AT2/1 Receptor

Proliferating cell nuclear antigen (PCNA) protein, which is increased by Ang II, is not expressed when Ang-(1–7) is inhibited, while A-779 by itself has little effect on causing VSMC migration and proliferation. A-779 blocks the effects of Ang-(1-7) on the VSMC inflammatory response, which is linked to the elevation of MCP-1, VCAM-1, and IL-1β production. Ang-(1-7) pretreatment can greatly delay this inflammatory response. But A-779 by itself was unable to cause VSMCs to become inflamed. VSMCs treated with Ang-(1-7) for 5 minutes prior to Ang II-induced activation of Akt and ERK1/2 was considerably reduced. A-779 also had this effect, although it did not cause Akt and ERK1 phosphorylation on its own. /2's function in VSMC[3].

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Results: (1) The results of Annexin V-FITC/PI double staining flow cytometry showed that the proportion of apoptotic cells was higher in ox-LDL group than in control group ((21.18±1.40)% vs. (1.59±0.26)%, P<0.01), lower in ox-LDL+ Ang-(1-7) group((7.42±1.07)%) and ox-LDL+ HTA125 group((9.19±1.01)%) than in ox-LDL group (both P<0.01), higher in ox-LDL+ Ang-(1-7)+ A-779 group ((19.91±1.30)%) and ox-LDL+ A-779 group((20.47±0.95)%) than in ox-LDL+ Ang-(1-7) group (both P<0.01). (2) The TUNEL results showed that the proportion of apoptotic cells was higher in ox-LDL group than in control group((10.83±0.77)% vs. (2.83±0.82)%, P<0.01), lower in ox-LDL+ Ang-(1-7) group ((3.66±0.54)%)and ox-LDL+ HTA125 group((4.97±0.60)%) than in ox-LDL group(both P<0.01), higher in ox-LDL+ Ang-(1-7)+ A-779 group((10.69±0.62)%) and ox-LDL+ A-779 group((11.43±0.42)%) than in ox-LDL+ Ang-(1-7) group (both P<0.01). (3) ROS level was higher in ox-LDL group than in control group(0.093±0.014 vs. 0.053±0.011, P<0.01), lower in ox-LDL+ Ang-(1-7) group (0.063±0.011, P<0.01)and ox-LDL+ HTA125 group(0.070±0.010, P<0.05)than in ox-LDL group, higher in ox-LDL+ Ang-(1-7)+ A-779 group(0.088±0.003) and ox-LDL+ A-779 group(0.095±0.005) than in ox-LDL+ Ang-(1-7) group (both P<0.01). (4) The mRNA expression level of NOX4 was higher in ox-LDL group than in control group(11.74±0.65 vs. 1.00±0.00, P<0.01), lower in ox-LDL+ Ang-(1-7) group (2.85±0.75)and ox-LDL+ HTA125 group(5.57±0.52) than in ox-LDL group(both P<0.01), higher in ox-LDL+ Ang-(1-7)+ A-779 group(10.51±0.54) and ox-LDL+ A-779 group (11.04±1.01) than in ox-LDL+ Ang-(1-7) group (both P<0.01), higher in ox-LDL group than in control group(27.60±1.86 vs. 1.00±0.00, P<0.01), lower in ox-LDL+ Ang-(1-7) group (8.00±1.03)and ox-LDL+ HTA125 group(14.83±0.97)than in ox-LDL group(both P<0.01), higher in ox-LDL+ Ang-(1-7)+ A-779 group(24.81±2.19) and ox-LDL+ A-779 group (26.64±0.65)than in ox-LDL+ Ang-(1-7) group (both P<0.01). (5)The protein expression level of NOX4 was higher in ox-LDL group than in control group (0.61±0.09 vs. 0.23±0.02, P<0.01), lower in ox-LDL+ Ang-(1-7) group(0.27±0.03) and ox-LDL+ HTA125 group(0.22±0.02) than in ox-LDL group(both P<0.01), higher in ox-LDL+ Ang-(1-7)+ A-779 group (0.58±0.06)and ox-LDL+ A-779 group(0.61±0.03) than in ox-LDL+ Ang-(1-7) group (both P<0.01). The protein expression level of TLR4 was higher in ox-LDL group than in control group(0.18±0.02 vs. 0.08±0.01, P<0.01), lower in ox-LDL+ Ang-(1-7) group(0.07±0.01) and ox-LDL+ HTA125 group(0.09±0.01) than in ox-LDL group(both P<0.01), higher in ox-LDL+ Ang-(1-7)+ A-779 group(0.18±0.02) and ox-LDL+ A-779 group(0.20±0.02) than in ox-LDL+ Ang-(1-7) group (both P<0.01). Conclusion: TLR4 mediated the ox-LDL induced injury in HUVECs, and Ang-(1-7) could attenuate ox-LDL induced injury in HUVECs by modulating the specific Mas receptors [3].

For six weeks, OVX rats receiving an infusion of Ang(1-7) and A-779 (400?ng/kg/min, sc) either alone or in combination did not stop uterine atrophy or inhibit weight gain. Serum levels of type I collagen telopeptide (CTX), tartrate-resistant acid phosphatase (TRAcP 5b), osteocalcin (OC), urine deoxypyridine phenylin (DPD), and bone-specific alkaline phosphatase (BALP) are all markedly elevated by A-779. Serum mineral concentrations in the OVX or sham groups were not significantly changed by the infusion of Ang(1–7) and/or A-779. In contrast to the OVX group, A-779 in OVX animals did not change the expression of AngII, Ang(1-7), AT1R, AT2R, ACE, ACE-2, Mas receptor, RANKL, or OPG protein. However, A-779 (400ng/kg/min) significantly reduced the protective effects of captopril on bone metabolism, mineralization, and microstructure. A-779 also Restored the effect of OVX on RANKL expression ACE-1/AngII/AT1R cascade and downregulated OPG expression and ACE-2/Ang1-7/Mas pathway [2].

Culture and Grouping of Human Umbilical Vein Endothelial Cells (HUVECs):
HUVECs were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a 37°C, 5% CO₂ incubator. Cell growth was monitored using an inverted phase-contrast microscope. When cell confluence reached ≥80%, the cells were washed with phosphate-buffered saline (PBS; containing 8.0 g NaCl, 0.2 g KCl, 3.4 g Na₂HPO₄·H₂O, and 0.2 g KH₂PO₄, pH 7.4) and digested with 0.25% trypsin and 0.04% EDTA for subculture. Cells were seeded in 6-well plates at a density of 5×10⁵ cells/mL. Upon reaching 80% confluence, they were synchronized in serum-free medium for 12 h before grouping.
Experimental Groups:
1. Control group (no intervention)
2. ox-LDL group (treated with 75 mg/L ox-LDL)
3. ox-LDL + Ang-(1-7) group (pretreated with 1 μmol/L Ang-(1-7) for 30 min, then treated with 75 mg/L ox-LDL)
4. ox-LDL + Ang-(1-7) + A-779 group (pretreated with 1 μmol/L A-779 [Mas receptor antagonist] for 30 min, followed by 1 μmol/L Ang-(1-7) for 30 min, then treated with 75 mg/L ox-LDL)
5. ox-LDL + A-779 group (pretreated with 1 μmol/L A-779 for 30 min, then treated with 75 mg/L ox-LDL)
6. ox-LDL + HTA125 group (pretreated with 10 μg/L HTA125 [TLR4-specific blocking antibody] for 30 min, then treated with 75 mg/L ox-LDL)

The experimental model and study protocol [1]
A bilateral OVX operation was use to induce postmenopausal osteoporosis in female Wistar albino rats. The animals were generally anesthetized using single IP injection of ketamine + xylazine combination (80 mg/kg and 5 mg/kg; respectively). After ligation, the ovaries were excised from a longitudinal incision made on the dorsolateral body region. Sham rats had the same procedure without the ligation and excision. The risk of postoperative infection was eliminated by applying fusidic acid topical antibiotic cream twice weekly for 4 weeks. Following the sham and OVX operations, animals were divided into eight groups (n = 8/group) including, Sham, Sham + Ang(1-7), Sham +A-779, Sham + Ang(1-7)+A-779, OVX, OVX + Ang(1-7), OVX + A-779 and OVX + Ang(1-7)+A-779. Eight weeks following sham and OVX operations, osmotic pumps were implanted subcutaneously to deliver Ang(1-7) (200 ng/kg/min), A-779 (400 ng/kg/min) or a mixture of Ang(1-7) and A-779 (200 ng/kg/min and 400 ng/kg/min; respectively) for 6 consecutive weeks. Body weights of all animals were monitored and recorded at the beginning and every week throughout the study. Animals’ general health was maintained during periods of treatments. At the end of treatments periods, animals were placed fasting in metabolic cages for 16 hr and urine samples were collected and frozen at −70 °C. Then, the blood samples were collected by cardiac puncture under ketamine + xylazine anesthesia and serum samples were obtain by centrifugation at 4000 RPM to. Animals were then sacrificed by decapitation and the uterine tissues and femoral bones were removed and cleaned. Uterine tissues were cleaned from fats and the wet weights were determined directly and expressed as g/100 g body weight. In each rat, both femurs were removed and cleaned from soft tissues. The left femoral bone samples were frozen at −70 °C until analyzed, while the right femoral bones were kept in 10% formalin for mico-CT and minerals analysis.

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Study design and animals grouping [2]
Animals were accumulated for 8 weeks after the OVX or sham procedure. Then, animals were grouped into six groups (n = 10) as follow: Group 1: Sham, Group 2: Sham + Cap, Group 3: Sham + Cap + A-779, Group 4: OVX, Group 5: OVX + Cap and Group 6: OVX + Cap + A-779. At the same time, Sham + Cap + A-779 and OVX + Cap + A-779 groups had a subcutaneous implantation of osmotic pumps (model 2006, Alzet, Durect Corporation, Minneapolis, USA) supplying the specific mass receptor antagonist (D-Ala7)-Angiotensin I/II (1–7) trifluoroacetate salt (A-779) at infusion rate 400 ng/kg/min for 6 weeks. Meanwhile, Sham + Cap, Sham + Cap + A-779, OVX + Cap and OVX + Cap + A-779 groups started the oral captopril treatment. Captopril was dissolved in distilled water and administered with gastric gavage in 40 mg kg−1 d−1 dose for 6 weeks. Animals general health and body weights were monitored during the treatment periods and the dose of captopril were weekly adjusted. All groups were provided controlled experimental conditions (temperature 22 ± 1 °C, 12/12 dark and light cycles and humidity 50–55%) as well as a free access to purina rat chow and water ad libitum. At the last day of the sixth week, all groups were located in metabolic cages fastened for 16hr and urine samples were obtained and kept at −70 °C. Blood was collected by cardiac puncture under ketamine and xylazine anesthesia and serum samples were provided at 4000 RPM. Next, the femoral bones samples were removed and cleaned from soft tissues and weighted, then, expressed as grams. The left femoral bone samples were preserved at −70 °C until analyzed, while the right femoral bones were kept in 10% formalin for mico-CT and minerals analysis. Moreover, uterine tissues were excised and cleaned from fats and their weights were measured and expressed as g/100 g final body weight.

[1]. Angiotensin (1-7) ameliorates the structural and biochemical alterations of ovariectomy-induced osteoporosis in rats via activation of ACE-2/Mas receptor axis. Sci Rep. 2017 May 23;7(1):2293.

[2]. ACE-2/Ang1-7/Mas cascade mediates ACE inhibitor, captopril, protective effects in estrogen-deficient osteoporotic rats. Biomed Pharmacother. 2017 Aug;92:58-68.

[3]. Effects and related mechanism of angiotensin-(1-7) on Toll-like receptor 4-mediated oxidative stress in human umbilical vein endothelial cells. Zhonghua Xin Xue Guan Bing Za Zhi. 2017 Mar 24;45(3):223-229.

The local and systemic renin angiotensin system (RAS) influences the skeletal system micro-structure and metabolism. Studies suggested angiotensin 1-7 (Ang(1-7)) as the beneficial RAS molecule via Mas receptor activation. This study examines the function of Ang(1-7) in bone micro-architecture and metabolism in an ovariectomized (OVX) rodent model of osteoporosis. OVX rats showed structural and bone metabolic degeneration in parallel with suppressed expressions of the angiotensin converting enzyme-2 (ACE-2)/Ang(1-7)/Mas components. The infusion of Ang(1-7) markedly alleviated the altered bone metabolism and significantly enhanced both trabecular (metaphyseal) and cortical (metaphyseal-diaphyseal) morphometry. Urinary and bones minerals were also improved in OVX rats by Ang(1-7). The infusion of the heptapeptide enhanced ACE-2/Mas receptor expressions, while down-regulated AngII, ACE, and AngII type-1 receptor (AT1R) in OVX animals. Moreover, Ang(1-7) markedly improved osteoprotegerin (OPG) and lowered receptor activator NF-κB ligand (RANKL) expressions. The defensive properties of Ang(1-7) on bone metabolism, structure and minerals were considerably eradicated after blockage of Mas receptor with A-779. Ang(1-7)-induced up-regulated ACE-2/Ang(1-7)/Mas cascade and OPG expressions were abolished and the expressions of ACE/AngII/AT1R and RANKL were provoked by A-779. These findings shows for the first time the novel valuable therapeutic role of Ang(1-7) on bone health and metabolism through the ACE-2/Mas cascade. [1]

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The local role of the renin angiotensin system (RAS) was documented recently beside its conventional systemic functions. Studies showed that the effector angiotensin II (AngII) alters bone health, while inhibition of the angiotensin converting enzyme (ACE-1) preserved these effects. The newly identified Ang1-7 exerts numerous beneficial effects opposing the AngII. Thus, the current study examines the role of Ang1-7 in mediating the osteo-preservative effects of ACEI (captopril) through the G-protein coupled Mas receptor using an ovariectomized (OVX) rat model of osteoporosis. 8 weeks after the surgical procedures, captopril was administered orally (40mgkg-1 d-1), while the specific Mas receptor blocker (A-779) was delivered at infusion rate of 400ngkg-1min-1 for 6 weeks. Bone metabolic markers were measured in serum and urine. Minerals concentrations were quantified in serum, urine and femoral bones by inductive coupled plasma mass spectroscopy (ICP-MS). Trabecular and cortical morphometry was analyzed in the right distal femurs using micro-CT. Finally, the expressions of RAS peptides, enzymes and receptors along with the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) were determined femurs heads. OVX animals markedly showed altered bone metabolism and mineralization along with disturbed bone micro-structure. Captopril significantly restored the metabolic bone bio-markers and corrected Ca2+ and P values in urine and bones of estrogen deficient rats. Moreover, the trabecular and cortical morphometric features were repaired by captopril in OVX groups. Captopril also improved the expressions of ACE-2, Ang1-7, Mas and OPG, while abolished OVX-induced up-regulation of ACE-1, AngII, Ang type 1 receptor (AT1R) and RANKL. Inhibition of Ang1-7 cascade by A-779 significantly eradicated captopril protective effects on bone metabolism, mineralization and micro-structure. A-779 also restored OVX effects on RANKL expression and ACE-1/AngII/AT1R cascade and down-regulated OPG expression and ACE-2/Ang1-7/Mas pathway. In line with the clinical observations of the bone-preservative properties following ACE-1 inhibition, local activation of ACE-2/Ang1-7/Mas signaling and suppressed osteoclastogenesis seem responsible for the osteo-preservative effect of captopril, which could offers a potential therapeutic value in treatment of disabling bone and skeletal muscular diseases.[2]

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Objective: To explore the role and related mechanisms of angiotensin-(1-7)(Ang-(1-7)) on Toll-like receptor 4 (TLR4) mediated oxidized low-density lipoprotein(ox-LDL)-induced oxidative stress in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were cultured in vitro and divided into six groups: the control group (normal medium), the ox-LDL group(treated with 75 mg/L ox-LDL), the ox-LDL+ Ang-(1-7) group (1 μmol/L Ang-(1-7) pretreated for 30 minutes, then intervened with 75 mg/L ox-LDL), the ox-LDL+ Ang-(1-7)+ A-779 group(1 μmol/L A-779 (Mas receptor) pretreated for 30 minutes, 1 μmol/L Ang-(1-7) pretreated for 30 minutes, then intervened with 75 mg/L ox-LDL), the ox-LDL+ A-779 group (1 μmol/L A-779 pretreated for 30 minutes, then intervened with 75 mg/L ox-LDL), the ox-LDL+ HTA125 group (10 μg/L HTA125 (TLR4-blocking antibody) pretreated for 30 minutes, then intervened with 75 mg/L ox-LDL ). The corresponding index was detected after 24 hours after intervention. Apoptosis of cells were detected by Annexin V-FITC/PI double staining flow cytometry and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL). The generation of reactive oxygen species (ROS), products in oxidative stress, were detected by DCFH-DA staining. The mRNA and protein expression levels of NADPH oxidase 4(NOX4) and TLR4 were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis respectively.[3]

C39H60N12O11

872.96700

872.45

C, 53.66; H, 6.93; N, 19.25; O, 20.16

159432-28-7

10169886

Asp-Arg-Val-Tyr-Ile-His-{d-Ala}; H-Asp-Arg-Val-Tyr-Ile-His-D-Ala-OH; L-alpha-aspartyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-D-alanine

DRVYIHA; DRVYIH-{d-Ala}

White to off-white solid powder

1.5±0.1 g/cm3

1.651

0.3

13

14

26

62

1570

8

CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@H](C)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N

GZSZZUXDAPDPOR-NGIFJXEWSA-N

InChI=1S/C39H60N12O11/c1-6-20(4)31(37(60)49-28(15-23-17-43-18-45-23)34(57)46-21(5)38(61)62)51-35(58)27(14-22-9-11-24(52)12-10-22)48-36(59)30(19(2)3)50-33(56)26(8-7-13-44-39(41)42)47-32(55)25(40)16-29(53)54/h9-12,17-21,25-28,30-31,52H,6-8,13-16,40H2,1-5H3,(H,43,45)(H,46,57)(H,47,55)(H,48,59)(H,49,60)(H,50,56)(H,51,58)(H,53,54)(H,61,62)(H4,41,42,44)/t20-,21+,25-,26-,27-,28-,30-,31-/m0/s1

(3S)-3-amino-4-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(1R)-1-carboxyethyl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-4-oxobutanoic acid

159432-28-7; A 779; (D-Ala7)-Angiotensin I/II (1-7); A-779; CHEMBL4578721; (2R,5S,8S,11S,14S,17S,20S)-5-((1H-imidazol-4-yl)methyl)-20-amino-8-((S)-sec-butyl)-17-(3-guanidinopropyl)-11-(4-hydroxybenzyl)-14-isopropyl-2-methyl-4,7,10,13,16,19-hexaoxo-3,6,9,12,15,18-hexaazadocosanedioic acid; (3S)-3-amino-4-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(1R)-1-carboxyethyl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-4-oxobutanoic acid; Ang(1-7) D-Ala7;

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)

0.1 M HCL : 25 mg/mL (~28.64 mM)
H2O : ~5 mg/mL (~5.73 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.)