Tight junctions (TJs)

The 6-mer synthetic peptide AT-1002 is a member of a newly discovered class of substances that reversibly promotes the paracellular transport of molecules across epithelial barriers. Cys-Cys dimerization is possible with AT-1002 [1]. Cellular ATP content was used to determine the viability of AT-1002 treatment (0 to 5 mg/mL, 3 or 24 hours) for undifferentiated Caco-2 cells. Cell viability was unaffected by AT-1002 treatment for up to three hours at any concentration. In instance, 5 mg/mL AT-1002 had no effect on the viability of Caco-2 cells. After 24 hours, AT-1002 at doses of 2.5 mg/mL and above decreases cell viability. Nevertheless, in the event that the cells were cleansed following a 3-hour exposure to AT-1002, they continued to be viable a day later, suggesting that AT-1002 does not cause irreversible harm to the cells [2].

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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]

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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.

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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 Cell
Tested Concentrations: 0 to 5 mg/mL
Incubation Duration: 3 or 24 hrs (hours)
Experimental Results: Treatment for up to 3 hrs (hours) did not affect cell viability at any concentration. At concentrations of 2.5 mg/mL or higher, cell viability diminished after 24 hrs (hours).

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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.

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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.

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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 a 6-mer synthetic peptide belongs to an emerging novel class of compounds that reversibly increase paracellular transport of molecules across the epithelial barrier. The aim of this project was to elaborate on the structure-activity relationship of this peptide with the specific goal to replace the P2 cysteine amino acid. Herein, we report the discovery of peptides that exhibit reversible permeability enhancement properties with an increased stability profile.[1]

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Tight junctions (TJs) are intercellular structures that control paracellular permeability and epithelial polarity. It is now accepted that TJs are highly dynamic structures that are regulated in response to exogenous and endogenous stimuli. Here, we provide details on the mechanism of action of AT-1002, the active domain of Vibrio cholerae's second toxin, zonula occludens toxin (ZOT). 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. In vivo, co-administration of salmon calcitonin with 1 mg of AT-1002 resulted in a 5.2-fold increase in AUC over the control group. Our findings provide a mechanistic explanation for AT-1002-induced tight junction disassembly, and demonstrate that AT-1002 can be used for delivery of other agents in vivo.[2]

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Drug absorption is thought to occur predominantly via passive transcellular and paracellular transport mechanisms (Behrens et al., 2001). Lipophilic drugs are transported primarily by the transcellular route and by means of transporters such as channels, pumps and carriers on the plasma membrane. However, the paracellular route is usually the main route of absorption for hydrophilic drugs (proteins, peptides, etc.). Here we confirm that AT-1002, a small peptide derived from ZOT, causes opening of the TJs of a Caco-2 cell monolayer (Motlekar et al., 2006, Song et al., 2008a) and show that this effect is reversible. Additional experiments showed that co-administration of AT-1002 with salmon calcitonin intratracheally increased the systemic exposure of salmon calcitonin by up to 5.2-fold suggesting that we can use AT-1002 to deliver antigens and other payloads systemically. In fact, previous studies have shown that AT-1002 enhances the delivery of small molecules (Motlekar et al., 2006, Song et al., 2008a, Song et al., 2008b). Another study showed that peptides from the extracellular loops of the TJ protein occludin could be used for TJ modulation (Tavelin et al., 2003) by increasing the permeability of the TJs without causing short-term toxicity. However, these peptides had an effect only when added to the basolateral side of the monolayer. Agents that enhance drug or antigen delivery have to be applied to the apical side of epithelial surfaces to be useful. Here we show that AT-1002 is such an agent and thus represents a prototype of a new class of TJ modulators.
Two main applications of TJ-opening molecules such as AT-1002 can be envisioned: classical drug delivery and antigen delivery for vaccination. Recently, a peptide from the capsid of rotaviruses was shown to facilitate insulin uptake in rats (Nava et al., 2004). Other TJ-modulating (TJM) peptides and peptide YY (PYY) improved drug transfer across epithelial tissues (Chen et al., 2006, Gonzalez-Mariscal and Nava, 2005). Therefore, compounds that enable efficient, non-toxic and non-invasive drug delivery would revolutionize the treatment of multiple diseases. Thus, a TJ-modulating peptide such as AT-1002 represents a promising advancement in mucosal drug delivery.[2]

C32H53N9O7S

707.88432

707.379

835872-35-0

AT-1002 TFA

11600115

Typically exists as solid at room temperature

5.074

10

10

22

49

1130

6

N[C@@H](CC1=CC=CC=C1)C(N[C@@H](CS)C(N[C@@H]([C@H](CC)C)C(NCC(N[C@@H](CCCNC(N)=N)C(N[C@@H](CC(C)C)C(O)=O)=O)=O)=O)=O)=O

LADIUXCZKYKVRR-PSEZLDIXSA-N

InChI=1S/C32H53N9O7S/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/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)/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

835872-35-0; AT-1002; L-Leucine, L-phenylalanyl-L-cysteinyl-L-isoleucylglycyl-L-arginyl-; (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; L-Phenylalanyl-L-cysteinyl-L-isoleucylglycyl-L-arginyl-L-leucine; CHEMBL500518;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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