NK1/neurokinin-1 receptor (IC50 = 330 nM)

Vapreotide reduces the impact of SP on calcium release in a concentration-dependent manner. Vapreotide needs a concentration that is roughly 100 times greater than that of the NK1R antagonist aprepitant in order to fully block the effects of SP. SSTR2 is the main mediator of Vapreotide's effect on cell proliferation. U373MG cells are pretreated with SSTR2 selective antagonist CYN, then incubated with Vapreotide and stimulated with SP to further demonstrate the NK1R antagonist effect of Vapreotide. The findings demonstrate that the inhibitory effect of vapreotide on SP-stimulated IL-8 mRNA expression is not reversed by pretreatment with CYN. Vapreotide decreases HIV-1 replication in MDM, as seen by the reduced expression of HIV gag mRNA in comparison to control MDM. Furthermore, SP treatment (10 μM) reverses the inhibitory effect of vapreotide on HIV-1 replication in MDM. According to this observation, vapreotide's inhibition of HIV-1 replication is most likely caused by its interaction with NK1R[1].

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Vapreotide dose-dependently inhibits SP-induced intracellular calcium increases [1]
SP induced an increase in calcium in U373MG cells in a dose-dependent manner with a maximum response at concentration of 0.1 μM, while the NK1R antagonist, aprepitant, completely antagonized this effect at 0.1 μM (Figure 1A). Vapreotide attenuated the effect of SP on calcium release in a concentration-dependent manner (Figure 1B). The concentration required for vapreotide to completely inhibit the effect of SP is about 100 times higher than that required for the NK1R antagonist aprepitant (Figure 1B).

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Vapreotide attenuates SP activation of NF-κB and inhibits SP-induced Gene Expression in HEK293-NK1R cells [1]
SP increased NF-κB-driven-luciferase gene expression in HEK293-NK1R (Figure 2A) and this enhancement was antagonized by aprepitant or CP-96,345 and partially inhibited by vapreotide pretreatment. SP treatment up-regulates the mRNA expression of IL-8, EGR-1 and c-FOS genes in HEK293-NK1R cells. The NK1R antagonist, aprepitant, inhibited these effects (Figure 2B and C). Vapreotide pretreatment also reduced SP-mediated increases in IL-8, EGR-1 and c-FOS expression.

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Vapreotide inhibits SP-induced cytokine and chemokine expression in U373MG cells [1]
In order to further establish the NK1R antagonist effect of vapreotide, we examined its effect in U373MG cells that express endogenous NK1R. SP induced IL-8 expression at both mRNA and protein levels while both vapreotide and aprepitant inhibited this SP-induced IL-8 up-regulation (Figure 3A and B). Similarly, SP enhanced MCP-1 secretion while both vapreotide and aprepitant antagonized this up-regulation (Figure 3C and D). The effect of vapreotide on cell proliferation is mediated primarily by SSTR2. In order to further establish the NK1R antagonist effect of vapreotide, U373MG cells were pretreated with SSTR2 selective antagonist CYN followed by incubation with vapreotide and SP stimulation. The data (Figure 3A) show that pretreatment with CYN did not reverse the inhibitory effect of vapreotide on SP-stimulated IL-8 mRNA expression, suggesting that inhibition by vapreotide was not mediated by SSTR2.

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Vapreotide Inhibits HIV-1 Infection of MDM In Vitro [1]
Vapreotide reduced HIV-1 replication in MDM as indicated by limited HIV gag mRNA expression compared to control MDM (Figure 4). In addition, SP treatment (10 μM) reversed vapreotide inhibition of HIV-1 replication in MDM (Figure 4). This observation indicates that the inhibition of HIV-1 replication by vapreotide is most likely due to its interaction with NK1R.

Oesophagogastric varices rupturing, which causes bleeding, is a serious side effect of portal hypertension in cirrhosis. When rats are given vapreotide acutely, their collateral circulation blood flow is reduced, and when they are given it chronically, its development is attenuated[4]. When administered subcutaneously at a dose of 100 μg/day/animal, RC-160 reduces tumor volumes and weights by approximately 40%. When medication is initiated early in the tumor's development, dipreotide can slow the growth of androgen-independent prostate cancer[5].

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The aim of this study was to assess the hemodynamic effects of acute and chronic administration of vapreotide, a somatostatin analog, in rats with intrahepatic portal hypertension induced by dimethylnitrosamine (DMNA) administration. Acute effects were evaluated at baseline and 30 min after placebo (N = 13) or vapreotide (8 /microg/kg/hr, N = 13) infusions in DMNA rats. Chronic hemodynamic effects were evaluated using subcutaneous implants for five weeks in anesthetized DMNA rats (placebo: N = 13, vapreotide: N = 13) and in sham rats (placebo: N = 10, vapreotide: N = 10). Hemodynamic measurements included splenorenal shunt blood flow (SRS BF) by the transit time ultrasound (TTU) method and cardiac output by the combined dilution-TTU method. Acute administration of vapreotide significantly decreased SRS BF (-17.3 +/- 19 vs - 1.1 +/- 14%, P < 0.05) and portal pressure (-8 +/- 9 vs 0 +/- 8%, p < 0.05) compared to placebo without systemic effects. Chronic administration of vapreotide significantly reduced the increase in SRS BF (2.4 +/- 1.5 vs 1.2 +/- 1.0 ml/min, P < 0.05) and cardiac index (50 +/- 15 vs 33 +/- 10 ml/min/100 g, P < 0.0001) while portal pressure and blood flow, and mean arterial pressure were not significantly changed compared to placebo. In conclusion, the acute administration of vapreotide decreased collateral circulation blood flow while chronic administration attenuated its development. Vapreotide seems to have a vasoconstrictive effect on collateral circulation.[4]

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Nude mice bearing xenografts of the androgen-independent human prostate-cancer cell line PC-3 were treated for 4 weeks with somatostatin analog RC-160, bombesin/gastrin-releasing peptide (GRP) antagonist (RC-3095), or the combination of both peptides. In the first experiment, treatment was started when the tumors measured approximately 10 mm3. Tumor volumes and weights were reduced by about 40% by RC-160 or RC-3095 administered by s.c. injections at doses of 100 micrograms/day/animal and 20 micrograms/day/animal respectively. The combination of RC-3095 with RC-160 did not further potentiate suppression of tumor growth, but histologically the ratio of apoptotic and mitotic indices was significantly higher in the groups treated with the combination than in the other groups. Serum gastrin levels were significantly reduced in all treated groups. Therapy with RC-160 or the combination also significantly decreased serum growth-hormone levels. Specific high-affinity binding sites for bombesin, somatostatin and epidermal growth factor (EGF) were found on the tumor membranes. Receptors for EGF were significantly down-regulated by treatment with RC-3095, RC-160 and a combination of both analogs. Tumors from mice treated with RC-160 showed a significant increase in maximal binding capacity for somatostatin as compared with control tumors, demonstrating the absence of down-regulation. In the second experiment, treatment was started when the tumors were well developed and measured approximately 90 mm3. No significant reduction in volume, weight and growth rate of tumors was found in the groups treated with RC-160 or RC-3095. Our results suggest that somatostatin analog RC-160 and bombesin/GRP antagonist RC-3095 can inhibit the growth of androgen-independent prostate cancer when the therapy is started at an early stage of tumor development[5].

For 10 minutes, either Vapreotide (0, 5, 10, 20 μM) or SP are added, and then the HEK293-NK1R and U373MG cells are incubated for three hours. In some experiments, cells are stimulated with SP for three hours after being incubated with CYN for ten minutes, after which they are added to vapreotide and left to incubate for an extra ten. There are controls in the form of mock-treated cells[1].

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Luciferase Assay for NF-κB Activation [1]
pNF-κB-luc plasmid (5 μg) was transfected into HEK293-NK1R (2 x106 cells per transfection) by Nucleofector II using the Cell Line Nucleofection Kit V following the manufacturer’s protocol. After transfection, the cells were cultured in 24-well plates (0.25 × 106 cells/well) in DMEM media containing 10% fetal bovine serum (FBS). 48 h post transfection, the cells were treated with SP (10−7M) in the presence or absence of NK1R antagonists (aprepitant or CP-96,345) (1 μM) or Vapreotide (10 μM) for 6 h. Mock treated cells were used as controls. Cell-free lysates were collected in 0.25 ml of 1 X Reporter Lysis Buffer and centrifuged at 10,000 × g for 1 min. The cell-free lysates were used to determine the NF-kB-driven luciferase activity by the Luciferase assay system and a luminometer. The luciferase activity is expressed as relative light units (RLU). The results are presented as fold-change in RLU compared to untreated controls, which are defined as 1.0.

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Vapreotide treatment [1]
The HEK293-NK1R cells and U373MG cells were incubated with or without Vapreotide for 10 minutes and then incubated with or without SP for 3 hours. In some experiments, cells were first incubated with CYN for 10 minutes, and then vapreotide was added and incubated for an additional 10 minutes, followed by stimulation with SP for 3 hours. Mock treated cells were used as controls.

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HIV Infection [1]
7–10 day-cultured MDM in 48-well plates (0.25 × 106 cells/well) were incubated with or without Vapreotide for 2 h before infection with HIV-1. Cells were first incubated with vapreotide for 10 min and then SP was added to the MDM and the cells were incubated for the additional 2 h. Mock treated MDM served as controls. After a 2 h incubation with or without vapreotide and/or SP, the cells were infected overnight with cell-free HIV based on p24 antigen content (16 ng/106 cells), and then extensively washed to remove unbound virus. Fresh medium supplemented with vapreotide and/or SP was added as described above. The culture medium and the reagents were replaced twice weekly. At day 7 post HIV infection, cellular RNA was extracted from the MDM for assessment of HIV gag mRNA expression using real time RT-PCR assays.

Rats: Acute effects in DMNA rats are assessed at baseline and 30 minutes after Vapreotide (8 μg/kg/hr) or placebo infusions. Subcutaneous implants are used to assess chronic hemodynamic effects in anesthetized DMNA rats and sham rats over a five-week period. The combined dilution–TTU method and the transit time ultrasound method are two methods used to measure hemodynamic parameters, including splenorenal shunt blood flow[4].
Mice: For four weeks, somatostatin analog vapreotide (20 μg/day/animal), bombesin/gastrin-releasing peptide (GRP) antagonist, or a combination of both peptides, are administered to nude mice containing xenografts of the androgen-independent human prostate-cancer cell line PC-3. Tumor weights and volumes are quantified [5].

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Study 1: Acute Hemodynamic Effects of Vapreotide. Acute changes in hemodynamics following vapreotide administration compared to placebo administration were assessed in rats with liver fibrosis induced by repeated injections of dimethylnitrosamine. DMNA diluted in a 1% saline solution was administered intraperitoneally at a dose of 1 ml/kg body weight (ie, 10 mg/kg) for three consecutive days per week, for five weeks, in 26 rats (initial body weight 281 ± 25 g, mean ± SD). After five weeks, hemodynamic measurements were performed at baseline and 30 min after a blind randomized bolus infusion of either vapreotide (8 µg/kg/hr, 0.6 ml over 30 min, in 13 rats) or placebo (saline 0.6 ml over 30 min, in 13 rats). At the time of hemodynamic measurements, animals weighed 323 ± 46 g.[4]

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Study 2: Chronic Effects of Vapreotide. Vapreotide was chronically released from subcutaneous implants (a 15-mmlong and 1.5-mm-diameter polylactic acid cylinder, Debiopharm SA, 1000-Lausanne 9, Switzerland) that contained 4.8 mg of vapreotide. Placebo implants contained only vehicle (Lpolylactic acid). The subcutaneous implants were placed on the back of the rat two days before the start of DMNA or saline injection in rats (mean body weight 234 ± 19 g) under ether anesthesia. Hemodynamic measurements were performed in four groups of rats: (1) sham + placebo (N = 10): rats with saline intraperitoneal injections and subcutaneous placebo implants. (2) sham + vapreotide (N = 10): rats with saline intraperitoneal injections and subcutaneous vapreotide implants; (3) DMNA + placebo (N = 13): rats with DMNA intraperitoneal injections and subcutaneous placebo implants; and (4) DMNA + vapreotide (N = 13): rats with DMNA intraperitoneal injections and subcutaneous vapreotide implants. There were more rats in the PHT groups to take into account the variability of SRS blood flow and response to somatostatin analog in PHT rats (12). DMNA or saline were administered as described above in the acute study. At the time of hemodynamic measurements, animals weighed 380 ± 37 g.[4]

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Rats: Studies were performed in adult male Sprague-Dawley rats. All rats had free access to food and water until 14–16 hr before the hemodynamic measurements, when food was withdrawn. Hemodynamic measurements were performed in rats anesthetized with pentobarbital (Nesdonal administered intraperitoneally, 5 mg in 1 ml/kg body weight) 30 min after manipulations, ie, when hemodynamic parameters were stabilized. During hemodynamic measurements, body temperature was maintained at 37◦C with a homeothermic blanket system. Observer Hemodynamic measurements were performed by an observer who was not aware of treatment allocation (placebo or Vapreotide).[4]

Absorption, Distribution and Excretion
Vapreotide is 76% eliminated in bile. The remainder is renally eliminated.

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Biological Half-Life
30 minutes

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Metabolism / Metabolites
Route of Elimination: Vapreotide is 76% eliminated in bile. The remainder is renally eliminated. Half Life: 30 minutes

Toxicity Summary
The exact mechanism of action is unknown, although one study has provided in vitro and in vivo evidence for a tachykinin NK1 receptor antagonist effect in the analgesic effects of vapreotide (A2442).

[1]. Analog of somatostatin vapreotide exhibits biological effects in vitro via interaction with neurokinin-1 receptor. Neuroimmunomodulation. 2013;20(5):247-55.

[2]. Relative potencies of the somatostatin analogs octreotide, BIM-23014, and RC-160 on theinhibition of hormone release by cultured human endocrine tumor cells and normal rat anterior pituitary cells. Endocrinology. 1994 Jan;134(1):301-6.

[3]. Inhibition of cell proliferation by the somatostatin analogue RC-160 is mediated by somatostatin receptor subtypes SSTR2 and SSTR5 through different mechanisms. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1580-4.

[4]. Hemodynamic effects of acute and chronic administration of vapreotide in rats with cirrhosis. Dig Dis Sci. 2003 Jan;48(1):154-61.

[5]. Effect of somatostatin analog RC-160 and bombesin/gastrin releasing peptide antagonist RC-3095 on growth of PC-3 human prostate-cancer xenografts in nude mice.

Vapreotide is a synthetic octapeptide somatostatin analog. It was being studied for the treatment of cancer.
Vapreotide is a synthetic cyclic octapeptide analogue of somatostatin with direct and indirect antitumor effects. Vapreotide binds to somatostatin receptors (SSTR), specifically SSTR-2 and to SSTR-5 with a lesser affinity, in the similar behaviors as other octapeptide somatostatin analogues. Like octreotide, this agent has direct and indirect antitumor effects via inhibiting the release of growth hormone and other peptides that regulate release of insulin, gastrointestinal hormones. Furthermore, vapreotide may also be useful for inducing hemostasis in cases of acute hemorrhage of the upper gastrointestinal tract.

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Drug Indication
For the treatment of esophageal variceal bleeding in patients with cirrhotic liver disease and has also shown efficacy in the treatment of patients with AIDS-related diarrhea.

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Mechanism of Action
The exact mechanism of action is unknown, although one study has provided in vitro and in vivo evidence for a tachykinin NK1 receptor antagonist effect in the analgesic effects of vapreotide (PMID: 7556407).

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Pharmacodynamics
Vapreotide is a somatostatin analog with a higher metabolic stability than the parent hormone. Vapreotide reduces splanchnic blood flow; inhibits growth hormone release, and inhibits the release of peptides and vasoactive compounds from neuroendocrine tumors.

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Objectives: Vapreotide, a synthetic analog of somatostatin, has analgesic activity most likely mediated through the blockade of neurokinin-1 receptor (NK1R), the substance P (SP)-preferring receptor. The ability of vapreotide to interfere with other biological effects of SP has yet to be investigated. Methods: We studied the ability of vapreotide to antagonize NK1R in three different cell types: immortalized U373MG human astrocytoma cells, human monocyte-derived macrophages (MDM) and a human embryonic kidney cell line, HEK293. Both U373MG and MDM express endogenous NK1R while HEK293 cells, which normally do not express NK1R, are stably transformed to express human NK1R (HEK293-NK1R). Results: Vapreotide attenuates SP-triggered intracellular calcium increases and nuclear factor-κB activation in a dose-dependent manner. Vapreotide also inhibits SP-induced interleukin-8 and monocyte chemotactic protein-1 production in HEK293-NK1R and U373MG cell lines. Vapreotide inhibits HIV-1 infection of human MDM in vitro, an effect that is reversible by SP pretreatment. Conclusions: Our findings indicate that vapreotide has NK1R antagonist activity and may have a potential application as a therapeutic intervention in HIV-1 infection.[1]

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In the present study we investigated the effects of the somatostatin (SS) analogs octreotide, RC-160, and BIM-23014 on GH release by cultured cells of human GH-secreting pituitary tumors, in normal rat anterior pituitary cells, and on gastrin release by cultured cells from a human gastrinoma. In all GH-secreting adenomas and in rat anterior pituitary cells, RC-160 was the most potent compound. RC-160 significantly inhibited GH-, PRL, and/or alpha-subunit release by human GH-secreting pituitary adenoma cells in concentrations as low as 10(-12)-10(-14) M, whereas at the same concentrations, octreotide and BIM-23014 did not inhibit or were significantly less effective in inhibiting GH release (P < 0.01, RC-160 vs. octreotide and BIM-23014). In rat anterior pituitary cell cultures, the IC50 values for inhibition of GH release were, in rank order of potency, 0.1, 5.3, 47, 48, and 99 pM for RC-160, SS-14, BIM-23014, octreotide, and SS-28, respectively. Maximal inhibitory effects by the three analogs were the same in the human GH adenoma cell cultures and the rat anterior pituitary cell cultures (-60%). On the basis of these data, RC-160 appears to be about 500 times more potent than octreotide and BIM-23014 in inhibiting GH release by rat anterior pituitary cells in vitro. Forskolin (100 microM) as well as pretreatment of the cells with pertussis toxin significantly diminished the inhibitory effects of the three SS analogs and those of SS-14 and SS-28 to the same extent. The latter data suggest that octreotide, RC-160, and BIM-23014 act mainly via a pertussis toxin-sensitive G-protein and an adenylyl cyclase-dependent mechanism. In the human gastrinoma culture, RC-160 inhibited gastrin release significantly more than octreotide at 10(-12)- and 10(-14)-M concentrations (P < 0.01). In conclusion, the SS analogs octreotide, RC-160, and BIM-23014 may have significant different potencies of inhibition of hormone release in vitro, with RC-160 being the most potent SS analog and octreotide and BIM-23014 having similar potencies. Depending on the pharmacokinetic properties of these three octapeptide SS analogs, these observations may have consequences for the medical therapy of patients with SS receptor-positive endocrine tumors.[2]

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Effects of the stable somatostatin analogue RC-160 on cell proliferation, tyrosine phosphatase activity, and intracellular calcium concentration were investigated in CHO cells expressing the five somatostatin receptor subtypes SSTR1 to -5. Binding experiments were performed on crude membranes by using [125I-labeled Tyr11] somatostatin-14; RC-160 exhibited moderate-to-high affinities for SSTR2, -3, and -5 (IC50, 0.17, 0.1 and 21 nM, respectively) and low affinity for SSTR1 and -4 (IC50, 200 and 620 nM, respectively). Cell proliferation was induced in CHO cells by 10% (vol/vol) fetal calf serum, 1 microM insulin, or 0.1 microM cholecystokinin (CCK)-8; RC-160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (EC50, 53 and 150 pM, respectively) but had no effect on growth of cells expressing SSTR1, -3, or -4. In SSTR2-expressing cells, orthovanadate suppressed the growth inhibitory effect of RC-160. This analogue inhibited insulin-induced proliferation and rapidly stimulated the activity of a tyrosine phosphatase in only this cellular clone. This latter effect was observed at doses of RC-160 (EC50, 4.6 pM) similar to those required to inhibit growth (EC50, 53 pM) and binding to the receptor (IC50, 170 pM), implicating tyrosine phosphatase as a transducer of the growth inhibition signal in SSTR2-expressing cells. In SSTR5-expressing cells, the phosphatase pathway was not involved in the inhibitory effect of RC-160 on cell growth, since this action was not influenced by tyrosine and serine/threonine phosphatase inhibitors. In addition, in SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium mobilization at doses (EC50, 0.35 nM) similar to those necessary to inhibit somatostatin-14 binding (IC50, 21 nM) and CCK-induced cell proliferation (EC50, 1.1 nM). This suggests that the inositol phospholipid/calcium pathway could be involved in the antiproliferative effect of RC-160 mediated by SSTR5 in these cells. RC-160 had no effect on the basal or carbachol-stimulated calcium concentration in cells expressing SSTR1 to -4. Thus, we conclude that SSTR2 and SSTR5 bind RC-160 with high affinity and mediate the RC-160-induced inhibition of cell growth by distinct mechanisms.[3]

C57H70N12O9S2

1131.3707

1130.483

C, 60.51; H, 6.24; N, 14.86; O, 12.73; S, 5.67

103222-11-3

Vapreotide acetate; 849479-74-9; 936560-75-7 (diacetate)

6918026

H-D-Phe-Cys(1)-Tyr-D-Trp-Lys-Val-Cys(1)-Trp-NH2; {d-Phe}-Cys-Tyr-{d-Trp}-Lys-Val-Cys-Trp-NH2 (Disulfide bridge: Cys2-Cys7); D-phenylalanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl-L-tryptophanamide (2->7)-disulfide

FCYWKVCW; {d-Phe}-CY-{d-Trp}-KVCW-NH2 (Disulfide bridge: Cys2-Cys7)

White to off-white solid powder

1.4±0.1 g/cm3

1540.9±65.0 °C at 760 mmHg

885.7±34.3 °C

0.0±0.3 mmHg at 25°C

1.699

3.47

13

13

18

80

2080

8

[FCYWKVCW-NH2(Disulfide bridge: Cys2-Cys7)]

SWXOGPJRIDTIRL-UHFFFAOYSA-N

InChI=1S/C57H70N12O9S2/c1-32(2)49-57(78)68-48(55(76)64-44(50(60)71)26-35-28-61-41-16-8-6-14-38(35)41)31-80-79-30-47(67-51(72)40(59)24-33-12-4-3-5-13-33)56(77)65-45(25-34-19-21-37(70)22-20-34)53(74)66-46(27-36-29-62-42-17-9-7-15-39(36)42)54(75)63-43(52(73)69-49)18-10-11-23-58/h3-9,12-17,19-22,28-29,32,40,43-49,61-62,70H,10-11,18,23-27,30-31,58-59H2,1-2H3,(H2,60,71)(H,63,75)(H,64,76)(H,65,77)(H,66,74)(H,67,72)(H,68,78)(H,69,73)

10-(4-aminobutyl)-N-[1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]-19-[(2-amino-3-phenylpropanoyl)amino]-16-[(4-hydroxyphenyl)methyl]-13-(1H-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-7-propan-2-yl-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxamide

RC160; BMY 41606; Vapreotide; Vapreotidum; Vapreotida; Sanvar IR; 103222-11-3; Vapreotidum [INN-Latin]; Vapreotida [INN-Spanish]; UNII-2PK59M9GFF; BMY-41606; BMY41606; Vapreotide; Sanvar IR; Vapreotidum; RC 160; RC-160; RC160

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 (~88.4 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.)