SST2 (IC50 = 0.26 nM); SST5 (IC50 = 6.92 nM)

When tritium is released on CHO hsst2 cells, angiopeptin (0.1 nM–10 μM; for 1 h) TFA functions as a partial agonist (pEC50=6.57), with a maximal response of 423% at 3 μM[2].

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1. Somatostatin and the stable octapeptide analogues, octreotide and Angiopepsin, were examined for their ability to stimulate the release of tritium from [(3)H]-arachidonic acid pre-loaded CHO-K1 cells expressing human recombinant sst(2), sst(3) or sst(5) receptors. 2. Somatostatin stimulated tritium release (pEC(50)) through the sst(2) (7.8+/-0.1) and sst(5) (7.3+/-0.2), but not the sst(3) receptor. Octreotide behaved as a full (sst(2) receptor) or partial agonist (sst(5) receptor), whereas angiopeptin behaved as a weak partial agonist at both receptor types. 3. Maximum responses to somatostatin through both receptor types were significantly reduced by pertussis toxin, whereas pEC(50) estimates were unaffected. 4. Inhibition of MEK1 or Src, but not PKA, PI 3-kinases or tyrosine kinases, by reportedly selective inhibitors reduced sst(2)-mediated responses by somatostatin, but not angiopeptin. A selective inhibitor of PKC (Ro-31-8220) reduced both somatostatin and angiopeptin responses. 5. These data provide further evidence for partial agonist activity of synthetic peptides of somatostatin. Furthermore, the somatostatin receptor signalling mechanisms which mediate arachidonic acid mobilization appear to be multiple and complex [2].

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Effects of somatostatin peptide analogues on tritium release from [3H]-arachidonic acidpreloaded CHO-K1 cells [2]
In CHO h sst2 cells, somatostatin stimulated the release of tritium in a concentration-dependent manner with a pEC50 of 7.8±0.13 and maximum stimulation of 985±91% over basal (Figure 1A). Octreotide did not reach a maximum response at 1 μM, although the response at this concentration (827±25%; estimated pEC50 value, 6.33±0.23) did not differ significantly (P>0.05) from that of somatostatin. In contrast, Angiopepsin (pEC50, 6.57±0.15) acted as a partial agonist, with a maximum response of 423±111% at 3 μM (Figure 1A). Neither somatostatin (31±13%), angiopeptin (13±22%) or octreotide (22±41%) were able to elicit the release of tritium significantly above that of basal (0%) from CHO h sst3 cells at concentrations up to 10 μM (Figure 1B). In CHO h sst5 cells, somatostatin stimulated tritium release (pEC50, 7.29±0.17; maximum response 221±8%). Octreotide also stimulated the release of tritium (pEC50, 7.44±0.29) but produced a lower (P<0.05) maximum stimulation than that of somatostatin (123±44%). Angiopeptin did not significantly stimulate the release of tritium above that of basal (42±27% at 10 μM; Figure 1C). The estimated pEC50 values, maximum stimulation and Hill slopes obtained with CHO h sst2 or CHO h sst5 cells are summarized in Table 1.

In order to determine whether the partial agonism of Angiopepsin in CHO h sst2 cells was due to its inability to stimulate coupling to specific G proteins, the effect of pertussis toxin on the ability of angiopeptin to release tritium was examined. The potency (pEC50 values) of angiopeptin was similar before and after pertussis toxin treatment (6.40±0.18 and 6.01±0.36, respectively), whereas the maximum response was significantly reduced (474±104% and 230±26%, respectively; P<0.05; Figure 2A).

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Mechanism of tritium release through the sst2 receptor [2]
The mechanism of somatostatin-stimulated tritium release via the sst2 receptor was further investigated. Quinacrine (1 or 10 μM), a non-selective inhibitor of PLA2, or PGE2 (1 nM to 10 μM) had no effect on the basal or somatostatin (1 μM)-stimulated release of tritium (see Table 2 for values). The selective MEK1 inhibitor, PD 98059 (40 μM), had no effect on the basal tritium release (8.5±1.1% and 9.3±1.2%, respectively; values are expressed as a per cent of the 1 μM somatostatin response), but reduced the somatostatin (1 μM)-stimulated release of tritium to 61.9±3.0% (Figure 3A). A higher concentration of PD 98059 (60 μM) had no further effect (data not shown). Surprisingly, the response to the partial agonist Angiopepsin in CHO h sst2 cells was unaffected by PD 98059 (42.5±10.4% and 50.1±3.4%, respectively). After pre-treatment of CHO h sst2 cells with pertussis toxin, the ability of somatostatin (34.1±2.5%) or angiopeptin (20.4±1.9%) to stimulate tritium release was unaffected by the PD 98059 compound (33.1±1.0% and 25.2±0.8%, respectively; Figure 3A).

Similar to PD 98059, a selective inhibitor of Src, PP1 (200 nM), had no effect on the basal release of tritium (7.1±0.9% and 11.0±3.7% before and after PP1, respectively), but inhibited somatostatin (1 μM)-stimulated release (100% and 64.0±1.7%, respectively; Figure 3B). In contrast, the responses to Angiopepsin (1 μM) were unaffected by PP1 (42.5±10.4% and 51.7±10.8%, respectively). In the presence of pertussis toxin, somatostatin- and angiopeptin-stimulated tritium release (26.8±3.7% and 17.7±2.2%, respectively) was unaffected by PP1 (34.0±1.7% and 18.4±0.8%, respectively; Figure 3B).

In the presence of PD 98059 and PP1 combined, somatostatin-stimulated tritium release was reduced to 65.6±3.4%, no different to the effect produced by either inhibitor alone on the somatostatin response (see above). As expected, the inhibitory effect of both inhibitors combined upon somatostatin was abolished after pertussis toxin pre-treatment (data not shown).

Genistein (10 μM), a non-selective inhibitor of protein tyrosine kinases, LY 294004 (1 μM), a selective PI 3-kinase inhibitor, the adenylate cyclase activator, forskolin (10 μM), and a PKA inhibitor amide 14 – 22 (1 μM), all had no effect on responses to either somatostatin or Angiopepsin (values shown in Table 2).

Basal tritium release before (5.6±0.4%) or after (6.8±0.2%) pertussis toxin pre-treatment was unaffected by the protein kinase C (PKC) inhibitor, Ro-31-8220 (1 μM; 5.1±0.1% and 4.3±0.2%, respectively). In both the absence and presence of pertussis toxin pre-treatment, the response to somatostatin (100% and 28.3±0.6, respectively) or Angiopepsin (43.2±1.0% and 17.1±1.3, respectively) were reduced by Ro-31-8220 (65.9±1.3% and 13.8±0.5% for somatostatin; 31.6±1.0% and 9.5±1.1% for angiopeptin, respectively; Figure 4).

Neointimal development is greatly inhibited by angiopeptin (20 and 50μg/kg; ih) TFA[1]. In cardiac allografts, angiopeptin (20 μg/kg; daily) TFA dramatically reduces the proliferation of coronary artery myointimal cells by roughly 50%[1].

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Vascular smooth muscle cell hyperplasia is a major component of atherogenesis in various animal models. Angiopepsin, a cyclic octapeptide analogue of somatostatin, markedly inhibits myointimal proliferation in response to endothelial cell injury in the rat carotid artery, rabbit aorta and iliac arteries and in coronary arteries of transplanted rabbit hearts. Angiopepsin does not affect serum lipid profiles in nonhuman primates. It is unlikely, therefore, that its antiproliferative effect is mediated by alterations in cholesterol metabolism. Angiopeptin and other peptide analogues of somatostatin are potent inhibitors of growth hormone release and insulin-like growth factor-1 production. However, inhibition of smooth muscle cell proliferation in vivo is not a property common to all somatostatin analogues. This suggests that plasma growth hormone and growth hormone-dependent insulin-like growth factor-1 production are not physiologic stimuli for myointimal proliferation in vivo. Angiopeptin inhibits 3H-thymidine incorporation into rat carotid artery explants, suggesting a local effect on autocrine or paracrine mechanisms regulating cell growth. In view of its potent inhibitory effect on smooth muscle cell replication, angiopeptin may have clinical utility in preventing restenosis after percutaneous transluminal coronary angioplasty and in preventing accelerated coronary atherosclerosis after cardiac transplantation.

[3H]-arachidonic acid assay [2] Cells were seeded into 24-well plates at a density of 5×104 and were incubated with 0.5 μCi ml−1 of [5,6,8,9,11,12,14,15-3H]-arachidonic acid (215 Ci mmol−1) in normal cell culture medium 18 h prior to experimental use. Arachidonic acid is rapidly taken up and almost completely stored in phospholipids (see Washizaki et al., 1994). Cells were then washed four times with 20 mM HEPES-buffered Krebs (mM : NaCl 125; KCl 5.4; NaHCO3 16.2; D-glucose 5.5; HEPES 20; NaH2PO4 1 and CaCl2 1.3, pH 7.4), supplemented with 0.1% protease-free BSA and left to equilibrate at 37°C for 10 min. The wash buffer was removed and replaced with fresh buffer (1 ml) containing varying agonist concentrations. An aliquot (600 μl) of the buffer was then removed and counted on a Canberra Packard 2500TR Liquid Scintillation Analyser following the addition of 2 ml of Ultima Gold XR scintillant (Packard). In experiments where the effects of pertussis toxin were to be determined, the toxin was added at the same time as the [3H]-arachidonic acid. Experiments to investigate the susceptibility of tritium release to a variety of enzyme inhibitors were modified to include a 120 min pre-incubation of the cells in the presence or absence (control) of the relevant enzyme inhibitor at 37°C. After pre-incubation, the buffer was removed and replaced with fresh buffer containing enzyme inhibitor±1 μM somatostatin (basal and agonist-stimulated tritium release for each enzyme concentration).

[1]. Peptide inhibition of myointimal proliferation by angiopeptin, a somatostatin analogue. J Am Coll Cardiol. 1991;17(6 Suppl B):132B-136B.

[2]. Somatostatin receptor-mediated arachidonic acid mobilization: evidence for partial agonism of synthetic peptides. Br J Pharmacol. 2001;132(3):760-766.

Although animal models of myointimal proliferation, such as balloon-induced endotheliat injury of normat arterial segments. are “artitkiat” by definition and do not exactly reproduce the clinical situation present at the time of angioplasty or atherectomy. they do provide valuable insights into potential mechanisms of cell gmwlh regulation. We have demonstrated that angiqeptin is a potent inhibitor of myw intimal proliferation in response to two extremely di&xent types of injury in viva (namely. balloon-induced eodothetial cell denudation in the rat and rabbit models and immune injury in the rabbit transplant model of atherosclerosis). Whether this success in the animal laboratory will translate into clinical success, preventing restenosis atIer ansioplarty or atherectomy. needs to be determined in a controlled clinical trial. A multicenter double-blind controlled clinical trial that will examine the e&t of angiopeptin on restenosis after coronary angioplasty has recently begun in the United States. It is hoped that the results of this trial will provide further insight into the phenomenon of myointimal proliieration. [1]

C59H74F6N10O14S2

1325.40

1325.468

2478421-60-0

Angiopeptin;113294-82-9

162678807

H-D-2Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2.2TFA; 3-(2-naphthyl)-D-alanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl-L-threoninamide trifluoroacetic acid;

{Nal}CYWKVCT-NH2; XCYWKVCT

White to off-white solid powder

17

24

29

91

2050

9

C1C=C(O)C=CC=1C[C@H](NC([C@@H](NC(=O)[C@H](N)CC1=CC2C=CC=CC=2C=C1)CS)=O)C(=O)N[C@@H](C(N[C@H](C(=O)N[C@H](C(N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(N)=O)=O)C(C)C)CCCCC)=O)CC1C2=CC=CC=C2NC=1.OC(=O)C(F)(F)F.OC(=O)C(F)(F)F

PLVCOVWXYIMTNV-XCNZXRHRSA-N

InChI=1S/C54H71N11O10S2.2C2HF3O2/c1-29(2)45(54(75)63-44(28-77)53(74)65-46(30(3)66)47(57)68)64-49(70)40(14-8-9-21-55)59-51(72)42(25-35-26-58-39-13-7-6-12-37(35)39)61-50(71)41(24-31-16-19-36(67)20-17-31)60-52(73)43(27-76)62-48(69)38(56)23-32-15-18-33-10-4-5-11-34(33)22-32;2*3-2(4,5)1(6)7/h4-7,10-13,15-20,22,26,29-30,38,40-46,58,66-67,76-77H,8-9,14,21,23-25,27-28,55-56H2,1-3H3,(H2,57,68)(H,59,72)(H,60,73)(H,61,71)(H,62,69)(H,63,75)(H,64,70)(H,65,74);2*(H,6,7)/t30-,38-,40+,41+,42-,43+,44+,45+,46+;;/m1../s1

(2S)-6-amino-N-[(2S)-1-[[(2R)-1-[[(2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl]amino]-1-oxo-3-sulfanylpropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-2-[[(2R)-2-[[(2S)-2-[[(2R)-2-[[(2R)-2-amino-3-naphthalen-2-ylpropanoyl]amino]-3-sulfanylpropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]hexanamide;2,2,2-trifluoroacetic acid

Angiopeptin TFA; Angiopeptin (TFA); 2478421-60-0;

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)

H2O: 10 mg/mL (7.54 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.)