gH625-liposomes as tool for pituitary adenylate cyclase-activating polypeptide brain delivery.

The blood-brain barrier (BBB) regulates the traffic of molecules into the central nervous system (CNS) and also limits the drug delivery. Due to their flexible properties, liposomes are an attractive tool to deliver drugs across the BBB. We previously characterized gH625, a peptide derived from Herpes simplex virus 1. The present study investigates the efficiency of liposomes functionalized on their surface with gH625 to promote the brain uptake of neuroprotective peptide PACAP (pituitary adenylate cyclase-activating polypeptide). Using a rat in vitro BBB model, we showed that the liposomes preparations were non-toxic for the endothelial cells, as assessed by analysis of tight junction protein ZO1 organization and barrier integrity. Next, we found that gH625 improves the transfer of liposomes across endothelial cell monolayers, resulting in both low cellular uptake and increased transport of PACAP. Finally, in vivo results demonstrated that gH625 ameliorates the efficiency of liposomes to deliver PACAP to the mouse brain after intravenous administration. gH625-liposomes improve both PACAP reaching and crossing the BBB, as showed by the higher number of brain cells labelled with PACAP. gH625-liposomes represent a promising strategy to deliver therapeutic agents to CNS and to provide an effective imaging and diagnostic tool for the brain.

Effect of controlled cortical impact on the passage of pituitary adenylate cyclase activating polypeptide (PACAP) across the blood-brain barrier.

Injuries to the central nervous system can affect the blood-brain barrier (BBB), including disruption and influencing peptide transport across the BBB. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a potent neurotrophic and neuroprotective peptide currently being investigated for its therapeutic role following injury to the central nervous system and can cross the BBB in a saturable manner. The goal of the current study was to investigate for the first time PACAP38 uptake by the brain following traumatic brain injury (TBI). Using radioactively labeled PACAP38, we measured the levels of PACAP38 present in the injured, ipsilateral cortex in Sham-treated mice compared to mice receiving a controlled cortical impact (CCI), a model of TBI. Experiments were conducted at 6 different time points (from 2h up to 4 weeks) following CCI to determine temporal changes in PACAP38 transport. PACAP38 uptake was increased at 2 and 72h post-CCI compared to Sham. We did not detect changes in PACAP38 uptake in the contralateral cortex and cerebellum between Sham and CCI-treatment. The rate of PACAP38 transport into the ipsilateral cortex following CCI was increased 3.6-fold 72h after compared to 2h post-CCI. In addition, the rate of transport into the cerebellum was greater than that of the cortices. The data presented here shows PACAP38 transport is temporally altered following CCI-treatment and PACAP38 uptake is greater in the cerebellum compared to the cortices.

Passage through the Ocular Barriers and Beneficial Effects in Retinal Ischemia of Topical Application of PACAP1-38 in Rodents.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has two active forms, PACAP1-27 and PACAP1-38. Among the well-established actions are PACAP's neurotrophic and neuroprotective effects, which have also been proven in models of different retinopathies. The route of delivery is usually intravitreal in studies proving PACAP's retinoprotective effects. Recently, we have shown that PACAP1-27 delivered as eye drops in benzalkonium-chloride was able to cross the ocular barriers and exert retinoprotection in ischemia. Since PACAP1-38 is the dominant form of the naturally occurring PACAP, our aim was to investigate whether the longer form is also able to cross the barriers and exert protective effects in permanent bilateral common carotid artery occlusion (BCCAO), a model of retinal hypoperfusion. Our results show that radioactive PACAP1-38 eye drops could effectively pass through the ocular barriers to reach the retina. Routine histological analysis and immunohistochemical evaluation of the Müller glial cells revealed that PACAP1-38 exerted retinoprotective effects. PACAP1-38 attenuated the damage caused by hypoperfusion, apparent in almost all retinal layers, and it decreased the glial cell overactivation. Overall, our results confirm that PACAP1-38 given in the form of eye drops is a novel protective therapeutic approach to treat retinal diseases.

Evaluation of a PACAP Peptide Analogue Labeled with (68)Ga Using Two Different Chelating Agents.

OBJECTIVE: The authors have conjugated chelating agents (DOTA and NODAGA) with a peptide (pituitary adenylate cyclase-activating peptide [PACAP] analogue) that has a high affinity for VPAC1 receptors expressed on cancer cells. To determine a suitable chelating agent for labeling with (68)Ga, they have compared the labeling kinetics and stability of these peptide conjugates. METHODS: For labeling, (68)GaCl3 was eluted in 0.1 M HCl from a [(68)Ge-(68)Ga] generator. The influences of peptide concentration, pH, and temperature on the radiolabeling efficiency were studied. The stability was evaluated in saline, human serum, DTPA, transferrin, and metallic ions (FeCl3, CaCl2, and ZnCl2). Cell binding assay was performed using human breast cancer cells (T47D). Tissue biodistribution was studied in normal athymic nude mice. RESULTS: Optimal radiolabeling (>95.0%) of the DOTA-peptide conjugates required a higher (50°C-90°C) temperature and 10 minutes of incubation at pH 2-5. The NODAGA-peptide conjugate needed incubation only at 25°C for 10 minutes. Both radiocomplexes were stable in saline, serum, as well as against transchelation and transmetallation. Cell binding at 37°C for 15 minutes of incubation with (68)Ga-NODAGA-peptide was 34.0% compared to 24.5% for (68)Ga-DOTA-peptide. Tissue biodistribution at 1 hour postinjection of both (68)Ga-labeled peptide conjugates showed clearance through the kidneys. CONCLUSIONS: NODAGA-peptide showed more convenient radiolabeling features than that of DOTA-peptide.

PACAP intraperitoneal treatment suppresses appetite and food intake via PAC1 receptor in mice by inhibiting ghrelin and increasing GLP-1 and leptin.

Pituitary adenylate cyclase-activating peptide (PACAP) is expressed within the gastroenteric system, where it has profound physiological effects. PACAP was shown to regulate food intake and thermogenesis centrally; however, PACAP peripheral regulation of appetite and feeding behavior is unknown. Therefore, we studied PACAP's effect on appetite and food intake control by analyzing feeding behavior and metabolic hormones in PAC1-deficient (PAC1-/-) and age-matched wild-type (WT) mice intraperitoneally injected with PACAP1-38 or PACAP1-27 before the dark phase of feeding. Food intake and feeding behavior were analyzed using the BioDAQ system. Active ghrelin, glucagon-like peptide-1 (GLP-1), leptin, peptide YY, pancreatic polypeptide, and insulin were measured following PACAP1-38 administration in fasted WT mice. PACAP1-38/PACAP1-27 injected into WT mice significantly decreased in a dose-dependent manner cumulative food intake and reduced bout and meal feeding parameters. Conversely, PACAP1-38 injected into PAC1-/- mice failed to significantly change food intake. Importantly, PACAP1-38 reduced plasma levels of active ghrelin compared with vehicle in WT mice. In PAC1-/- mice, fasting levels of active ghrelin, GLP-1, insulin, and leptin and postprandial levels of active ghrelin and insulin were significantly altered compared with levels in WT mice. Therefore, PAC1 is a novel regulator of appetite/satiety. PACAP1-38/PACAP1-27 significantly reduced appetite and food intake through PAC1. In PAC1-/- mice, the regulation of anorexigenic/orexigenic hormones was abolished, whereas active ghrelin remained elevated even postprandially. PACAP significantly reduced active ghrelin in fasting conditions. These results establish a role for PACAP via PAC1 in the peripheral regulation of appetite/satiety and suggest future studies to explore a therapeutic use of PACAP or PAC1 agonists for obesity treatment.

VPAC1 receptors for imaging breast cancer: a feasibility study.

UNLABELLED: VPAC1 encodes G-protein-coupled receptors expressed on all breast cancer (BC) cells at the onset of the disease, but not on benign lesions. Our extensive preclinical studies have shown that (64)Cu-TP3805 has a high affinity for VPAC1, is stable in vivo, and has the ability to distinguish spontaneously grown malignant BC masses from benign lesions. Our long-term goal is to develop (64)Cu-TP3805 as an agent to perform in vivo histology, to distinguish malignant lesions from benign masses noninvasively and thereby avoid patient morbidity and the excess economic costs of benign biopsies. METHODS: (18)F-FDG obtained commercially served as a control. (64)Cu-TP3805 was prepared using a sterile kit containing 20 µg of TP3805. Radiochemical purity and sterility were examined. Nineteen consenting women with histologically proven BC were given 370 MBq of (18)F-FDG. One hour later, 6 of these patients were imaged with PET/CT and 13 with positron emission mammography (PEM). Two to 7 d later, 6 PET/CT patients received 111 MBq (± 10%) (n = 2), 127 MBq (± 10%) (n = 2), or 148 MBq (± 10%) (n = 2) of (64)Cu-TP3805 and were imaged 2 and 4 h later. Thirteen PEM patients received 148 MBq (± 10%) of (64)Cu-TP3805 and were imaged 15 min, 1 h, 2 h, and 4 h later. Standardized uptake value (SUV) was calculated for PET/CT patients, and PUV/BGV (PEM uptake value/background value) was calculated for PEM patients. Tumor volume was also calculated. RESULTS: The radiochemical purity of (64)Cu-TP3805 was 97% ± 2%, and specific activity was 44.4 GBq (1.2 Ci)/µmol. In 19 patients, a total of 24 lesions were imaged (15 invasive ductal carcinoma, 1 high-grade mammary carcinoma, 3 lobular carcinoma, 1 invasive papilloma, and 4 sentinel lymph nodes). All lesions were unequivocally detected by (64)Cu-TP3805 and by (18)F-FDG. The average tumor volume as determined by PET/CT with (64)Cu-TP3805 was 90.6% ± 16.1% of that with (18)F-FDG PET/CT, and the average SUV was 92% ± 26.4% of that with (18)F-FDG. For PEM, the tumor volume with (64)Cu-TP3805 was 113% ± 37% of that with (18)F-FDG and the PUV/BGV ratio was 97.7% ± 24.5% of that with (18)F-FDG. CONCLUSION: (64)Cu-TP3805 is worthy of further investigation in patients requiring biopsy of suggestive imaging findings, to further evaluate its ability to distinguish malignant lesions from benign masses noninvasively.

The TAT peptide endows PACAP with an enhanced ability to traverse bio-barriers.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic neuropeptide. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to deliver protein cargoes across the cell membrane and the blood-brain barrier. A novel fusion protein PACAP-TAT, containing TAT at the C-terminus of PACAP was therefore produced and studied for the ability to cross blood barriers. The gene encoding PACAP-TAT was cloned into the expression vector pKYB, and the target peptide PACAP-TAT was purified using the Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system. The results of cell assays showed that PACAP-TAT stimulated the cell viability of PAC1-CHO cells with the same potency as PACAP, which indicated that the fusion of TAT did not affect the ability of PACAP-TAT to activate the PACAP-specific receptor PAC1. The transfer efficiencies of PACAP-TAT and PACAP across the blood-brain barrier (BBB), blood-air barrier (BAB) and blood-testis barrier (BTB) were assayed using peptides labeled with fluorescein isothiocyanate (FITC). The results showed that PACAP-TAT traversed blood barriers with an efficiency approximately 2.5-fold greater than PACAP. Fluorescence microscopic examination showed that PACAP-TAT traversed the BBB significantly more efficiently than PACAP. Furthermore, intraperitoneal (i.p.) injection of PACAP-TAT induced a stronger inhibitory effect on food intake than PACAP (p<0.01, PACAP-TAT vs. PACAP), which indicated that TAT helped to increase the localization of PACAP-TAT in the brain. Preparation of PACAP-TAT with the enhanced ability to cross biological barriers will improve its route of administration and expand its scope of application.

Intranasal administration of PACAP: uptake by brain and regional brain targeting with cyclodextrins.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent neurotrophic and neuroprotectant that is transported across the blood-brain barrier in amounts sufficient to affect brain function. However, its short half-life in blood makes it difficult to administer peripherally. Here, we determined whether the radioactively labeled 38 amino acid form of PACAP can enter the brain after intranasal (i.n.) administration. Occipital cortex and striatum were the regions with the highest uptake, peaking at levels of about 2-4% of the injected dose per gram of brain region. Inclusion of unlabeled PACAP greatly increased retention of I-PACAP by brain probably because of inhibition of the brain-to-blood efflux transporter for PACAP located at the blood-brain barrier. Sufficient amounts of PACAP could be delivered to the brain to affect function as shown by improvement of memory in aged SAMP8 mice, a model of Alzheimer's disease. We found that each of three cyclodextrins when included in the i.n. injection produced a unique distribution pattern of I-PACAP among brain regions. As examples, ß-cyclodextrin greatly increased uptake by the occipital cortex and hypothalamus, α-cyclodextrin increased uptake by the olfactory bulb and decreased uptake by the occipital cortex and striatum, and (2-hydropropyl)-ß-cyclodextrin increased uptake by the thalamus and decreased uptake by the striatum. These results show that therapeutic amounts of PACAP can be delivered to the brain by intranasal administration and that cyclodextrins may be useful in the therapeutic targeting of peptides to specific brain regions.

Thiolated chitosans: design and in vivo evaluation of a mucoadhesive buccal peptide drug delivery system.

PURPOSE: Intravenous application of pituitary adenylate cyclase-activating polypeptide (PACAP) has been identified as a promising strategy for the treatment of type 2 diabetes. To generate a more applicable formulation, it was the aim of this study to develop a sustained buccal delivery system for this promising therapeutic peptide. METHODS: 2-Iminothiolane was covalently bound to chitosan to improve the mucoadhesive and permeation-enhancing properties of chitosan used as drug carrier matrix. The resulting chitosan-4-thiobutylamidine conjugate was homogenized with the enzyme inhibitor and permeation mediator glutathione (gamma-Glu-Cys-Gly), Brij 35, and PACAP (formulation A). The mixture was lyophilized and compressed into flat-faced discs (18 mm in diameter). One formulation was additionally coated on one side with palm wax (formulation B). Tablets consisting of unmodified chitosan and PACAP (formulation C) or of unmodified chitosan, Brij 35, and PACAP (formulation D) served as controls. Bioavailability studies were performed in pigs by buccal administration of these test formulations. Blood samples were analyzed via an ELISA method. RESULTS: Formulations A and B led to an absolute bioavailability of 1%, whereas PACAP did not reach the systemic circulation when administered via formulations C and D. Moreover, in the case of formulations A and B, a continuously raised plasma level of the peptide drug being in the therapeutic range could be maintained over the whole period of application (6 h). Formulations A and B were removed by moderate force from the buccal mucosa after 6 h, whereas formulations C and D detached from the mucosa 4 h after application. CONCLUSION: The study reveals this novel mucoadhesive delivery system to be a promising approach for buccal delivery of PACAP.

In vitro evaluation of various buccal permeation enhancing systems for PACAP (pituitary adenylate cyclase-activating polypeptide).

PURPOSE: Buccal administration of pituitary adenylate cyclase-activating polypeptide (PACAP) could represent a new possibility for the treatment of type 2 diabetes. In this study the effect of various buccal permeation enhancers on PACAP and FD-4 was evaluated. METHODS: The permeation-enhancing properties of the well-established permeation enhancers sodium deoxycholate (Na DOC) and cetrimide on the permeation of PACAP were investigated on freshly excised porcine buccal mucosa in Ussing chambers. Furthermore, the effect of chitosan and that of chitosan-4-thiobutylamidine conjugate (chitosan-TBA) optionally in combination with reduced glutathione (GSH) on the permeation of PACAP across the buccal mucosa was studied. RESULTS: The apparent permeability coefficient (P(app)) of PACAP in buffer only was 5.7 +/- 3.1x10(-8) cm/s. In the presence of 5% (m/v) Na DOC, the enhancement of the permeation was 18.6-fold, whereas due to the addition of 5% (m/v) cetrimide an enhancement ratio of 46.5 was obtained. In the presence of the chitosan-TBA conjugate (1%), a 38.9-fold increased permeation was achieved, whereas unmodified chitosan (1%) did not show any effect. The combination of chitosan-TBA conjugate (1%) with GSH (2%) led to an increase in P(app) up to 441.7 +/- 89.9x10(-8) cm/s, which represents a 77.5-fold improvement. The P(app) of GSH per se was only 1.0 +/- 0.2x10(-9) cm/s, showing that GSH remains concentrated on the surface of the buccal mucosa. Results were confirmed by additional permeation studies performed with FD-4 used as hydrophilic macromolecular test compound. CONCLUSIONS: Based on their permeation-enhancing properties, chitosan-TBA conjugates represent a promising tool for the buccal administration of peptide drugs, e.g., PACAP.