Mas-related G protein-coupled receptor, member D (MrgD). Alamandine is the endogenous ligand of MrgD. This receptor is distinct from the two known vasodilator receptors of the RAS (Mas and angiotensin II type 2). Alamandine binds to MrgD, initiating intracellular signaling pathways that lead to vasodilation, anti-hypertensive effects, and organ protection (antifibrotic, cardioprotective, renoprotective). Alamandine's activity is similar to Ang-(1-7) rather than that of angiotensin II or angiotensin A, but it acts at a different receptor. By activating MrgD, alamandine counteracts the vasoconstrictive and pro-fibrotic effects of angiotensin II, contributing to the protective arm of the renin-angiotensin system. The MrgD receptor is also known as Mas-related receptor, and its activation by alamandine leads to nitric oxide (NO) release and reduction of oxidative stress.

Alamandine is made either directly from angiotensin 1-7 (Ang-(1-7)) or via ACE2 catalyzing Ang A. It is produced from angiotensin II (Ang II) by the Ang II converting enzyme 2 (ACE2), and because of this, it has protective (vasodilatory) qualities. It is possible to decarboxylate Ang (1–7) to produce the peptide alamandine. Another endogenous peptide that can be found in human blood is alamandine [1]. In primary endothelial and mesangial cells, alamandine raises the concentration of cAMP, which also suggests Gs coupling [2]. Leptin secretion, expression, and blood levels are all reduced by amandine. In both isolated adipocytes and adipose tissue, alamandine stimulates the expression of plasminogen activator inhibitor-1 (PAI-1) and iNOS [2].

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Alamandine is an endogenous heptapeptide that binds to the Mas-related receptor MrgD with high affinity. In vitro studies have shown that alamandine causes vasodilation in isolated blood vessels. The peptide acts as a vasoactive peptide, relaxing pre-constricted arteries through MrgD activation. Alamandine's activity is similar to Ang-(1-7) rather than angiotensin II or angiotensin A, but it acts at a different receptor. The peptide exhibits antifibrotic activity in cell-based assays, reducing collagen synthesis and fibroblast proliferation. It also protects against oxidative stress in cultured cells, reducing reactive oxygen species levels and increasing cell survival under stress conditions. Alamandine's effects are mediated through MrgD-dependent signaling pathways involving nitric oxide (NO) and cyclic GMP. The peptide has also been shown to protect kidney cells from injury and to reduce inflammatory responses in vitro.

After six weeks of treatment with alamandine (0.15 μL/h; given by mini-osmotic pump), hypertension was improved and left ventricular (LV) function was compromised in patients with SHR. Additionally, it slowed the growth of the cross-sectional area of myocardial cells, decreased the mass increase of the heart and lungs in SHR, and decreased the levels of brain and atrial natriuretic peptide mRNA[3].

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Alamandine has demonstrated significant in vivo efficacy in animal models of hypertension and organ damage. In spontaneously hypertensive rats (SHR), a novel orally active formulation of alamandine produced a long-term antihypertensive effect, reducing blood pressure over extended periods. The peptide also exhibited cardioprotective effects, reducing cardiac fibrosis and improving cardiac function in hypertensive animals. Alamandine protects the kidney and heart through anti-hypertensive actions, reducing renal injury and proteinuria in models of hypertensive nephropathy. The peptide has antifibrotic effects in the heart and kidney, reducing collagen deposition and extracellular matrix remodeling. Alamandine has also shown anti-hyperalgesic and anti-allodynic activities via suppression of inflammatory signaling in animal models. These in vivo effects confirm the therapeutic potential of alamandine and its receptor MrgD as targets for the treatment of hypertension, heart failure, chronic kidney disease, and fibrotic disorders. Alamandine targets to protect the kidney and heart through anti-hypertensive actions.

MrgD receptor binding assay: Membranes from cells expressing recombinant MrgD are incubated with varying concentrations of Alamandine (0.1 nM-10 microM) and a radiolabeled tracer (e.g., ¹2⁵I-alamandine or 3H-alamandine if available) in binding buffer for 1-2 hours at room temperature or 4degC. Bound radioactivity is separated by filtration through glass fiber filters (e.g., using a cell harvester) and counted by liquid scintillation or gamma counter. Non-specific binding is determined in the presence of excess unlabeled alamandine or a competing MrgD ligand. Saturation binding assays are performed to determine Kd (dissociation constant) and Bmax (maximum binding). Competitive binding assays with increasing concentrations of unlabeled alamandine are used to determine Ki values. Alamandine binds specifically to MrgD with high affinity. The binding assay protocol should include appropriate positive and negative controls, including known MrgD agonists/antagonists. The assay buffer typically contains 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM EDTA, and protease inhibitors. The binding should be performed in a volume of 100-200 microL in 96-well plates. Incubation time and temperature need to be optimized based on the stability of the ligand and receptor. Filtration and washing steps should be rapid to minimize dissociation of bound ligand. The filters are typically washed 3 times with ice-cold binding buffer containing 0.1% BSA to reduce non-specific binding. This type of assay has been used to characterize the binding of alamandine to MrgD in published literature.

For vasodilation studies, isolated rat or mouse arteries (e.g., aorta or mesenteric arteries) are mounted in a wire myograph or pressure myograph system. Arterial segments are equilibrated in physiological salt solution (PSS) at 37degC and bubbled with 95% O2/5% CO2. Arteries are pre-constricted with a vasoconstrictor such as phenylephrine (1-10 microM) or U46619 (thromboxane A2 analog). Once a stable contraction is achieved, alamandine (0.1 nM-10 microM) is added cumulatively, and the relaxation response is recorded. For studies investigating the role of MrgD, arteries are pre-incubated with a selective MrgD antagonist or with inhibitors of signaling pathways (e.g., L-NAME to inhibit NO synthase, ODQ to inhibit guanylyl cyclase). Alamandine-induced relaxation is expressed as a percentage of the pre-constriction tone. The EC₅0 for relaxation and maximal relaxation (Emax) are calculated from concentration-response curves. This protocol has been used to demonstrate the vasodilator activity of alamandine. For cell-based studies, cells (e.g., cardiac fibroblasts, renal mesangial cells, or vascular smooth muscle cells) are treated with Alamandine (10-1000 nM) for 1-48 hours. Cell proliferation is assessed by BrdU incorporation or MTT assay. Collagen synthesis is measured by hydroxyproline assay, Sirius red staining, or by qPCR for collagen type I and III. Apoptosis is assessed by caspase-3/7 activity, Annexin V/PI staining, or TUNEL assay. Inflammatory markers (TNF-alpha, IL-6, IL-1beta, MCP-1) are measured by ELISA or qPCR. Reactive oxygen species (ROS) are measured using DCFH-DA probe. All experiments are performed in triplicate. Western blotting is performed to assess activation of signaling pathways (e.g., AKT, ERK, eNOS). Positive controls (e.g., Ang-(1-7) or other MrgD agonists) should be included.

Animal/Disease Models: Male spontaneously hypertensive rats (SHRs, 50weeks old)[3]
Doses: 0.15 μL/h (~50 μg/kg/day)
Route of Administration: Administered by mini-osmotic pumps; for 6 weeks
Experimental Results: Attenuated hypertension , alleviated cardiac hypertrophy, and improved LV function.

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For in vivo antihypertensive and cardioprotective studies in spontaneously hypertensive rats (SHR), male SHR (12-16 weeks old) are used. Alamandine is administered orally as an orally active formulation (novel formulation developed for this purpose) at doses ranging from 10-100 mg/kg/day for 2-4 weeks. Blood pressure is measured by tail-cuff plethysmography or telemetry at baseline and at regular intervals during treatment. At the end of the study, heart and kidney tissues are collected. Cardiac function is assessed by echocardiography (e.g., ejection fraction, fractional shortening). Tissue fibrosis is assessed by Masson‘s trichrome staining, picrosirius red staining, and hydroxyproline assay. Markers of oxidative stress (MDA, SOD, GSH) and inflammation (TNF-alpha, IL-6, NF-kappaB) are measured in tissue homogenates. Protein expression (e.g., MrgD, collagen, fibronectin) is assessed by immunohistochemistry or Western blotting. For studies in other animal models (e.g., myocardial infarction, renal ischemia-reperfusion injury), alamandine treatment is started immediately after surgery or 24-48 hours before surgery. Endpoints include reduction in infarct size, improvement in renal function (serum creatinine, BUN), and reduction in proteinuria. Dosages, routes of administration, and treatment durations should be optimized based on the specific model and formulation. Always follow institutional animal care and use guidelines. In diabetic rats, alamandine has been shown to protect against diabetic nephropathy and cardiomyopathy. The compound should be stored as a lyophilized powder at -20degC or -80degC and reconstituted in sterile water or saline before use. Avoid repeated freeze-thaw cycles. The stability of alamandine in solution should be tested, as peptides can degrade over time. For oral administration, a specific formulation that protects the peptide from degradation in the gastrointestinal tract is required. The orally active formulation of alamandine has been described in the literature.

Alamandine has a molecular weight of 855.01 or 855.00 (depending on the form) and molecular formula C40H₆2N12O₉ (trifluoroacetate salt may be used for research). The peptide sequence is L-Alanyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-proline. Solubility: alamandine is soluble in water and in DMSO. For in vitro studies, stock solutions are prepared in sterile water or PBS at concentrations of 1-10 mM. For in vivo studies, the peptide should be dissolved in sterile saline or PBS. A novel orally active formulation has been developed that produces long-term antihypertensive effects in spontaneously hypertensive rats. Storage: Alamandine should be stored as a lyophilized powder at -20degC or -80degC, protected from light and moisture. Once reconstituted, the solution should be aliquoted and stored at -20degC or -80degC and used within 1-3 months to avoid degradation. Avoid repeated freeze-thaw cycles. Pharmacokinetic properties: As a peptide, alamandine is expected to be rapidly degraded by proteases in the gastrointestinal tract and in the bloodstream. The development of an orally active formulation that protects the peptide from degradation and enhances absorption is a significant advance. Detailed PK parameters (half-life, Cmax, AUC, oral bioavailability) for the formulated alamandine have been reported in the literature . The peptide is stable in solution under appropriate storage conditions. The half-life of alamandine in plasma is likely short (minutes to hours), requiring frequent dosing or sustained-release formulations for chronic use. The orally active formulation improves bioavailability and duration of action. Further PK studies are needed to fully characterize the absorption, distribution, metabolism, and excretion of alamandine.

Based on its endogenous nature as a heptapeptide that is part of the renin-angiotensin system, alamandine is expected to have low toxicity at physiological concentrations. In animal studies, alamandine has been well-tolerated at the doses tested (10-100 mg/kg/day, orally or intraperitoneally for up to 4 weeks). No significant adverse effects have been reported in the available literature. Alamandine is endogenous, and its administration supplements the natural peptide, which is expected to be safe within a certain dose range. However, comprehensive toxicological studies (including genotoxicity, reproductive toxicity, and chronic toxicity) have not been extensively performed. The orally active formulation may contain excipients that could have their own toxicity profiles. As with all research compounds, standard safety precautions should be followed when handling. Alamandine is not approved for human therapeutic use and should be used only for research purposes. Clinical trials have not been conducted, so the safety profile in humans is unknown. It is important to note that interfering with the renin-angiotensin system can have significant effects on blood pressure, electrolyte balance, and kidney function, so alamandine should be used with caution in research settings. However, alamandine is part of the protective arm of the RAS, and its effects are generally beneficial (vasodilation, antifibrotic, antihypertensive). Doses should be carefully selected based on the experimental model and desired outcomes. Monitoring of blood pressure and renal function is recommended in animal studies. Long-term safety studies would be required before clinical development. The compound is not intended for human use.

[1]. Alamandine: a new member of the angiotensin family. Curr Opin Nephrol Hypertens. 2014 Mar;23(2):130-4.

[2]. Johanna Schleifenbaum. Alamandine and Its Receptor MrgD Pair Up to Join the Protective Arm of the Renin-Angiotensin System. Front Med (Lausanne). 2019 Jun 11;6:107.

[3]. Alamandine attenuates hypertension and cardiac hypertrophy in hypertensive rats. Amino Acids. 2018 Aug;50(8):1071-1081.

Alamandine is a peptide.

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Alamandine is an endogenous vasoactive heptapeptide that was discovered in 2013 as a new component of the renin-angiotensin system (RAS). It is considered part of the “protective arm” of the RAS, along with Ang-(1-7) and the Mas receptor, counterbalancing the vasoconstrictive, pro-inflammatory, and pro-fibrotic effects of angiotensin II. Alamandine exerts its effects through the MrgD receptor, which is distinct from the Mas receptor used by Ang-(1-7). This dual protective system (Ang-(1-7)/Mas and alamandine/MrgD) offers multiple targets for therapeutic intervention in cardiovascular and renal diseases. Alamandine is not a marketed drug and is currently available for research use only. It is sold as a research-grade peptide (typically as the trifluoroacetate salt) for in vitro and in vivo studies. Alamandine is used as a tool to study the physiology and pathophysiology of the RAS and to investigate the therapeutic potential of MrgD activation. The development of an orally active formulation of alamandine is a significant advance that could facilitate its use in chronic disease models and potentially in clinical applications. Alamandine has been shown to have anti-hypertensive, cardioprotective, renoprotective, antifibrotic, and anti-inflammatory effects. It may also have anti-hyperalgesic and anti-allodynic activities. The peptide can serve as a model peptide, e.g., in the development and evaluation of analytical methods for peptide quantification in biological samples. The amino acid sequence of alamandine is L-Alanyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-proline. The peptide is sensitive to degradation by proteases; therefore, care should be taken when handling and storing solutions. For long-term storage, the lyophilized powder should be stored at -20degC or -80degC, desiccated, and protected from light. Reconstituted solutions should be stored at -20degC or -80degC in small aliquots to avoid repeated freeze-thaw cycles. The compound is available from multiple research chemical suppliers. Always check the certificate of analysis (COA) for purity (typically >95-98% by HPLC) and storage recommendations. Alamandine is not for human use. Additional research is ongoing to explore its full therapeutic potential.

C40H62N12O9

855.00

854.476

1176306-10-7

44192273

White to off-white solid powder

1.4±0.1 g/cm3

1.657

0.12

11

12

23

61

1540

8

CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N2CCC[C@H]2C(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](C)N

QVMYBFKDLPBYSO-GIGALADGSA-N

InChI=1S/C40H62N12O9/c1-6-22(4)32(37(58)49-29(18-25-19-44-20-46-25)38(59)52-16-8-10-30(52)39(60)61)51-35(56)28(17-24-11-13-26(53)14-12-24)48-36(57)31(21(2)3)50-34(55)27(47-33(54)23(5)41)9-7-15-45-40(42)43/h11-14,19-23,27-32,53H,6-10,15-18,41H2,1-5H3,(H,44,46)(H,47,54)(H,48,57)(H,49,58)(H,50,55)(H,51,56)(H,60,61)(H4,42,43,45)/t22-,23-,27-,28-,29-,30-,31-,32-/m0/s1

(2S)-1-[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-aminopropanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carboxylic acid

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: 25 mg/mL (29.24 mM)

Solubility in Formulation 1: 25 mg/mL (29.24 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication.

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