JP2006232818A - Inclusion compound of porphyrin metal complex and albumin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new compound as an oxygen carrier containing a porphyrin metal complex as an oxygen-adsorbing and desorbing site. <P>SOLUTION: The new inclusion compound is obtained by including in albumin a tetraphenylporphyrin metal complex represented by general formula (1) (R<SB>1</SB>is a cyclohexyl group-containing substituent group; R<SB>2</SB>is a substituent group; M is a transition metal atom or metal ion belonging to the group 6 to the group 10 of the periodic table; X<SP>-</SP>is a halogen ion; the number of X<SP>-</SP>is a number obtained subtracting 2 from the valence of the metal ion). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention is used in the medical field for oxygen supply to ischemic sites and tumor tissues, for blood transfusion of patients with massive bleeding, organ preservation perfusate, extracorporeal circulation fluid, cell culture fluid, and reversibly absorbs and desorbs oxygen. It relates to an oxygen carrier that can be used.
More specifically, the present invention relates to a novel compound in which a porphyrin metal complex is included with albumin as an oxygen adsorption / desorption site, and an oxygen carrier containing the novel compound as an active ingredient.

  In order to recover a patient's hemorrhagic shock, a technique of replenishing a plasma expander having a colloid osmotic pressure almost equal to that of living blood has been employed. However, when there is bleeding of 30% or more of the circulating blood volume, oxygen supply to peripheral tissues becomes insufficient, and in addition to the administration of the plasma expander, the administration of an oxygen carrier is further required.

  Conventionally, natural oxygen or erythrocyte concentrate containing natural red blood cells has been used as such an oxygen carrier. However, in order to avoid clotting due to antigen-antibody reaction, it is necessary to match the blood types of the donor and recipient, and to perform a cross suitability test at the time of use, and such natural blood or erythrocyte concentrate is The effective storage period is as short as 3 weeks (4 ° C.), and frozen blood that can be stored for a long time by cryopreservation has a problem of high cost and easy hemolysis due to osmotic shock during use. Furthermore, there were concerns about the occurrence of infectious diseases such as hepatitis and AIDS.

  As an oxygen carrier for solving such problems, porphyrin iron (II) complexes existing in hemoglobin and myoglobin have attracted attention because they can absorb and desorb oxygen molecules reversibly. Many studies on oxygen carriers using synthetic complexes similar to such natural porphyrin iron (II) complexes have been reported (see, for example, Non-Patent Documents 1 and 2).

By the way, using a synthetic compound (especially a synthetic porphyrin metal complex), its function is reproduced under physiological conditions (in a physiological salt aqueous solution (pH 7.4), room temperature to 37 ° C.), and used for medical purposes such as artificial erythrocytes, The following conditions are required when using it as an organ preservation solution or an oxygen supply solution for an artificial lung or the like.
(I) The presence of an axial base ligand is generally required to increase the oxygen binding capacity, but some imidazole derivatives that are widely used as such ligands have pharmacological action, Some of them are highly toxic in vivo, so the concentration should be kept to a minimum.
(Ii) Not only solubilizing the porphyrin metal complex in water but also embedding and immobilizing it in a microscopic hydrophobic environment, through oxidation of the central metal by protons and via the μ-oxo dimer To keep the oxygen coordination complex stable by preventing oxidation.

For the above condition (i), a porphyrin compound in which an imidazolyl group is covalently bonded to a porphyrin ring, that is, a substituted porphyrin compound having an axial base in the molecule (porphyrin / imidazole molar ratio of 1: 1 is required minimum) It has been clarified that this forms a stable oxygen coordination complex (see Patent Document 1).
In this porphyrin complex, particularly when an axial base is bound by an ester bond, it is highly biodegradable and extremely advantageous for in vivo administration, and is useful as a highly safe oxygen binding site (porphyrin metal complex). It is known.

  Regarding the condition (ii), it is known to use a bilayer membrane vesicle composed of a micelle composed of a surfactant or a phospholipid. However, micelles are dynamic in form, are inferior in stability compared to bilayer vesicles, and are less hydrophobic in the hydrophobic environment they form. Therefore, bilayer vesicles that are relatively stable in shape and can provide sufficient hydrophobic regions are often used as carriers to provide a hydrophobic environment for the complex, and between the bilayers of phospholipid vesicles. The development of oxygen carriers that can transport oxygen stably even under physiological conditions by dispersing porphyrin metal complexes with high orientation has been continuously promoted.

From the idea that by introducing a terminal hydrophilic alkyl substituent on the porphyrin ring and adding an amphiphilic structure to the porphyrin, it can be embedded in the hydrophobic environment of the phospholipid bilayer membrane with high orientation. A series of oxygen carriers effective in an aqueous phase system have been developed by embedding and orienting these porphyrin complexes in phospholipid bilayers (for example, Patent Document 2).
However, since these oxygen carriers use a large amount of phospholipid, there are problems in terms of production on an industrial scale and biocompatibility including metabolism.

  On the other hand, as an oxygen carrier that does not use phospholipids, an inclusion compound of albumin and heme (albumin-heme) has been studied as an oxygen carrier. As an example of albumin-heme, tetraphenylporphyrin iron derivative 2- [8- [N- (2-methylimidazolyl)] octanoyloxymethyl] -5,10,15,20-tetrakis (α, α, (α, α-o-pivalamide) phenylporfinato iron complex and the like encapsulated in a hydrophobic pocket of albumin (see Non-Patent Document 3).

  However, these oxygen carriers still have room for improvement in terms of biocompatibility including metabolism and production on an industrial scale.

JP-A-6-271777 JP 58-213777 A J. et al. P. Collman, Accounts of Chemical Research, 10, 265 (1977) F. Basolo, B.B. M.M. Hoffman, J.M. A. Ibers, Accounts of Chemical Research, 8, 384 (1975) E. Tsuchida, et al., Bioconjugate Chemistry, vol.8, 534-538, 1997

  The present invention seeks to provide a novel compound that is superior in biocompatibility and relatively easy to produce on an industrial scale as an oxygen carrier containing a porphyrin metal complex as an oxygen adsorption / desorption site, and a method for producing the same. is there.

In order to solve the above problems, the present inventors provide a molecular design and functional expression of an oxygen carrier (porphyrin metal complex) capable of stably transporting oxygen under physiological conditions and a hydrophobic environment for the porphyrin metal complex. Research was conducted on the carrier with high biocompatibility to be obtained.
As a result, a tetraphenylporphyrin metal complex having a 2-position side chain represented by the following general formula (1), and albumin that accounts for 50 to 55% of plasma proteins and is responsible for transporting various compounds in vivo An inclusion compound was produced, and it was found that this compound is useful as an oxygen carrier excellent in biocompatibility.
General formula (1)

（式中、Ｒ₁ はシクロヘキシル基を含む置換基、Ｒ₂ は置換基、Ｍは元素周期表第６〜１０族の遷移金属原子または金属イオン、Ｘ^- はハロゲンイオンを表わし、Ｘ^- の個数は金属イオンの価数から２を差し引いた数を示す。）

(In the formula, R ₁ represents a substituent containing a cyclohexyl group, R ₂ is a substituted group, M is an element of the periodic table Groups 6-10 transition metal atom or a metal ion, X ^- represents the halogen ions, X ^- number of Indicates the number obtained by subtracting 2 from the valence of the metal ion.)

That is, the present invention
(1): a novel inclusion compound obtained by inclusion of a tetraphenylporphyrin metal complex having a two-position side chain represented by the general formula (1) with albumin,
(2): The following general formula (2)

（式中、Ｒ₂ は置換基、Ｍは元素周期表第６〜１０族の遷移金属原子または金属イオン、Ｘ^- はハロゲンイオンを表わし、Ｘ^- の個数は金属イオンの価数から２を差し引いた数を示す。）で示される、上記（１）記載の包接化合物、
（３）： Ｒ₂ が一般式（３）

(In the formula, R ₂ represents a substituent, M represents a transition metal atom or metal ion of Groups 6 to 10 of the periodic table, X ⁻ represents a halogen ion, and the number of X ⁻ subtracts 2 from the valence of the metal ion. The inclusion compound according to the above (1),
(3): R ₂ is the general formula (3)

［式中、Ｒ₃ はＣ₁ 〜Ｃ₁₈のアルキル基、または一般式（４）

[Wherein R ₃ is a C _{1 to} C ₁₈ alkyl group, or a group represented by the general formula (4)

（式中、Ｒ₄ はこれが結合しているイミダゾールの中心金属への配位を阻害しない基、
Ｒ₅ はアルキレン基を示す。）］
で示されるものである、上記（１）記載の包接化合物、
（４）： Ｍが鉄、コバルトまたはクロムである、上記（１）記載の包接化合物、
（５）： Ｍが＋２価の鉄、＋３価の鉄または＋２価のコバルトである、上記（１）記載の包接化合物、
（６）： Ｒ₄ が水素、メチル基、エチル基またはプロピル基である、上記（３）記載の包接化合物、
（７）： Ｒ₅ がＣ₁ 〜Ｃ₁₀のアルキレン基である、上記（３）記載の包接化合物。
（８）： アルブミンがヒト血清アルブミン又は組換えヒト血清アルブミンである、上記（１）記載の包接化合物、
（９）： 上記（１）記載の包接化合物を有効成分として含む酸素運搬体、および
（１０）： 上記（９）記載の酸素運搬体を含む医薬組成物
に関する。

Wherein R ₄ is a group that does not inhibit the coordination of the imidazole to which it is attached to the central metal,
R ₅ represents an alkylene group. ]]
An inclusion compound as described in (1) above,
(4): The inclusion compound according to (1), wherein M is iron, cobalt, or chromium,
(5): The inclusion compound according to the above (1), wherein M is + 2-valent iron, + 3-valent iron, or + 2-valent cobalt,
(6): The clathrate compound according to the above (3), wherein R ₄ is hydrogen, methyl group, ethyl group or propyl group,
(7) The inclusion compound according to (3), wherein R ₅ is a C _{1 to} C ₁₀ alkylene group.
(8) The inclusion compound according to (1) above, wherein the albumin is human serum albumin or recombinant human serum albumin,
(9): It relates to an oxygen carrier containing the inclusion compound described in (1) as an active ingredient, and (10): a pharmaceutical composition containing the oxygen carrier described in (9) above.

The oxygen carrier containing the inclusion compound of the porphyrin metal complex of the present invention and albumin (hereinafter abbreviated as porphyrin-albumin inclusion compound) has excellent biocompatibility and excellent oxygen adsorption / desorption ability even under physiological conditions. Indicates.
In addition, the porphyrin-albumin inclusion compound of the present invention and the oxygen carrier containing the compound as an active ingredient can be relatively easily produced on an industrial scale.

  Further, in the porphyrin-albumin inclusion compound of the present invention, since the porphyrin metal complex which is an oxygen adsorbing site is fixed in the internal hydrophobic environment of albumin, oxidation by the proton of the central metal, and μ-oxo dimer As a result of completely inhibiting the oxidative degradation process via, the inclusion compound of the present invention can retain a stable oxygen coordination complex even in an aqueous phase system.

  Furthermore, the porphyrin-albumin inclusion compound of the present invention quickly forms a stable oxygen coordination complex when it comes into contact with oxygen. The oxygen coordination complex can adsorb and desorb oxygen according to the oxygen partial pressure. This oxygen adsorption / desorption can be repeated reversibly and stably due to the difference in oxygen partial pressure. In addition, oxygen bond dissociation is rapid, and the inclusion compound of the present invention is a semi-artificial oxygen carrier that can efficiently carry oxygen even in the bloodstream in the living body, and when recombinant human serum albumin is used, It can function as a total synthetic oxygen carrier.

  Furthermore, the porphyrin-albumin inclusion compound of the present invention is easy to produce, and is particularly suitable for production on an industrial scale when recombinant human serum albumin is used.

The porphyrin metal complex used in the present invention is a tetraphenylporphyrin metal complex having a two-position side chain in which a central metal is coordinated to a porphyrin ring derivative as shown in the general formula (1). The porphyrin ring is a macrocyclic compound in which four pyrrole rings are alternately bonded to four methine groups at the α-position and derivatives thereof.
The porphyrin metal complex of the present invention is characterized in that, in the general formula (1), R ₁ is a substituent containing a cyclohexyl group. As the substituent R ₁ , a substituent as shown in the general formula (2) is preferable.

In the general formula (1), examples of the 2-position substituent (R ₂ ) include those represented by the following general formula (3).
General formula (3)

（式中、Ｒ₃ はＣ₁ 〜Ｃ₁₈のアルキル基、または下記一般式（４）で示されるものである。）
一般式（４）

(In the formula, R ₃ is a C _{1 to} C ₁₈ alkyl group, or one represented by the following general formula (4).)
General formula (4)

（式中、Ｒ₄ はこれが結合しているイミダゾールの中心金属への配位を阻害しない基、Ｒ₅ はアルキレン基を示す。）

(In the formula, R ₄ represents a group that does not inhibit the coordination of the imidazole to which it is bonded to the central metal, and R ₅ represents an alkylene group.)

In the general formula (4), R ₄ is preferably hydrogen or a lower alkyl group such as a methyl group, an ethyl group, or a propyl group, and R ₅ is preferably a C _{1 to} C ₁₀ alkylene group.

In the tetraphenylporphyrin metal complex represented by the general formula (1) of the present invention, the complex that can specifically bind oxygen is such that the central metal (M) is in a + 2-valent state and one base is coordinated. These are complexes, and these are preferably those represented by the following general formula (5) or general formula (6).
General formula (5)

なお、前記一般式（５）において、Ｌはイミダール誘導体などの窒素系軸配位子を示す。
一般式（６）

In the general formula (5), L represents a nitrogen-based axial ligand such as an imidazole derivative.
General formula (6)

  In the tetraphenylporphyrin metal complex represented by the general formula (1) of the present invention, the effective oxygen adsorbing / desorbing agent or oxygen carrier as described above has a structure in which the central metal (M) is a divalent complex. is there. In the case of an imidazole derivative in which the substituent at the 2-position itself functions as an axial base, the intramolecular imidazole can be coordinated to the porphyrin central metal (M), so that it does not add a large excess of the axial base externally. With this, oxygen binding ability can be demonstrated. In other words, in the porphyrin compound in which the axial base is bound intramolecularly, it is not necessary to add a large amount of the imidazole derivative as the axial base, and when considering in vivo administration, the useful material in which the axial base concentration is greatly reduced. It becomes.

The central metal (M in the above general formula (1)) in the porphyrin metal complex of the present invention is a transition metal atom of Groups 6 to 10 of the periodic table, and may be in the form of a metal ion. Of these, iron, cobalt, chromium and the like are preferable, and +2 valent iron (iron (II)), +3 valent iron (iron (III)), or +2 valent cobalt (cobalt (II)) is more preferable. When the central metal is iron, the iron has oxygen adsorption / desorption ability when it is in a +2 state.
Among the porphyrin metal complexes of the present invention, when the iron (III) complex is in the form, a suitable reducing agent (sodium dithionite, ascorbic acid, etc.) is used, and central iron is trivalent to divalent by a conventional method. If reduced, oxygen binding activity can be imparted.

As the porphyrin metal complex of the present invention, a tetraphenylporphyrin metal complex in which a transition metal atom is coordinated to a porphyrin ring as shown in the following general formula (7) is particularly preferable.
In the formula, M represents a transition metal atom or metal ion of Groups 6 to 10 of the periodic table.
General formula (7)

（式中、Ｍは元素周期表第６〜１０族の遷移金属原子または金属イオン、Ｒ₄ はこれが結合しているイミダゾールの中心金属への配位を阻害しない基、Ｒ₅ はアルキレン基を示す。）

(In the formula, M is a transition metal atom or metal ion of Groups 6 to 10 of the periodic table, R ₄ is a group that does not inhibit the coordination of the imidazole to which it is bonded to the central metal, and R ₅ is an alkylene group. .)

Among these porphyrin metal complexes, 2- [8- (2-methyl-1-imidazolyl) octanoyloxymethyl] -5,10,15,20-tetrakis [α, α represented by the following general formula (8) , Α, α-o- (1-methylcyclohexanoylamino) phenylporphyrin] is preferably a complex in which iron is coordinated at the center.
General formula (8)

  In the porphyrin metal complex represented by the general formula (8), it is important that the alkylimidazolyl group is bonded to the 2-position of the porphyrin ring. According to the study by the present inventors, a porphyrin complex having no imidazolyl group in the molecule can be added in the aqueous phase system even if a small excess of imidazole derivative (for example, 1-methylimidazole) is added from the outside. It deteriorated immediately without producing a stable oxygen complex.

  The inclusion compound of a porphyrin metal complex and albumin of the present invention is a compound in which a porphyrin metal complex is embedded and fixed (inclusion) in an internal hydrophobic region formed by albumin. The number of binding of the porphyrin metal complex to 1 mol of albumin (for example, human serum albumin) is usually about 1-8. The number of porphyrin metal complexes included / bound in 1 mol of albumin is, for example, a Scatchard plot (CJ Halfman, T. Nishida, Biochemistry, 11, 3493 (1972)). Can be determined.

  The albumin used in the present invention is not limited in its origin such as human serum albumin, recombinant human serum albumin, bovine serum albumin and the like. When considering application to humans, human serum albumin or human serum albumin produced by gene recombination technology (hereinafter referred to as recombinant human serum albumin) is preferable, and recombinant human serum albumin is particularly preferable. .

In recent years, with the development of genetic recombination technology, high-purity recombinant human serum albumin having the same structure, composition and physicochemical characteristics as human serum albumin has been developed (Yokoyama, Omura, Clinical Molecular Medicine, 1, page 939 (1993)), a porphyrin metal complex-recombinant human serum albumin clathrate compound in which this recombinant human serum albumin is clathrated with a substituted porphyrin metal complex can be provided as a total synthesis system. Manufacture on an industrial scale is also relatively easy.
In addition, since albumin is a plasma protein, it is much more advantageous than a system using phospholipid endoplasmic reticulum for application to a living body, particularly for use as a substitute for red blood cells.

  Albumin is a simple protein whose main role is to control colloid osmotic pressure in blood, but also functions as a transport protein for nutrient substances, their metabolites, drugs, and the like. The present invention has been completed by paying attention to such non-specific binding ability of albumin which is not found in other proteins.

The oxygen carrier of the present invention is a compound or structure containing an inclusion compound obtained by inclusion of the porphyrin metal complex in albumin, an erythrocyte substitute at the time of blood transfusion, a tissue disorder inhibitor for ischemic sites, It is used as a therapeutic agent for ischemic diseases such as myocardial infarction and a sensitizer in tumor radiotherapy.
In addition, various pharmaceutical compositions containing such an oxygen carrier and added with a stabilizer and the like are also included in the scope of the present invention.

  The tetraphenylporphyrin metal complex represented by the general formula (1) of the present invention can be produced by various known production methods. For example, 2- [8- (2-methyl-1-imidazolyl) octanoyloxymethyl] -5,10,15,20-tetrakis [α, α, α, α-o- represented by the above general formula (8) The (1-methylcyclohexanoylamino) phenyl] porfinato iron complex can be produced by the production method described in T. komatsu et al. Bioconjugate Chemistry 13, p.397-402, 2002.

  Next, in order to prepare the inclusion compound of the porphyrin metal complex and albumin of the present invention, first, the porphyrin metal complex is dissolved in a solvent (for example, ethanol, methanol, etc.), and albumin (for example, human serum albumin) is dissolved therein. ) Solution (for example, water, phosphate buffer (pH 5-9), physiological saline, Craves-Ringer solution, etc.) as a solvent, and then shake gently. The obtained aqueous dispersion is concentrated to about 10% of the total amount by ultrafiltration (for example, using an ultrafiltration membrane having an ultramolecular weight of 20,000 to 40,000), then water is added again, and ultrafiltration is performed. When the operation of filtration is repeated, a porphyrin metal complex-albumin inclusion compound is obtained. This dispersion is stable with no precipitation or aggregation even after storage at 4-35 ° C. for several months.

When the central metal of the porphyrin metal complex is iron (III), the central iron is trivalent to divalent by a conventional method such as adding a reducing agent (for example, an aqueous solution of sodium dithionite, ascorbic acid, etc.). It is desirable to impart oxygen binding activity by reduction to
This reduction can be performed not only by addition of a reducing agent, but also by palladium carbon / hydrogen gas. For example, it is possible to reduce central iron by dissolving porphyrin iron (III) complex in dry dichloromethane, benzene, toluene, etc., adding a small amount of palladium carbon, and then thoroughly blowing hydrogen gas into the mixture at room temperature. it can. After the reduction, the palladium carbon can be filtered off and the filtrate can be used after vacuum drying.
The above reduction is preferably performed before the inclusion reaction.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

(Production of porphyrin metal complex)
2- [8- (2-methyl-1-imidazolyl) octanoyloxymethyl] -5,10,15, produced by the method described in T. komatsu et al. Bioconjugate Chemistry 13, p.397-402, 2002 20-tetrakis [α, α, α, α-o- (1-methylcyclohexanoylamino) phenyl] porfinato iron complex was obtained.

(Inclusion of porphyrin metal complex and albumin)
In a carbon monoxide atmosphere, 2- [8- (2-methyl-1-imidazolyl) octanoyloxymethyl] -5,10,15,20-tetrakis [α, α, α, α-o- (1-methyl Cyclohexanoylamino) phenyl] porfinato iron complex (1.07 mM) in ethanol solution (1.5 L) is added with 1.5 M of 0.6 M L-ascorbic acid aqueous solution to reduce the solution, and this solution is genetically modified human. The solution was added to 6.5 L of a phosphate buffer aqueous solution (pH 7.4, 1/30 mM) containing 0.27 mM of serum albumin (hereinafter rHSA) and stirred. Constant volume ultrafiltration dialysis using an ultrafiltration device (Millipore ultrafiltration membrane: ultramolecular weight 30,000) while adding 60 L of phosphate buffered water (pH 7.4, 1/30 mM) to the mixture. The ethanol contained in the mixed solution was removed. This mixture was concentrated to 300 mL to obtain a desired porphyrin metal complex-albumin inclusion compound dispersion (hereinafter abbreviated as rHSA-FecycP). rHSA-FecycP (CO conjugate) was extremely stable with no precipitation or aggregation even when stored for several months at room temperature or 4 ° C.

  When rHSA-FecycP (CO conjugate) was diluted to 1/50 and an ultraviolet-visible absorption spectrum was measured, the absorption maximum was shown at a wavelength of 427 nm.

When oxygen gas was passed through rHSA-FecycP (CO conjugate) while irradiating light (500 W), the spectrum immediately changed and the absorption maximum was shown at a wavelength of 424 nm. This indicates that the porphyrin iron (II) complex included in albumin is an oxygenated complex. When nitrogen was bubbled through rHSA-FecycP (O ₂ conjugate), the spectrum immediately changed and showed a maximum absorption at a wavelength of 443 nm. This indicates that oxygen was removed from porphyrin iron (II) included in albumin. When oxygen was passed through rHSA-FecycP (Deoxy isomer), the visible absorption spectrum changed to an oxygenated spectrum, and it was confirmed that the adsorption and desorption of oxygen occurred reversibly. In addition, adsorption / desorption of oxygen was able to be repeatedly performed by repeating alternate aeration of oxygen and nitrogen.

The oxygen affinity of rHSA-FecycP (P _1/2 (O ₂ ): oxygen partial pressure necessary to maintain a state in which oxygen is bonded to a half of the porphyrin metal complex) is 35 Torr (37 ° C. )Met. The oxygen transport efficiency between the lung (110 Torr) and the peripheral tissue (40 Torr) estimated from the oxygen bond dissociation equilibrium curve is about 20% (37 ° C.), and this inclusion compound effectively acts as an oxygen transporter for erythrocyte replacement. Was shown to do. In addition, the enthalpy / entropy change of the oxygen bond revealed that the behavior of the oxygen coordination complex in this inclusion compound was almost the same as that of hemoglobin in erythrocytes.

(Experimental example 1)
About the rHSA-FecycP obtained in Example 1, the rat blood exchange model was used, and the in vivo safety | security was evaluated using the body weight change and the blood biochemical value as a parameter | index.

(Test substance and control substance)
RHSA-FecycP obtained in Example 1 was used as a test substance, and genetically modified human serum albumin (25% preparation, hereinafter rHSA, manufactured by Baifa Corporation) was used as a control substance. The test substance was oxygenated, stored at 4 ° C., and used within 8 hours. In addition, 25% rHSA was diluted 5-fold with a physiological saline solution (Otsuka Pharmaceutical Factory Co., Ltd., Otsuka Raw Food Injection) and prepared at the time of use to obtain a concentration of 5% to obtain an administration solution.

(Used drugs and reagents)
The drugs and reagents used in addition to the test substance and the control substance are as follows. Diethyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), sevoflurane (manufactured by Maruishi Pharmaceutical Co., Ltd., trade name: Sevofurene), heparin (manufactured by Aventis Pharma, trade name: Novo Heparin Injection 1000) and physiological saline (manufactured by Otsuka Pharmaceutical Factory, trade name: Otsuka raw food note).

(Animal used)
Crj: Wistar male rats (7 weeks old) were purchased from Charles River, Japan, and 36 animals with good growth were used for the test. The animals consisted of a total of 3 groups: a control group (no blood exchange), an rHSA group (blood exchange using rHSA) and a cyc group (blood exchange using rHSA-FecycP). 12 were assigned to each. However, 6 out of 12 animals in each group were collected from the surviving rats 1 day after the completion of blood exchange and the remaining 6 animals were collected 7 days after the completion of blood exchange.

(Surgery)
After induction anesthesia with ether, the rat was fixed to the dorsal position under anesthesia with sevoflurane (trade name: sevoflurane, 2.0% inhalation), and the surgical site was shaved with a clipper for animals. The right thigh was incised and the tip of a catheter filled with 50 U / mL of heparin was inserted into the right femoral vein to obtain a blood removal route. The animals performed a 20% blood exchange for the rHSA and cyc groups. That is, after the determination of 1 mL from the right femoral vein, 1 mL of the test substance and the control substance were administered from the right femoral vein, and these operations were performed once and repeated 4 times in total. Blood was collected from the abdominal vena cava 1 and 7 days after the completion of blood exchange.

(Observation of general symptoms)
General symptoms were observed before blood exchange and 1, 3, and 7 days after completion of blood exchange. There was no change in general symptoms in all three groups.

(Weight change)
Body weight was measured before blood exchange and 1, 3, and 7 days after completion of blood exchange. The weight change (g) was calculated by subtracting the weight before blood exchange from the weight after 1, 3 and 7 days. FIG. 1 shows the change in body weight of each group up to 7 days after blood exchange. Observation was conducted until 7 days after the end of blood exchange, but no significant difference was observed among the control group, the rHSA group and the cyc group.

(Blood biochemistry)
Quantification was performed for AST, ALT, LDH, LAP, CRN, and BUN in plasma. The results 1 and 7 days after the end of blood exchange for each group are shown in FIG. The collected venous blood was centrifuged and then plasma was collected and immediately stored frozen (set temperature: −20 ° C.).

(1 day after the end of blood exchange)
In all three groups, no significant difference was observed in the amounts of AST, ALT, LDH, LAP, CRN and BUN in plasma.

(7 days after blood exchange)
In all three groups, no significant difference was observed in the amounts of AST, ALT, LDH, LAP, CRN and BUN in plasma.

  As is apparent from Experimental Examples 1 and 2 above, it was shown that rHSA-FecycP obtained in Example 1 exhibited excellent oxygen adsorption / desorption ability even under physiological conditions and high biocompatibility.

  The inclusion compound of the present invention can be used as an oxygen carrier for various medical uses, such as an erythrocyte substitute during blood transfusion, a tissue damage inhibitor at an ischemic site, an ischemic disease such as myocardial infarction. It can be used for applications such as therapeutic agents and sensitizers in tumor radiotherapy.

It is a figure which shows transition of the body weight after the blood exchange in Experimental example 1. FIG. It is a figure which shows the blood biochemical test value after the blood exchange in Experimental example 1. FIG.

Claims (10)

The following general formula (1)

(In the formula, R ₁ represents a substituent containing a cyclohexyl group, R ₂ is a substituted group, M is an element of the periodic table Groups 6-10 transition metal atom or a metal ion, X ^- represents the halogen ions, X ^- number of Indicates the number obtained by subtracting 2 from the valence of the metal ion.)
A novel inclusion compound obtained by inclusion of a tetraphenylporphyrin metal complex having a 2-position side chain represented by formula (II) with albumin. The following general formula (2)

(In the formula, R ₂ represents a substituent, M represents a transition metal atom or metal ion of Groups 6 to 10 of the periodic table, X ⁻ represents a halogen ion, and the number of X ⁻ subtracts 2 from the valence of the metal ion. The inclusion compound according to claim 1, which is represented by: R ₂ represents the general formula (3)

[Wherein R ₃ is a C _{1 to} C ₁₈ alkyl group, or a group represented by the general formula (4)

Wherein R ₄ is a group that does not inhibit the coordination of the imidazole to which it is attached to the central metal,
R ₅ represents an alkylene group. ]]
The clathrate compound according to claim 1, which is represented by:   The inclusion compound according to claim 1, wherein M is iron, cobalt or chromium.   The clathrate compound according to claim 1, wherein M is +2 valent iron, +3 valent iron, or +2 valent cobalt. The inclusion compound according to claim 3, wherein R ₄ is hydrogen, a methyl group, an ethyl group or a propyl group. R ₅ is an alkylene group of C ₁ -C _10, claim 3 clathrate compound according.   The inclusion compound according to claim 1, wherein the albumin is human serum albumin or recombinant human serum albumin.   An oxygen carrier comprising the clathrate compound according to claim 1 as an active ingredient. A pharmaceutical composition comprising the oxygen carrier according to claim 9.

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