivery of drugs.

Protein transduction domains (PTDs) are recognized as promising vehicles for the delivery of macromolecular drugs. We have previously shown that a region in the N-terminus (residues 1-10) of translationally controlled tumor protein (TCTP) contains a PTD (TCTP-PTD), MIIYRDLISH, which can serve as a vehicle for the delivery of macromolecules into the cells and tissues. In the current study, we evaluated the potential and safety of TCTP-PTD and its three mutant analogs as nasal absorption enhancers for delivery of drugs. We conducted this evaluation employing insulin as test drug. We examined the degree to which insulin was absorbed in nasal mucosa and also if any mucosal damage occurs following such nasal delivery of insulin using TCTP-PTDs as a vehicle. The systemic delivery of insulin was assessed by measuring the changes in blood glucose levels after nasal coadministration insulin and four PTDs. Of the three TCTP-PTD analogs examined, one, TCTP-PTD analog (MIIFRALISHKK) significantly enhanced the nasal absorption of insulin in both normal and streptozotocin-induced diabetic mice. The relative pharmacological bioavailability of insulin nasally coadministered with the TCTP-PTD analog was 21.3% relative to the subcutaneous route. Molecular association between insulin and the TCTP-PTD analog was observed by fluorescence resonance energy transfer measurements. The binding between the TCTP-PTD analog and insulin may enable the penetration of insulin through the nasal mucosa. Histological examination of mice nasal mucosa 7 days after repeated nasal administration showed no evidence of toxicity at the site of nasal administration. In this study using insulin as a test system we demonstrate that the TCTP-PTD analog offers a promising approach for nasal peptides and protein-drugs delivery.

Microscopic detection of IgY-Fc binding signal in the inner layers of ovarian follicular tissue in quail.

In avian species, it has been assumed that an Fc receptor in the ovarian follicles mediates immunoglobulin Y (IgY) transport into the yolk. However, no such receptor responsible for IgY has been identified to date. To examine potential IgY binding activity in the entire ovarian follicle, whole-mount sections of quail ovarian follicle were incubated with the Fc fragment of chicken IgY (cIgY). Whole-mount frozen sections of the second largest ovarian follicle were prepared, and then the sections were incubated with digoxigenin-labeled Fc or Fab fragments of cIgY. Microscopic observation revealed that incubation with the cIgY-Fc fragment produced a binding signal in the inner layer of the ovarian follicular tissues, most likely in the granulosa cell layer. However, no such signal was detected when the sections were incubated with cIgY-Fab. Coincubation of the ovarian sections with Alexa488-labeled cIgY-Fc and antiserum raised against ZP1, an envelope protein specifically localized in the perivitelline layer, demonstrated that the source of the Fc binding signals partly coincided with the perivitelline layer. In conclusion, our data suggest that potential IgY binding substances interacting with the Fc domain are present in the inner layers of ovarian follicular tissues, most likely in the granulosa cell layer and/or in the perivitelline layer.

Identification of the amino acid residues involved in human IgG transport into egg yolks of Japanese quail (Coturnix japonica).

In avian species, maternal immunoglobulin (Ig) Y is selectively incorporated into the yolks of maturing oocytes, although the relevance of receptor-mediated uptake is unclear. When administered to birds, several mammalian Igs, including human IgG (hIgG), are also incorporated into the yolks. In the current study, to gain insight into selective Ig transport into yolks, we intended to identify the amino acid residues critical for Ig uptake into egg yolks using alanine and glycine-scanning mutagenesis of 16 residues located along the C(H)2 and C(H)3 domains of hIgG1. Wild-type hIgG1-Fc (WT) and its mutants were synthesized, and their uptakes into the egg yolks of Japanese quail (Coturnix japonica) were determined. The triple mutation of loop MIS252-254 to GGG resulted in a 40% decrease in Fc uptake in comparison to that of the WT. Furthermore, quartet substitution of HEAL429-432 to GGGG located in an exposed loop at the C(H)3 domain completely abolished Fc uptake into egg yolks. Next, the residues HEAL429-432 were individually substituted with either alanine or glycine. Regardless of the glycine and alanine substitution, single mutations of H (429), E (430) and L (432) significantly reduced Fc uptake compared with WT uptake. Notably, the blood clearance rates of these mutants were equivalent to that of the WT. These results suggest that the clustered residues HEAL429-432 in the C(H)3 domain are important for the hIgG1 transport into the egg yolks. The sequence HEAL is conserved in chicken IgY at positions 550-553 within the C(H)4 domain, which might be involved in its uptake into the egg yolks by receptor-mediated endocytosis.