Arginase
The primary target of nor-NOHA di-acetate is arginase, a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. Arginase exists in two isoforms: arginase I (cytosolic, primarily in the liver) and arginase II (mitochondrial, in various tissues). By inhibiting arginase, nor-NOHA increases the availability of L-arginine for nitric oxide synthase (NOS), thereby enhancing the production of nitric oxide (NO). This mechanism has potential therapeutic applications in conditions where NO signaling is impaired, such as cardiovascular diseases, erectile dysfunction, and pulmonary hypertension. nor-NOHA is a potent and selective arginase inhibitor with Ki values in the nanomolar range.

nor-NOHA (0.1-1 mM, 72 h) induces apoptosis of K562 cells in a dose-dependent manner under hypoxia (1.5% O2)[1]. nor-NOHA (1 mM, 72 h) can attenuate hypoxia-mediated imatinib resistance in K562 or KCL22 cells[1].
In vitro, nor-NOHA di-acetate is a potent and selective inhibitor of arginase. Its activity is typically measured using arginase enzyme assays that monitor the production of urea from L-arginine. IC50 or Ki values are determined from dose-response curves. The compound's selectivity for arginase over other enzymes, including nitric oxide synthase (NOS), is assessed to confirm its specificity. The compound's ability to enhance NO production is confirmed in cell-based assays that measure NO levels.

or-NOHA (100 mg/kg, intravenous injection, once) can significantly reduce infarct size in male Sprague-Dawley rats [2]. nor-NOHA (100 mg/kg IV once) increases plasma citrulline and nitrite levels and decreases plasma ornithine levels in male Sprague-Dawley rats [2]
In vivo, nor-NOHA di-acetate has been studied for its potential therapeutic applications in cardiovascular diseases, erectile dysfunction, and other conditions where NO signaling is impaired. By inhibiting arginase and enhancing NO production, the compound can improve vasodilation, reduce blood pressure, and improve erectile function. The compound has also been studied for its effects on immune function and wound healing. While specific in vivo efficacy data are not extensively detailed in the available literature, its mechanism of action supports its potential therapeutic applications.

Arginase activity assay[1]
Arginase activity was analysed as described with modifications. Cells were counted, and equal numbers of cells were lysed in 50μl of lysis buffer (PBS with 1mM EDTA, 0.1% Triton X−100 and protease Inhibitors) and centrifuged for 15 minutes at 14,000g at 4°C. The supernatants were mixed with 50μl of freshly prepared activation buffer (10mM MnCl2, 50mM Tris-HCl pH7.5) and 50μl of 0.5M arginine, and heated for 10 minutes at 56°C. Thereafter, 800μl of acidic solution (H2SO4 (96%)/H3PO4 (85%)/H2O, 1/3/7, v/v/v) and 25μl of 9% α–isonitrosopropiophenone (in ethanol) were added to the mixture and heated for 15 minutes at 100°C. The mixture was allowed to develop colour in the dark. Finally, 250μl was transferred to a 96-well plate for OD measurements at 550nm.
In vitro enzyme/receptor binding assays for nor-NOHA di-acetate involve arginase inhibition assays using purified arginase enzyme (arginase I or II). The compound's inhibitory activity is measured by monitoring the production of urea from L-arginine in the presence of the enzyme and varying concentrations of the inhibitor. Urea production is typically measured using colorimetric assays (e.g., the diacetyl monoxime method). IC50 and Ki values are determined from dose-response curves. Selectivity profiling against nitric oxide synthase (NOS) and other enzymes is performed to assess the compound's specificity.

Measurement of cellular respiration by Seahorse Analyzer[1]
0.1x106 K562 cells were plated per well in poly–L–lysine-coated XF–24 well cell culture microplates in XF Assay media supplemented with 4.5 g/L glucose and 1mM sodium pyruvate. The cells were spin–immobilized to the microplates at 200g for 1 minute. The cellular oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and photon production rate (PPR) were obtained using an XF24 Analyzer from Seahorse Bioscience. The measurements were performed according to the manufacturer’s instructions, using Oligomycin, Carbonyl cyanide–4–(trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone & antimycin A (R/A; all from Sigma−Aldrich) at the specified concentrations. Data was analysed using the Seahorse XF software.
In vitro cellular assays for nor-NOHA di-acetate are conducted in cell lines that express arginase, such as macrophages, endothelial cells, or smooth muscle cells. Cells are treated with varying concentrations of nor-NOHA, and arginase activity is measured by assessing urea production. NO production is measured using the Griess assay or by using fluorescent NO indicators. The compound's effects on cell proliferation, migration, and other functional responses are also assessed. These assays confirm that nor-NOHA engages its target in a cellular context and produces the expected enhancement of NO production.

Sprague-Dawley rats were subjected to 30 min of coronary artery ligation, followed by 2 h of reperfusion. The animals were given either saline, or the arginase inhibitor N-omega-hydroxy-nor-l-arginine (nor-NOHA) with or without the NO scavenger carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) or the NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) iv 15 min before ischaemia. The infarct size was 79 +/- 4% of the area at risk in the control group. Nor-NOHA treatment reduced the infarct size to 39 +/- 7% (P < 0.001). Administration of cPTIO or l-NMMA completely abolished the protective effect of nor-NOHA. Expression of arginase I was significantly (P < 0.05) increased in ischaemic myocardium. Nor-NOHA treatment resulted in higher plasma levels of nitrite (P < 0.05) and a 10-fold increase in the citrulline/ornithine ratio (P < 0.001), indicating a shift in arginine utilization towards NOS.
Conclusion: Inhibition of arginase protects from myocardial infarction by a mechanism that is dependent on NOS activity and bioavailability of NO by shifting arginine utilization from arginase towards NOS. These findings suggest that targeting of arginase is a promising future therapeutic strategy for protection against myocardial IR injury.[2]
In vivo animal studies for nor-NOHA di-acetate are conducted in animal models of cardiovascular diseases, erectile dysfunction, and other conditions. Animals are administered the compound orally or intraperitoneally, and outcomes are assessed based on the specific disease model. In cardiovascular models, blood pressure and vascular function are assessed. In erectile dysfunction models, erectile function is assessed. Pharmacokinetic studies are performed to determine the compound's bioavailability, half-life, and tissue distribution.

Pharmacokinetic properties of nor-NOHA di-acetate indicate that it has a molecular weight of 336.30 and a molecular formula of C12H20N4O8. The compound is a di-acetate salt of nor-NOHA, which enhances its solubility and stability. It is soluble in water and DMSO. For storage, the powder should be kept under appropriate conditions to maintain stability. Its physicochemical properties suggest that it has reasonable drug-like characteristics.

Toxicological information for nor-NOHA di-acetate is primarily derived from its use as a research compound in preclinical studies. As an arginase inhibitor, potential on-target effects could include enhanced NO production, which may lead to hypotension and other cardiovascular effects. Comprehensive toxicology studies would be required for therapeutic development, including assessments of cardiovascular, renal, and immune function.

[1]. The arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target. PLoS One. 2018 Oct 11;13(10):e0205254.

[2]. Arginase inhibition mediates cardioprotection during ischaemia-reperfusion. Cardiovasc Res. 2010 Jan 1;85(1):147-54.

ω-hydroxy-n-L-arginine is an L-α-amino acid. N-hydroxy-n-L-arginine (nor-NOHA) is being investigated in the clinical trial NCT02009527 (arginase inhibitors in ischemia-reperfusion injury). Cancer cells, including chronic myeloid leukemia (CML), rely on hypoxia to persist in the host and evade immunotherapy. Therefore, drug development targeting cancer-specific hypoxia responses has attracted considerable interest. However, a major challenge in leukemia research is identifying differentially expressed, targetable hypoxia responses between leukemia cells and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme in the urea cycle, is overexpressed in CML cells but not in normal progenitor cells. ARG2 is a target of hypoxia-inducible factors (HIF1-α and HIF2-α) and is essential for polyamine production required for cell growth. Therefore, we investigated whether the clinically validated arginase inhibitor Nω-hydroxy-norarginine (nor-NOHA) was effective against leukemia cells under hypoxic conditions. Notably, nor-NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxic conditions, but was ineffective under normoxic conditions. Combination therapy with a BCR-ABL1 kinase inhibitor overcame hypoxia-mediated resistance. While nor-NOHA itself shows promise in targeting the hypoxic response in leukemia, we unexpectedly found that its antileukemic activity was independent of ARG2 inhibition. ARG2 gene knockout using CRISPR/Cas9 had no effect on the viability of leukemia cells or their sensitivity to nor-NOHA. The different effects of ARG2 knockout and nor-NOHA on cellular respiration further confirmed this difference. In conclusion, we found that nor-NOHA possesses significant, but non-targeted, antileukemic activity in hypoxic cells expressing ARG2. Since nor-NOHA has been used in clinical trials and is widely used in studies of endothelial dysfunction, immunosuppression, and metabolism, its various biological effects must be carefully evaluated before attributing its activity to ARG inhibition. [1]
nor-NOHA di-acetate is a potent and selective inhibitor of arginase, an enzyme that competes with nitric oxide synthase for L-arginine. By inhibiting arginase, nor-NOHA enhances nitric oxide production. The compound has potential applications in cardiovascular diseases and erectile dysfunction. nor-NOHA di-acetate is not approved for clinical use and is available from research chemical suppliers for preclinical studies.

C9H20N4O7

296.277702331543

296.133

C, 36.49; H, 6.80; N, 18.91; O, 37.80

1140844-63-8

189302-40-7; 1140844-63-8 (acetate); 291758-32-2 (HCl); 2250019-93-1 (nor-NOHA monoacetate)

16760508

White to off-white solid powder

7

9

5

20

213

1

OC([C@H](CC/N=C(\N)/NO)N)=O.OC(C)=O.OC(C)=O

PQFYTZJPYBMTCT-QTNFYWBSSA-N

InChI=1S/C5H12N4O3.2C2H4O2/c6-3(4(10)11)1-2-8-5(7)9-12;2*1-2(3)4/h3,12H,1-2,6H2,(H,10,11)(H3,7,8,9);2*1H3,(H,3,4)/t3-;;/m0../s1

acetic acid;(2S)-2-amino-4-[[amino-(hydroxyamino)methylidene]amino]butanoic acid

nor-NOHA acetate; 1140844-63-8; nor-NOHA (acetate); acetic acid;(2S)-2-amino-4-[[amino-(hydroxyamino)methylidene]amino]butanoic acid; 2S-amino-4-[[(hydroxyamino)iminomethyl]amino]-butanoic acid, diacetate; Nor-NOHA Dihydrochloride; N-Omega-hydroxy-l-norarginine diacetate salt; nor-NOHA acetate?;

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 : ~200 mg/mL (~675.04 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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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