Metalporphyrin,

Hematin (0.01 mg/ml) prevents human thrombin from hydrolyzing synthetic substrates and prevents bovine thrombin (0.12 U/ml) from coagulating bovine fibrinogen (1.3 to 2.6 mg/ml) [2]. VIII:C activity is lowered from 0.88 U/ml to 0.40 U/ml by hematin (0.035 mg/ml)[2]. Heme (0.05 mg/ml) stimulates VIII:C and inhibits thrombin (0.04 U/ml) [2]. Substrates for plasmin production are not hydrolyzed by hematurin (0.09 mg/ml) [2].

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Prolonged clotting times and reduced levels of clotting factors have been reported in Hematin-treated patients. This effect persists for up to 5 hr after hematin infusion, associated with plasma levels ranging from 0.01 to 0.04 mg/ml. Therefore we performed in vitro studies to investigate the effects of hematin on fibrinogen, thrombin, factor VIII:C, and plasmin. Hematin in a final concentration of 0.01 mg/ml inhibited the clotting of bovine fibrinogen (1.3 to 2.6 mg/ml) by bovine thrombin (0.12 U/ml) and inhibited the hydrolysis of a synthetic substrate by human thrombin. However, if the hematin was first mixed with albumin (25 mg/ml), fourfold higher concentrations were required to prolong the thrombin clotting time. Hematin, 0.035 mg/ml, reduced VIII:C activity from 0.88 to 0.40 U/ml as measured by two-stage assay. Hematin (0.05 mg/ml) also inhibited the activation of VIII:C by thrombin (0.04 U/ml): baseline activity, 0.84 U/ml; thrombin-activated, 2.94 U/ml; with hematin added, 1.33 U/ml. Hematin also inhibited clot lysis. The inclusion of hematin (0.03 mg/ml) in the diluting buffer reduced the lysis of whole blood clots from 86% +/- 5 to 23% +/- 5 (p less than 0.001, mean +/- S.D. of four determinations) and decreased the lysis of 125I-fibrin clots induced by plasmin (0.02 CTA U/ml) from 100% to 27%. In concentrations as low as 0.09 microgram/ml, hematin inhibited the hydrolysis of a synthetic substrate by plasmin. Hematin was mixed with fibrinogen, albumin, or thrombin, and the mixtures applied to Sephadex G-200 columns. Adherence of the hematin to Sephadex was prevented by either prerinsing the column with albumin or using borate buffer at pH 9.2. Hematin co-eluted with each protein applied to the column and, in the case of fibrinogen, altered its electrophoretic mobility and markedly prolonged the thrombin clotting time of the eluted fibrinogen. We conclude that Hematin binds to a variety of hemostatic proteins, inhibiting their biologic activity.[2]

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Targeted nanomedicine for cancer therapy has gained widespread popularity and is being extensively explored. Porphyrins have intrinsic tumor localizing ability and have been studied for photodynamic therapy. However, they have not been used as cancer targeting agents for nanomedicines. In this study, PLGA nanoparticles were formulated and an iron-containing blood porphyrin, Hematin was conjugated to the surface of the nanoparticles to investigate selectivity towards cancer cell and cellular internalization. Hematin was previously shown to facilitate growth and proliferation of cancer cells. PLGA nanoparticles were characterized by FE-SEM, AFM, DLS, and Zeta potential analyzer. The conjugation of hematin was confirmed by FTIR. HeLa cells were used to study tumor selectivity and uptake. Hematin conjugated particles (ζ potential: -15.19mV) showed higher affinity towards the cancer cells than the control particles. The result indicated that the particles were internalized by heme carrier protein-1. Together these data suggest that hematin is a promising cancer targeting material for nanotherapeutics[3].

Heme (intravenous; single injection) suppresses porphyrin synthesis in SD rats (body weight 160-205 g) [4].

Conjugation of Hematin on the surface of nanoparticles [3]
Hematin is insoluble in water at neutral pH, but soluble at alkaline pH. Hematin was dissolved in 1 M sodium hydroxide giving a dark greenish black solution. pH of the solution was decreased to 8.3 by slowly adding hydrochloric acid solution. EDC and sulfo-NHS (at a molar ratio of 1:1) were added to the hematin solution to activate the carboxylic group of hematin. The reaction was conducted for 15 min under magnetic stirring. Amine modified PLGA nanoparticle suspension was added to an excess amount of carboxyl activated hematin solution, and the reaction was conducted for 3 h. Unconjugated hematin was removed by centrifugation, changing the solvent pH slightly. Hematin functionalized nanoparticles were washed three times with deionized water and stored at − 20 °C for subsequent experiments.

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Determination of surface conjugation [3]
Fourier transform infrared (FTIR) spectroscopy was employed to confirm the conjugation of molecules on the surface of nanoparticles. Unconjugated PLGA nanoparticles, l-arginine conjugated particles, and Hematin functionalized nanoparticles were separately analyzed, and spectra were taken in attenuated total reflection (ATR) mode by an infrared spectrophotometer (JASCO FT/IR-4000). The infrared range across 4000–600 cm− 1 was measured.

Cellular uptake of nanoparticles [3]
HeLA cells were seeded at a density of 1 × 106 cells/mL on a 12 well cell culture plate. After 24 h, the media was removed carefully, and serum free media containing nanoparticles at a concentration of 200 μg/mL was added. Cells were treated with l-arginine-PLGA (control) and Hematin-PLGA nanoparticles, and were incubated for 3 h and 5 h. After treatment, the media was removed carefully and the cells were rinsed and washed three times with ice-cold PBS. The cells were fixed with 4% paraformaldehyde and air dried. Samples were analyzed using a fluorescence microscope to evaluate the uptake of nanoparticles.

Introduction: Intravenous heme has been used to treat acute intermittent porphyria (AIP) since the early 1970s and was marketed in 1983 under the brand name Panhematin (heme for injection; Ovation Pharmaceuticals, USA). However, to date, no literature has summarized the known pharmacodynamic and toxicological effects of heme and its impact on treatment. This review aims to identify, integrate, and summarize existing scientific literature on the physicochemical properties, pharmacokinetics, toxicology, and hemostatic effects of intravenous heme. Methods: A comprehensive literature search was conducted, and the results were summarized. Previously unpublished toxicological data extracted from original new drug applications were also included. Results: Heme reconstituted with sterile water degrades rapidly, and its degradation products are presumed to cause complications such as thrombophlebitis, thrombocytopenia, and transient anticoagulation. Reconstitution with human serum albumin yields well-tolerated heme formulations and significantly improves their stability. The clearance of intravenously infused heme follows a two-compartment model, which consists of a rapid initial clearance phase and a slower, longer second phase. This model is supported by evidence that heme binds to heme-binding proteins first, and then to albumin after saturation. The highest reported intravenous dose of human heme is 12.2 mg/kg (1000 mg), which resulted in acute gastrointestinal pain, paresthesia, and acute tuberculous necrosis. Renal function returned to normal within the following 15 hours. Conclusion: Heme is generally well tolerated at doses approved by the U.S. Food and Drug Administration. Reconstitution with albumin can produce more stable formulations and may result in better tolerability and less interference with hemostasis compared to reconstitution with sterile water. Heme is cleared by the liver after administration, and its pharmacokinetics best conform to a two-compartment model, which consists of a rapid initial phase and a slower second phase. [1]

[1]. Siegert SW, et al. Physicochemical properties, pharmacokinetics, and pharmacodynamics of intravenous hematin: a literature review. Adv Ther. 2008 Sep;25(9):842-57.
[2]. Green D, et al. The inactivation of hemostatic factors by hematin. J Lab Clin Med. 1983;102: 361-369.
[3]. Amin ML, et al. Development of hematin conjugated PLGA nanoparticle for selective cancer targeting. Eur J Pharm Sci. 2016 Aug 25;91:138-43.
[4]. Goetsch C, et al. Instability of hematin used in the treatment of acute hepatic porphyria. N Engl J Med. 1986;315: 235-238.
[5]. Quadros HC, et al. The Role of the Iron Protoporphyrins Heme and Hematin in the Antimalarial Activity of Endoperoxide Drugs. Pharmaceuticals (Basel). 2022;15(1):60. Published 2022 Jan 4.

Chloro(7,12-divinyl-3,8,13,17-tetramethyl-21H,23H-porphyrin-2,18-dipropionic acid(4-)-N(21),N(22),N(23),N(24))iron(2-)dihydro. Plasmodium has evolved the ability to regulate iron protoporphyrin IX (Fe-PPIX) levels and oxidative states. Antimalarial internal peroxides, such as 1,2,4-trioxane artemisinin and 1,2,4-trioxane atropine, undergo a bioreductive activation step mediated by heme (FeII-PPIX) rather than heme (FeIII-PPIX), generating free radicals. These free radicals can alkylate proteins essential for parasite survival and alkylate heme into heme-drug adducts. Heme alkylation is prevalent and accompanied by interconversions from the ferrous to the ferric state, which can lead to an imbalance in iron redox homeostasis. Furthermore, heme-artemisinin adducts antagonize the spontaneous biomineralization of heme to form heme crystals, unlike artemisinin-based drugs, which do not directly inhibit heme biomineralization. These heme-drug adducts, despite lacking the peroxide bonds required for radical-induced alkylation, are potent antimalarial agents. This article reviews our current understanding of Fe-PPIX as a bioreduction activator and molecular target. A compelling pharmacological model suggests that endoperoxide drugs, through alkylation of heme, can cause an imbalance in iron homeostasis; the resulting heme-drug adducts may then exert strong cytotoxicity by replicating some of the toxic effects of free Fe-PPIX. The antimalarial phenotype and mode of action of heme-drug adducts open new possibilities for elucidating the mechanisms of action of endoperoxide drugs and malaria intervention. [5]

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Plasmodium parasites inhibit the free flow of Fe-PPIXs across the dorsal bursa membrane through a synchronizing system. Multiple studies have shown that antimalarial endoperoxides, such as 1,2,4-trioxane artemisinin and 1,2,4-trioxane atropine, act through heme-mediated radical-induced alkylation. Many molecules can be alkylated, so a plausible pharmacological model is that the antimalarial activity of endoperoxides can also be achieved through alkylation of heme. Evidence supporting this view is that heme-drug adducts are abundant, have strong antimalarial activity, and are potent antagonists of heme biomineralization. These alkylated heme can recognize both soluble and insoluble heme pools, but as modified Fe-PPIX compounds, they do not mineralize to form heme crystals, but rather block heme biomineralization. Furthermore, heme alkylation alters the heme/heme ratio, leading to an imbalance in the redox homeostasis of iron species. Similar to other metalloporphyrins, heme-artemisinin adducts irreversibly bind to growing β-heme crystals, thereby inhibiting the detoxification effect of heme. These adducts, as a modified Fe-PPIX structure, contain two pharmacophores: Fe-PPIX recognizes and inhibits the formation of β-heme, while the highly lipophilic sesquiterpene lactone from artemisinin helps overcome the poor lipophilicity of metalloporphyrins. In addition, it may interact with parasitic targets. Based on this reasoning, heme-artemisinin adducts can be considered to have heterodivalent properties and be able to recognize multiple molecular targets. Heterodivalent properties are one of the principles of molecular hybridization for designing hybrid drugs, which reinforces the concept of Fe-PPIX as a component of antimalarial pharmacophores and provides a new potential theoretical basis for hybrid drug design. [5] In this study, rhodamine-loaded PLGA nanoparticles were prepared and heme was coupled to their surface. These particles had a slightly negatively charged surface and were successfully internalized by HeLa cells. The results also showed that these particles were internalized via heme carrier protein-1. In summary, these data suggest that heme is a promising nanomedicine targeting molecule. [3]

C34H33FEN4O5

633.5

633.18

C, 64.46; H, 5.25; Fe, 8.82; N, 8.84; O, 12.63

15489-90-4

44237360

Purple to black solid powder

1128.5ºC at 760 mmHg

180ºC

636.3ºC

0mmHg at 25°C

3.432

1

9

6

44

1570

0

C=CC1=C(C)C2=CC3=NC(=CC4=NC(=CC5=C(C=C)C(=C(C=C1[N-]2)N5)C)C(=C4CCC(=O)[O-])C)C(=C3C)CCC(=O)[O-].[Fe+3].O

XLKTVQRBSKAXKW-UHFFFAOYSA-J

InChI=1S/C34H34N4O4.Fe.H2O/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);;1H2/q;+5;/p-4

3-[18-(2-carboxylatoethyl)-7,12-bis(ethenyl)-3,8,13,17-tetramethylporphyrin-21,23-diid-2-yl]propanoate;iron(5+);hydrate

Ferriheme; Ferrihemate; Hematin porcine; 15489-90-4; Hematin

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)

DMSO : ~1.43 mg/mL (~2.26 mM ()

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.)