Tight junctions (TJs)

The 6-mer synthetic peptide AT-1002 is part of a newly discovered class of drugs that can reversibly enhance the paracellular transport of molecules over the epithelial barrier. Cys-Cys dimerization is possible for AT-1002[1]. Cell viability is assessed by measuring the cellular ATP concentration, and undifferentiated Caco-2 cells are assessed using AT-1002 (0 to 5 mg/mL, 3 or 24 hours). Cell viability at any concentration is unaffected by treatment as measured by AT-1002 for up to three hours. Specifically, 5 mg/mL of AT-1002 had no effect on the viability of Caco-2 cells. At doses of 2.5 mg/mL and above, AT-1002 decreases cell viability after 24 hours. However, washing the cells after they have been exposed to AT-1002 for three hours does not cause irreversible damage to the cells, as the cells still function after twenty-four hours[2].

---

AT-1002, a hexamer peptide, caused the redistribution of ZO-1 away from cell junctions as seen by fluorescence microscopy. AT-1002 also activated src and mitogen activated protein (MAP) kinase pathways, increased ZO-1 tyrosine phosphorylation, and rearrangement of actin filaments. Functionally, AT-1002 caused a reversible reduction in transepithelial electrical resistance (TEER) and an increase in lucifer yellow permeability in Caco-2 cell monolayers.[2]

---

The effect of AT-1002 on cell viability was measured using the CellTiter-Glo® cell viability assay. After 3 h of treatment, AT-1002 did not significantly reduce cell viability compared to untreated control cells (Fig. 2). These data suggests that this compound enhance LY permeability due to their permeability modulation activity and not due to cell viability reduction[1].

In vivo activities [2]
It was of great interest to determine if the in vitro effects of AT-1002 described above could be translated into in vivo effects. Previously it has been shown that AT-1002 was able to increase the delivery of payloads administered into the gastrointestinal system in vivo (Motlekar et al., 2006, Song et al., 2008a, Song et al., 2008b). Here we wanted to determine if AT-1002 could enhance delivery of payloads applied to the airway epithelia. Thus, we tested whether AT-1002 could increase the systemic exposure of salmon calcitonin (sCT), which by itself has very low bioavailability. Intratracheal instillation of 10 μg of sCT with increasing amounts of AT-1002 into rats resulted in increased pulmonary absorption and higher systemic concentrations of sCT in the treatment groups with the highest amount of AT-1002 (Fig. 8). When sCT was administered 2 h after the AT-1002 administration, no enhanced absorption was observed indicating that the effect of AT-1002 was transient (data not shown). No differences were observed among pharmacokinetic parameters below a delivered dose of 300 μg of AT-1002 (data not shown). The sCT AUC0–240 min was significantly increased over the control group (0 μg dose) at 300 and 1000 μg of AT-1002 (Table 2). Co-administration of sCT with 300 and 1000 μg of AT-1002 resulted in a 1.6-fold (161.8%) and 5.2-fold (522.5%) increase in AUC over the control group, respectively. Cmax, which is a measure of the highest concentration achieved, was also 2.3-fold higher when 1000 μg of AT-1002 was co-administered with sCT relative to control.

TEER and lucifer yellow permeability assays [2]
Details of this method and modifications have been described previously (Artursson, 1990, Ginski and Polli, 1999). For transepithelial electrical resistance (TEER) and lucifer yellow (LY) permeability assays, Caco-2 cells were seeded onto 12-well Transwells™ (pore size 0.4 μm) at a density of 100,000 cells/cm2 and grown for 21–28 days until fully differentiated. The apical and basolateral compartments of the Caco-2 cell monolayers were pre-incubated in HBSS at 37 °C for 30 min. Treatment solutions containing a range of concentrations of AT-1002 from 0.4 to 5 mg/ml AT-1002 or 5 mg/ml scrambled peptide in HBSS were added to the apical compartment of each monolayer and then incubated at 37 °C, 50 rpm for 180 min. TEER was measured using a MilliCell-ERS at 0, 30, 60, 120 and 180 min. At 180 min, AT-1002 was replaced in the apical compartment by 7.5 mM lucifer yellow. After 1 h incubation at 37 °C, samples were removed from the basolateral compartment and analyzed for LY in a Tecan Spectrofluor fluorescence plate reader at excitation wavelength of 485 nm and emission wavelength of 535 nm. The decrease in TEER and increase in LY permeability was calculated for each treatment and expressed relative to lucifer yellow untreated control. The permeability is calculated as follows: Papp = [(dC/dt) × Vr]/(Co × A), where dC/dt is the permeability rate, Vr is the volume of the receiver, A is the surface area of the membrane filter, and Co is the initial concentration in the donor chamber, and the enhancement ratio is defined as Papp AT-1002/Papp HBSS.

---

Reversibility of AT-1002 effects on Caco-2 cells [2]
Caco-2 cells were seeded on transwell membranes as described above and grown in DMEM for a period of 21 days at 37 °C, 5% CO2 and 95% humidity with medium change every other day. At the end of the growth period the medium from the upper (apical) and lower (basolateral) compartments was removed. The cells were incubated in pre-warmed (37 °C) HBSS (with Ca and Mg) with 10 mM HEPES pH 7.4. The transwells were treated apically with or without AT-1002 at a concentration of 5 mg/ml in HBSS for various times. The AT-1002 was either replaced by HBSS after 15, 30, 45 and 60 min or not removed at all. TEER readings were monitored using an Ohm meter at different time points.

Cell Viability Assay[2]
Cell Types: Caco-2 cells
Tested Concentrations: 0 to 5 mg/mL
Incubation Duration: 3 or 24 hrs (hours)
Experimental Results: Treatment for up to 3 h did not affect cell viability at any concentration. decreased cell viability after 24 h at concentrations of 2.5 mg/mL and higher.

---

In vitro cytotoxicity assays [2]
Cell viability was determined by measuring the amount of ATP in cells using a luminescence ATP assay. The concentration of ATP is determined by the amount of light emitted when beetle luciferin is mono-oxygenated by luciferase in a reaction that is Mg2+ and ATP-dependent. A range of concentrations of AT-1002 from 0 to 5 mg/ml in 100 μl of Hank’s Balanced Salt Solution (HBSS) was added to 30,000 Caco-2 cells grown on 96-well tissue culture plates after removal of the growth media. An equal volume of Cell titer-Glo reagent was added to the wells after 3 h and the chemiluminescence was measured after 15 min incubation in a Tecan Spectrafluor plus plate reader. A standard curve was generated for ATP and used to calculate the concentration of ATP after treatment with AT-1002.

---

Immunofluorescence [2]
IEC6 cells were plated on 8-chamber slides at 60,000 cells per chamber. At 24 h post-plating, cells were washed in serum-free medium and incubated with AT-1002 (5 mg/ml) diluted in serum-free medium for 60 min at 37 °C. Following treatment, cells were washed in PBS and fixed in PBS containing 4% paraformaldehyde for 15 min at room temperature. Cells were washed in PBS, permeabilized in PBS containing 0.5% Triton X-100 for 5 min at room temperature, and blocked in PBS containing 2% goat serum for 30 min at room temperature. Cells were then incubated with primary antibodies diluted in blocking buffer (pMLC (1:50)) for 1–2 h at 37 °C. Cells were washed in PBS and incubated with FITC tagged anti-rabbit antibody for 45 min at room temperature. Actin and ZO-1 were detected using Alexa Fluor555-phalloidin and FITC labeled anti-ZO-1 antibodies, respectively. Slides were washed and mounted in Vectashield containing DAPI and imaged on a Nikon-TE2000 fluorescence microscope. Caco-2 BBE cells were treated apically with AT-1002 (5 mg/ml) for 3 h at 37 °C. Following treatment cells were fixed in methanol:acetone (1:1) and blocked in PBS containing 2% goat serum. Filters were incubated with FITC labeled anti-ZO-1 antibodies for 1 h at room temperature, washed and mounted on slides as described above.

---

Flow cytometry [2]
Caco-2 BBE cells were treated apically with AT-1002 (5 mg/ml) for 3 h at 37 °C. Following treatment cells were detached from filters using trypsin. Detached cells were washed in PBS, fixed in PBS containing 4% paraformaldehyde for 15 min at room temperature, permeabilized in PBS containing 0.5% Triton X-100 for 5 min at room temperature, and blocked in PBS containing 2% goat serum for 30 min at room temperature. Cells were incubated with Alexa Fluor555-phalloidin for 1 h at room temperature, washed in PBS and analyzed by flow cytometry using FACSCAN.

Intratracheal delivery of salmon calcitonin [1]
Male Sprague–Dawley rats were used for this study and were approximately 12 weeks of age at the initiation of the study. All rats were instilled intratracheally with 10 μg of sCT in 200 μl of saline containing 0, 300 or 1000 μg of AT-1002 (n = 6 per dose group). Blood samples (200 μl) were collected and placed into EDTA coated tubes prior to dosing and at 2.5, 5, 10, 15, 30, 60, 120 and 240 min following dosing. Plasma was harvested and stored at ≤−70 °C until assayed for sCT. The DSL 10–3600 ACTIVE® Salmon Calcitonin Enzyme-Linked Immunosorbent (ELISA) kit was used with slight modifications to determine concentrations of sCT in rat plasma. This assay is an enzymatically amplified “two-step” sandwich-type immunoassay involving the biotin-streptavidin bridging detection system. For these studies standards were prepared in a rat serum matrix and the curve ranged from 15.6 to 1000 pg/ml. The LLOQ was 31.3 pg/ml. The sample volume used was 50 μl. When necessary, samples (concentrations expected or measured above 1000 pg/ml) were diluted with the rat serum matrix. Standard curves were calculated using a four parameter fit model using the KC4 software available on a BioTek Plate reader. Assay performance did not appear to be influenced by the difference between the sample (rat plasma) and standard (rat serum) matrix. Microsoft Excel® was used for calculation of AUC using the linear trapezoidal rule and data were plotted using GraphPad Prism version 4.01. Cmax and Tmax for each condition were also determined.

[1]. Structure-activity relationship studies of permeability modulating peptide AT-1002. Bioorg Med Chem Lett. 2008 Aug 15;18(16):4584-6.

[2]. Mechanism of action of ZOT-derived peptide AT-1002, a tight junction regulator and absorption enhancer. Int J Pharm. 2009 Jan 5;365(1-2):121-30.

AT-1002 is a hexapeptide synthetic peptide belonging to a new class of novel compounds that can reversibly enhance the transepithelial bypass transport of molecules across the epithelial barrier. This project aims to elucidate the structure-activity relationship of this peptide and focuses on the substitution of its P2 cysteine residue. This article reports the peptide we discovered that has reversible permeability enhancement properties and higher stability. [1] Tight junctions (TJs) are intercellular structures that control transepithelial bypass permeability and epithelial polarity. It is generally believed that TJs are highly dynamic structures whose activity is regulated by endogenous and exogenous stimuli. This article elaborates on the mechanism of action of AT-1002, which is the active domain of the second toxin of Vibrio cholerae—tight junction toxin (ZOT). AT-1002 is a hexapeptide, and fluorescence microscopy shows that it can redistribute ZO-1 from cell junctions. AT-1002 can also activate the Src and mitogen-activated protein kinase (MAP) pathways, increase ZO-1 tyrosine phosphorylation levels, and cause actin filament rearrangement. Functionally, AT-1002 reversibly reduces the transepithelial resistance (TEER) of the Caco-2 cell monolayer and increases the permeability of fluorescein yellow. In vivo experiments showed that, compared with the control group, the AUC value of salmon calcitonin combined with 1 mg AT-1002 increased by 5.2 times. Our results provide a mechanistic explanation for the AT-1002-induced tight junction disintegration and demonstrate that AT-1002 can be used to deliver other drugs in vivo. [2] Drug absorption mainly occurs through passive transcellular and paracellular transport mechanisms (Behrens et al., 2001). Lipophilic drugs are mainly transported through transcellular pathways and transport proteins such as channels, pumps and carriers on the plasma membrane. However, the paracellular pathway is usually the main absorption pathway for hydrophilic drugs (proteins, peptides, etc.). We demonstrate that the small peptide AT-1002 derived from ZOT can lead to the opening of tight junctions in Caco-2 cell monolayers (Motlekar et al., 2006; Song et al., 2008a), and show that this effect is reversible. Other experiments have shown that combined intratracheal administration of AT-1002 and salmon calcitonin increased systemic exposure to salmon calcitonin by up to 5.2-fold, suggesting the potential use of AT-1002 for systemic delivery of antigens and other payloads. Indeed, previous studies have shown that AT-1002 can enhance the delivery of small molecules (Motlekar et al., 2006; Song et al., 2008a; Song et al., 2008b). Another study has shown that peptides from the extracellular loop of the tight junction protein occludin can be used to modulate tight junctions (Tavelin et al., 2003) by increasing tight junction permeability without causing short-term toxicity. However, these peptides are only effective when added to the basal outer layer of monolayer cells. Agents that enhance drug or antigen delivery must be applied to the apex of the epithelial cell surface to function. This paper shows that AT-1002 is such an agent and thus represents a prototype of a new class of tight junction (TJ) modulators. TJ-opening molecules like AT-1002 can be envisioned for two main applications: conventional drug delivery and antigen delivery for vaccination. Recently, studies have shown that a peptide in the rotavirus capsid can promote insulin absorption in rats (Nava et al., 2004). Other TJ-regulating (TJM) peptides and peptide YY (PYY) can also improve drug transport across epithelial tissues (Chen et al., 2006; Gonzalez-Mariscal and Nava, 2005). Therefore, compounds that can achieve efficient, non-toxic, and non-invasive drug delivery will revolutionize the treatment of a variety of diseases. Thus, TJ-regulating peptides like AT-1002 represent a promising advance in the field of mucosal drug delivery. [2]

C34H54F3N9O9S

821.91

821.37172

AT-1002;835872-35-0

138319696

L-phenylalanyl-L-cysteinyl-L-isoleucyl-glycyl-L-arginyl-L-leucine trifluoroacetic acid; H-Phe-Cys-Ile-Gly-Arg-Leu-OH.TFA

FCIGRL

White to off-white solid powder

11

15

22

56

1210

6

CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CC=CC=C1)N.C(=O)(C(F)(F)F)O

JDVTZXDTPSAPFV-CACDTQBQSA-N

InChI=1S/C32H53N9O7S.C2HF3O2/c1-5-19(4)26(41-29(45)24(17-49)40-27(43)21(33)15-20-10-7-6-8-11-20)30(46)37-16-25(42)38-22(12-9-13-36-32(34)35)28(44)39-23(31(47)48)14-18(2)3;3-2(4,5)1(6)7/h6-8,10-11,18-19,21-24,26,49H,5,9,12-17,33H2,1-4H3,(H,37,46)(H,38,42)(H,39,44)(H,40,43)(H,41,45)(H,47,48)(H4,34,35,36);(H,6,7)/t19-,21-,22-,23-,24-,26-;/m0./s1

(2S)-2-[[(2S)-2-[[2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-amino-3-phenylpropanoyl]amino]-3-sulfanylpropanoyl]amino]-3-methylpentanoyl]amino]acetyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-4-methylpentanoic acid;2,2,2-trifluoroacetic acid

AT-1002 (TFA); AT1002 TFA;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~33.33 mg/mL (~40.55 mM)
H2O :~1 mg/mL (~1.22 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.04 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (3.04 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (3.04 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 8.33 mg/mL (10.13 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

---

 (Please use freshly prepared in vivo formulations for optimal results.)