Phosphoramidon Disodium is a metalloprotease inhibitor. Phosphoramidon inhibits endothelin-converting enzyme (ECE), neutral endopeptidase (NEP), and angiotensin-converting enzyme (ACE) with IC₅₀ values of 3.5, 0.034, and 78 μM, respectively.

IC50: 3.5 μM (ECE), 34 nM (NEP), 78 μM (ACE)^[1]

Phosphoramidon is a naturally occurring glycopeptide first isolated from a strain of Streptomyces tanashiensis. It has a unique chemical structure featuring a phosphoramidate linkage between a-L-rhamnose and L-leucineL-tryptophan. As a microbial metabolite, phosphoramidon exhibits potent inhibitory activity against thermolysin, a zinc endopeptidase isolated from Bacillus thermoproteolyticus (K_i=32 nM)^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Intranasal administration of phosphoramidon produces significantly elevated cerebral Aβ levels in wild-type mice. Furthermore, intranasal phosphoramidon administration in double knockout mice lacking NEP and NEP2 also shows increased levels of Aβ40^[3]. Phosphoramidon blocks the formation of endothelin-1 (ET-1), a proinflammatory mediator implicated in the pathogenesis of a variety of lung diseases. Phosphoramidon significantly reduces LPS-induced pulmonary inflammation as measured by lung histology, neutrophil content of bronchoalveolar lavage (BAL) fluid, percent tumor necrosis factor receptor 1 (TNFR1)-labeled BAL macrophages, and alveolar septal cell apoptosis^[4]. Phosphoramidon significantly decreased ET-1 levels, causing a concomitant big ET-1 increase and dose-dependently attenuated indomethacin-induced gastric mucosal damage^[5].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

587.47

C₂₃H₃₂N₃Na₂O₁₀P

164204-38-0

磷酰胺二钠

Room temperature in continental US; may vary elsewhere.

4deg;C, sealed storage, away from moisture

*In solvent : -80deg;C, 6 months; -20deg;C, 1 month (sealed storage, away from moisture)

In Vitro:;

DMSO : 100 mg/mL (170.22 mM; Need ultrasonic)

H₂O : 100 mg/mL (170.22 mM; Need ultrasonic)

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂：;10% DMSO ;; 40% PEG300 ;; 5% Tween-80 ;; 45% saline

  Solubility: ≥ 2.5 mg/mL (4.26 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.26 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂：;10% DMSO ;; 90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (4.26 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.26 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂：;10% DMSO ;; 90% corn oil

  Solubility: ≥ 2.5 mg/mL (4.26 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.26 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Kukkola PJ, et al. Differential structure-activity relationships of phosphoramidon analogues for inhibition of three metalloproteases: endothelin-converting enzyme, neutral endopeptidase, and angiotensin-converting enzyme.J CardiovascPharmacol. 1995;26Suppl 3:S65-8.

  [2]. Sun Q, et al. Synthesis and enzymatic evaluation of phosphoramidon and its β anomer: Anomerization of α-l-rhamnose triacetate upon phosphitylation.Bioorg Med Chem. 2013 Nov 1;21(21):6778-87.

  [3]. Hanson LR, et al. Intranasal phosphoramidon increases beta-amyloid levels in wild-type and NEP/NEP2-deficient mice.J MolNeurosci. 2011 Mar;43(3):424-7.

  [4]. Bhavsar TM, et al. Phosphoramidon, an endothelin-converting enzyme inhibitor, attenuates lipopolysaccharide-induced acute lung injury.Exp Lung Res. 2008 Mar;34(3):141-54.

  [5]. Matsumaru K, et al. Phosphoramidon, an inhibitor of endothelin-converting enzyme, prevents indomethacin-induced gastric mucosal damage in rats.Life Sci. 1998;62(7):PL79-84.

The K_i values are determined in a 50 mM Tris-HCl, 10 mM CaCl₂ buffer (pH 7.5) with FA-Gly-Leu-NH2 (FAGLA) as a substrate by using an Agilent 8453 UV-vis spectrophotometer in triplicate. Henderson plots are employed for the calculation of K_i values. A mixture of buffer (970 μL), phosphoramidon (0-80 nM, 20 μL), and thermolysin (40 nM, 10 μL) is incubated at 25 C for 15 min in a cuvette (2 mL). A solution of FAGLA (0.1-0.5 mM, 1.0 mL) in Tris buffer pH 7.5 is added into the cuvette. The absorbance decrease upon cleavage of FAGLA by thermolysinis recorded at 340 nm wavelength for 5 min. The concentration of thermolysinis determined from the molar extinction coefficient^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
Phosphoramidon is dissolved in phosphate-buffered saline (PBS+1 mM ascorbic acid) at a concentration of 30 mM. Anesthetized mice are placed on their backs and eight 3-μL drops of phosphoramidon solution are administered to alternating nares every 2 min. This is done once per day for 5 days. Mice are euthanized under anesthesia for tissue collection 2 h post phosphoramidon administration on day 5. Control mice are treated with intranasal PBS vehicle solution alone. Brains are removed and dissected into the desired brain regions before being homogenized in 5 M guanidine HCl to extract total Aβ. After centrifugation (16,000×g), the supernatants are diluted tenfold and Aβ (1-42 and 1-40) is quantified by specific ELISA.
Rats^[4]
Animals are treated with phosphoramidon either intraperitoneally or intratracheally via nebulization. To examine the effects of intraperitoneal administration, animals are injected with 0.5 mg of phosphoramidon dissolved in 0.5 mL of phosphatebuffered saline (PBS). For the nebulization studies, animals are placed in an exposure chamber and treated for 1 hour with an aerosol composed of a 0.1% solution of phosphoramidon dissolved in distilled water. The aerosolized phosphoramidonis delivered through a ceiling port via a Misty-Ox nebulizer attached to an air compressor. Negative pressure is applied by a blower attached to a secondary outflow port to insure proper circulation of the aerosol.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Kukkola PJ, et al. Differential structure-activity relationships of phosphoramidon analogues for inhibition of three metalloproteases: endothelin-converting enzyme, neutral endopeptidase, and angiotensin-converting enzyme.J CardiovascPharmacol. 1995;26Suppl 3:S65-8.

  [2]. Sun Q, et al. Synthesis and enzymatic evaluation of phosphoramidon and its β anomer: Anomerization of α-l-rhamnose triacetate upon phosphitylation.Bioorg Med Chem. 2013 Nov 1;21(21):6778-87.

  [3]. Hanson LR, et al. Intranasal phosphoramidon increases beta-amyloid levels in wild-type and NEP/NEP2-deficient mice.J MolNeurosci. 2011 Mar;43(3):424-7.

  [4]. Bhavsar TM, et al. Phosphoramidon, an endothelin-converting enzyme inhibitor, attenuates lipopolysaccharide-induced acute lung injury.Exp Lung Res. 2008 Mar;34(3):141-54.

  [5]. Matsumaru K, et al. Phosphoramidon, an inhibitor of endothelin-converting enzyme, prevents indomethacin-induced gastric mucosal damage in rats.Life Sci. 1998;62(7):PL79-84.