PDD4091

Glucose-6-phosphate dehydrogenase (G6PD)

PDD4091是一种新型的G6PD活性选择性抑制剂。

为了确定G6PD抑制是否会降低PH，我们首先确定了G6PD抑制剂的最大耐受剂量（PDD4091）。Hx小鼠的最大耐受剂量为15 mg kg - 1 day - 1，超过该剂量PDD4091可抑制左室功能。更重要的是，G6PD抑制剂PDD4091对Hx小鼠的治疗以剂量依赖的方式降低了升高的RVSP和RVEDP（图1，B和C，顶部面板）。PDD4091具有相当宽的治疗窗（0.01-15 mg kg - 1 day - 1）， EC50为0.26±0.10,0.58±0.36 mg kg - 1 day - 1均可降低RVSP和RVEDP。此外，PDD4091 （1.5 mg kg - 1 day - 1）对Hx和Hx + SU小鼠均有效降低了升高的RVSP和RVEDP（图1，B和C，底部面板）。与Nx和Nx + SU组相比，Hx和Hx + SU组富尔顿指数升高。G6PD抑制剂降低Hx和Hx + SU组富尔顿指数升高（表1）。此外，PDD4091 （1.5 mg kg−1天−1）治疗预先存在Hx + SU诱导PH的小鼠（n = 6）降低RVSP(从Hx + SU: 74±5.3降至Hx + SU + PDD4091: 41±3.3 mm Hg；P < 0.05)， RVEDP(从Hx + SU: 13±3到Hx + SU + PDD4091: 7±1 mm Hg；P < 0.05)，富尔顿指数(从Hx + SU: 0.4±0.01到Hx + SU + PDD4091: 0.3±0.01；P < 0.05)。PDD4091 （1.5 mg kg−1 day−1）使G6PD活性降低20%。[1]

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G6PD抑制Hx + SU小鼠的PA预收缩，减少PASMC生长，减少PA重塑。[1]
PA重塑是严重ph的标志。增生和抗凋亡的PA内皮细胞和pasmc有助于高血压重塑（Morrell等，2009）。之前，我们和其他人提出高血压患者和动物的PA中SMCs从分化表型转变为去分化表型，并有助于PA重塑(Zhou et al., 2009；Chettimada et al., 2015；Sahoo et al., 2016)。去分化的SMCs具有高增殖、迁移和分泌功能（Frismantiene et al., 2018）。既往研究表明，Hx + SU模型PH小鼠的PA重塑比Hx小鼠更严重（Vitali et al., 2014）。因此，我们在离体研究中确定G6PD抑制是否使PA松弛，在细胞培养中抑制暴露于Hx和SU的PASMCs的生长，并减少Hx + SU小鼠中PA的重塑。我们的结果显示PDD4091剂量依赖性松弛PA与KCl预收缩（图2A）。PDD4091 [1 μmol/l， EC50剂量（Hamilton et al., 2012）]作用于缺氧培养的PASMCs 48小时后，细胞数量减少（图2B）， Hx和Hx + SU诱导的细胞生长也减弱（图2C）。PDD4091 （1.5 mg kg - 1 day - 1）治疗Hx + SU小鼠3周后，闭塞性肺血管重构消失（图2D）。

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G6PD抑制Hx + SU对小鼠和人PASMCs肺中Cyp1a1和Sufu基因表达的影响。[1]
接下来,我们决定是否抑制G6PD活性降低的Cyp1a1和Sufu的表达,增加在肺高血压小鼠(图3 c)和Hx Hx +苏老鼠和人类PASMCs暴露在Hx +苏。治疗PDD4091(1.5毫克公斤−−1天1)为3周的老鼠和应用PDD4091(1μM)对人类PASMCs 48小时取消Hx + SU-induced Cyp1a1和Sufu表达在肺(图4,A和B)和在人类PASMCs(图4 c)。

人肺动脉平滑肌细胞在添加生长因子（SMGM-2平滑肌单股试剂盒）的平滑肌基础培养基中，于37℃、5% CO2下保存。当细胞达到约70%的汇合时，用0.05%的胰蛋白酶- edta在六孔板中传代培养，每孔约3 × 105个细胞。

Animal Models and Experimental Protocols. [1]
Male and female C57BL/6J mice (18–32 g) were purchased from the Jackson Laboratory and were randomly divided into four groups: normoxia (Nx), normoxia + Sugen5416 (Nx + SU), hypoxia (Hx), and hypoxia + Sugen5416 (Hx + SU) groups. Mice in the Nx group were placed in a normoxic (21% O2) environment, and mice in the Hx group were placed in a normobaric hypoxic chamber (10% O2) for 6 weeks (Joshi et al., 2020). Mice in the Nx + SU and Hx + SU groups received subcutaneous injection of SU5416 (20 mg/kg) once weekly during 3 weeks of Nx (21% O2) or Hx (10% O2) as previously described (Vitali et al., 2014). Mice in the drug treatment groups received daily subcutaneous injection of a novel G6PD inhibitor, N-[(3β,5α)-17-oxoandrostan-3-yl]sulfamide (PDD4091; 1.5 mg kg−1 day−1) (Hamilton et al., 2012), for 3 weeks. To determine whether PDD4091 reduces PH in a dose-dependent manner, mice were randomized to receive low-dose (0.15 mg kg−1 day−1), medium-dose (1.5 mg kg−1 day−1), or high-dose (15 mg kg−1 day−1) injections of PDD4091. Hx and Hx + SU mice are preclinical models of PH (Stenmark et al., 2009). At the end of the treatment period, hemodynamic measurements were performed, tissue (lungs and arteries) was harvested, and blood samples were collected. Data analysis was performed in a blinded fashion.

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Reduced Representation Bisulfite Sequencing. [1]
To determine DNA methylation status in lungs of Nx, Hx, Hx + SU, and Hx + PDD4091 mice, genomic DNA was isolated from lungs using the Qiagen All Prep DNA/RNA/miRNA Universal kit according to the manufacturer’s instructions. DNA was quantified using the NanoDrop and Qubit Fluorometer. Genomic DNA quality was assessed using the Agilent TapeStation. Reduced representation bisulfite sequencing library construction was performed with the Premium Reduced Representation Bisulfite Sequencing Kit following the manufacturer’s instructions. Libraries were sequenced on the HiSeq2500 with paired-end reads of 125 nt. Raw reads generated from the Illumina HiSeq2500 sequencer were de-multiplexed using bcl2fastq version 2.19.0. Quality filtering and adapter removal are performed using Trim Galore version 0.4.4_dev with the following parameters: “–paired–clip_R1 3–clip_R2 3–three_prime_clip_R1 2–three_prime_clip_R2 2” (http://www.bioinformatics.babraham.ac.uk/projects/ trim_galore/). Processed and cleaned reads were then mapped to the mouse reference genome (mg38) using Bismark version 0.19.0 with the following parameters: “–bowtie2–maxins 1000.” Differential methylation analysis was performed using methylKit version 1.4.0 within an R version 3.4.1 environment. Bismark alignments were processed via methylKit in the CpG context with a minimum quality threshold of 10. Coverage was normalized after filtering for loci with a coverage of at least five reads and no more than the 99.9th percentile of coverage values. The coverage was then normalized across samples, and the methylation counts were aggregated for 500-nt windows spanning the entire genome. A unified window set across samples was derived such that only windows with coverage by at least one sample per group were retained. Differential methylation analysis between conditional groups was performed using the χ2 test and applying a q-value (SLIM) threshold of 0.05 and a methylation difference threshold of 25%.

[1]. Inhibition of Glucose-6-Phosphate Dehydrogenase Activity Attenuates Right Ventricle Pressure and Hypertrophy Elicited by VEGFR Inhibitor + Hypoxia. J Pharmacol Exp Ther. May 1, 2021, 377 (2) 284-292.

Pulmonary hypertension (PH) is a disease of hyperplasia of pulmonary vascular cells. The pentose phosphate pathway (PPP)—a fundamental glucose metabolism pathway—is vital for cell growth. Because treatment of PH is inadequate, our goal was to determine whether inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, prevents maladaptive gene expression that promotes smooth muscle cell (SMC) growth, reduces pulmonary artery remodeling, and normalizes hemodynamics in experimental models of PH. PH was induced in mice by exposure to 10% oxygen (Hx) or weekly injection of vascular endothelial growth factor receptor blocker [Sugen5416 (SU); 20 mg kg−1] during exposure to hypoxia (Hx + SU). A novel G6PD inhibitor (N-[(3β,5α)-17-oxoandrostan-3-yl]sulfamide; 1.5 mg kg−1) was injected daily during exposure to Hx. We measured right ventricle (RV) pressure and left ventricle pressure-volume relationships and gene expression in lungs of normoxic, Hx, and Hx + SU and G6PD inhibitor–treated mice. RV systolic and end-diastolic pressures were higher in Hx and Hx + SU than normoxic control mice. Hx and Hx + SU decreased expression of epigenetic modifiers (writers and erasers), increased hypomethylation of the DNA, and induced aberrant gene expression in lungs. G6PD inhibition decreased maladaptive expression of genes and SMC growth, reduced pulmonary vascular remodeling, and decreased right ventricle pressures compared with untreated PH groups. Pharmacologic inhibition of G6PD activity, by normalizing activity of epigenetic modifiers and DNA methylation, efficaciously reduces RV pressure overload in Hx and Hx + SU mice and preclinical models of PH and appears to be a safe pharmacotherapeutic strategy. [1]

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In addition to arresting maladaptive gene expression in vascular cells of the PA wall and reducing cell growth in occlusive pulmonary arterial disease, PDD4091—a novel and selective inhibitor of G6PD activity (Hamilton et al., 2012)—dose-dependently relaxed precontracted PAs. We have previously shown that inhibition of G6PD activity with nonspecific inhibitors, such as 17-ketosteroids [dehydroepiandrosterone (DHEA) and epiandrosterone, a DHEA metabolite], and siRNA-mediated knockdown of G6pd elicit relaxation of precontracted pulmonary artery (Gupte et al., 2002) and reduce RV pressures in hypertensive rats (Chettimada et al., 2012, 2015). Therefore, these studies and our current findings collectively suggest that G6PD inhibition reduces the elevated RV pressures and PH in Hx and Hx + SU mice by dilating PAs and reducing PA remodeling. In conclusion, our results collectively demonstrate that G6PD activity is an important contributor to differential DNA methylation, maladaptive gene expression, and remodeling of PA in Hx and Hx + SU mice. The inhibition of G6PD activity by pharmacologic manipulations abrogated pulmonary vascular remodeling and improved the hemodynamics in mouse models of PH. Therefore, G6PD inhibitor N-[(3β,5α)-17-oxoandrostan-3-yl]sulfamide might be employed in the future as a pharmacotherapeutic agent to treat different forms of PH. [1]

C19H32N2O3S

368.533984184265

368.21

C, 61.92; H, 8.75; N, 7.60; O, 13.02; S, 8.70

1373651-41-2

1373651-41-2;

Solid powder

C[C@@]12[C@@]3([C@]([C@]4([C@](C)(CC3)C(=O)CC4)[H])(CC[C@]1(C[C@@H](NS(N)(=O)=O)CC2)[H])[H])[H]

QLIJDTTVUNSTPX-LUJOEAJASA-N

InChI=1S/C19H32N2O3S/c1-18-9-7-13(21-25(20,23)24)11-12(18)3-4-14-15-5-6-17(22)19(15,2)10-8-16(14)18/h12-16,21H,3-11H2,1-2H3,(H2,20,23,24)/t12-,13-,14-,15-,16-,18-,19-/m0/s1

N-[(3beta,5alpha)-17-Oxoandrostan-3-yl]sulfamide

PDD4091; PDD 4091; PDD-4091

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。