CGP-42112 (≥1 nM) significantly reduces the basal value of cGMP production. From the basal value, CGP-42112 (≥1 nM) significantly inhibits TH-enzyme activity. PD123319 (an AT(2)-R antagonist) completely eliminates the inhibitory effects of CGP-42112 on TH-enzyme activity and cGMP production, whereas CV-11974 (an AT(1)-R antagonist) has no effect[1]. [¹²⁵5I] CGP-42112 selectively binds to the subtype of angiotensin II receptor known as AT2. [¹²⁵5I] Compared to the adrenal, the brain is where CGP-42112 binds more strongly. Although it does not change its binding in the adrenal gland, beta-Mercaptoethanol increased the binding of [¹²⁵5I]CGP-42112 in the brain[2]. High affinity binding is observed for [¹²⁵5I]CGP-42112 (Kd = 0.07-0.3 nM, depending on the region studied). [¹²⁵5I]CGP-42112 binding is selective for AT2 receptors, as shown by the non-selective peptides Ang II and angiotensin III (Ang III) as well as competition from the AT2 ligands PD 123177, unlabeled CGP-42112, and losartan, the AT1 ligand[4].

CGP-42112 (≥1 nM) significantly reduces the basal value of cGMP production. From the basal value, CGP-42112 (≥1 nM) significantly inhibits TH-enzyme activity. PD123319 (an AT(2)-R antagonist) completely eliminates the inhibitory effects of CGP-42112 on TH-enzyme activity and cGMP production, whereas CV-11974 (an AT(1)-R antagonist) has no effect[1]. [¹²⁵5I] CGP-42112 selectively binds to the subtype of angiotensin II receptor known as AT2. [¹²⁵5I] Compared to the adrenal, the brain is where CGP-42112 binds more strongly. Although it does not change its binding in the adrenal gland, beta-Mercaptoethanol increased the binding of [¹²⁵5I]CGP-42112 in the brain[2]. High affinity binding is observed for [¹²⁵5I]CGP-42112 (Kd = 0.07-0.3 nM, depending on the region studied). [¹²⁵5I]CGP-42112 binding is selective for AT2 receptors, as shown by the non-selective peptides Ang II and angiotensin III (Ang III) as well as competition from the AT2 ligands PD 123177, unlabeled CGP-42112, and losartan, the AT1 ligand[4].

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In cultured porcine adrenal medullary chromaffin cells, CGP-42112 (at concentrations ≥1 nM) significantly inhibited intracellular cGMP production from the basal level in a dose-dependent manner (e.g., 1 nM inhibited by 18%, 100 nM by 41%, and 1 μM by 52%). This inhibitory effect was abolished by the AT₂-R antagonist PD123319 (100 nM) but not by the AT₁-R antagonist CV-11974 (100 nM) [1]
.
CGP-42112 (at concentrations ≥1 nM) significantly inhibited tyrosine hydroxylase (TH) enzyme activity in a dose-dependent manner (e.g., 1 nM inhibited by 14%, 100 nM by 35%, and 1 μM by 52%). This inhibition was blocked by PD123319 (100 nM) but not by CV-11974 (100 nM) [1]
.
Pretreatment of cells with 1 mM 8-Br-cGMP (a membrane-permeable cGMP analogue) prevented the inhibitory effect of CGP-42112 (100 nM) on TH enzyme activity, whereas pretreatment with 1 mM 8-Br-cAMP did not [1]
.
CGP-42112 (at concentrations ≥1 nM) significantly reduced tyrosine hydroxylase (TH) mRNA levels in a dose-dependent manner (e.g., 1 nM inhibited by 18%, 100 nM by 54%, and 1 μM by 65%) after 8 hours of incubation. This effect was abolished by PD123319 (100 nM) but not by CV-11974 (100 nM) [1]
.
CGP-42112 (at concentrations ≥1 nM) significantly decreased tyrosine hydroxylase (TH) protein levels in a dose-dependent manner (e.g., 1 nM inhibited by 12%, 100 nM by 48%, and 1 μM by 74%) after 24 hours of incubation. This effect was abolished by PD123319 (100 nM) but not by CV-11974 (100 nM) [1]
.
Radioligand binding competition experiments using [¹²⁵I]-Ang II as a tracer showed that both CGP-42112 and the AT₂-R antagonist PD123319 strongly competed for binding sites on porcine adrenal medullary cells, whereas the AT₁-R antagonist CV-11974 competed weakly, indicating predominant expression of AT₂-R in these cells [1]
.

The upper limit of CBF autoregulation was shifted toward higher blood pressures by intravenous infusions of PD 123319 (0.36 and 1 mg kg-1 min-1) and CGP-42112 (0.1 and 1 mg kg-1 min-1) without changing baseline CBF[3].

Tyrosine Hydroxylase (TH) Enzyme Activity Assay: Cells were incubated with test substances in HEPES-buffered Krebs buffer at 37°C for 10 minutes. After incubation, cells were homogenized in sucrose solution. The enzyme reaction mixture contained tissue homogenate, sodium acetate buffer (pH 6.0), L-tyrosine, a cofactor solution (6-methyl-5,6,7,8-tetrahydropterine in 2-mercaptoethanol), and catalase. The mixture was incubated at 37°C for 30 minutes, and the reaction was stopped with perchloric acid containing an internal standard. The product, DOPA, was extracted using an aluminum oxide method. The extracted DOPA was then separated by HPLC with electrochemical detection, and TH activity was calculated as the amount of DOPA formed per milligram of protein per minute [1]
.

Cell Culture: Primary dissociated chromaffin cells from porcine adrenal medulla were prepared and purified by differential plating. Cells were plated and maintained as monolayer cultures in DMEM supplemented with fetal bovine serum, penicillin, streptomycin, and an antifungal agent. Cultures were kept in a humidified atmosphere of 5% CO₂/95% O₂ at 37°C for 2–3 days before experiments [1]
.
cGMP Production Measurement: Cells were washed and preincubated in Eagle's minimal essential medium containing 0.2 mmol/L 3-isobutyl-1-methylxanthine (IBMX) for 5 minutes. The medium was then replaced with HEPES-buffered Krebs buffer containing test substances and 0.2 mmol/L IBMX, and cells were incubated at 37°C for 30 minutes. The reaction was terminated by adding HCl. The acid extract was used to measure cGMP levels with a commercial cGMP assay kit [1]
.
Northern Blot Analysis for TH mRNA: Total RNA was extracted from cells. RNA samples were electrophoresed on formaldehyde-agarose gels, transferred to a nitrocellulose membrane, and hybridized with a ³²P-labeled cDNA probe for human tyrosine hydroxylase (TH). Rat G3PDH cDNA was used as an internal control. Hybridization signals were quantified using an image analyzer [1]
.
Western Blot Analysis for TH Protein: Cells were solubilized in a buffer containing SDS, Triton X-100, sodium deoxycholate, and Tris-HCl. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked. Membranes were incubated with a monoclonal anti-tyrosine hydroxylase antibody, followed by incubation with horseradish peroxidase-labeled Protein A. Signals were detected using enhanced chemiluminescence (ECL) reagents and analyzed by densitometry [1]
.
Receptor Binding Studies: Cells were incubated with a fixed concentration of [¹²⁵I]-Ang II tracer and increasing concentrations of unlabeled competitors (CGP42112, PD123319, or CV-11974) in a binding buffer at 20°C for 90 minutes. Cells were then washed, lysed, and cell-bound radioactivity was measured with a γ-counter to determine binding competition [1]
.

[1]. Angiotensin-II subtype 2 receptor agonist (CGP-42112) inhibits catecholamine biosynthesis in cultured porcine adrenal medullary chromaffin cells. Biochem Biophys Res Commun. 2000 Jun 7;272(2):544-50.

[2]. [125I]CGP 42112 binding reveals differences between rat brain and adrenal AT2 receptor binding sites. Regul Pept. 1993 Mar 19;44(2):189-97.

[3]. Angiotensin II AT2 receptor stimulation extends the upper limit of cerebral blood flow autoregulation: agonist effects of CGP 42112 and PD 123319. J Cereb Blood Flow Metab. 1994 Jan;14(1):38-44.

[4]. Quantitative autoradiography of angiotensin II AT2 receptors with [125I]CGP 42112. Brain Res. 1995 Apr 17;677(1):29-38.

CGP-42112A is a hexapeptide composed of L-tyrosine, L-lysine, L-histidine, L-proline, and L-isoleucine amino acid residues linked in sequence. The amino group of the L-tyrosine residue is replaced by a (pyridin-3-ylcarbonyl)nitrile group, and the side-chain amino group of the L-lysine residue is replaced by a {N(2)-[(benzyloxy)carbonyl]-L-argininoyl}nitrile group. It is a potent angiotensin II receptor type 2 (AT2 receptor) agonist. It possesses multiple functions, including angiotensin receptor agonist, vasodilator, antitumor agent, anti-inflammatory agent, and neuroprotective agent. It is an oligopeptide, benzyl ester, and pyridine carboxamide.

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Studies have shown that stimulation of AT₂ receptors by CGP-42112 can inhibit the biosynthesis of catecholamines in adrenal chromaffin cells. The mechanism involves a decrease in intracellular cGMP levels, which leads to a reduction in the activity and synthesis of the rate-limiting enzyme tyrosine hydroxylase (TH), both at the enzyme activity and gene/protein expression levels [1]. In multiple systems, CGP-42112 has been described as a complete AT₂ receptor agonist [1].

C52H69N13O11

1052.19

1051.523

C, 59.36; H, 6.61; N, 17.31; O, 16.73

127060-75-7

123794

White to off-white solid powder

1.4±0.1 g/cm3

1.659

2.38

11

14

30

76

1930

7

CC[C@H](C)[C@@H](C(O)=O)NC([C@H]1N(C([C@H](CC2=CNC=N2)NC([C@H](CCCCNC([C@H](CCCNC(N)=N)NC(OCC3=CC=CC=C3)=O)=O)NC([C@H](CC4=CC=C(C=C4)O)NC(C5=CC=CN=C5)=O)=O)=O)=O)CCC1)=O

UXGNARZDONUMMK-LRMQDCNJSA-N

InChI=1S/C52H69N13O11/c1-3-32(2)43(50(73)74)64-48(71)42-17-11-25-65(42)49(72)41(27-36-29-56-31-59-36)62-46(69)39(60-47(70)40(26-33-18-20-37(66)21-19-33)61-44(67)35-14-9-22-55-28-35)15-7-8-23-57-45(68)38(16-10-24-58-51(53)54)63-52(75)76-30-34-12-5-4-6-13-34/h4-6,9,12-14,18-22,28-29,31-32,38-43,66H,3,7-8,10-11,15-17,23-27,30H2,1-2H3,(H,56,59)(H,57,68)(H,60,70)(H,61,67)(H,62,69)(H,63,75)(H,64,71)(H,73,74)(H4,53,54,58)/t32-,38-,39-,40-,41-,42-,43-/m0/s1

(2S,3S)-2-[[(2S)-1-[(2S)-2-[[(2S)-6-[[(2S)-5-(diaminomethylideneamino)-2-(phenylmethoxycarbonylamino)pentanoyl]amino]-2-[[(2S)-3-(4-hydroxyphenyl)-2-(pyridine-3-carbonylamino)propanoyl]amino]hexanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylpentanoic acid

2934.99.03.00

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)

Solubility in Formulation 1: 2.5 mg/mL (2.38 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (2.38 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: 2.5 mg/mL (2.38 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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