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 (ZPP) is the ionized form of tetrazolium protoporphyrin IX, containing zinc in its metal ion-binding pocket. ZPP is formed in circulating erythrocytes (RBCs) under conditions including the presence of lead or iron deficiency, as both inhibit ferrous ion insertion into protoporphyrin IX to form heme. Therefore, elevated ZPP levels are associated with heme deficiency. An elevated molar ratio of ZPP to heme in erythrocytes suggests a variety of pathological conditions, including lead poisoning, iron deficiency, protoporphyria, and various types of anemia.

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Phloroglucinol (PG) is a component of phloroglucinol tannins, which are abundant in marine brown algae. Recent studies have shown that PG helps protect cells from oxidative stress damage. This study evaluated the protective effect of PG on HaCaT human skin keratinocytes stimulated by oxidative stress (hydrogen peroxide, H₂O₂). The results showed that PG significantly inhibited H₂O₂-induced growth inhibition in HaCaT cells, which was associated with increased heme oxygenase-1 (HO-1) expression due to Nrf2 activation. PG significantly reversed H₂O₂-induced excessive reactive oxygen species (ROS) production, DNA damage, and apoptosis. Furthermore, H₂O₂-induced mitochondrial dysfunction was associated with decreased ATP levels, and PG significantly inhibited these changes. In addition, PG pretreatment significantly inhibited H₂O₂-induced increases in cytochrome c cytoplasmic release, elevated Bax/Bcl-2 ratio, and activation of caspase-9 and caspase-3. However, the protective effect of PG against H₂O₂ was significantly weakened after inhibiting HO-1 function with the HO-1 inhibitor zinc protoporphyrin. Overall, our results indicate that PG can protect HaCaT keratinocytes from oxidative stress-induced DNA damage and apoptosis by activating the Nrf2/HO-1 signaling pathway. [1] Background: Iron deficiency erythropoiesis leads to excessive production of zinc protoporphyrin (ZPP), which can be detected rapidly and at low cost using a portable hemoglobin fluorometer. ZPP can be used as a screening biomarker for iron deficiency in pregnant women and children, and can also be combined with hemoglobin concentration to assess iron status in populations. We investigated the association between ZPP and common diseases in Africa. In addition, we evaluated the utility of ZPP (measured in whole blood and erythrocytes) alone or in combination with hemoglobin concentration in diagnosing iron deficiency (plasma ferritin concentration <15 μg/L). Methods: We collected single blood samples from 470 women with singleton pregnancies (gestational age 13 to 23 weeks, hemoglobin concentration ≥90 g/L) in rural Kenya. We used linear regression analysis to assess the association between ZPP and iron markers (including anemia), known or suspected iron status-related factors, inflammatory markers (plasma C-reactive protein and α1-acid glycoprotein concentrations), infections (Plasmodium infection, HIV infection), and other diseases (α(+)-thalassemia, plasma total bilirubin, and lactate dehydrogenase concentrations). Subsequently, in subjects without inflammation, Plasmodium infection, or HIV infection, we used logistic discriminant analysis and examined receiver operating characteristic (ROC) curves and their corresponding areas under the curves (AUC) to evaluate the diagnostic performance of ZPP alone and in combination with hemoglobin concentration. Results: Individually, whole blood ZPP, erythrocyte ZPP, and erythrocyte protoporphyrin have limited ability to distinguish between iron-deficient and non-iron-deficient pregnant women. Combined detection of these markers with hemoglobin concentration did not provide additional diagnostic value. The conventional cutoff value for whole blood ZPP (>70 μmol/mol heme) leads to a significant overestimation of iron deficiency prevalence. Conclusion: In disease screening with low prevalence (e.g., 10%), erythrocyte ZPP has limited value in ruling out iron deficiency. The diagnostic utility of ZPP in differentiating between iron-deficient and non-iron-deficient pregnant women is unreliable. Based on these findings, guidelines for using ZPP to assess the iron status of pregnant women individually or in groups need to be re-examined. [2] Background: HO-1 is involved in the degradation of heme. Its products can play a unique cytoprotective role. Many tumors highly express HO-1, suggesting that this enzyme may be a potential therapeutic target. This study aimed to evaluate the potential cytosolic/cytotoxic effects of the selective HO-1 inhibitor zinc protoporphyrin IX (Zn(II)PPIX) and to assess its antitumor activity in combination with chemotherapeutic drugs. Methods: The cytosolic/cytotoxic effects of Zn(II)PPIX were assessed by crystal violet staining and colony formation assays. Protein expression was assessed by Western blotting. The effects of Zn(II)PPIX on apoptosis induction and reactive oxygen species generation were assessed by flow cytometry. HO-1 expression was knocked down using siRNA. The antitumor effects of Zn(II)PPIX, alone or in combination with chemotherapeutic agents, were investigated in xenograft models. Results: Zn(II)PPIX induced a significant accumulation of reactive oxygen species (ROS) in tumor cells. This effect was partially reversed by administration of exogenous bilirubin. Furthermore, Zn(II)PPIX exhibited potent cell-inhibitory/cytotoxic effects on both human and mouse tumor cell lines. Although cyclin D expression in Zn(II)PPIX-treated cells showed a significant time- and dose-dependent decrease, no accumulation of tumor cells in the G1 phase of the cell cycle was observed. However, incubation of C-26 cells with Zn(II)PPIX increased the proportion of cells in the sub-G1 phase. Flow cytometry combined with propidium iodide and annexin V staining, along with Western blotting detection of cleaved caspase 3, indicated that Zn(II)PPIX induced tumor cell apoptosis. When B16F10 melanoma cells overexpressing HO-1 were transplanted into syngeneic mice, they developed resistance to the antitumor effects of Zn(II)PPIX or cisplatin. In three different tumor models, Zn(II)PPIX did not enhance the antitumor effects of 5-fluorouracil, cisplatin, or doxorubicin, but it significantly enhanced the toxicity of 5-fluorouracil and cisplatin. Conclusion: Inhibition of HO-1 has antitumor effects, but unless a selective tumor-targeting HO-1 inhibitor is developed, it should not be used to enhance the antitumor effects of anticancer chemotherapeutic drugs. [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.)