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 (i.v.) hematin has been used in the treatment of acute intermittent porphyria (AIP) since the early 1970s and commercially available as Panhematin (hemin for injection; Ovation Pharmaceuticals, Inc., USA) since 1983, yet no publication to date has attempted to summarize the known pharmacodynamics and toxicological actions of hematin and the implications on treatment. It is the objective of this literature review to identify, consolidate, and summarize the available scientific literature regarding the physicochemical properties, pharmacokinetics, toxicology, and hemostatic effects of i.v. hematin injections.
Methods: A comprehensive search of the available literature was performed and resulting data were summarized. Furthermore, previously unpublished toxicology data extracted from the original New Drug Application were included.
Results: Hematin, reconstituted with sterile water, rapidly degrades and it is hypothesized that the degradation products lead to morbidities such as thrombophlebitis, thrombocytopenia, and transient anticoagulation. Reconstitution with human serum albumin produces a well-tolerated hematin preparation and improves its stability significantly. The clearance of i.v. hematin infusions are shown to fit a two-compartment model consisting of a rapid initial rate followed by a slower and prolonged second phase. This model is supported by the evidence demonstrating that hematin is first bound by hemopexin and, upon saturation, second by albumin. The highest i.v. human hematin dose reported in the literature was 12.2 mg/kg (1000 mg) and resulted in acute gastrointestinal pain, paresthesia, and acute tubercular necrosis. The patient's renal function returned to normal over the following 15 hours.
Conclusion: Hematin, at doses approved by the US Food and Drug Administration, is generally well tolerated. Reconstitution with albumin produces a significantly more stable preparation than reconstitution with sterile water and may lead to a more tolerable administration with less hemostatic interference. Hematin, once administered, is cleared hepatically and is best represented pharmacokinetically by a two-compartment model comprised of a rapid initial phase followed by 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-diethenyl-3,8,13,17-tetramethyl-21H,23H-porphine-2,18-dipropanoato(4-)-N(21),N(22),N(23),N(24)) ferrate(2-) dihydrogen.

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Plasmodium has evolved to regulate the levels and oxidative states of iron protoporphyrin IX (Fe-PPIX). Antimalarial endoperoxides such as 1,2,4-trioxane artemisinin and 1,2,4-trioxolane arterolane undergo a bioreductive activation step mediated by heme (FeII-PPIX) but not by hematin (FeIII-PPIX), leading to the generation of a radical species. This can alkylate proteins vital for parasite survival and alkylate heme into hematin-drug adducts. Heme alkylation is abundant and accompanied by interconversion from the ferrous to the ferric state, which may induce an imbalance in the iron redox homeostasis. In addition to this, hematin-artemisinin adducts antagonize the spontaneous biomineralization of hematin into hemozoin crystals, differing strikingly from artemisinins, which do not directly suppress hematin biomineralization. These hematin-drug adducts, despite being devoid of the peroxide bond required for radical-induced alkylation, are powerful antiplasmodial agents. This review addresses our current understanding of Fe-PPIX as a bioreductive activator and molecular target. A compelling pharmacological model is that by alkylating heme, endoperoxide drugs can cause an imbalance in the iron homeostasis and that the hematin-drug adducts formed have strong cytocidal effects by possibly reproducing some of the toxifying effects of free Fe-PPIX. The antiplasmodial phenotype and the mode of action of hematin-drug adducts open new possibilities for reconciliating the mechanism of endoperoxide drugs and for malaria intervention. [5]

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Plasmodium operates a synchronized system to mitigate a free flow of Fe-PPIXs across the DV membrane. Multiple studies indicate that antimalarial endoperoxides, such as 1,2,4-trioxane artemisinin and 1,2,4-trioxolane arterolane, operate a mechanism of radical-induced alkylation mediated by heme. A plethora of molecules can be alkylated, and a plausible pharmacological model is that the antimalarial activity of endoperoxides can be achieved by alkylating heme as well. In support of this notion is the fact that hematin–drug adducts are highly abundant, strong antimalarials, and potent antagonists of hematin biomineralization. These alkylated heme species recognize soluble and insoluble hematin pools, but as modified Fe-PPIX species, they do not mineralize into Hz crystals but, rather, block hematin biomineralization. Furthermore, heme alkylation alters the heme/hematin ratio and can thereby induce an imbalance in the redox homeostasis of iron species. Similar to other metalloporphyrins, hematin–artemisinin adducts bind irreversibly to growing β-hematin crystals, and this initiates the suppression of heme detoxification. These adducts act as a modified Fe-PPIX structure containing two pharmacophores: Fe-PPIX recognizes and inhibits β-hematin formation while the highly lipophilic sesquiterpene lactone from artemisinin contributes to overcome the poor lipophilicity of metalloporphyrin, in addition to its plausible interaction with parasite targets. Via this reasoning, hematin–artemisinin adducts could be seen as being heterobivalent with respect to their recognition of multiple molecular targets. Heterobivalency is one of the principles of the molecular hybridization approach for designing hybrid-based drugs, which reinforces the notion of Fe-PPIX as an antimalarial pharmacophore component, and adds a new potential rationale for hybrid-based drug design.[5]

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In this study, rhodamine loaded PLGA nanoparticles were formulated and hematin was coupled to the surface. The particles had a slightly negative surface charge and demonstrated successful internalization by HeLa cells. The result also implied that the particles were internalized by heme carrier protein-1. Collectively, these data suggest that hematin is a promising targeting molecule for nanomedicine.[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.)