Iron(II)porphyrin–Cyclodextrin Supramolecular Complex as a Carbon Monoxide-Depleting Agent in Living Organisms

Abstract

The specific intermolecular interaction between an anionic tetraarylporphyrin and per-O-methylated β-cyclodextrin (TMe-β-CD) paved the way to produce a functional supramolecule that works as a strong carbon monoxide (CO)-depleting agent in living organisms. The supramolecular complex, hemoCD, that is composed of meso-tetrakis(4-sulfonatophenyl)porphinatoiron(II) and a TMe-β-CD dimer linked by a pyridine linker, captured internal CO from carboxyhemoglobin during its circulation in the blood of animals. HemoCD thus produced the pseudo-knockdown (loss-of-functional) state of endogenous CO in the animals. This unique property led us to investigate the biological function of endogenous CO as a gaseous signal mediator in living systems. In this paper, we introduce our recent study on the hemoCD complex as a biological CO-depleting agent.

1. Introduction

Carbon monoxide (CO) is a well-known toxic gas that can strongly bind to hemoglobin (Hb) circulating in the blood. Inhaled CO from the lung causes interruption of oxygen delivery by Hb in the red blood cells, leading to hypoxia in the whole body.^1–4) Despite the feared toxicity, vertebrate animals continuously produce CO in every cell.⁵⁾ Approximately 10 mL of CO gas is produced per day in the human body.⁶⁾ The main source of endogenous CO is the metabolic degradation of hemin,^5–8) an essential cofactor for heme proteins such as Hb. Redundant free hemin is immediately decomposed by a heme oxygenase (HO) enzyme to produce iron, biliverdin, and CO in the cells (Fig. 1). Most of the endogenous CO (ca. 80%) produced by this process binds to Hb and circulates in the blood as CO-bound Hb (CO-Hb).^9,10) Therefore, a fraction of CO-Hb is normally contained in the blood.¹¹⁾ The other endogenous CO (ca. 20%) might function as a gaseous messenger in the intracellular signaling pathways. The biological function of endogenous CO is considered similar to nitric oxide (NO)¹²⁾ and hydrogen sulfide (H₂S).¹³⁾ In addition, CO has been proven to have unique protective effects in cells, such as anti-inflammation, anti-apoptosis, and anti-proliferation.^14–18) However, different from NO and H₂S, the detailed mechanism for the physiological functions have not been studied, because it is difficult to prepare knockdown (loss-of-function) phenotypes of CO without interrupting the metabolic processes caused by HO. Therefore, for elucidation of the physiological functions of CO, selective depleting agents for endogenous CO are needed.

Fig. 1. Endogenous Production of CO in Mammalian Cells and Its Physiological Functions

2. Porphyrin–Cyclodextrin (CD) Supramolecular Complexes

It is known that cyclodextrins (CDs) form inclusion complexes with water-soluble tetraarylporphyrins in aqueous solutions. Per-O-methylated CD derivatives strongly interact with anionic porphyrins such as meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) to form 2 : 1 inclusion complexes in aqueous solution.^19–21) In 1996, Kano et al. first reported the 1 : 2 inclusion complex of TPPS with 2,3,6-tri-O-methyl-β-cyclodextrin (TMe-β-CD, Fig. 2). Their important finding was that TMe-β-CD provided a microscopic hydrophobic environment around the porphyrin ring, as confirmed by comparing the pK_a values of TPPS in the absence and presence of TMe-β-CD. The center nitrogen atoms of TPPS were hardly protonated even in acidic solution when it complexed with TMe-β-CD.¹⁹⁾ The microscopic hydrophobic environment around the porphyrin created by TMe-β-CD is quite similar to that of a globin protein of Hb. A hydrophobic heme pocket provided by the globin protein is essential to form stable O₂ complexes of ferrous heme in aqueous media.

Fig. 2. Intermolecular Interaction between Per-O-methylated β-Cyclodextrins (TMe-β-CD) and a Water-Soluble Porphyrin (TPPS) in Aqueous Solution

Inspired by the unique nature of the TPPS/TMe-β-CD complex, we synthesized a per-O-methylated β-CD dimer linked by a pyridine moiety (Py3CD, Fig. 3) that was introduced to mimic an axial coordination to Fe(II) in the globin protein of myoglobin (Mb) or Hb. For the stable O₂ complex formation of iron(II)porphyrin in aqueous solution similar to Mb or Hb,^22,23) the axial coordination to Fe(II) by a nitrogenous atom and a hydrophobic environment around Fe(II) are both essential. Without the hydrophobic pocket, the O₂ complex is easily decomposed by a water-catalyzed autoxidation reaction to form the inactive iron(III)porphyrin.²⁴⁾ Interestingly, the 1 : 1 inclusion complex of Py3CD with Fe(II)TPPS (hemoCD, Fig. 3) formed a stable O₂ complex in aqueous solution at an ambient temperature, in which the half life-time for the autoxidation of the O₂ complex was 30 h at 25°C.²²⁾ Although many synthetic Hb/Mb model compounds have been reported so far, most of them formed the O₂ complexes in anhydrous organic solvents.^25–29) To the best of our knowledge, hemoCD is the only synthetic Hb/Mb model compound that forms a stable O₂ complex in 100% aqueous solution at an ambient temperature. Therefore, hemoCD must be a potential candidate as an artificial O₂ carrier in the blood of mammals.

Fig. 3. Supramolecular Complex HemoCD That Is Composed of the Cyclodextrin Dimer (Py3CD) and Fe(II)TPPS

HemoCD functions as an Hb/Mb model compound in aqueous media.

3. HemoCD as a CO-Depleting Agent in Vivo

To investigate the pharmacokinetic behavior of hemoCD, a solution of hemoCD in phosphate buffered-saline (PBS) was injected into the vein of a rat³⁰⁾ (Fig. 4). The infused hemoCD did not cause any toxicity or vital changes. Due to its high water solubility and small molecular size, hemoCD circulating in the bloodstream is easily filtered at the glomerulus. Therefore, the intravenously infused hemoCD was quantitatively excreted in the urine within 30 min. In addition, because of its much higher CO affinity than Hb,^23,25) hemoCD removed endogenous CO from CO-Hb in the blood and was excreted in the urine as the CO-bound form (CO-hemoCD) as confirmed by spectroscopic analysis of the urine.^30,31) The binding affinity of hemoCD to CO is the strongest among the reported values of the heme proteins in aqueous solutions. Therefore, hemoCD can remove endogenous CO from the CO-bound proteins existing in living organisms. Spectroscopic analysis of the urine quantified that 5.1×10⁻⁷ mol kg⁻¹ h⁻¹ of CO was removed from the rat body by hemoCD. This value is consistent with the reported one for endogenous CO production in the human body.^6,32) The results indicate that hemoCD has the ability to almost completely remove internal CO that is continuously produced in the rat body.

Fig. 4. Intravenously Injected HemoCD Captures Endogenous CO and Is Excreted in the Urine as the CO-Bound Form

Focusing on the unique property of hemoCD in vivo, we then utilized hemoCD as a CO-depleting agent to investigate the biological roles of endogenous CO in living organisms.³³⁾ Quantitative analysis of the CO-Hb content in the blood of hemoCD-administered mice revealed that hemoCD completely removed CO from CO-Hb, leading to an almost zero level of CO-Hb in the blood (Fig. 5a). Notably, the temporarily reduced CO-Hb in the blood was smoothly returned to the normal level. This indicated that the reduced CO-Hb level was compensated by the acceleration of endogenous CO production to maintain the CO-Hb homeostasis. In fact, the expression level of heme oxygenase-1 (HO-1) was significantly enhanced (ca. 9-fold) in the liver of the hemoCD-administered mice (Fig. 5b). The overexpressed HO-1 could be cable of producing further endogenous CO via the decomposition of hemin.

Fig. 5. The Effect of Endogenous CO-Depletion by HemoCD

(a) Time–course of the fraction of CO-Hb (%) in blood and (b) relative mRNA expression levels of HO-1 (Hmox-1) in the liver of mice after the administration of hemoCD. The control data were obtained by administration of PBS. Each bar represents the mean±standard error (S.E.) (n=3 mice per time point). An asterisk denotes statistical significance: * p<0.05, ** p<0.01, as compared to the controls.

The feedback mechanism for endogenous CO production was studied. An in vitro analysis using cultured HepG2 cells revealed that the addition of hemoCD to the culture medium did not induce HO-1 in the cells. This indicated that hemoCD did not directly affect the intracellular event to induce the expression of HO-1. Blood plasma analysis in the hemoCD-administered mice revealed that the removal of CO from CO-Hb in the blood significantly increased the amount of free hemin and bilirubin in the blood plasma. Therefore, we conclude that the removal of CO from CO-Hb causes the increase of free hemin, which triggers the induction of HO-1. The oxygen complex of Hb (oxy-Hb) must be formed by removal of CO from CO-Hb, where oxy-Hb is more oxidation-susceptible than CO-Hb and can be easily oxidized to the ferric complex of Hb (met-Hb).³⁴⁾ The free hemin, a potent inducer for the expression of HO-1, can be readily released from met-Hb.³⁵⁾ The proposed feedback mechanism for the depletion of endogenous CO is summarized in Fig. 6. The newly produced HO-1 can decompose the excessively released free hemin to compensate the endogenous CO. Therefore, it can be said that endogenous CO has a role in maintaining the homeostatic balance of free hemin concentration in blood plasma via the coordination to Hb. The homeostatic feedback response for endogenous CO was first revealed by our supramolecular complex hemoCD as a CO-depleting agent in vivo.

Fig. 6. Proposed Feedback Mechanism for Endogenous CO Production Induced by CO-Depletion in Blood

4. Supramolecular Complexes with Cell-Penetrating Ability

HemoCD is a hydrophilic molecule that cannot penetrate the hydrophobic cell membrane.³⁶⁾ Meanwhile, some endogenous CO functions as a gaseous transmitter in the signaling pathways occurring inside the cells. To apply hemoCD as a CO-depleting agent inside the cells, we have been trying to deliver the hemoCD molecule into the cells by using cell-penetrating peptides (CPPs). CPP has been regarded as a powerful chemical tool to afford cell penetrating ability to various types of molecules including small drugs and proteins.³⁷⁾ The molecular structures of the hemoCD analogues (porphyrin–CD supramolecular complexes) bearing CPP are shown in Fig. 7. Among reported CPPs, Futaki et al. have showed that the arginine octamer (R8) is the simplest and most promising CPP that can deliver a wide variety of cargo molecules.³⁸⁾ Thus, we chose an R8 peptide to be introduced into the tetraarylporphyrin³⁹⁾ (R8-TPP, Fig. 7a), or TMe-β-CD⁴⁰⁾ (R8-CD, Fig. 7b). These supramolecular complexes were successfully internalized inside the cells.^39,40) Interestingly, the structures of the supramolecular complexes were maintained even after decomposition of the cells, as confirmed by spectroscopic analysis of the cell lysates. The intracellular delivery of the hemoCD analogues was achieved by its covalent conjugation with R8. Thus it should be possible to produce the pseudo-knockdown state of intracellular CO by intracellular delivery of hemoCD. This project is now under progress in our research laboratory.

Fig. 7. Octaarginine (R8) Conjugated Supramolecular Porphyrin–CD Complexes for Intracellular Delivery

5. Summary

We have developed a selective CO-depletion system in vivo using the iron(II)porphyrin–CD supramolecular complex, hemoCD. The extremely high CO-binding affinity of hemoCD enabled us to produce the pseudo-knockdown state of CO in mice. Since administration of an appropriate amount of CO has been proven to exhibit a drastic protective effect against inflammation, several research groups have extensively studied the pharmaceutical use for CO in vivo. Thus, the biological role of CO needs to be fully understood before its use. The pseudo-knockdown method would be a powerful approach with which to perform such a study. We believe that the various biological functions of endogenous CO in vivo will be revealed by our hemoCD system.

Conflict of Interest

The authors declare no conflict of interest.

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