Arginase

nor-NOHA（0.1-1 mM，72 h）在缺氧（1.5% O2）条件下以剂量依赖性方式诱导 K562 细胞凋亡[1]。nor-NOHA（1 mM，72 h）可以减弱 K562 或 KCL22 细胞中缺氧介导的伊马替尼耐药性[1]。

nor-NOHA（100 mg/kg，静脉注射，一次）可显著减少雄性 Sprague-Dawley 大鼠的梗死面积 [2]。nor-NOHA（100 mg/kg，静脉注射，一次）可增加雄性 Sprague-Dawley 大鼠血浆瓜氨酸和亚硝酸盐水平，并降低血浆鸟氨酸水平 [2]

精氨酸酶活性测定[1]
精氨酸酶活性经修饰后按描述进行分析。计数细胞，等量细胞在50μl裂解缓冲液（PBS加1mM EDTA， 0.1% Triton X−100和蛋白酶抑制剂）中裂解，4℃下14000 g离心15分钟。将上清液与50μl新配制的活化缓冲液（10mM MnCl2, 50mM Tris-HCl pH7.5）和50μl 0.5M精氨酸混合，在56℃下加热10分钟。然后，加入800μl酸性溶液(H2SO4 （96%)/H3PO4 (85%)/H2O, 1/3/7, v/v/v）和25μl 9% α -异硝基丙烯酮（乙醇），在100℃下加热15 min。让这种混合物在黑暗中显色。最后将250μl转移到96孔板上，在550nm处测量外径。

Seahorse分析仪测量细胞呼吸[1]
0.1x106 K562细胞每孔镀于聚L -赖氨酸包被的XF - 24孔细胞培养微孔板上，XF Assay培养基中添加4.5 g/L葡萄糖和1mM丙酮酸钠。将细胞自旋固定在200g的微孔板上1分钟。细胞耗氧率（OCR）、细胞外酸化率（ECAR）和光子产生率（PPR）采用Seahorse Bioscience公司的XF24分析仪进行测定。根据制造商的说明进行测量，使用低霉素，羰基氰化物- 4 -（三氟甲氧基）苯腙（FCCP）和鱼藤酮&抗霉素A (R/A；所有来自Sigma - Aldrich)在指定的浓度。数据分析使用Seahorse XF软件。

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]

[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.

Nomega-hydroxy-nor-l-arginine is a L-alpha-amino acid. N-Hydroxy-nor-L-arginine (nor-NOHA) is under investigation in clinical trial NCT02009527 (Arginase Inhibition in Ischemia-reperfusion Injury).

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Cancer cells, including in chronic myeloid leukemia (CML), depend on the hypoxic response to persist in hosts and evade therapy. Accordingly, there is significant interest in drugging cancer-specific hypoxic responses. However, a major challenge in leukemia is identifying differential and druggable hypoxic responses between leukemic and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme of the urea cycle, is overexpressed in CML but not normal progenitors. ARG2 is a target of the hypoxia inducible factors (HIF1-α and HIF2-α), and is required for the generation of polyamines which are required for cell growth. We therefore explored if the clinically-tested arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) would be effective against leukemic cells under hypoxic conditions. Remarkably, nor-NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxia but not normoxia. Co-treatment with nor-NOHA overcame hypoxia-mediated resistance towards BCR-ABL1 kinase inhibitors. While nor-NOHA itself is promising in targeting the leukemia hypoxic response, we unexpectedly found that its anti-leukemic activity was independent of ARG2 inhibition. Genetic ablation of ARG2 using CRISPR/Cas9 had no effect on the viability of leukemic cells and their sensitivity towards nor-NOHA. This discrepancy was further evidenced by the distinct effects of ARG2 knockouts and nor-NOHA on cellular respiration. In conclusion, we show that nor-NOHA has significant but off-target anti-leukemic activity among ARG2-expressing hypoxic cells. Since nor-NOHA has been employed in clinical trials, and is widely used in studies on endothelial dysfunction, immunosuppression and metabolism, the diverse biological effects of nor-NOHA must be cautiously evaluated before attributing its activity to ARG inhibition.[1]

C7H16N4O5

236.225741386414

236.112

C, 35.59; H, 6.83; N, 23.72; O, 33.86

2250019-93-1

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

131648256

White to off-white solid powder

171

6

7

5

16

213

1

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

RYUGHGOGNIYFKU-DFWYDOINSA-N

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

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

Nor NOHA monoacetate; 2250019-93-1; nor-NOHA (monoacetate); nor-NOHA monoacetate; N-OMega-hydroxy-L-norarginine acetate salt; AKOS032962868; HY-112885B; N-OMega-hydroxy-L-norarginineacetatesalt;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~423.32 mM)
H2O : ~50 mg/mL (~211.66 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (10.58 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (10.58 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (10.58 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 100 mg/mL (423.32 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶 (<60°C).

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。