Endogenous Metabolite; Metalloporphyrin

Zinc protoporphyrin (Zn(II)-protoporphyrin IX; 5 μM; 72 hours) promotes late hypoxia and a rise in the number of mid-cell cells from 10.9% in the mold to 30.4% after 72 hours [3]. Zinc protoporphyrin (2.5, 5 μM; 48 or 72 hours) exhibits an inhibitory/cytotoxic effect on tumor cells [3]. Zinc protoporphyrin (2.5, 5 μM; 48 or 72 hours) induces a decrease in the quantity and time of cells in the G1 phase of the cell cycle [3]. Zinc protoporphyrin (1.25-40 μM; 48 ) induces the accumulation of azo (active) caspase-3 [3].

A dose-dependent anti-tumor action is effected by zinc protoporphyrin (12.5, 25, 50 mg/kg intraperitoneally; 12.5, 50 mg/kg intraperitoneally; from day 7 to day 19) which delays the growth of tumors [3].

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Zn(II)PPIX induces antitumor effects in a murine C-26 model [3]
BALB/c mice inoculated with C-26 cells were treated with Zn(II)PPIX for 7 consecutive days. HO-1 inhibitor was administered either intraperitoneally (i.p.) or per os and the tumor volume was monitored every second day, starting from day 7 after inoculation of tumor cells. Zn(II)PPIX exerted dose-dependent antitumor effects manifested by the retardation of tumor growth. A statistical significance was reached on days 17 and 19 for Zn(II)PPIX administered at a dose of 25 mg/kg either i.p. or orally (Fig. 4A and 4B). A stronger effect was observed when Zn(II)PPIX was administered i.p. at a dose of 50 mg/kg, where a statistically significant retardation of tumor growth was observed on days 13–19, as compared with controls (Fig. 4A).

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Zn(II)PPIX does not affect antitumor effects of chemotherapeutics [3]
In further studies the influence of Zn(II)PPIX (50 mg/kg) on the in vivo antitumor effects of chemotherapeutics was evaluated. Three different cell lines syngeneic with BALB/c or C57Bl/6 mice were used, namely C-26, B16F10 melanoma and EMT6 breast adenocarcinoma. The following chemotherapeutics were used in these studies: 5-fluorouracil at a dose of 50 mg/kg (5-FU, for C-26), cisplatin at a dose of 5 mg/kg (for B16F10) and doxorubicin at a dose of 7,5 mg/kg (for EMT6 cells). Although in in vivo studies administration of Zn(II)PPIX (at a dose of 50 mg/kg) resulted in retardation of tumor growth (although in EMT6 tumors the effect was only modest) there was no further potentiation of the antitumor effects by concomitant administration of Zn(II)PPIX together with chemotherapeutics (Fig. 6A–C). Only for Zn(II)PPIX and 5-FU a slightly stronger effect was observed for the combination treatment, but the difference between the combination and single drug-treated tumors did not reach statistical significance (Fig. 5A). Remarkably, the combined administration of Zn(II)PPIX with either cisplatin or 5-FU resulted in significant weight loss (Fig. 6D and 6E). This effects was not observed in mice treated with Zn(II)PPIX and doxorubicin (Fig. 6F). No treatment-related mortality was observed in these experiments.

Apoptosis analysis [3]
Cell Types: C-26 Cell
Tested Concentrations: 5 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: After 72 hrs (hours), the proportion of late apoptotic and necrotic cells increased from 10.9% in the control group to 30.4%.

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Cytotoxicity assay [3]
Cell Types: C-26 and MDA-MB231 Cell
Tested Concentrations: 1.25, 2.5, 5, 10, 20, 40 μM
Incubation Duration: 48 or 72 hrs (hours)
Experimental Results: Cytostatic/cytotoxic effects on tumor cells .

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Cell cycle analysis[3]
Cell Types: C-26 Cell
Tested Concentrations: 2.5, 5 μM
Incubation Duration: 48 or 72 hrs (hours)
Experimental Results: Dose- and time-dependent reduction of cells resulting in G1 phase of the cell cycle.

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Western Blot Analysis[3]
Cell Types: C-26 Cell
Tested Concentrations: 1.25, 2.5, 5, 10, 20, 40 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Result in accumulation of cleaved (active) caspase-3.

Animal/Disease Models: balb/c (Bagg ALBino) mouse were inoculated with C-26 cells [3]
Doses: intraperitoneal (ip) injection 12.5, 25, 50 mg/kg; 12.5, 50 mg/kg, oral
Route of Administration: intraperitoneal (ip) injection or oral administration; from day 7 to Results on day 19: exerted a dose-dependent anti-tumor effect, manifested by delayed tumor growth.

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Tumor treatment and monitoring [3]
For assessment of antitumor activity of Zn(II)PPIX in vivo, exponentially growing C-26 were harvested, re-suspended in PBS medium to the appropriate concentration, and injected at the dose of 1 × 105 cells per mouse into the footpad of the right hind limb of experimental mice. Tumor cell viability measured by trypan blue exclusion was always above 95%. For in vivo treatment Zn(II)PPIX was dissolved in DMSO and further diluted in 0.9% NaCl to required concentrations. Final DMSO concentration was always less then 0.1%. Zn(II)PPIX was distributed intraperitoneally at doses from 12.5 to 50 mg per kg of body weight or orally at doses from 11 to 22 mg per kg of body weight. Control animals received 0.1% DMSO solution in 0.9% NaCl i.p. or orally.
For in vivo experiments evaluating the effectiveness of combine treatment using Zn(II)PPIX and chemotherapeutics, exponentially growing C-26, EMT6 and B16F10 cells were injected at the dose of 1 × 105, 1 × 105 and 1 × 106 cells per mouse, respectively into the footpad of the right hind limb of experimental mice. Zn(II)PPIX treatment (50 mg/kg i.p.) was started on the day 7 after inoculation of tumor cells and continued for 7 consecutive days. First dose of HO-1 inhibitor was administered 1 h before each of the chemotherapeutics to eliminate any possible interactions (such as neutralization) between drugs. Cisplatin at the dose of 7.5 mg/kg i.p., 5-FU – 50 mg/kg i.p., or doxorubicin – 7.5 mg/kg i.p. were administered at a single dose on the day 7th after inoculation of tumor cells.
For in vivo experiments exponentially growing B1 and B5E cells were injected at a dose of 1 × 106 cells per mouse into the footpad of the right hind limb of experimental mice. Cisplatin was administered i.p. at a single dose of 7.5 mg/kg on the 7th day after inoculation of tumor cells. Zn(II)PPIX treatment (50 mg/kg i.p.) was started on the day 7 after inoculation of tumor cells and continued for 7 consecutive days. First dose of HO-1 inhibitor was administered 1 h before the chemotherapeutic.

[1]. Protective Effect of Phloroglucinol on Oxidative Stress-Induced DNA Damage and Apoptosisthrough Activation of the Nrf2/HO-1 Signaling Pathway in HaCaT Human Keratinocytes. Mar Drugs. 2019 Apr 13;17(4).

[2]. Diagnostic utility of zinc protoporphyrin to detect iron deficiency in Kenyan pregnant women. BMC Med. 2014 Nov 26;12:229.

[3]. Zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, demonstrates potent antitumor effects but is unable to potentiate antitumor effects of chemotherapeutics in mice. BMC Cancer. 2008 Jul 11;8:197.

Zinc Protoporphyrin is an ionized form of the tetrazole protoporphryin IX that contains zinc in the metal ion binding pocket. Zinc protoporphyrin (ZPP) is formed in circulating red blood cells (RBC) either in the presence of lead or in the absence of sufficient iron, as both of these conditions inhibit the insertion of a ferrous ion into protoporphryin IX to form heme. Therefore, elevated levels of ZPP is associated with heme deficiency. Elevated RBC molar ratios of ZPP to heme are indicative of multiple pathologies including lead poisoning, iron deficiency, protoporphyria, and various types of anemia.

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Phloroglucinol (PG) is a component of phlorotannins, which are abundant in marine brown alga species. Recent studies have shown that PG is beneficial in protecting cells from oxidative stress. In this study, we evaluated the protective efficacy of PG in HaCaT human skin keratinocytes stimulated with oxidative stress (hydrogen peroxide, H2O2). The results showed that PG significantly inhibited the H2O2-induced growth inhibition in HaCaT cells, which was associated with increased expression of heme oxygenase-1 (HO-1) by the activation of nuclear factor erythroid 2-related factor-2 (Nrf2). PG remarkably reversed H2O2-induced excessive ROS production, DNA damage, and apoptosis. Additionally, H2O2-induced mitochondrial dysfunction was related to a decrease in ATP levels, and in the presence of PG, these changes were significantly impaired. Furthermore, the increases of cytosolic release of cytochrome c and ratio of Bax to Bcl-2, and the activation of caspase-9 and caspase-3 by the H2O2 were markedly abolished under the condition of PG pretreatment. However, the inhibition of HO-1 function using zinc protoporphyrin/zinc protoporphyrin, a HO-1 inhibitor, markedly attenuated these protective effects of PG against H2O2. Overall, our results suggest that PG is able to protect HaCaT keratinocytes against oxidative stress-induced DNA damage and apoptosis through activating the Nrf2/HO-1 signaling pathway.[1]

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Background: Iron-deficient erythropoiesis results in excess formation of zinc protoporphyrin (ZPP), which can be measured instantly and at low assay cost using portable haematofluorometers. ZPP is used as a screening marker of iron deficiency in individual pregnant women and children, but also to assess population iron status in combination with haemoglobin concentration. We examined associations between ZPP and disorders that are common in Africa. In addition, we assessed the diagnostic utility of ZPP (measured in whole blood and erythrocytes), alone or in combination with haemoglobin concentration, in detecting iron deficiency (plasma ferritin concentration <15 μg/L).
Methods: Single blood samples were collected from a population sample of 470 rural Kenyan women with singleton pregnancies, gestational age 13 to 23 weeks, and haemoglobin concentration ≥90 g/L. We used linear regression analysis to assess associations between ZPP and iron markers (including anaemia), factors known or suspected to be associated with iron status, inflammation markers (plasma concentrations of C-reactive protein and α 1-acid glycoprotein), infections (Plasmodium infection, HIV infection), and other disorders (α(+)-thalassaemia, plasma concentrations of total bilirubin, and lactate dehydrogenase). Subsequently, in those without inflammation, Plasmodium infection, or HIV infection, we used logistic discriminant analysis and examined receiver operating characteristics curves with corresponding area-under-the-curve to assess diagnostic performance of ZPP, alone and in combination with haemoglobin concentration.
Results: Individually, whole blood ZPP, erythrocyte ZPP, and erythrocyte protoporphyrin had limited ability to discriminate between women with and without iron deficiency. Combining each of these markers with haemoglobin concentration had no additional diagnostic value. Conventional cut off points for whole blood ZPP (>70 μmol/mol haem) resulted in gross overestimates of the prevalence of iron deficiency.
Conclusions: Erythrocyte ZPP has limited value to rule out iron deficiency when used for screening in conditions with a low prevalence (e.g., 10%). ZPP is of unreliable diagnostic utility when discriminating between pregnant women with and without iron deficiency. Based on these findings, guidelines on the use of ZPP to assess iron status in individuals or populations of pregnant women need review.[2]

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Background: HO-1 participates in the degradation of heme. Its products can exert unique cytoprotective effects. Numerous tumors express high levels of HO-1 indicating that this enzyme might be a potential therapeutic target. In this study we decided to evaluate potential cytostatic/cytotoxic effects of zinc protoporphyrin IX (Zn(II)PPIX), a selective HO-1 inhibitor and to evaluate its antitumor activity in combination with chemotherapeutics.
Methods: Cytostatic/cytotoxic effects of Zn(II)PPIX were evaluated with crystal violet staining and clonogenic assay. Western blotting was used for the evaluation of protein expression. Flow cytometry was used to evaluate the influence of Zn(II)PPIX on the induction of apoptosis and generation of reactive oxygen species. Knock-down of HO-1 expression was achieved with siRNA. Antitumor effects of Zn(II)PPIX alone or in combination with chemotherapeutics were measured in transplantation tumor models.
Results: Zn(II)PPIX induced significant accumulation of reactive oxygen species in tumor cells. This effect was partly reversed by administration of exogenous bilirubin. Moreover, Zn(II)PPIX exerted potent cytostatic/cytotoxic effects against human and murine tumor cell lines. Despite a significant time and dose-dependent decrease in cyclin D expression in Zn(II)PPIX-treated cells no accumulation of tumor cells in G1 phase of the cell cycle was observed. However, incubation of C-26 cells with Zn(II)PPIX increased the percentage of cells in sub-G1 phase of the cells cycle. Flow cytometry studies with propidium iodide and annexin V staining as well as detection of cleaved caspase 3 by Western blotting revealed that Zn(II)PPIX can induce apoptosis of tumor cells. B16F10 melanoma cells overexpressing HO-1 and transplanted into syngeneic mice were resistant to either Zn(II)PPIX or antitumor effects of cisplatin. Zn(II)PPIX was unable to potentiate antitumor effects of 5-fluorouracil, cisplatin or doxorubicin in three different tumor models, but significantly potentiated toxicity of 5-FU and cisplatin.
Conclusion: Inhibition of HO-1 exerts antitumor effects but should not be used to potentiate antitumor effects of cancer chemotherapeutics unless procedures of selective tumor targeting of HO-1 inhibitors are developed.[3]

C34H32N4O4-2.ZN+2

626.03228

624.171

C, 65.23; H, 5.15; N, 8.95; O, 10.22; Zn, 10.44

15442-64-5

455799

Brown to black solid powder

1128.5ºC at 760mmHg

636.3ºC

0mmHg at 25°C

3.609

2

8

8

43

1010

0

CC1=C(C2=CC3=NC(=CC4=C(C(=C([N-]4)C=C5C(=C(C(=N5)C=C1[N-]2)C)C=C)C)C=C)C(=C3CCC(=O)O)C)CCC(=O)O.[Zn+2]

FUTVBRXUIKZACV-UHFFFAOYSA-L

InChI=1S/C34H34N4O4.Zn/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);/q;+2/p-2

zinc;3-[18-(2-carboxyethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoic acid

Zinc protoporphyrin; 15442-64-5; PROTOPORPHYRINATO ZINC; Zinc protoporphyrin-9; PROTOPORPHYRIN IX CONTAINING ZN; 3-[(2Z,7Z,11Z,16Z)-5-(2-carboxyethyl)-15,20-diethenyl-4,10,14,19-tetramethyl-21,23,24,25-tetraaza-22-zincahexacyclo[9.9.3.1(3),?.1(1)(3),(1)?.0?,(2)(3).0(1)?,(2)(1)]pentacosa-1(20),2,4,6(25),7,9,11,13(24),14,16,18-undecaen-9-yl]propanoic acid; MFCD00011612;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~20.83 mg/mL (~33.27 mM)

Solubility in Formulation 1: 1.67 mg/mL (2.67 mM) in 50% PEG300 +50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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