Biochemical; metalloporphyrin

棉酚会阻止氧合血红蛋白释放氧气，并对红细胞产生溶血作用。在过量服用棉酚的情况下，由于血液的携氧能力降低，呼吸和循环器官会承受极大的负担。铬原卟啉（CrPP）已被证明可以竞争性抑制或显著改善动物和人类中各种自然发生或实验诱导的黄疸形式。在这篇文章中，描述了一种对棉酚（50微摩尔/千克体重）和与CrPP（50微毫升/千克重量）相关的棉酚的新的组织依赖性反应。我们的研究结果表明，棉酚刺激了肝脏、脾脏和肾脏的δ-氨基酮戊酸合酶（ALA-S）活性，血红素生物合成酶，同时服用CrPP和棉酚会协同棉酚介导的ALA-S活性的增加。发现棉酚在不同程度上是大鼠肝脏和肾脏血红素加氧酶（HMOX）活性的强效刺激剂。这种组织反应与脾脏的反应形成鲜明对比，在脾脏中，棉酚降低了酶的活性。随着肝脏和肾脏HMOX活性的增加，棉酚治疗的大鼠血清总胆红素浓度显著增加。当大鼠同时服用CrPP和棉酚时，棉酚介导的肝脏和肾脏HMOX活性的增加被有效阻断。此外，酶活性的增加是通过服用棉酚后微粒体总蛋白含量的下降来实现的。这些发现强调了棉酚通过刺激肝脏和肾脏中的HMOX活性引发血红素降解增加的毒性作用，以及CrPP在发生高胆红素血症的实验和临床条件下的潜在用途[1]。

Experimental Animals [1]
Male Wistar Rats of weight range 150–200 g from our laboratory maintained colony were used as experimental models in the investigation. Only healthy animals were taken in individual cages having raised wire mesh floors. The animals were kept on fasting for 20 h but had free access to water. After 20 h the animals were divided into four groups with eight animals per group.

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Animal Treatment [1]
Group I: Animals of this group were treated as control and were administered equivalent amount of saline subcutaneously.
Group II: 50 µmol/kg bw of gossypol was given subcutaneously to animals in this group.
Group III: Animals in this group were administered 50 µmol/kg bw of CrPP subcutaneously.
Group IV: Animals in this group were given 50 µmol/kg bw of gossypol along with 50 µmol/kg bw of CrPP subcutaneously.
The solutions of gossypol and CrPP for administration were prepared fresh in small volumes, in dark, because of their photosensitivity and unstable nature. Metalloporphyrins require an alkaline media for dissolving i.e., for making 1 mL solution, the porphyrin was dissolved in 0.2 mL of 0.02 N NaOH and the volume was then made up by potassium phosphate buffer (pH 7.4). Stock solution of gossypol was prepared in 95% ethanol. The gossypol concentration was determined by measuring absorbance at 372 nm and using a value of € = 1.48 × 104 L mol−1cm−1 (Finaly et al., Citation[[1993]]).

[1]. Effect of gossypol in association with chromium protoporphyrin on heme metabolic enzymes. Artif Cells Blood Substit Immobil Biotechnol. 2004 Feb;32(1):159-72.
[2]. Protoporphyrin IX: the Good, the Bad, and the Ugly. J Pharmacol Exp Ther. 2016;356(2):267-275.

An important concept, which has emerged from these studies, is that ALA-S activity is regulated in a tissue specific manner. The biochemical basis for the observed differences in regulation of the enzyme may, atleast be partially apparent in terms of the physiology of these tissues. We envision that heme synthesis in liver cells must be responsive to external stimuli, since the detoxification or metabolism of xenobiotics and endogenous steroids require the synthesis of hemo protein such as cytochrome P-450. Under our conditions, we have observed that gossypol increases the activity of ALA-S in the liver, spleen, and kidney. We have unravelled that the combined effect of gossypol and CrPP results in a further enhancement of intracellular heme concentration i.e., co-administration causes a further substantial enhancement of the activity. It has been suggested that there is a competition for intracellular heme for the synthesis of various hemo proteins, including the microsomal cytochromes, P-450 and b5, the mitochondrial cytochromes, catalase and tryptophan pyrrolase, and that the remaining uncommitted to apoprotein serves either to increase the synthesis of ALA-S (and thus enhance net heme synthesis) or is catabolized to bile pigments via heme oxygenase. We have observed that the activity of ALA-S is stimulated in the tissues under our experimental conditions. The mechanisms through which xenobiotics induce elevations in ALA-S activity have not yet been determined. An inducer may act directly on the gene to increase the rate of its transcription. It may be interpreted that gossypol appears to increase the activity of the enzyme by lowering the concentration of heme. It might induce de novo synthesis of ALA-S rather than activate the existing enzyme system. Given the wide spectrum activity of gossypol, the finding reported here that gossypol exerts stimulatory effects on the activity of HMOX, which is considerably antagonized when CrPP is co-administered, may have significant biological implications. Gossypol exhibits a potent ability to increase HMOX activity in the liver and kidney of treated rats. This finding suggests that, for the most part, the biological basis for the development of hyperbilirubinemia by gossypol may be related to the enhanced rate of enzymic conversion of hemoglobin to bilirubin. The cellular basis for the gossypol-mediated increase in the HMOX activity is not clear. It may involve any of the following factors: (a) the intermediate action of the hemoglobin released in the course of hemolysis, (b) the direct action of the parent compound, (c) the activity of the reactive metabolites of gossypol. The present study, however, does not permit a preference for any of the suggested possibilities. Thus biochemical effects underlying the observation remain obscure. Because of the hemolytic effect of gossypol in erythrocytes, clearly a potential exists for the stimulatory action of hemoglobin released in the course of the hemolysis of erythrocytes on the activity of microsomal HMOX. This, in turn, could significantly contribute to an increase in the serum levels of bilirubin. These concepts are consistent with the findings of the following investigations; the induction of HMOX activity in the rat kidney by hemoglobin infusion (Pimstone et al., Citation[[1971]]); the promotion of an increase in HMOX activity by methemalbumin in the rat liver (Tenhunen et al., Citation[[1970]]); and the development of postparturition hyperbilirubinemia which accompanies increased HMOX activity in the liver and the kidney in rats (Maines and Kappas, Citation[[1978b]]). Similarly, the possibility of a direct HMOX inducing action of gossypol and/or its metabolites cannot be dismissed. The effectiveness of CrPP in subsiding the gossypol mediated increase in activity of HMOX suggests the utility of this metalloporphyrin for the suppression of the enzyme activity in various hemolytic conditions. The gossypol mediated induction of hepatic and renal HMOX activity is accompanied by the development of hyperbilirubinemia. Thus, we may postulate that, provided that the xenobiotic material administered reaches the target organs in sufficient quantities, the ability of these substances to act as stimulators or inhibitors of the enzyme is probably contingent upon their metabolism by the cells of the tissue, wherein, the effect on HMOX activity might be explored in terms of the degree to which a particular tissue can maintain its metabolism in response to its being subjected to such alterations. [1]

C34H32CLCRN4O4

648.1

647.152

C, 63.01; H, 4.98; Cl, 5.47; Cr, 8.02; N, 8.64; O, 9.87

41628-83-5

120389-54-0

134129038

Typically exists as solid at room temperature

1.998

133.66

2

8

8

44

1020

0

C=CC1=C2C=C3C(C)=C(CCC(=O)O)C4=CC5=NC(=CC6=NC(=CC(=C1C)N2[Cr](Cl)N34)C(C=C)=C6C)C(C)=C5CCC(=O)O CC1C(C=C)=C2C=C3C(C)=C(CCC(O)=O)C4=CC5=NC(=CC6=NC(=CC=1N2[Cr](Cl)N43)C(C=C)=C6C)C(C)=C5CCC(O)=O |c:20,24,t:16|CopyCopied

KGPQQEWHGUXPBK-UHFFFAOYSA-K

InChI=1S/C34H34N4O4.ClH.Cr/c1-7-21-17(3)25-13-26-19(5)23(9-11-33(39)40)31(37-26)16-32-24(10-12-34(41)42)20(6)28(38-32)15-30-22(8-2)18(4)27(36-30)14-29(21)35-25;;/h7-8,13-16H,1-2,9-12H2,3-6H3,(H4,35,36,37,38,39,40,41,42);1H;/q;;+3/p-3

3-[18-(2-carboxyethyl)-7,12-bis(ethenyl)-3,8,13,17-tetramethylporphyrin-21,22-diid-2-yl]propanoic acid;chlorochromium(2+)

Cr(III) Protoporphyrin IX Chloride; 41628-83-5; 3-[18-(2-carboxyethyl)-7,12-bis(ethenyl)-3,8,13,17-tetramethylporphyrin-21,22-diid-2-yl]propanoic acid;chlorochromium(2+); Cr(III)ProtoporphyrinIXChloride

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;
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。