The present invention relates to a carbon monoxide removal agent and particularly to a carbon monoxide removal agent that uses a porphyrin complex.
Carbon monoxide (hereinafter abbreviated as “CO”) is a toxic gas produced by incomplete combustion of carbon and when inhaled, it binds strongly with hemoglobin (hereinafter abbreviated as “Hb”) in blood in place of oxygen (hereinafter abbreviated as “O2”), depriving Hb of its inherent O2 transport ability and causing an entire body to fall into an oxygen deprived state. Consequently, symptoms of so-called CO poisoning, such as headache, nausea, vomiting, physical deconditioning, confusion, loss of consciousness, chest pain, shortness of breath, coma, etc., occur.
The binding of Hb with O2 or CO is reversible and an affinity of CO to Hb is approximately 250 times greater than that of O2. Thus even when a small amount of CO is present in air, Hb is rapidly converted from an O2 bound form to a CO bound form.
Presently, a CO poisoning remedy that reconverts the CO bound form of Hb to the O2 bound form and thereby cure CO poisoning symptoms has yet to be developed. Thus as methods for recovery from poisoning, there were only methods of gradually shifting an equilibrium of Hb in blood from the CO bound form to the O2 bound form by using a facemask to make high concentration oxygen be inhaled or by placing a CO poisoning patient under a high concentration O2 atmosphere (see Non-Patent Document 1).
However, these methods require certain facilities and in a large-scale fire or other case where a large number of CO patients occurs simultaneously, the patients cannot be treated efficiently and in some cases, this leads to deaths of patients. Also with these treatment methods, although a large part of the CO bound to Hb can be removed, it is difficult to remove CO that has spread widely to intricate parts of the body, and aftereffects of CO poisoning are a serious problem.
Meanwhile, the inventors have been conducting research from before on an inclusion complex formed by inclusion of a water-soluble metalloporphyrin by a cyclodextrin dimer, and have discovered that the inclusion complex has high affinities to O2 and CO and that the affinity to CO is no less than 100 times that of Hb (see Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3).
Thus an object of the present invention is to provide a carbon monoxide removal agent that can be easily administered to a patient by injection or orally.
The inventors noted that there is a possibility for the inclusion complex to be used as a carbon monoxide removal agent and have thereby come to complete present invention.
That is, a carbon monoxide removal agent according to a first aspect of the present invention contains, as an active ingredient, an inclusion complex in which a cyclodextrin dimer represented by chemical formula (1) includes a water-soluble metalloporphyrin.
(In the formula, m represents either of number 1 or 2 and n represents any of number 1, 2, or 3.)
A carbon monoxide removal agent according to a second aspect is the carbon monoxide removal agent according to the first aspect where m=1 and n=2.
A carbon monoxide removal agent according to a third aspect is the carbon monoxide removal agent according to the first or second aspect where the water-soluble metalloporphyrin is represented by either of chemical formula (2) or (3).
(In the formulae, each of R1 and R2 represents any of a carboxyl group, a sulfonyl group, or a hydroxyl group and M represents any of Fe2+, Mn2+, Co2+, or Zn2+.)
A carbon monoxide removal agent according to a fourth aspect is the carbon monoxide removal agent according to the third aspect where the water-soluble metalloporphyrin is 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (II) iron complex.
The inclusion complex contained in the carbon monoxide removal agent according to the present invention has a higher affinity to CO than Hb and deprives Hb of CO contained in blood or peripheral tissue of a patient. The carbon monoxide removal agent according to the present invention is thus high in ability to treat CO poisoning. Thus, when the carbon monoxide removal agent according to the present invention is used clinically, many poisoning patients can be treated to save their lives.
The inclusion complex contained in the carbon monoxide removal agent according to the present invention can also absorb CO that is produced inside a body. Meanwhile, it is known that CO is produced by a decomposition reaction of hemoglobin and is related to biological reactions in functioning as a regulatory factor for gene expression, etc., (see, for example, S. Aono, Acc. Chem. Res., 36, 825-831 (2003)). The carbon monoxide removal agent according to the present invention can thus contribute not only to treatment of CO poisoning but also to research of such biological reactions.
The carbon monoxide removal agent according to the present invention contains, as an active ingredient, an inclusion complex formed by inclusion of a water-soluble metalloporphyrin by a specific cyclodextrin dimer. The respective components shall now be described in detail. The inclusion complex can be manufactured by mixing the cyclodextrin dimer and the water-soluble metalloporphyrin in an aqueous solvent.
1. Cyclodextrin Dimer
As shown in chemical formula (1), in the cyclodextrin dimer, two cyclodextrin molecules, with which all hydroxyl groups are methylated, are bound via 3,5-di(mercaptomethyl)pyridine, which is a linker molecule.
The cyclodextrin dimer is manufactured, for example, by tosylating and then epoxidizing cyclodextrin, thereafter methylating the hydroxyl groups of the cyclodextrin, and then binding the methylated cyclodextrins with the linker molecule as described in the prior art documents. The hydroxyl groups of cyclodextrin are methylated in advance to prevent difficulty of inclusion of the water-soluble metalloporphyrin in inner holes of the cyclodextrin dimer due to hardening of the inner holes of the cyclodextrins by hydrogen bonds formed by the hydroxyl groups.
The cyclodextrin that is the raw material of the cyclodextrin dimer is any of a-cyclodextrin, β-cyclodextrin (m=1 and n=2), or γ-cyclodextrin, and among these, the use of β-cyclodextrin as the raw material is preferable because it readily includes the water-soluble metalloporphyrin.
2. Water-Soluble Metalloporphyrin
The water-soluble metalloporphyrin is a porphyrin-based compound that is soluble in water and has a metal ion coordinated at a center and is not restricted in particular as long as it can be included by the cyclodextrin dimer represented by the chemical formula (1).
Among water-soluble metalloporphyrins, a compound represented by the chemical formula (2) or the chemical formula (3) can be cited from a point of being reliably capable of adsorption and desorption of O2, and more specifically, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (II) iron complex (hereinafter abbreviated as “FeTPPS”), 5,15-bis(3,5-dicarboxylatophenyl)-10,20-diphenylporphyrin (II) iron complex (hereinafter abbreviated as “Fe-trans-2DC”), etc., can be cited. These compounds may, for example, be synthesized by a known method or a commercially available product (for example products of Frontier Scientific Inc., Tokyo Chemical Industry Co., Ltd., etc.) may be used as it is.
3. Dosage Form, Etc.
The carbon monoxide removal agent according to the present invention may be administered to a human or other animal in the form of the inclusion complex alone or by forming a medical composition with a known pharmaceutical carrier. A dosage form of the medical composition is not restricted in particular and may be selected appropriately as needed. Oral preparations, such as pills, capsules, granular agents, fine grain agents, powdered agents, and non-oral preparations, such as injectable agents, suppositories, embrocations, etc., can be cited as specific examples. A quantity of the carbon monoxide removal agent in the medical composition and a dosage amount of the medical composition for a patient may be selected freely according to the dosage form and age, weight, and degree of disorder of the patient.
In a case where the carbon monoxide removal agent according to the present invention is to be manufactured as a pill or other oral preparation, it can be manufactured by a known manufacturing method and using together a known diluent, binding agent, disintegrating agent, surfactant, lubricant, fluidity promoter, etc.
The carbon monoxide removal agent according to the present invention can also be orally administered as a suspension, an emulsion agent, a syrup agent, an elixir agent, etc. In this case, a flavoring agent, an odor improving agent, colorant, etc., may be contained.
In a case where the carbon monoxide removal agent according to the present invention is to be manufactured as a non-oral preparation, such as an injectable agent, drip, etc., it can be manufactured by a known manufacturing method and using together a known diluent, such as distilled water for injection, physiological saline diluent, glucose aqueous solution, etc. Also, a disinfectant, preservative, or stabilizer may be added as necessary. From a point of stability, the non-oral preparation may be frozen after being filled in a vial, etc., removed of water by an ordinary lyophilization process, and reconstituted as a liquid agent from the lyophilized product immediately before use. Further, a tonicity agent, stabilizer, preservative, or soothing agent may be added as necessary.
As other examples of non-oral preparations of the carbon monoxide removal agent according to the present invention, embrocations, such as liquid agents for external use, ointments, etc., suppositories for intrarectal administration, etc., can be cited, and these can also be manufactured according to known methods.
The carbon monoxide removal agent according to the present invention may be administered internally by a known DDS technique, for example, by sealing the carbon monoxide removal agent according to the present invention in a liposome or other carrier. In this case, by using a carrier that specifically recognizes cells of a target site, the carbon monoxide removal agent according to the present invention can be carried efficiently to the target site.
The present invention shall now be described in more detail based on examples. However, the scope of claims of the present invention is by no means restricted by the following examples.
A carbon monoxide removal agent according to the present invention was prepared and a CO removal ability thereof was examined. Specifically, an experiment was performed as follows.
(1) Reagents, Etc.
As reagents, such as FeTPPS (made by Frontier Scientific Inc.), etc., commercially available products were used as they were. As Py3CD, that synthesized by the inventors according to Patent Document 1, Non-Patent Document 2, and Non-Patent Document 2 was used. As rats, Wister male rats (obtained from Shimizu Laboratory Supplies) were used. Ultraviolet-visible absorption spectra were measured using spectrophotometers (UV-2450 and MaltiSpec-1500, made by Shimadzu Corporation).
(2) Preparation of the Carbon Monoxide Removal Agent
FeTPPS and Py3CD were respectively weighed out to a molar ratio of 1/1.2 using an electronic balance, placed in a beaker, and dissolved by adding 0.5 mL of a PBS buffer (pH 7.0) to the beaker. An excess amount (10 to 50 mg) of Na2S2O4 was then added to the beaker to reduce the central iron in FeTPPS from Fe (III) to Fe (II).
The solution in the beaker was desalted by a HiTrap Desalting Column (made by GE Healthcare) (eluent: PBS buffer) to remove the excess Na2S2O4. In this process, the reduced FeTPPS/Py3CD complex (hemoCD) becomes oxy-hemoCD because the Fe(II) that is the central ion binds with O2 in air. After measuring the ultraviolet-visible absorption spectrum of the column-purified oxy-hemoCD solution to determine the concentration, the PBS buffer was used to adjust the solution to a predetermined concentration (0.2 to 3.5 mM) as the carbon monoxide removal agent.
(3) Administration of the Carbon Monoxide Removal Agent To Animal (Rat) and Aspiration of Urine
A rat was put to sleep using a urethane anesthetic and a femoral region was exfoliated. Thereafter, the carbon monoxide removal agent was administered at a fixed rate (1.0 mL/h) from the femoral vein using a syringe pump. At every 30 minutes from the start of administration, urine was sampled from a vesicular portion, the interior of the bladder was then washed with physiological saline, and the urine and the physiological saline were put together and used as the urine in quantitative analysis (the same applies hereinafter).
(4) Measurement of Ultraviolet-Visible Absorption Spectra
Ultraviolet-visible absorption spectra of the carbon monoxide removal agent prepared in (2) and the urine obtained in (3) were measured. The results are shown in
From each of the ultraviolet-visible absorption spectra in
(5) Experimental Results
From
It is thus considered that when administered, the hemoCD exchanges the ligand from the original O2 to CO, which is higher in affinity, in a process of being circulated through the entire body and is then excreted as urine. This suggests that there is a possibility for use of hemoCD as a carbon monoxide removal agent.
The influence that the cyclodextrin dimer has on the excretion of the carbon monoxide removal agent in urine was examined. Specifically, each of the porphyrins was administered solitarily into a rat and absorbance at 420 nm, at which each administered porphyrin has a strong absorption band, was measured to examine the amount of the porphyrin excreted into urine. Also, after administration of each porphyrin, the cyclodextrin dimer was administered and the influence thereof was examined. Specifically, an experiment was performed as follows.
(1) Preparation of Medical Agents, Etc.
Commercially available FeTPPS (made by Frontier Scientific Inc.) and TPPS (made by Tokyo Chemical Industry Co., Ltd.) with the central metal removed from FeTPPS were respectively dissolved separately to a concentration of 0.5 mM in a PBS buffer (pH 7.4) and thereby prepared as medical agents. Also, a Py3CD solution was prepared by dissolving Py3CD to a concentration of 0.6 mM in the PBS buffer (pH 7.4). Further, ultraviolet-visible absorption spectra were measured using the same apparatuses as in EXAMPLE 1.
(2) Administration to Animal and Measurement of Ultraviolet-Visible Absorption Spectra
The medical agent containing FeTPPS was administered continuously to a rat by the syringe pump (1 mL/min). By the same method as in EXAMPLE 1, urine was sampled every 30 minutes from the start of administration and the change with time of the absorbance at 420 nm was examined. Also after 120 minutes from the start of administration of the medical agent, the administration of the medical agent containing FeTPPS was stopped, the Py3CD solution was administered by the syringe pump (1 mL/min) continuously for 180 minutes, urine was sampled every 30 minutes from the start of administration, and the change with time of the absorbance at 420 nm was examined. The results are shown in
Also, the same experiment was performed using the medical agent containing TPPS. However, the administration of the Py3CD solution was started 150 minutes after the start of administration of the medical agent, and the administration time of the Py3CD solution was also 150 minutes. The results are also shown in
Further, with the urine at the time of start of administration of the Py3CD solution among the urine sampled in the experiment using the medical agent containing TPPS, the ultraviolet-visible absorption spectrum was compared with the ultraviolet-visible absorption spectra of a separately prepared medical agent containing TPPS (2.5 μM) and a medical agent containing a TPPS (2.5/Py3CD (3.0 μM) complex. The results are shown in
(3) Experimental Results
From
Also from
Serum albumin (RSA) is a protein component of the highest content in blood and is known to have a property of taking in various hydrophobic molecules. FeTPPS is not an exception and is known to bind strongly to serum albumin (see, for example, V. E. Yushmanov et al, Mag. Res. Imaging, 14, 255-261 (1996), T. T. Tominaga et al, J. Inorg. Biochem., 65, 235-244 (1997), etc.). The effect of inducing urinary excretion of FeTPPS by Py3CD was thus compared with that by RSA. Specifically, an experiment was performed as follows.
(1) Preparation of Medical Agents, Etc.
FeTPPS was dissolved in a PBS buffer (pH 7.4) to prepare a 5 μM solution, and Py3CD and RSA (made by SIGMA. Co., Ltd.) were separately dissolved in the PBS buffer (pH 7.4) to prepare respective stock solutions. Ultraviolet-visible absorption spectra were measured with the same apparatuses as those used in EXAMPLE 1.
(2) Measurements of Ultraviolet-Visible Absorption Spectra
The FeTPPS solution was placed in a cuvette, ultraviolet-visible absorption spectra were measured while adding fixed amounts of the RSA solution to the cuvette, and at a point at which changes reached saturation, ultraviolet-visible absorption spectra were measured while adding the Py3CD solution in place of the RSA solution. The results are shown in
Oppositely, the FeTPPS solution was placed in a cuvette, ultraviolet-visible absorption spectra were measured while adding fixed amounts of the Py3CD solution to the cuvette, and at a point at which changes reached saturation, ultraviolet-visible absorption spectra were measured while adding the RSA solution in place of the Py3CD solution. The results are shown in
(3) Experimental Results
From
Further, from
Meanwhile, from
The mechanism by which the carbon monoxide removal agent according to the present invention is excreted from blood into urine via kidneys was examined by way of a kidney model using an ultrafiltration membrane. Specifically, an experiment was performed as follows.
(1) Preparation of Medical Agents, Etc.
FeTPPS, RSA, and Py3CD (procured from the same firms as in EXAMPLE 3) were respectively dissolved in a PBS buffer (pH 7.4) to prepare 0.22 μM solutions. Also, ultraviolet-visible absorption spectra were measured using the same apparatuses as in EXAMPLE 1. As the ultrafiltration membrane, a stirred cell (Model 8050 made by Millipore Corp.) with an ultrafiltration membrane with a molecular weight cutoff of 30,000 attached was used.
(2) Diafiltration Test
Equivalent amounts of the FeTPPS solution and the RSA solution were mixed to prepare an equimolar mixture, and the equimolar mixture solution was placed in the stirred cell that was set above a recovery beaker. While pressurizing with nitrogen gas, a filtrate was recovered at every fixed amount and subject to ultraviolet-visible absorption spectral analysis.
At a point at which 20 mL of the filtrate was recovered, the Py3CD solution was added so that the molar amount of Py3CD was equal to the molar amount of each of FeTPPS and RSA in the residual solution. Thereafter, while pressurizing with nitrogen gas, a filtrate was recovered at every fixed amount and subject to ultraviolet-visible absorption spectral analysis. The results are shown in
(3) Experimental Results
From
The molecular weight of RSA is 68,000 and the molecular weight of Py3CD is approximately 3,000. The experimental results thus suggest that whereas while FeTPPS is held by RSA, it cannot pass through the ultrafiltration membrane, when FeTPPS is released from RSA by addition of Py3CD and becomes included in Py3CD, it becomes capable of passing through the ultrafiltration membrane.
In the same manner as in the principle of ultrafiltration, filtration membranes of the kidneys also perform screening according to molecular size and serum albumin is not excreted in urine. In consideration of this and the above experimental results, it is considered that the FeTPPS excretion induction effect of Py3CD is due to a change of molecular size.
To examine the dynamics of the carbon monoxide removal agent inside the body, changes with time of the concentration of hemoCD excreted into urine, mol % of CO-hemoCD in hemoCD, and CO amount were examined. Specifically, an experiment was performed as follows.
(1) Preparation of Medical Agents, Etc.
An oxy-hemoCD solution was prepared by the method described for EXAMPLE 1 and used as the carbon monoxide removal agent. Procurement of the experimental animal (rat), administration of the medical agent, sampling of urine, and ultraviolet-visible absorption spectral analysis were performed in the same manner as in EXAMPLE 1.
(2) Progressive Changes of HemoCD Concentration In Urine, Mol % of CO-HemoCD in HemoCD, and CO Amount
Urine was sampled every 30 minutes from the start of administration of the medical agent, the ultraviolet-visible absorption spectra of the urine were measured, and the hemoCD concentration in urine, the mol % of CO-hemoCD in hemoCD, and the CO amount excreted into urine were computed. The results are shown in
The molar concentration of hemoCD in the sampled urine was determined by adding suitable amounts of Na2S2O4 and CO gas to the urine to convert all of the hemoCD in the urine to the CO bound form, measuring the ultraviolet-visible absorption spectrum of the converted urine to determine the absorbance at the maximum absorption wavelength, and computing the concentration from the absorbance and the molar extinction coefficient indicated in the prior art documents, etc.
The mol % of CO-hemoCD in hemoCD contained in the sampled urine was determined by measuring the ultraviolet-visible absorption spectra of the urine as it is and the urine with all of the hemoCD therein converted to the CO bound form and computing the concentration from the absorbance difference at the maximum absorption wavelength. Also, the CO amount in urine was computed from the molar concentration of hemoCD contained in urine, the mol % of CO-hemoCD in hemoCD, and the urine amount.
(3) Experimental Results
a) shows that the excretion of hemoCD starts immediately after the start of administration and the amount of excreted hemoCD decreases extremely at nearly the same time as the end of administration. These suggest that the internal retention time of hemoCD is considerably short. Also from
Influences of differences in the concentration of oxy-hemoCD in the carbon monoxide removal agent administered on the mol of CO-hemoCD in the hemoCD excreted into urine and the amount of CO excreted into urine were examined. Specifically, the following was performed.
(1) Experimental Method
After preparing carbon monoxide removal agents of different oxy-hemoCD concentrations, the same experiment as in EXAMPLE 5 was performed to compute the mol % of CO-hemoCD in hemoCD and the excreted CO amount. The results are shown in
(2) Experimental Results
From
Number | Date | Country | Kind |
---|---|---|---|
2009-043632 | Feb 2009 | JP | national |
This is a continuation Application of International Application No. PCT/JP2010/053072, filed on Feb. 26, 2010, which claimed the priority of Japanese Application No. 2009-043632 filed Feb. 26, 2009, the entire content of each of which is incorporated herein by reference.
Number | Date | Country | |
---|---|---|---|
Parent | PCT/JP2010/053072 | Feb 2010 | US |
Child | 13218100 | US |