Diamine oxidase; ROS; NOS/nitric oxide synthase

In A549 cells, Aminoguanidine (100–1000 μM, 24 h) can lessen DOX-induced cellular inflammation and DNA damage [1]. In AR42J cells, aminoguanidine (100 μM, 30 min) can activate ERK and aid in cell rebuilding [1].

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Doxorubicin (DOX) is a broad-spectrum anthracycline that has cardiotoxicity as a major side effect. Reactive oxygen species (ROS) and reactive nitrogen species generations have been proposed to be an important mechanism of DOX-induced cardiotoxicity and cardiomyocyte apoptosis, which may be mediated by p53 protein. Aminoguanidine (AG) is an effective antioxidant due to its free radical scavenger activity. A549 lung cell line was incubated with various concentrations of AG (100-1,000 μM) wit/without 0.25 μM DOX for 24 h. The expression of p53 and its transcriptional target p21 were analyzed by Western blot. Apoptosis was analyzed with Annexin V assay. JC1 and H2AX immunofluorescence were used to assess mitochondrial and nuclear DNA damage, respectively. This study demonstrated that AG has a dose-dependent antiapoptotic effect on DOX-induced apoptosis. Thus, these data further identify AG as a potential chemopreventive agent to reduce ROS and nitric oxide synthase damage generated by DOX.[1]

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MDA concentration in cells treated with Aminoguanidine/AG was not different from untreated cells. However, treatment with H2O2 either alone or in combination with AG increased MDA significantly (p<0.05). AG treatment alone induced 3.5 fold activation of pERK-1/2, as compared to 2.5 fold increase with H2O2 alone (p<0.05) as compared to untreated control. The results of ERK activation were confirmed further by its co-localization employing FITC-conjugated ERK antibody. AG -induced maximal cell proliferation occurred at 48 hr. incubation (p<0.05); these values were not significantly different from that of H2O2 treated and control cells. Cell function (CCK-stimulated amylase release) was significantly enhanced by AG (p<0.05). Conclusion: These data suggest that in an in-vitro system, Aminoguanidine/AG acts as a pro-oxidant on AR42J cell proliferation and possibly affects the resulting function [2].

A 50 mg/kg intraperitoneal dose of aminoguanidine shields coils from the hepatotoxic effects of CCl4 [3]. Aminoguanidine (200 mg/kg, intraperitoneal, one dose) in coil to prevent hepatotoxicity caused by cyclophosphamide (CP) [3].

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The present study was undertaken to evaluate the effect of Aminoguanidine (AG) on carbon tetrachloride (CCl4)-induced hepatotoxicity. Treatment of mice with CCl4 (20 microl/kg, i.p.) resulted in damage to centrilobular regions of the liver, increase in serum aminotransferase and rise in lipid peroxides level 24 hours after CCl4 administration. Pretreatment of mice with AG (50 mg/kg, i.p.) 30 minutes before CCl4 was found to protect mice from the CCl4-induced hepatic toxicity. This protection was evident from the significant reduction in serum aminotransferase, inhibition of lipid peroxidation and prevention of CCl4-induced hepatic necrosis revealed by histopathology. Aminoguanidine, a relatively specific inhibitor of inducible nitric oxide synthase, did not inhibit the in vitro lipid peroxidation. Taken together, these data suggest a potential role of nitric oxide as an important mediator of CCl4-induced hepatotoxicity.[3]

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Aim: To find out whether Aminoguanidine (AG) protects against CP-induced oxidative stress and renal damage. Method: Renal damage was induced in the rats by administration of a single injection of CP at a dose of 150 mg/kg body weight intraperitoneally. For the Aminoguanidine/AG pretreatment studies, the rats were injected intraperitoneally with AG at a dose of 200 mg/kg body weight 1 hour before administration of CP. The control rats received AG or saline alone. All the rats were killed 16 hours after the administration of CP or saline. The kidneys were used for histological examination by light microscopy and biochemical assays--malondialdehyde, protein carbonyl content, reduced glutathione (GSH), and the activities of antioxidant enzymes including glutathione peroxidase (GPx), glutathione S transferase (GSTase), catalase, glutathione reductase, and myeloperoxidase (MPO), a marker of neutrophil infiltration. Results: Pretreatment with AG attenuated CP-induced renal damage histologically. Pretreatment with AG prevented CP-induced lipid peroxidation, protein oxidation, depletion of reduced GSH, and loss of activities of the antioxidant enzymes including GPx, catalase, and GSTase and also MPO activity. Conclusion: The results of the present study reveal that Aminoguanidine/AG can prevent CP-induced renal damage by inhibiting oxidative stress. Thus, AG may be useful for prevention of the nephrotoxicity of CP [4].

Apoptosis analysis [1]
Cell Types: A549
Tested Concentrations: 100-1000 μM
Incubation Duration: 24 h
Experimental Results: Shows a protective effect on DOX-induced DNA damage and reduces DOX-induced apoptosis. 2].

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Cell proliferation assay[2]
Cell Types: AR42J
Tested Concentrations: 100 μM
Incubation Duration: 24-96 hrs (hours)
Experimental Results: Cell proliferation increased Dramatically after 48 hrs (hours) of incubation.

Animal/Disease Models: Male Swiss albino mouse [3]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection 30 minutes before CCl4 administration
Experimental Results: Inhibited serum AST levels, protected hepatotoxin-oxidation intermediate and renal damage, and had a protective effect [ 4]. Induces lipid peroxidation.

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Animal/Disease Models: Adult male Wistar rats [4]
Doses: 200 mg/kg
Route of Administration: intraperitoneal (ip) injection 1 hour before CP injection, and sacrificed 16 hrs (hrs (hours)) after CP injection.
Experimental Results: Attenuated CP-induced MDA elevation and prevented CP-induced protein oxidation. Restored GSH levels and attenuated CP-induced increase in MPO activity.

The subcutaneous LD50 of 2146 in rats was 1258 mg/kg. Journal of Pharmacology and Experimental Therapeutics, 119(444), 1957 [PMID:13417100]
The subcutaneous LD50 of 2146 in mice was 963 mg/kg. Journal of Pharmacology and Experimental Therapeutics, 119(444), 1957 [PMID:13417100]

[1]. Sabuncuoglu S. Antiapoptotic effect of aminoguanidine on doxorubicin-induced apoptosis. Mol Cell Biochem. 2014 Sep;394(1-2):129-35.

[2]. Chowdhury P. Aminoguanidine (AG) Induces Induced both Pro- and Antioxidant Effect in AR42J Cells, a Rat Pancreatic Tumor Cell Line. Ann Clin Lab Sci. 2017 Sep;47(5):572-580. PMID: 29066484.

[3]. Protective effect of aminoguanidine, a nitric oxide synthase inhibitor, against carbon tetrachloride induced hepatotoxicity in mice. Life Sci. 2000;66(3):265-70.

[4]. Protective effect of aminoguanidine against cyclophosphamide-induced oxidative stress and renal damage in rats. Redox Rep. 2011;16(1):8-14.

Aminoguanidine is a single-carbon compound whose unique structure allows it to function as a derivative of hydrazine, guanidine, or formamide. It is an EC 1.4.3.4 (monoamine oxidase) inhibitor and an EC 1.14.13.39 (nitric oxide synthase) inhibitor. It belongs to the guanidine family and is also a single-carbon compound. Pimageline, developed by Synvista Therapeutics, is used to treat diabetic nephropathy. It is an inhibitor of advanced glycation end products (AGEs) and can be used alone or in combination with other therapies for the treatment of diabetic nephropathy. It is beneficial for the treatment of patients with diabetic nephropathy.

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Drug Indications
It has been studied for the treatment of diabetic nephropathy.

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Mechanism of Action
Pimageline has been reported to inhibit the formation of glycosylated proteins (AGEs) and has other effects, including inhibition of aldose reductase.

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Aminoguanidine (AG) is an inhibitor of diamine oxidase and nitric oxide synthase, and has been used in research on diabetes, follicular thyroid carcinoma, hepatocellular carcinoma, pancreatic xenografts, and breast cancer. The effects of AG on these pathological conditions may be related to its regulation of cell proliferation, angiogenesis, and antioxidant enzyme expression. However, its effects as a pro-oxidant and/or antioxidant on signal transduction and function in pancreatic tumor cell lines have not been investigated. This study aimed to verify whether aminoguanidine exposure to AR42J cells induces a pro-oxidative effect, thereby promoting the proliferation and growth of these cells. Methods: AR42J cells were cultured at 37°C in 5% CO₂ F-12 medium until confluence exceeded 90%, and then treated with 20 μM hydrogen peroxide (H₂O₂) for 20 min, or with 100 μM aminoguanidine (AG) for 30 min, or a combination of both. Cell lysates were collected, and the generation of lipid peroxides was detected by malondialdehyde (MDA) assay, and the activation of phosphorylated ERK1/2 signaling pathway was detected by Western blotting. The activation of ERK signaling pathway was further confirmed by immunohistochemical analysis. The effect of ERK1/2 on cell proliferation after AG and H₂O₂ treatment was assessed by MTT assay, and the functional status of AR42J cells was detected by the release of amylase after CCK-8 stimulation. [2] Cyclophosphamide (CP) is widely used to treat tumors and B-cell malignancies, such as lymphoma, myeloma, chronic lymphocytic leukemia and Waldenström macroglobulinemia. Kidney injury is one of the dose-limiting side effects of CP. Oxidative stress has been reported to play an important role in CP-induced kidney injury. [4]

HN:C(NH2)NHNH2.H2CO3

74.08510

74.059

C, 16.21; H, 8.16; N, 75.62

79-17-4

1937-19-5 (hydrochloride); 996-19-0

2146

Typically exists as solid at room temperature

1.72g/cm3

261.4ºC at 760 mmHg

111.9ºC

1.666

0.234

3

2

0

5

41.6

0

N=C(NN)N

HAMNKKUPIHEESI-UHFFFAOYSA-N

InChI=1S/CH6N4/c2-1(3)5-4/h4H2,(H4,2,3,5)

2-aminoguanidine

aminoguanidine; Pimagedine; Hydrazinecarboximidamide; 2-aminoguanidine; Pimagedine [INN]; 2-azanylguanidine; pimagedina; ...; 79-17-4;

2934.99.9001

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)