Biochemical; metalloporphyrin

Gossypol prevents the liberation of oxygen from oxyhemoglobin and exerts a hemolytic effect on erythrocytes. In excessive dosages of gossypol, an extreme burden is placed upon the respiratory and circulatory organs owing to the reduced oxygen carrying capacity of blood. Chromium protoporphyrin (CrPP) has been shown to either competitively suppress or to significantly ameliorate a variety of naturally occurring or experimentally induced forms of jaundice in animals and man. In this communication, a novel tissue dependent response to gossypol (50 micromol/kg bw) and gossypol in association with CrPP (50 micromol/kg bw) is described. Our results revealed that gossypol stimulated the hepatic, splenic, and renal delta-aminolevulinic acid synthase (ALA-S) activity, the heme biosynthetic enzyme, and simultaneous administration of CrPP and gossypol synergized the gossypol-mediated increase of ALA-S activity. Gossypol was found to be a potent stimulator of heme oxygenase (HMOX) activity in rat liver and kidney to varying degrees. This tissue response contrasted with that of the spleen, where gossypol decreased the activity of the enzyme. In consonance with the increased hepatic and renal HMOX activity, a marked increase was observed in total serum bilirubin concentration in gossypol treated rats. When rats were given CrPP simultaneously with gossypol, the gossypol mediated increase in hepatic and renal HMOX activity was effectively blocked. Furthermore, the increase in enzymatic activity was accomplished by a decline in the total microsomal protein content on gossypol administration. These findings emphasize the toxic effect of gossypol in eliciting increased heme degradation by stimulating HMOX activity in the liver and the kidney and the potential usefulness of CrPP in experimental and perhaps clinical conditions in which hyperbilirubinemia occurs [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

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

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.

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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).

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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)

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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).

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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

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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.

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