Dipeptidyl peptidase 4 (DPP-4)

At 100 μM, 30 minutes following CXCR4 blocker or Src oxidation treatment, human endothelial cells exposed to normoxic and H/R conditions experience phosphorylation of Src [Tyr 416] and VE-cadherin [Tyr731]. This oxidizes and breaks down CXCR4 (SDF-1α receptor) foaming agents or Src-coupled endothelial cell-to-endothelial cell junctions [1].

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Disruption of integrity and increased permeability of endothelial cells by DPP4-inhibitor [1]
To test whether DPP4-inhibitors would increase vascular permeability, we treated hECs with Diprotin A (DipA; Ile-Pro-Ile) and assessed the phosphorylation of Src and VE-cadherin. H/R on hECs induced the phosphorylation of Src [Tyr 416] and VE-cadherin [Tyr731] (Fig. 3a–c). The phosphorylation of Src [Tyr416] and VE-cadherin [Tyr731] was further increased by DPP4-inhibitor. To investigate whether the phosphorylation of VE-cadherin [Tyr731] by DPP4-inhibitor is mediated by SDF-1α/CXCR4 ligand/receptor interaction and the downstream signaling molecule Src kinase, we assessed the inhibitory effects of CXCR4-blocker (AMD3100) and Src-inhibitor (PP2) on the phosphorylation of Src and VE-cadherin. Both CXCR4-blocker and Src-inhibitor reduced the DPP4-inhibitor-induced phosphorylation of Src [Tyr416] and VE-cadherin [Tyr731] in hECs (Fig. 3a–c). We then performed immuno-fluorescence staining for VE-cadherin in hECs to determine whether the DPP4-inhibitor-induced phosphorylation of VE-cadherin actually led to a disruption of endothelial integrity. DPP4-inhibitor disrupted endothelial cell-to-cell junctions labeled with VE-cadherin. This disruption of junctions was prevented by CXCR4-blocker or Src-inhibitor (Fig. 3d,e). Step by step, we evaluated the correlation of disrupted endothelial cell-to-cell junctions on immuno-fluorescence images and the ‘actual leakage’ of the endothelial monolayer, using an in-vitro transwell endothelial permeability assay. Endothelial permeability was determined by measuring FITC-dextran (fluorescein isothiocyanate conjugated-dextran; 40 kDa) passage through the endothelial monolayer from the upper to lower chamber (Fig. 3f; see Supplementary Fig. S3 for detailed experimental scheme). The FITC-dextran content in the lower chamber, which represents endothelial permeability, was significantly increased after adding DPP4-inhibitor to the upper chamber, which was prevented by CXCR4-blocker or Src-inhibitor treatment (Fig. 3g).

Massive vascular leakage and increased phosphorylation of Src and VE-cadherin are the results of using diprotin A (ip; 70 μg/kg; twice daily; 7 days). In summary, SDF-1α/CXCR4/Src are enhanced by diprotin A.

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Endothelial permeability in multiple doses of Diprotin A (DipA; Ile-Pro-Ile) and sitagliptin [1]
To explore the dose-response relationship, we tested multiple doses of Diprotin A (DipA; Ile-Pro-Ile) (1, 10, 100 μM). Since DPP4 inhibition is achieved through gliptins in diabetic patients, we also investigated the effects of sitagliptin on the phosphorylation of Src and VE-cadherin and endothelial permeability. Sitagliptin concentrations of 0.1, 1, and 10 μM was used to reflect the actual plasma concentrations in human volunteers taking sitagliptin 25, 100, and 600 mg, respectively32, DipA and sitagliptin increased the phosphorylation of Src [Tyr 416] and VE-cadherin [Tyr731] in a dose-dependent manner (Fig. 5a,b). Endothelial permeability was also increased in a dose-dependent manner (p < 0.001 for both DipA and sitagliptin; Fig. 5c). The p values of 6 possible pair-wise comparisons were all <0.01 for both groups.

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In-vivo endothelial leakage by DPP4-inhibitor [1]
In order to evaluate whether DPP4-inhibitors increase vascular permeability in-vivo, we performed the Miles permeability assay, using the ears of mice (Supplementary Fig. S4). The ears into which SDF-1α was injected turned blue owing to the extravasation of Evans blue dye, which was systemically administered (Fig. 6a,b). The leakage induced by SDF-1α was aggravated by the intra-peritoneal administration of DPP4-inhibitor (Diprotin A (DipA; Ile-Pro-Ile)) for 5 days. However, vascular leakage by DPP4-inhibitor and SDF-1α was significantly diminished by CXCR4-blocker (AMD3100) or Src-inhibitor (PP2) (Fig. 6a,b).

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Retinal capillary leakage due to DPP4-inhibitor: retinopathy of prematurity model [1]
We tested the in-vivo effects of DPP4-inhibitor on retinal vascular permeability, using a retinopathy of prematurity mouse model. As shown in the experimental scheme (Fig. 7a), neonatal mouse pups were exposed to high oxygen (75%) from day 7 to 12 (situation of hyperoxia) and then were returned to normal air (20%) for 5 days (situation of relative hypoxia). Two different dyes were systemically administered before the harvesting of eyes; TRITC-conjugated lectin from Bandeiraea simplicifolia (BS-1 lectin) for vascularity examination, and FITC-dextran for vascular leakage examination. The retinas of postnatal day 17 mice under relative hypoxia showed increased neovascularization, compared to neonatal or postnatal day 12 retina under hyperoxia (Supplementary Fig. S5). Systemic administration of DPP4-inhibitor (Diprotin A (DipA; Ile-Pro-Ile)) increased not only vascularity (Supplementary Fig. S6) but also vascular leakage (Fig. 7b–d), which was prevented by CXCR4-blocker (AMD3100).

In-vitro permeability assay [1]
FITC-dextran (40 kDa) is an easily detectable tracer. The permeability of the endothelial membrane was assessed by the passage of FITC-dextran through the hECs monolayer. Two days before the experiment, hECs were seeded onto fibronectin-coated 0.4 μm pore 24-well size cell culture inserts. The cells were cultured in EBM supplemented with 0.5% fetal bovine serum for starvation under standard culture conditions (37 °C, 95% humidified air and 5% CO2) for 18 hours. At the start of the experiment, the culture medium was pre-treated with CXCR4-blocker (AMD3100; 1 μg/ml) or Src-inhibitor (PP2; 1 μM). DPP4-inhibitor (Diprotin A (DipA; Ile-Pro-Ile), Ile-Pro-Ile; 100 μM) was applied 30 minutes after CXCR4-blocker or Src-inhibitor treatment. FITC-dextran at a concentration of 20 μg/ml was added to the upper chamber 60 minutes after DPP4-inhibitor treatment (drug treatment time of Diprotin A (DipA; Ile-Pro-Ile): 60 minutes). After incubation at 37 °C for 20 minutes, 100 μl of the medium were drawn from the lower chamber (time for permeance: 20 minutes). In order to explore dose-response relationship, multiple doses of Diprotin A (DipA; Ile-Pro-Ile) (1, 10, 100 μM) and sitagliptin (0.1, 1, 10 μM) was added to the upper chamber. After 30 minutes, FITC-dextran was added to the upper chamber (drug treatment time: 30 minutes). Lower chamber medium was drawn 5 minutes after FITC-dextran treatment (time for permeance: 5 minutes). The fluorescence of the lower chamber was determined by a fluorescence spectro-fluorometer.

Western Blot Analysis[1]
Cell Types: Human endothelial cells[1]
Tested Concentrations: 100 μM
Incubation Duration: 30 minutes after treatment with CXCR4 blocker or Src inhibitor
Experimental Results: Induction of Src [Tyr 416] and VE-cadherin in hEC [Tyr731] Phosphorylation.

Animal/Disease Models: Streptozotocin-induced wild-type C57/BL6 mouse diabetic retinopathy model [1]
Doses: 70 μg/kg
Route of Administration: intraperitoneal (ip) injection; /VE-cadherin signaling induces leaky blood vessels [1]. twice (two times) daily; 7 days
Experimental Results: Induces vascular leakage by enhancing SDF-1α/CXCR4/Src/VE-cadherin signaling pathway.

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In-vivo permeability assay [1]
Miles assay was performed in wild-type C57/BL6 mice. Three groups of mice received an intra-peritoneal injection of DPP4-inhibitor (Diprotin A (DipA; Ile-Pro-Ile); 70 μg/kg twice daily), and another group received vehicle for 5 days. The specific dose of DPP4-inhibitor used in this study has been shown to mediate therapeutic effects in murine models20,51. After 5 days, 2 groups of mice injected with DPP4-inhibitor received an intra-peritoneal injection of CXCR4-blocker (AMD3100; 7.5 mg/kg, once per day) or Src-inhibitor (PP2; 1 mg/kg, once per day). Thirty minutes later, each mouse was injected with PBS into its right ear and SDF-1α (250 ng) to its left ear, and this was followed by an injection of intra-cardiac 0.5% Evans blue dye. Photographs of the ears were obtained, and the mice were euthanized 30 minutes later. Mouse ear tissue was collected with an 8 mm skin punch and was incubated in 300 μl of formamide at 56 °C for 48 hours. The quantity of Evans blue dye in the tissues and standards was determined by assessing the optical density at 600 nm.

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Retinopathy of prematurity model [1]
The oxygen exposure protocol placed oxygen-exposed mouse pups with their nursing mothers in the same covered plastic box with 75% oxygen from postnatal day 7 through postnatal day 12 as previously described42. The oxygen was delivered at 75 ± 2%, and it was monitored at least three times daily during the oxygen exposure period. Oxygen concentrations were measured with an oxygen monitor. On postnatal day 12, the animals were returned to room air and were subsequently sacrificed by a lethal intra-peritoneal injection of chloral hydrate (360 mg/kg) on postnatal day 17. DPP4-inhibitor injection (Diprotin A (DipA; Ile-Pro-Ile); 70 μg/kg, twice daily) was administered from postnatal day 12 to 17. The control mice were injected with PBS in the same manner as DPP4-inhibitor. CXCR4-blocker (AMD3100; 7.5 mg/kg, once per day) was also injected in the same manner as that for the DPP4-inhibitor. BS-1 Lectin (Sigma-Aldrich, St. Louis, MO, USA) was infused systemically for vascularity examination and FITC-dextran or permeability examination. Both eyes of each animal were used for examination of the retinal vascular pattern after flat mounting of the retina.

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Streptozotocin-induced diabetic retinopathy model [1]
To induce diabetes mellitus, 180 mg/kg of intra-peritoneal STZ was injected into 7-week-old C57/BL6 mice. Blood sugar levels from tail vein blood samples and body weight were monitored weekly. Before the mice were sacrificed, 250 μl of whole blood were drained from the heart for hemoglobin A1c (HbA1c) examination. Two weeks after STZ injection, the mice were confirmed to be diabetic if the BST level was greater than 500 mg/dl. These mice were divided into 4 groups: STZ only; STZ + DPP4-inhibitor; STZ + DPP4-inhibitor + CXCR4-blocker; and STZ + DPP4-inhibitor + Src-inhibitor. Intra-peritoneal DPP4-inhibitor (Diprotin A (DipA; Ile-Pro-Ile); 70 μg/kg twice daily) was injected for 7 days beginning from 6 weeks after STZ injection. Single doses of intra-peritoneal CXCR4-blocker (AMD3100; 7.5 mg/kg, once per day) and Src-inhibitor (PP2; 1 mg/kg, once per day) were also injected in the same manner as that for the DPP4-inhibitor. TRITC-conjugated BS-1 Lectin was infused for vascularity examination and FITC-dextran for permeability examination. Both eyes of each mouse were used for examination of the retinal vascular pattern after flat mounting of the retina. Images were obtained with a Nikon DS-Qi2 CMOS camera head mounted on a Nikon Ti-E motorized inverted microscope. To quantify the fluorescence intensity of FITC-dextran, captured images from each experiment were analyzed using NIS-Elements and ROI statistics software. Using the black-green interface, we created region of interest on each captured image. By ROI statistics, we calculated the mean intensity of FITC-dextran in the region of interest of each captured image. After the retinal vascularity examination, western blot analysis for phosphorylated Src and VE-cadherin was performed using the retinal tissue.

mouse LD intravenous >250 mg/kg Journal of Antibiotics., 37(422), 1984 [PMID:6427168]

[1]. Dipeptidyl Peptidase-4 Inhibitor Increases Vascular Leakage in Retina through VE-cadherin Phosphorylation. Sci Rep. 2016 Jul 6;6:29393.

(2S,3S)-2-[[[(2S)-1-[(2S,3S)-2-amino-3-methyl-1-oxopentyl]-2-pyrrolidinyl]-oxomethyl]amino]-3-methylpentanoic acid is a peptide.
diprotin A has been reported in Bacillus cereus with data available.

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The inhibitors of CD26 (dipeptidyl peptidase-4; DPP4) have been widely prescribed to control glucose level in diabetic patients. DPP4-inhibitors, however, accumulate stromal cell-derived factor-1α (SDF-1α), a well-known inducer of vascular leakage and angiogenesis both of which are fundamental pathophysiology of diabetic retinopathy. The aim of this study was to investigate the effects of DPP4-inhibitors on vascular permeability and diabetic retinopathy. DPP4-inhibitor (Diprotin A (DipA; Ile-Pro-Ile)or sitagliptin) increased the phosphorylation of Src and vascular endothelial-cadherin (VE-cadherin) in human endothelial cells and disrupted endothelial cell-to-cell junctions, which were attenuated by CXCR4 (receptor of SDF-1α)-blocker or Src-inhibitor. Disruption of endothelial cell-to-cell junctions in the immuno-fluorescence images correlated with the actual leakage of the endothelial monolayer in the transwell endothelial permeability assay. In the Miles assay, vascular leakage was observed in the ears into which SDF-1α was injected, and this effect was aggravated by DPP4-inhibitor. In the model of retinopathy of prematurity, DPP4-inhibitor increased not only retinal vascularity but also leakage. Additionally, in the murine diabetic retinopathy model, DPP4-inhibitor increased the phosphorylation of Src and VE-cadherin and aggravated vascular leakage in the retinas. Collectively, DPP4-inhibitor induced vascular leakage by augmenting the SDF-1α/CXCR4/Src/VE-cadherin signaling pathway. These data highlight safety issues associated with the use of DPP4-inhibitors.

C17H31N3O4

341.44574

341.231

C, 59.80; H, 9.15; N, 12.31; O, 18.74

90614-48-5

Diprotin A TFA;209248-71-5

94701

White to off-white solid powder

1.14 g/cm3

583.1ºC at 760 mmHg

306.5ºC

1.517

1.995

3

5

8

24

469

5

CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N

JNTMAZFVYNDPLB-PEDHHIEDSA-N

InChI=1S/C17H31N3O4/c1-5-10(3)13(18)16(22)20-9-7-8-12(20)15(21)19-14(17(23)24)11(4)6-2/h10-14H,5-9,18H2,1-4H3,(H,19,21)(H,23,24)/t10-,11-,12-,13-,14-/m0/s1

(2S,3S)-2-[[(2S)-1-[(2S,3S)-2-amino-3-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-3-methylpentanoic acid

diprotin A; 90614-48-5; Ile-Pro-Ile; isoleucylprolylisoleucine; isoleucyl-prolyl-isoleucine; l-isoleucyl-l-prolyl-l-isoleucine; N-(1-L-Isoleucyl-L-prolyl)-L-isoleucine; MFCD00038707;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O : ≥ 100 mg/mL (~292.87 mM)
DMSO : ~100 mg/mL (~292.87 mM)

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.)