JP5020525B2 - Ligand substitution infusion formulation - Google Patents
Ligand substitution infusion formulation Download PDFInfo
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- JP5020525B2 JP5020525B2 JP2006095901A JP2006095901A JP5020525B2 JP 5020525 B2 JP5020525 B2 JP 5020525B2 JP 2006095901 A JP2006095901 A JP 2006095901A JP 2006095901 A JP2006095901 A JP 2006095901A JP 5020525 B2 JP5020525 B2 JP 5020525B2
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Description
本発明は、医療分野において、輸血を必要とする患者に対して使用するための輸液製剤に係り、より詳しくは、血管拡張作用または細胞保護効果を示す配位子置換型輸液製剤に関する。本発明は、特に、虚血状態からの回復において虚血再灌流障害を防止し得る輸液製剤に関する。 The present invention relates to an infusion preparation for use in patients who require blood transfusion in the medical field, and more particularly to a ligand-substituted infusion preparation exhibiting a vasodilating action or a cytoprotective effect. In particular, the present invention relates to an infusion preparation that can prevent ischemia-reperfusion injury in recovery from an ischemic state.
現行の献血／輸血システムは医療に不可欠の技術として確立している。しかし諸問題（感染、保存期限、献血者数の減少等）も抱え、輸血に代わる方法の探索が緊急課題になっており、特に酸素運搬機能を司る赤血球の代替物の実現が求められている。 The current blood donation / transfusion system has been established as an essential medical technology. However, there are various problems (infection, shelf life, decrease in the number of blood donors, etc.), and the search for alternatives to blood transfusion has become an urgent issue, and in particular, the realization of an alternative to red blood cells that control oxygen transport functions is required. .
赤血球に高濃度で含まれるヘモグロビン（Ｈｂ）が酸素結合蛋白質であることから、赤血球の代替物として、このＨｂを利用した人工赤血球の開発が進められている。Ｈｂは、厳密な精製を経れば、病原体や血液型物質を完全に除去することが可能である。Ｈｂを利用した人工赤血球としては、化学修飾Ｈｂ（水溶性高分子結合型、分子内架橋型、重合型）のほか、リン脂質小胞体の内水相に高純度Ｈｂを内包したＨｂ小胞体等が知られている。これらのうち、世界的に見て、修飾Ｈｂ溶液の開発が先行し、臨床試験の最終段階に到達したものがある（特許文献１、特許文献２、特許文献３、特許文献４）。しかし、予期せぬ副作用のために開発を中断するケースが相次いでいる。これは、赤血球の生理学的意義に基づいた構造の欠如による。赤血球は直径約８μｍの中窪み円盤状粒子であり、Ｈｂ（分子量６４５００）の高濃度溶液（３５％）を赤血球膜で内包した構造を持っている。Ｈｂ溶液が赤血球膜で覆われている理由は、３５％濃厚Ｈｂ溶液の高い粘度と膠質浸透圧の抑制、Ｈｂ機能維持のための各種リン酸化合物や酵素類等機能物質の保持の役割、また、本来毒性のあるＨｂの逸脱の抑制である。遊離したＨｂは血管内皮弛緩因子である一酸化窒素（ＮＯ）を強く結合し、投与に際し血圧の異常亢進や食道の蠕動運動の障害等の副作用が明らかになっている。また、血管壁との直接的な接触により血管壁に損傷を与えることも指摘されている。従って、赤血球と同様の小胞体（細胞型）構造が重要である。 Since hemoglobin (Hb) contained in erythrocytes at a high concentration is an oxygen-binding protein, development of artificial erythrocytes using Hb as an alternative to erythrocytes is underway. Hb can completely remove pathogens and blood group substances through strict purification. Artificial erythrocytes using Hb include chemically modified Hb (water-soluble polymer binding type, intramolecular crosslinking type, polymerization type), Hb endoplasmic reticulum encapsulating high-purity Hb in the inner aqueous phase of phospholipid endoplasmic reticulum, etc. It has been known. Among these, the development of modified Hb solutions precedes the world, and some have reached the final stage of clinical trials (Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4). However, there are many cases where development is interrupted due to unexpected side effects. This is due to the lack of structure based on the physiological significance of red blood cells. Red blood cells are medium-dented disk-like particles having a diameter of about 8 μm, and have a structure in which a high-concentration solution (35%) of Hb (molecular weight 64500) is enclosed in a red blood cell membrane. The reason why the Hb solution is covered with the erythrocyte membrane is that the high viscosity of 35% concentrated Hb solution and the suppression of colloid osmotic pressure, the role of holding functional substances such as various phosphate compounds and enzymes for maintaining Hb function, It is the suppression of the departure of Hb, which is inherently toxic. The liberated Hb strongly binds nitric oxide (NO), which is a vascular endothelial relaxing factor, and side effects such as abnormal increase in blood pressure and disturbance of esophageal peristalsis have been revealed upon administration. It has also been pointed out that the blood vessel wall is damaged by direct contact with the blood vessel wall. Therefore, an endoplasmic reticulum (cell type) structure similar to that of red blood cells is important.
両親媒性分子であるリン脂質が水中で自己集合して二分子膜を形成しこれが小胞構造（リポソーム）になることが１９６０年代後半に報告され、このリン脂質小胞体にＨｂを内包する試みがなされてきたが、粒径制御等調製の困難さと、血漿蛋白質との相互作用に起因する凝集を阻止するための有効な手段が見いだせなかったため、成功しなかった（非特許文献１）。しかし、その後、リン脂質二分子膜で高濃度Ｈｂ溶液を被覆する技術が開発され、特に毛細血管を容易に通過することを可能とする粒径の制御と、血中分散安定性の向上が達成されたことにより、Ｈｂ小胞体が完成した（非特許文献２）。また、ポリエチレングリコールをリン脂質小胞体粒子の表面に配することによりＨｂ小胞体粒子間の凝集の抑制と分散安定性の向上が達成され、Ｈｂ小胞体分散溶液から酸素を実質的に完全に除去することにより、その分散溶液のまま室温にて長期間の保存が可能となった（特許文献５）。 It was reported in the late 1960s that phospholipids, which are amphipathic molecules, self-assembled in water to form bilayers that became vesicular structures (liposomes), and attempts to encapsulate Hb in these phospholipid vesicles. However, it has been unsuccessful because an effective means for preventing aggregation caused by difficulty in preparation such as particle size control and interaction with plasma proteins has not been found (Non-patent Document 1). However, technology for coating a high-concentration Hb solution with a phospholipid bilayer was later developed, and in particular, control of the particle size enabling easy passage through capillaries and improvement in blood dispersion stability were achieved. As a result, the Hb endoplasmic reticulum was completed (Non-patent Document 2). In addition, by disposing polyethylene glycol on the surface of the phospholipid vesicle particles, the suppression of aggregation between the Hb vesicle particles and the improvement of dispersion stability are achieved, and oxygen is substantially completely removed from the Hb vesicle dispersion solution. As a result, the dispersion solution can be stored for a long time at room temperature (Patent Document 5).
Ｈｂ小胞体粒子は分子状のＨｂ修飾体に比較して粒子径が大きいため、希薄分散溶液状態でのその酸素解離速度定数は、ストップドフロー法で計測すると、Ｈｂ小胞体の方が極めて遅い（非特許文献３）。さらにＨｂ小胞体の濃厚溶液の微小細管内における酸素の放出速度も、分子状Ｈｂよりも遅い。これは、分子状Ｈｂはそれ自体が拡散することにより酸素の移動が促進される効果（促進輸送効果）があるためである（非特許文献４）。 Since Hb endoplasmic reticulum particles have a larger particle size than molecular Hb modified bodies, the oxygen dissociation rate constant in a dilute dispersion solution state is much slower for Hb endoplasmic reticulum when measured by the stopped flow method. (Non-Patent Document 3). Furthermore, the oxygen release rate in the microtubules of the concentrated solution of Hb endoplasmic reticulum is also slower than that of molecular Hb. This is because molecular Hb has the effect of promoting oxygen movement (facilitated transport effect) by diffusing itself (Non-Patent Document 4).
Ｈｂ小胞体粒子分散液は膠質浸透圧を持たないので、血液交換率が高い場合には循環血液量を維持するために、血漿増量剤の添加が必要となる。例えばＨｂ小胞体を５％アルブミン水溶液に分散させた場合の膠質浸透圧は２０ｍｍＨｇとなり、粘度と浸透圧は血液とほぼ同等になり、循環動態の恒常性に寄与する。Ｈｂ小胞体の粒径は赤血球の約１／３０と小さく、血漿中に均一分散できる。Ｈｂ小胞体の動物投与試験では、臨床現場で想定される最大の交換率４０％の交換輸血においてもラットは全例が生存し、ヘマトクリットは１週間で完全に回復する。Ｈｂ小胞体は最終的に細網内皮系に捕捉され、安全に分解、排泄される。従って、Ｈｂ小胞体は、臨床では術前血液希釈、術中出血分の補給、さらに胸部外科手術における人工心肺（ＥＣＭＯ）体外循環回路の補充液としての利用が十分に期待できる。 Since the Hb endoplasmic reticulum particle dispersion does not have colloid osmotic pressure, it is necessary to add a plasma expander in order to maintain the circulating blood volume when the blood exchange rate is high. For example, when the Hb endoplasmic reticulum is dispersed in a 5% albumin aqueous solution, the colloid osmotic pressure is 20 mmHg, the viscosity and the osmotic pressure are almost the same as blood, and contribute to the constancy of circulatory dynamics. The particle size of the Hb endoplasmic reticulum is as small as about 1/30 that of red blood cells, and can be uniformly dispersed in plasma. In the animal administration test of Hb endoplasmic reticulum, all rats survived even in the exchange transfusion with the maximum exchange rate of 40% assumed in the clinical field, and the hematocrit is completely recovered in one week. The Hb endoplasmic reticulum is finally captured by the reticuloendothelial system, and is safely degraded and excreted. Therefore, Hb endoplasmic reticulum can be sufficiently expected to be used as a replenisher for an artificial cardiopulmonary (ECMO) extracorporeal circuit in thoracic surgery as well as preoperative hemodilution, replenishment of bleeding during surgery, and chest surgery.
Ｈｂ小胞体は、出血性ショック時の蘇生液としても検討され、赤血球と同等の酸素運搬機能が実証されている。例えば、麻酔下、ラットの循環血液量の５０％を脱血して１５分後にＨｂ小胞体を５％アルブミン溶液に分散させて投与し６時間観察した実験では、アルブミン単独の投与では８匹中２匹が死亡したのに対し、Ｈｂ小胞体の投与では循環動態も血液ガス組成も脱血液の投与と同等に推移し全例が生存した（非特許文献２）。これらの結果は、Ｈｂ小胞体が医療現場で使用できるようになれば、特に救急医療や外科的手術において、血液型の不一致や感染の心配をせずに、何時でも要求に応じて投与し、同種血輸血の回避、または必要輸血量の低減が可能となることを示唆する。 Hb endoplasmic reticulum has also been investigated as a resuscitation fluid at the time of hemorrhagic shock, and an oxygen transport function equivalent to that of red blood cells has been demonstrated. For example, in an experiment in which 50% of the circulating blood volume of a rat was exsanguinated under anesthesia and Hb vesicles were dispersed and administered in a 5% albumin solution 15 minutes later and observed for 6 hours. Whereas 2 animals died, the administration of Hb endoplasmic reticulum changed the circulatory dynamics and blood gas composition to the same level as that of blood removal (Non-Patent Document 2). These results show that once the Hb endoplasmic reticulum can be used in the medical field, it can be administered at any time, without worrying about blood type mismatches or infections, especially in emergency medicine and surgery. This suggests that it is possible to avoid allogeneic blood transfusions or to reduce the required transfusion volume.
一方、一酸化窒素（ＮＯ）が血管内皮細胞から産生され、血管弛緩因子として作動することが報告されて以来、生体内ＮＯが情報伝達分子として様々な機能を果すことが明らかにされている。また、一酸化炭素（ＣＯ）についても、ヘムの代謝の過程で微量に産生され、これが極めて重要な役割を果していることが解ってきた。例えば肝臓では、肝実質細胞に存在する酵素ヘムオキシゲナーゼ−２（ＨＯ−２）がヘム分解酵素として存在し、この酵素によるヘム１分子の分解で、１分子のＣＯガスが放出される。このＣＯは、肝類洞血管内皮の伊東細胞に対し、血管弛緩因子として作動することが明らかになっている（非特許文献５）。また、内因性のＣＯガスは、抗炎症作用を示すことも明らかになっている（非特許文献６）。これらの事実から、ＣＯの薬効を積極的に活用するため、ＣＯガス吸入器が報告された（特許文献６）。また、静脈内投与可能な化合物としてルテニウムのＣＯ錯体等を合成し、そのＣＯ放出薬としての効果を調べる研究も行われるようになってきた（非特許文献７、特許文献７）。さらに、一酸化炭素中毒で知られるように、不完全燃焼等で生じるＣＯガスの吸入は極めて危険であるが、出血性ショックモデルラットに対し、低濃度で吸入させることにより、多臓器不全の改善が見られることが明らかになっている（非特許文献８）。 On the other hand, since it has been reported that nitric oxide (NO) is produced from vascular endothelial cells and operates as a vasorelaxant, it has been clarified that in vivo NO plays various functions as a signaling molecule. Carbon monoxide (CO) is also produced in a trace amount in the process of heme metabolism, and it has been found that this plays an extremely important role. For example, in the liver, the enzyme heme oxygenase-2 (HO-2) present in hepatocytes is present as a heme-degrading enzyme, and one molecule of CO gas is released by the decomposition of one molecule of heme by this enzyme. This CO has been shown to act as a vasorelaxant factor on Ito cells of hepatic sinusoidal endothelium (Non-patent Document 5). It has also been clarified that endogenous CO gas exhibits an anti-inflammatory effect (Non-patent Document 6). From these facts, a CO gas inhaler has been reported to actively utilize the medicinal effects of CO (Patent Document 6). Studies have also been conducted to synthesize ruthenium CO complexes and the like as compounds that can be administered intravenously and to examine their effects as CO releasing agents (Non-patent Documents 7 and 7). Furthermore, as is known for carbon monoxide poisoning, inhalation of CO gas generated by incomplete combustion is extremely dangerous, but improvement of multiple organ failure by inhalation of hemorrhagic shock model rats at low concentrations (Non-Patent Document 8).
Ｈｂ分子は、酸素だけでなく、一酸化窒素（ＮＯ）や一酸化炭素（ＣＯ）とも結合するので、これまでは、Ｈｂを用いる人工赤血球の投与においてはこれら内因性のガス状分子を捕捉しない分子設計が重要であるとされてきた。 Since Hb molecules bind not only to oxygen, but also to nitric oxide (NO) and carbon monoxide (CO), until now, these endogenous gaseous molecules have not been captured in the administration of artificial red blood cells using Hb. Molecular design has been considered important.
ところで、Ｈｂ小胞体の調製において、一酸化炭素（ＣＯ）を使用する工程が取り入れられている（特許文献８、特許文献９、特許文献１０、特許文献１１）。すなわち、ＨｂのＣＯに対する親和性は酸素の約２００倍といわれ、ＨｂＣＯ体（カルボニルヘモグロビン）は酸化劣化に対する耐性があるので、Ｈｂ精製工程においてＨｂをＨｂＣＯ体として加熱処理（６０℃、１０時間）している。Ｈｂ小胞体の調製の最終工程で、酸素気流下、可視光の照射によってＣＯは容易に解離除去され、それにより得られた酸素錯体を体内に投与している。 By the way, in the preparation of Hb endoplasmic reticulum, steps using carbon monoxide (CO) are incorporated (Patent Document 8, Patent Document 9, Patent Document 10, and Patent Document 11). That is, the affinity of Hb for CO is said to be about 200 times that of oxygen, and the HbCO isomer (carbonyl hemoglobin) is resistant to oxidative degradation. Therefore, in the Hb purification step, heat treatment is performed using Hb as the HbCO isomer (60 ° C., 10 hours). is doing. In the final step of the preparation of the Hb endoplasmic reticulum, CO is easily dissociated and removed by irradiation with visible light in an oxygen stream, and the resulting oxygen complex is administered into the body.
しかし、Ｈｂ小胞体にＣＯが結合したままの投与は従来知られていなかった。それはＣＯが有毒ガスであるという認識に基づいている。 However, administration in which CO remains bound to the Hb endoplasmic reticulum has not been conventionally known. It is based on the recognition that CO is a toxic gas.
また、アルブミン分子に合成ヘム誘導体を包接させたアルブミン−ヘム包接体が、全合成系のヘム蛋白質として開発され、その酸素運搬効果が明らかになっている。このアルブミン−ヘム包接体のヘムに一酸化炭素を結合させることにより、自動酸化が抑制され、長期間の保存安定度が得られ（非特許文献９）、また、ヘムに結合したＣＯを光照射によって排除し、酸素を結合させた後に動物に投与した場合の酸素運搬機能が確認されている。 An albumin-heme inclusion body in which a synthetic heme derivative is included in an albumin molecule has been developed as a fully synthetic heme protein, and its oxygen carrying effect has been clarified. By binding carbon monoxide to the heme of the albumin-heme clathrate, auto-oxidation is suppressed, and long-term storage stability is obtained (Non-patent Document 9). Oxygen transport function has been confirmed when administered to animals after being eliminated by irradiation and combined with oxygen.
しかし、ＣＯを結合したままでの投与は従来知られていなかった。
人工赤血球の臨床的適応の一つに、出血性ショック状態における緊急投与が想定され、赤血球投与と同等の血行動態の回復と蘇生効果が確認されている。しかし、急性出血性ショック状態に陥ると、カテコールアミン等の血管収縮因子が産生され、それにより末梢血管が収縮しているため、人工赤血球の投与は輸血と同様に、臓器によっては末梢血流の迅速な回復には繋がらない場合があるので、何らかの血管拡張剤の併用が求められていた。 One of the clinical indications for artificial erythrocytes is emergency administration in the state of hemorrhagic shock, and hemodynamic recovery and resuscitation effects equivalent to erythrocyte administration have been confirmed. However, when an acute hemorrhagic shock occurs, vasoconstrictors such as catecholamine are produced, which causes peripheral blood vessels to contract. In some cases, it may not lead to complete recovery, and some vasodilators have been required to be used in combination.
また、出血性ショックからの蘇生は、全身的な虚血再灌流を意味し、活性酸素の産生による様々な障害、いわゆる虚血再灌流障害、さらには多臓器不全に繋がる可能性がある。一般的に虚血状態では細胞内ＡＴＰが代謝されてヒポキサンチンになり、これが組織内に高濃度で蓄積する。ここに再灌流により酸素が供給されるとキサンチンオキシダーゼの作用により活性酸素（スーパーオキサイド）が多量に発生する。また、マクロファージが活性化されて炎症性サイトカインを産生し、これに反応して好中球等が遊走される。各種接着因子も誘導され、好中球は血管内皮細胞に接着し、さらに血管外組織にまで浸潤する。ここに再灌流により酸素が供給されると、好中球細胞膜にあるＮＡＤＰＨ−オキシダーゼが大量の活性酸素を産生する。従って、出血性ショックに対する蘇生液として輸血または酸素運搬体を投与することは、初期の状態においては生体に対し酸素を過剰に供給して活性酸素産生を促し、虚血再灌流障害を助長させる。特に小腸は虚血の影響を強く受け、この虚血再灌流は小腸障害のみでなく、肺、肝、腎等の遠隔臓器の障害をもたらす。従って、酸素運搬機能が投与直後は抑制されているが、次第に増大する蘇生液が理想的である。 In addition, resuscitation from hemorrhagic shock means systemic ischemia / reperfusion and may lead to various disorders due to the production of active oxygen, so-called ischemia / reperfusion disorders, and even multi-organ failure. In general, in an ischemic state, intracellular ATP is metabolized to hypoxanthine, which accumulates at a high concentration in the tissue. When oxygen is supplied here by reperfusion, a large amount of active oxygen (superoxide) is generated by the action of xanthine oxidase. In addition, macrophages are activated to produce inflammatory cytokines, and in response, neutrophils and the like are migrated. Various adhesion factors are also induced, and neutrophils adhere to vascular endothelial cells and further invade extravascular tissues. When oxygen is supplied here by reperfusion, NADPH-oxidase in the neutrophil cell membrane produces a large amount of active oxygen. Therefore, administration of blood transfusion or oxygen carrier as resuscitation fluid for hemorrhagic shock promotes active oxygen production by supplying oxygen excessively to the living body in the initial state, and promotes ischemia-reperfusion injury. In particular, the small intestine is strongly affected by ischemia, and this ischemia reperfusion not only damages the small intestine, but also damages remote organs such as the lung, liver, and kidney. Therefore, the oxygen carrying function is suppressed immediately after administration, but a resuscitation fluid that gradually increases is ideal.
また、ＣＯガスまたはＮＯガスを直接的に吸入させ、肺胞を経由して血中に移行させる方法が検討されている。しかし、緊急時に大掛かりな吸入設備を用いたガス吸入は医療施設内では可能であるものの、受傷現場では不可能である。さらに、吸入濃度を調節しても、体内血中への移行量は肺のガス交換に依存しており、呼吸機能が低下している場合には使用できない。また、出血性ショックの蘇生手順は一般的に循環血液量の補給が先決であり、ＣＯガスまたはＮＯガスを輸液と一緒に投与できる方法が求められていた。 In addition, a method in which CO gas or NO gas is directly inhaled and transferred to the blood via the alveoli has been studied. However, gas inhalation using a large inhalation facility in an emergency is possible in a medical facility, but not at the site of injury. Furthermore, even if the inhalation concentration is adjusted, the amount transferred to the blood in the body depends on the gas exchange of the lungs and cannot be used when the respiratory function is lowered. In addition, the resuscitation procedure for hemorrhagic shock is generally preceded by the replenishment of circulating blood volume, and a method that can administer CO gas or NO gas together with an infusion is required.
また、静脈内投与可能なルテニウムのＣＯ錯体等の化合物が開発されているものの、分子量が小さいためＣＯを放出する前に血中から消失することが予想され、血中滞留時間の長いＣＯガス担体が求められていた。 In addition, although compounds such as ruthenium CO complex that can be administered intravenously have been developed, it is expected to disappear from the blood before releasing CO due to its low molecular weight, and the CO gas carrier has a long residence time in the blood. Was demanded.
以上のことを背景にして、本発明は、血管拡張効果および細胞保護効果を示す輸液製剤、特に虚血状態にある患者をその虚血状態から回復させ、しかも虚血状態からの回復において虚血再灌流障害を防止し得る輸液製剤を提供することを課題とする。 Against the background described above, the present invention provides an infusion preparation exhibiting a vasodilatory effect and a cytoprotective effect, in particular, recovering a patient in an ischemic state from the ischemic state and ischemic in the recovery from the ischemic state. It is an object to provide an infusion preparation that can prevent reperfusion injury.
発明者らは上述の背景と課題を熟慮し、鋭意研究を重ねた結果、人工赤血球の構成成分の配位子結合部位（ヘム）に予めＣＯガスまたはＮＯガスが結合した状態で血管内投与することによって血管拡張効果および細胞保護効果を得る手法を見いだした。特に、配位子結合部位に予めＣＯガスが結合した状態で出血性ショック状態の動物に対し血管内投与することで、虚血再灌障害を防止し得ることを見いだした。ヘムに対しＣＯガスは極めて強力に結合するが、血管内投与後は酸素との配位子交換反応により、徐々にＣＯを放出し、血管弛緩因子として作用し、血管を拡張して末梢血流を促進させる。予めＣＯを結合させたヘムは、投与直後では酸素運搬効果は低いものの、虚血再灌流による活性酸素の産生量を低減させ、組織細胞を保護する効果が期待できるし、輸液として循環血液量の増大にも貢献する。また、Ｈｂ分子は４個のサブユニットから構成されているが、このうち一部にＣＯが結合した状態では酸素配位のアロステリック効果が消失し、残りのサブユニットの酸素親和性が極めて高い状態になるので、ＣＯを放出して末梢血管を拡張しながら低酸素領域に陥った末梢組織に効率的に酸素を運搬することが可能となる。ヘムは、ＣＯを放出し終えると完全な酸素運搬体として機能し、赤血球と同様に、全身に必要量の酸素を供給することができるのである。 The inventors have considered the above-mentioned background and problems, and have conducted extensive research. As a result, they are administered intravascularly in a state in which CO gas or NO gas is bound in advance to the ligand binding site (heme) of the component of artificial red blood cells. Thus, a technique for obtaining a vasodilator effect and a cytoprotective effect was found. In particular, it has been found that ischemia-reperfusion injury can be prevented by intravascular administration to a hemorrhagic shocked animal with CO gas previously bound to the ligand binding site. CO gas binds very strongly to heme, but after intravascular administration, it gradually releases CO by ligand exchange reaction with oxygen, acts as a vascular relaxation factor, dilates blood vessels, and peripheral blood flow To promote. Heme pre-coupled with CO has a low oxygen transport effect immediately after administration, but can reduce the amount of active oxygen produced by ischemia-reperfusion, and can be expected to protect tissue cells. Contributes to growth. The Hb molecule is composed of four subunits. When all of these subunits are bound to CO, the allosteric effect of oxygen coordination disappears, and the remaining subunits have extremely high oxygen affinity. Therefore, it is possible to efficiently transport oxygen to the peripheral tissue that has fallen into the hypoxic region while releasing the CO and dilating the peripheral blood vessels. Heme functions as a complete oxygen carrier when it has released CO, and it can supply the necessary amount of oxygen to the whole body, like red blood cells.
すなわち、本発明は、配位子結合部位に一酸化炭素が結合したヘムを包含し、虚血状態の生体の血管内に投与された後に一酸化炭素を徐放し、その後酸素運搬体として機能することにより虚血再灌流障害を防止することを特徴とする輸液製剤を提供する。 That is, the present invention includes a heme having carbon monoxide bonded to a ligand binding site, and releases carbon monoxide after being administered into a blood vessel of an ischemic living body, and then functions as an oxygen carrier. Thus, an infusion preparation characterized by preventing ischemia-reperfusion injury is provided.
本発明の輸液製剤の、血管内投与された後のＣＯの徐放は、虚血再灌流障害を防止することに繋がり、特に肝臓組織および心臓組織の保護効果が顕著に得られる。 The sustained release of CO after intravascular administration of the infusion preparation of the present invention leads to prevention of ischemia-reperfusion injury, and in particular, the protective effect of liver tissue and heart tissue is remarkably obtained.
また、本発明の輸液製剤がＨｂ小胞体により代表される細胞型Ｈｂ製剤のように粒子径が１００〜５００ｎｍに調節されている場合は、粒子自体の血中滞留時間が長く、また非細胞型のＨｂを利用する輸液製剤に比較してＣＯ保持効果が長く適度なＣＯ放出速度が得られる。 In addition, when the infusion preparation of the present invention has a particle diameter adjusted to 100 to 500 nm as in a cell-type Hb preparation typified by Hb endoplasmic reticulum, the particles themselves have a long residence time in the blood, and the non-cell type Compared with the infusion preparation using Hb, the CO retention effect is long and an appropriate CO release rate can be obtained.
さらに、本発明の輸液製剤は、血管内投与後、ＣＯガスを放出した後は、静脈を経て肺胞に到達すると速やかに酸素が結合し、動脈を経て末梢組織に到達し、結合した酸素を放出するようになる。 Furthermore, the infusion preparation of the present invention, after intravascular administration and release of CO gas, oxygen quickly binds when it reaches the alveoli via veins, reaches peripheral tissues via arteries, and binds oxygen. It will be released.
本発明によれば、臨床現場で、ＣＯガスまたはＮＯガスを結合したヘムを含む輸液製剤を投与することにより、血管拡張効果および細胞保護効果を得ることができる。また、出血性ショック状態の患者に対し、ＣＯガスを結合したヘムを含む輸液製剤を投与することにより、投与直後に遊離ＣＯが末梢血管を拡張させて血流を速やかに回復させるとともに、次第に酸素運搬体として作動するようになり、赤血球と同等の酸素運搬機能を得て、正常な酸素代謝状態を維持することができる。従って、虚血再灌流障害が有効に防止される。 According to the present invention, a vasodilator effect and a cytoprotective effect can be obtained by administering an infusion preparation containing heme combined with CO gas or NO gas at a clinical site. In addition, by administering an infusion preparation containing heme combined with CO gas to patients with hemorrhagic shock, free CO expands peripheral blood vessels immediately after administration to quickly restore blood flow, and gradually increases oxygen. It comes to operate as a carrier, can obtain an oxygen carrying function equivalent to that of red blood cells, and can maintain a normal oxygen metabolic state. Therefore, ischemia reperfusion injury is effectively prevented.
以下、本発明をより詳しく説明する。 Hereinafter, the present invention will be described in more detail.
本発明の輸液製剤は、ヘムを活性成分として包含し、その配位子結合部位に一酸化炭素（ＣＯ）または一酸化窒素（ＮＯ）が配位している。 The infusion preparation of the present invention includes heme as an active ingredient, and carbon monoxide (CO) or nitric oxide (NO) is coordinated at the ligand binding site.
本発明のヘム（Ｈｂ）を含む輸液製剤としては、Ｈｂに化学的修飾を加え分子量や粒子径を増大させて、血管外漏出とＨｂの毒性を低減させた修飾Ｈｂ溶液（重合型、水溶性高分子結合型）のほか、赤血球と類似の構造をした細胞型Ｈｂ（H. Sakai et al., Bioconjug. Chem. 2000; 11:56-64参照）等のいわゆる輸液製剤が含まれる。重合型Ｈｂには、グルタルアルデヒド等の架橋剤でＨｂの表面のリジン残基と結合させたＨｂ重合体（S.A. Gould et al., J. Am. Coll. Surg., 2002;195:445-52参照）、Ｈｂのグロビン鎖の末端カルボン酸とリジン残基を分子間で直接結合させたＨｂ重合体（B. Matheson et al., J. Appl. Physiol., 2002; 93:1479-86参照）がある。水溶性高分子結合型としては、ヒドロキシエチルスターチと共重合させたもの（T. Hu et al., Biochem. J. 2005; 392:555-64参照）、Ｈｂの表面に片末端メトキシ基ポリエチレングリコール（ＰＥＧ）を結合させたＰＥＧ−Ｈｂ（T.M. Chang, Appl. Biochem. Biotechnol., 1984;10:5-24参照）等が利用できる。細胞型Ｈｂとしては、ナイロン、ポリ乳酸、ゼラチン、シリコンゴム等高分子薄膜から形成されるカプセルの中にＨｂが高濃度で内包されている形態や（L. Djordjevich & I.F. Miller, Fed. Proc., 1977; 36:567）、リン脂質二分子膜から構成されるリン脂質小胞体（リポソーム）の中にＨｂが内包されている形態（Ｈｂ小胞体）もある。また、親水性高分子と疎水性高分子が共重合した両親媒性高分子が形成するカプセル（ポリマーソーム）の中にＨｂを内包した形態も利用できる（D.R. Arifin, & A.F. Palmer. Biomacromolecules, 2005; 6:2172-81参照）。しかし、細胞膜と類似の脂質成分から構成され、生体適合性に優れるＨｂ小胞体が好ましい。一般的に修飾Ｈｂは粒子径が小さく、配位子促進輸送効果が現れるため、配位子の放出速度が極めて速くなり、配位子の保持効果が低下する。従って、細胞型Ｈｂのように粒子径はできるだけ大きいものがよく、５０ｎｍ以上であることが好ましい。しかし、製造工程におけるフィルタ滅菌の容易性、血中での均一分散性、および毛細管を塞栓させないことを考慮すると、その粒子径は１０００ｎｍ以下、好ましくは３００ｎｍ以下である。Ｈｂ小胞体は、脂質成分（リン脂質、コレステロール、負電荷脂質、ポリエチレングリコール結合脂質）が分子間相互作用により自発的に集合した形態にあるので、粒子径を極めて容易に制御できるという利点を有する。粒子径の厳密な調節には、エクストルージョン法が最適であるが（前出H. Sakai et al., Bioconjug. Chem., 2000; 11:56-64参照）、高速撹拌法によって剪断応力を与えることによってもある程度の調節可能である。さらに、その粒子内部には３５ｇ／ｄＬもの高濃度Ｈｂ（約３万個のＨｂ）が充填でき、一粒子当たりの配位子運搬数は１２万個になる。 As an infusion preparation containing heme (Hb) according to the present invention, a modified Hb solution (polymerization type, water-soluble) in which extravasation and Hb toxicity are reduced by chemically modifying Hb to increase molecular weight and particle size. In addition to the polymer-bound type, so-called infusion preparations such as cell type Hb (see H. Sakai et al., Bioconjug. Chem. 2000; 11: 56-64) having a structure similar to erythrocytes are included. The polymerized Hb includes an Hb polymer (SA Gould et al., J. Am. Coll. Surg., 2002; 195: 445-52) bound to a lysine residue on the surface of Hb with a crosslinking agent such as glutaraldehyde. Hb polymer in which terminal carboxylic acid and lysine residue of globin chain of Hb are directly bonded between molecules (see B. Matheson et al., J. Appl. Physiol., 2002; 93: 1479-86) There is. The water-soluble polymer-bonded type includes those copolymerized with hydroxyethyl starch (see T. Hu et al., Biochem. J. 2005; 392: 555-64), and one-end methoxy group polyethylene glycol on the surface of Hb. PEG-Hb conjugated with (PEG) (see TM Chang, Appl. Biochem. Biotechnol., 1984; 10: 5-24) and the like can be used. Examples of cell-type Hb include a form in which Hb is encapsulated in a high concentration in a capsule formed from a polymer thin film such as nylon, polylactic acid, gelatin, or silicon rubber (L. Djordjevich & IF Miller, Fed. Proc. 1977; 36: 567), there is also a form (Hb endoplasmic reticulum) in which Hb is encapsulated in a phospholipid endoplasmic reticulum (liposome) composed of a phospholipid bilayer membrane. Also, a form in which Hb is encapsulated in a capsule (polymersome) formed by an amphiphilic polymer obtained by copolymerization of a hydrophilic polymer and a hydrophobic polymer can be used (DR Arifin, & AF Palmer. Biomacromolecules, 2005). ; See 6: 2172-81). However, Hb endoplasmic reticulum composed of lipid components similar to the cell membrane and excellent in biocompatibility is preferred. In general, the modified Hb has a small particle diameter and a ligand-enhanced transport effect appears, so that the release rate of the ligand becomes extremely fast and the retention effect of the ligand decreases. Accordingly, the particle size should be as large as possible, such as cell type Hb, and is preferably 50 nm or more. However, considering the ease of filter sterilization in the manufacturing process, the uniform dispersibility in blood, and the fact that capillaries are not embolized, the particle diameter is 1000 nm or less, preferably 300 nm or less. Hb endoplasmic reticulum has the advantage that the particle size can be controlled very easily because the lipid components (phospholipids, cholesterol, negatively charged lipids, polyethylene glycol-binding lipids) are spontaneously assembled by intermolecular interactions. . Extrusion method is optimal for precise particle size control (see H. Sakai et al., Bioconjug. Chem., 2000; 11: 56-64), but shear stress is applied by high-speed stirring method. Can be adjusted to some extent. Further, the inside of the particles can be filled with a high concentration Hb of about 35 g / dL (about 30,000 Hb), and the number of ligands transported per particle is 120,000.
輸血用赤血球（血液から取り出された赤血球）に含有されるＨｂにＣＯまたはＮＯを結合させて投与すれば、これもＣＯまたはＮＯの徐放効果が得られる。しかし、赤血球はその構造が脆弱なので、ＣＯまたはＮＯガスのバブルにより容易に溶血する可能性があるので、輸液製剤としては、構造的により安定な輸液製剤（例えば、ヘモグロビンを内包したリン脂質小胞体粒子の形態にある輸液製剤等）を用いることがより好ましい。 If CO or NO is bound to Hb contained in red blood cells for blood transfusion (red blood cells extracted from blood) and administered, this also provides a sustained release effect of CO or NO. However, since the structure of erythrocytes is fragile, it may be easily hemolyzed by CO or NO gas bubbles. Therefore, as an infusion preparation, a structurally more stable infusion preparation (for example, a phospholipid vesicle encapsulating hemoglobin is used. It is more preferable to use an infusion preparation or the like in the form of particles.
いうまでもなく、本発明の輸液製剤は、人工赤血球（特に細胞型Ｈｂ）、血液から取り出された赤血球等の小胞体が生理学的に許容され得る分散媒に分散した形態にあり得る。生理学的に許容され得る分散媒としては、等張の生理食塩水を好適に用いることができるが、さらにその高張溶液でもよい。また、ヒト血清由来アルブミン、リコンビナントアルブミン、ヒドロキシエチルスターチ、デキストラン、修飾ゼラチン等代用血漿剤の水溶液を併用することもできる。いずれの形態においても、本発明の輸液製剤は、電解質や代用血漿剤を用いて、生理学的に許容される晶質浸透圧（２００ｍＯｓｍ／Ｌ〜２５００ｍＯｓｍ／Ｌ、膠質浸透圧（０〜８０ｍｍＨｇ）、ｐＨ（４〜９）に調節される。 Needless to say, the infusion preparation of the present invention can be in a form in which erythrocytes such as artificial red blood cells (particularly cell type Hb) and red blood cells extracted from blood are dispersed in a physiologically acceptable dispersion medium. As a physiologically acceptable dispersion medium, isotonic saline can be preferably used, but a hypertonic solution thereof may also be used. In addition, an aqueous solution of a blood plasma substitute such as human serum-derived albumin, recombinant albumin, hydroxyethyl starch, dextran, and modified gelatin can be used in combination. In any form, the infusion preparation of the present invention uses physiologically acceptable crystal osmotic pressure (200 mOsm / L to 2500 mOsm / L, colloid osmotic pressure (0 to 80 mmHg), using an electrolyte or a plasma substitute. Adjust to pH (4-9).
Ｈｂを用いる輸液製剤の保存形態としては従来、酸素が結合した状態でガラス容器内に充填され、これを冷蔵保存する方法、またはプラスチック製バッグに充填されて冷凍保存する方法が知られている。また、充填容器内に存在する酸素を実質的に完全に除去し、酸素流入を完全に遮断し、溶液状態で室温保存する方法が知られている。本発明では、いずれの形態においてもＨｂの酸素結合部位であるヘムの全てまたは一部にＣＯまたはＮＯが結合した状態に変換して保存をすればよい。輸液製剤にＣＯガスを通気すれば、オキシヘモグロビン（ＨｂＯ2）は次第にカルボニルヘモグロビン（ＨｂＣＯ）に変換されるし、配位子が結合していないデオキシモグロビン（デオキシＨｂ）の状態にある輸液製剤にＣＯガスを通気すれば、極めて速やかにＣＯが結合し、ＨｂＣＯに変換される。ＣＯの結合反応を促進するため、気−液界面積を増大させる目的で、液膜を形成してＣＯガスと接触させたり、市販の膜型人工肺を使用してもよい。ＣＯは酸素よりも結合力が２００倍強く耐熱性が向上し、また自動酸化が抑制され、保存安定度が向上する利点を有する。ＮＯガスの場合も同様に、配位子が結合していないデオキシＨｂの状態にある輸液製剤にＮＯガスを通気すれば極めて速やかにＮＯが結合し、ニトロシルヘモグロビン（ＨｂＮＯ）に変換される。ＨｂＣＯまたはＨｂＮＯの含量は、可視吸収スペクトルにおいてソレー帯またはＱ帯におけるデオキシＨｂとの吸光度比から容易に算出できる。 As a storage form of an infusion preparation using Hb, conventionally, a method of filling a glass container in a state where oxygen is bound and refrigerated storage or a method of storing it in a plastic bag and storing it frozen is known. In addition, a method is known in which oxygen present in the filled container is substantially completely removed, oxygen inflow is completely blocked, and the solution is stored at room temperature. According to the present invention, in any form, it may be stored after being converted into a state in which CO or NO is bound to all or part of the heme which is the oxygen binding site of Hb. If CO gas is passed through the infusion preparation, oxyhemoglobin (HbO 2 ) is gradually converted to carbonyl hemoglobin (HbCO), and the infusion preparation in the form of deoxymoglobin (deoxyHb) to which no ligand is bound. If CO gas is ventilated, CO is bound very quickly and converted to HbCO. In order to promote the CO binding reaction, a liquid film may be formed and contacted with CO gas for the purpose of increasing the gas-liquid interface area, or a commercially available membrane oxygenator may be used. CO has the advantage that its binding strength is 200 times higher than that of oxygen and heat resistance is improved, and auto-oxidation is suppressed, and storage stability is improved. Similarly, in the case of NO gas, if NO gas is passed through an infusion preparation in the form of deoxy Hb to which no ligand is bound, NO is bound very quickly and converted to nitrosylhemoglobin (HbNO). The content of HbCO or HbNO can be easily calculated from the absorbance ratio with deoxy Hb in the Sore band or Q band in the visible absorption spectrum.
使用に際し、配位座の全てまたは一部にＣＯまたはＮＯが結合されヘムを含む本発明の輸液製剤（通常、上記生理学的に許容され得る分散媒に分散された形態、または同分散媒に溶解した溶液の形態にある）を静脈内投与する。Ｈｂ小胞体のように粒子径が大きい場合は膠質浸透圧を持たないので、大量に投与する際にはアルブミンやヒドロキシエチルスターチ等代用血漿剤の投与も必要になる。いうまでもなくＣＯ、ＮＯガスは毒性も示すので、投与に際しては血行動態や血液ガス組成を継続してモニタリングする。また、投与されたＣＯガスは体内で薬理効果を示した後、最終的には呼気として排出されるのみであるので、呼吸状態の維持管理が重要である。血中のＨｂＣＯ量またはＨｂＮＯ量の推移は、採血液の可視吸収スペクトルにおいてソレー帯またはＱ帯におけるデオキシＨｂとの吸光度比から容易に算出できる。本発明の輸液製剤の投与量としては、ＣＯとして、０．００１〜０．２５ミリモル／ｋｇ（体重）となるような量である。ＮＯとしては、ＣＯとほぼ同程度か、それよりも少ない。 In use, the infusion preparation of the present invention comprising CO or NO bonded to all or part of the coordination position and containing heme (usually dispersed in the above physiologically acceptable dispersion medium or dissolved in the same dispersion medium) (In the form of a solution). When the particle size is large such as Hb endoplasmic reticulum, it does not have colloid osmotic pressure. Therefore, when a large amount is administered, it is also necessary to administer a plasma substitute such as albumin or hydroxyethyl starch. Needless to say, since CO and NO gas also show toxicity, hemodynamics and blood gas composition are continuously monitored during administration. In addition, since the administered CO gas only shows a pharmacological effect in the body and is finally discharged as expiration, maintenance of the respiratory state is important. The transition of the amount of HbCO or HbNO in the blood can be easily calculated from the absorbance ratio with deoxy Hb in the Soray band or Q band in the visible absorption spectrum of the collected blood. The dosage of the infusion preparation of the present invention is such an amount that it is 0.001 to 0.25 mmol / kg (body weight) as CO. NO is about the same as or less than CO.
ＣＯが結合したヘムを含む本発明の輸液製剤を投与することにより、先ず血管拡張効果が期待できる。そのメカニズムとしては次のことが考えられる。（ｉ）ＣＯガスが血管壁平滑筋にあるグアニル酸シクラーゼ（ＧＣ）に直接結合して血管拡張し、末梢血流を改善する。（ｉｉ）正常組織では血管弛緩因子であるＮＯがミオグロビン（Ｍｂ）と反応して失活する経路が存在するが、このとき本発明の輸液製剤から放出されるＣＯがＭｂに結合することにより、ＮＯの失活が抑制される。ＧＣに対する作用はＣＯよりもＮＯの方が数千倍強力であり、血管拡張により末梢血流を改善する。（ｉｉｉ）末梢血流の改善は、代謝産物の除去と酸素供給を促進する。 By administering the infusion preparation of the present invention containing heme to which CO is bound, first, a vasodilatory effect can be expected. The following can be considered as the mechanism. (I) CO gas directly binds to guanylate cyclase (GC) in the vascular wall smooth muscle to vasodilate and improve peripheral blood flow. (Ii) In normal tissues, there is a pathway in which NO, which is a vasorelaxant, reacts with myoglobin (Mb) and deactivates, and at this time, CO released from the infusion preparation of the present invention binds to Mb, Inactivation of NO is suppressed. The effect on GC is thousands of times stronger than CO and improves peripheral blood flow by vasodilation. (Iii) Improvement of peripheral blood flow promotes metabolite removal and oxygen supply.
本発明の輸液製剤による虚血再灌流障害の低減のメカニズムとしては、（ｉｖ）ヘムにＣＯを結合させることにより酸素含量を低下させ、投与直後の酸素負荷を低減でき、ヒポキサンチン−キサンチンオキシダーゼの反応による活性酸素の産生量を低減させる。（ｖ）活性化好中球の細胞膜に存在するヘム蛋白質ＮＡＤＰＨ−オキシダーゼが活性酸素を産生するが、本発明の輸液製剤から放出されるＣＯガスがＮＡＤＰＨ−オキシダーゼのヘム活性部位に結合することによって、活性酸素の産生が抑制され、細胞障害を抑制する。これらのメカニズムは類推の域を脱していないが、内因性ＣＯおよび吸入ＣＯガスの効果は最近では多くの研究者が報告しているところである。 The mechanism of reduction of ischemia-reperfusion injury by the infusion preparation of the present invention is as follows: (iv) The oxygen content can be reduced by binding CO to heme, the oxygen load immediately after administration can be reduced, and hypoxanthine-xanthine oxidase Reduce the amount of active oxygen produced by the reaction. (V) Although the heme protein NADPH-oxidase present in the cell membrane of activated neutrophils produces active oxygen, CO gas released from the infusion preparation of the present invention binds to the heme active site of NADPH-oxidase. , Production of active oxygen is suppressed and cell damage is suppressed. Although these mechanisms are not in analogy, many researchers have recently reported the effects of endogenous CO and inhaled CO gas.
本発明のＣＯが結合したヘムを包含する輸液製剤は、血管内投与後ＣＯを放出すると、代わって酸素が可逆的にＨｂのヘムに結合解離するようになり、赤血球と同等の酸素運搬機能を呈する。すなわち、本発明のＣＯが結合したヘムを包含する輸液製剤は、血管内に投与すると、初期にはＣＯ運搬体として機能し、その後酸素運搬体として機能する。 In the infusion preparation containing heme to which CO is bound according to the present invention, when CO is released after intravascular administration, oxygen is reversibly bound to and dissociated from heme of Hb instead. Present. That is, the infusion preparation containing heme to which CO of the present invention is bound, when administered intravascularly, initially functions as a CO carrier and then functions as an oxygen carrier.
以下、本発明を実施例等により説明する。 Hereinafter, the present invention will be described with reference to examples.
比較例１
Ｈｂ小胞体（ＨｂＶ）は、既報（H. Sakai et al., J. Pharmacol. Exp. Ther., 2004; 311:874-84）に従って調製した。具体的には、Ｈｂ溶液に一酸化炭素を結合させて６０℃にて１０時間の加熱処理を行い、さらに限外濾過膜処理により高純度高濃度Ｈｂ溶液を得た。これにアロステリック因子としてピリドキサル５’−リン酸を添加した。次いで、混合脂質粉末（ジパルミトイルホスファチジルコリン／コレステロール／負電荷脂質／ポリエチレングリコール結合型ジステアロイルホスファチジルエタノールアミン）を添加して形成された多重層のＨｂ小胞体を、エクストルージョン法を用いて平均粒径を２５０ｎｍに制御した。酸素気流下、可視光の照射によりＨｂに結合した一酸化炭素を光解離させ、酸素と置換させた。最後に、ガラス容器に封入して、窒素ガスを通気して完全に酸素を除去して保存した。使用直前に空気を通気して酸素を結合したＨｂ小胞体とした。
Comparative Example 1
Hb endoplasmic reticulum (HbV) was prepared according to a previous report (H. Sakai et al., J. Pharmacol. Exp. Ther., 2004; 311: 874-84). Specifically, carbon monoxide was bonded to the Hb solution, heat treatment was performed at 60 ° C. for 10 hours, and a high-purity high-concentration Hb solution was obtained by ultrafiltration membrane treatment. To this was added pyridoxal 5′-phosphate as an allosteric factor. Subsequently, a multilayered Hb vesicle formed by adding a mixed lipid powder (dipalmitoylphosphatidylcholine / cholesterol / negatively charged lipid / polyethylene glycol-linked distearoylphosphatidylethanolamine) was averaged using an extrusion method. Was controlled to 250 nm. Under a stream of oxygen, carbon monoxide bonded to Hb was photodissociated by irradiation with visible light and substituted with oxygen. Finally, it was sealed in a glass container and nitrogen gas was passed through to completely remove oxygen and store it. Immediately before use, air was aerated to form oxygen-bound Hb vesicles.
ウィスター系ラット（雄、体重約２５０ｇ）を、１．５％セボフルレンの吸入により、麻酔状態とし、自発呼吸を維持させた。ラットの頸動脈と頸静脈にカテーテルを留置し、頸動脈より５０％脱血した（全血液量を５６ｍＬ／ｋｇとすると、脱血量は２８ｍＬ／ｋｇ、約８．４ｍＬ）。脱血速度は１ｍＬ／分とした。脱血後、１５分間放置し、試料を投与した。投与試料は、上記酸素を結合したＨｂＶの分散液と２５％アルブミン（ＨＳＡ）を混合したＨｂＶ／ＨＳＡ（ＨｂＶ／２５％ｒＨＳＡ＝８．６／１．４（ｖｏｌ／ｖｏｌ）、［Ｈｂ］＝８．６ｇ／ｄＬ、ｎ＝８）、脱血した自己血（ＳＡＢ群、ｎ＝８）、ヒト血清アルブミン（ＨＳＡ群、ｎ＝８）、および洗浄赤血球をＨＳＡに分散させ、Ｈｂ濃度を８．６ｇ／ｄＬに調整した溶液（ＲＢＣ／ＨＳＡ、ｎ＝８）、およびＨＳＡ単独であった。測定ポイントは、脱血前、脱血後、投与直後、投与１時間後、投与３時間後、および投与６時間後（犠牲死）とした。投与６時間後、血液を６ｍＬ程度採取、採血管に入れて遠心分離し血球成分を除去。血清を超遠心分離機にかけてＨｂＶを除去し、血液生化学的検査を実施し、肝機能を反映する項目としてアラニンアミノトランスフェラーゼ（ＡＬＴ）およびアスパラギン酸アミノトランスフェラーゼ（ＡＳＴ）を測定した。また、心筋機能を反映する項目として、乳酸脱水素酵素アイソエンザイム−１（ＬＤＨ−１）を測定した。 Wistar rats (male, body weight approximately 250 g) were anesthetized by inhalation of 1.5% sevoflurane and maintained spontaneous breathing. A catheter was placed in the carotid artery and jugular vein of the rat, and 50% blood was removed from the carotid artery (when the total blood volume was 56 mL / kg, the blood removal amount was 28 mL / kg, approximately 8.4 mL). The blood removal rate was 1 mL / min. After blood removal, the sample was administered for 15 minutes. The administration sample was HbV / HSA (HbV / 25% rHSA = 8.6 / 1.4 (vol / vol), [Hb] = HbV / HSA) in which the HbV dispersion combined with oxygen was mixed with 25% albumin (HSA). 8.6 g / dL, n = 8), deblooded autologous blood (SAB group, n = 8), human serum albumin (HSA group, n = 8), and washed erythrocytes are dispersed in HSA, and the Hb concentration is 8 The solution was adjusted to 6 g / dL (RBC / HSA, n = 8), and HSA alone. The measurement points were before blood removal, after blood removal, immediately after administration, 1 hour after administration, 3 hours after administration, and 6 hours after administration (sacrifice death). Six hours after administration, about 6 mL of blood was collected, placed in a blood collection tube, and centrifuged to remove blood cell components. The serum was subjected to ultracentrifugation to remove HbV, blood biochemical tests were performed, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured as items reflecting liver function. In addition, lactate dehydrogenase isoenzyme-1 (LDH-1) was measured as an item reflecting myocardial function.
ＨＳＡ群のみ８例中、２例が投与３時間後から６時間後の間に死亡した。他は生存した。血圧（ＭＡＰ）は、脱血前に１００ｍｍＨｇ程度であったが、脱血後に３０ｍｍＨｇ程度にまで低下した。ＳＡＢ群は投与直後に１１０ｍｍＨｇにまで上昇したが、１時間後に９０ｍｍＨｇになり、そのまま６時間推移した。ＨＳＡ群は投与直後に９０ｍｍＨｇに上昇したが、１時間後に７４ｍｍＨｇにまで低下した。生存したラットの６時間後の血圧は７１ｍｍＨｇでありＳＡＢ群に比較して有意に低い値であった。一方でＨｂＶは投与後に９８ｍｍＨｇにまで回復、１時間後には９３ｍｍＨｇになり、そのまま６時間後まで推移し、ＨＳＡ群よりも有意に高い値であり、ＳＡＢ群と同等であった。ヘマトクリット（Ｈｃｔ）は、脱血前約４３％であるが、脱血後に約３５％に低下した。ＳＡＢ群では投与後に４３％程度にまで回復した。一方、ＨＳＡ群とＳＡＢ群では、投与直後に血液が希釈されるため、約２０％に低下した。血液ガス組成については、脱血後に代償機能によりＰａＯ2の上昇、ＰａＣＯ2の低下、またｐＨの低下と乳酸値の上昇が見られた。蘇生液の投与直後に、全群に関してＰａＯ2、ＰａＣＯ2が初期値にまで回復した。ｐＨ値は投与直後に最低値を示した。また乳酸値も投与直後には回復しなかったが、１時間後には初期値まで回復し、６時間まで推移した。ｐＨ、乳酸値の回復の遅れは、投与直後に末梢微小循環が回復に向い、蓄積していた代謝産物が洗い出されたために見られた現象である。全群ともに同様の傾向を示したが、ｒＨＳＡ投与死亡群では、ＰａＯ2が低値傾向を示した。従って、酸素運搬の低下による低血圧と、麻酔条件では呼吸に依る代償機能が不十分であったことが死因と考えられる。従って、Ｈｂ小胞体の出血性ショック蘇生効果は、赤血球と同等であることが解る。 Of the 8 cases in the HSA group alone, 2 cases died between 3 and 6 hours after administration. Others survived. The blood pressure (MAP) was about 100 mmHg before blood removal, but dropped to about 30 mmHg after blood removal. The SAB group rose to 110 mmHg immediately after administration, but became 90 mmHg after 1 hour and remained unchanged for 6 hours. The HSA group increased to 90 mmHg immediately after administration but decreased to 74 mmHg after 1 hour. The blood pressure of the surviving rats after 6 hours was 71 mmHg, which was significantly lower than that in the SAB group. On the other hand, HbV recovered to 98 mmHg after administration, became 93 mmHg after 1 hour, remained unchanged until 6 hours, was significantly higher than the HSA group, and was equivalent to the SAB group. Hematocrit (Hct) was about 43% before blood removal but dropped to about 35% after blood removal. In the SAB group, it recovered to about 43% after administration. On the other hand, in the HSA group and the SAB group, the blood was diluted immediately after administration, and thus decreased to about 20%. Regarding the blood gas composition, an increase in PaO 2 , a decrease in PaCO 2, a decrease in pH and an increase in lactic acid level were observed due to the compensation function after blood removal. Immediately after administration of the resuscitation fluid, PaO 2 and PaCO 2 recovered to the initial values for all groups. The pH value showed the lowest value immediately after administration. The lactic acid level also did not recover immediately after administration, but recovered to the initial value after 1 hour and changed to 6 hours. The delay in the recovery of pH and lactic acid level is a phenomenon observed because the peripheral microcirculation is suitable for recovery immediately after administration, and accumulated metabolites are washed out. All groups showed the same trend, but the rOSA-administered death group showed a low value for PaO 2 . Therefore, the cause of death is considered to be hypotension due to a decrease in oxygen transport and insufficient compensation function due to respiration under anesthesia conditions. Therefore, it can be seen that the hemorrhagic shock resuscitation effect of the Hb endoplasmic reticulum is equivalent to that of red blood cells.
投与６時間経過後の血液生化学検査の結果、ＨｂＶ群、ＳＡＢ群、ＲＢＣ群のＡＳＴ値はそれぞれ２４９±１０４、２２８±９７、２１１±１０９Ｕ／Ｌとなり、またＡＬＴ値はそれぞれ９７±５４、１０８±６０、９５±７３Ｕ／Ｌであり、正常値（ＡＳＴ：６４±１３Ｕ／Ｌ；ＡＬＴ：３２±８Ｕ／Ｌ）に比較して極めて高い値になっていた。一般的にＡＳＴは、心筋、肝、脳に高濃度に存在し、ついで骨格筋、腎等に多く含まれる。また、ＡＬＴは肝臓に最も多く含まれ、以下は腎、心筋、骨格筋の順番である。臨床的には、ＡＳＴ、ＡＬＴの上昇は主に肝疾患の時に見られる。従って、ＡＳＴ値およびＡＬＴ値が著明に増大した事実から判断して、出血性ショックによる虚血と人工赤血球投与による再灌流の結果、肝臓に虚血再灌流障害が生起したことを意味する。また、ＨｂＶ群、ＳＡＢ群、ＲＢＣ群のＬＤＨ−１値は、それぞれ、３０８±１１１、２８７±１５７、２０７±１２９Ｕ／Ｌであり、正常値（３２±１７Ｕ／Ｌ）に比較して極めて高い値になった。一般的にＬＤＨ−１は心筋に多く存在し、臨床的には心筋梗塞などの障害があるときに高い値になる。従って、ＬＤＨ−１が顕著に増大した事実から判断して、出血性ショックによる虚血と人工赤血球投与による再灌流の結果、心筋に虚血再灌流障害が生起したことを意味する。 As a result of blood biochemical examination 6 hours after administration, the AST values of the HbV group, SAB group, and RBC group were 249 ± 104, 228 ± 97, 211 ± 109 U / L, respectively, and the ALT value was 97 ± 54, 108 ± 60 and 95 ± 73 U / L, which were extremely high values compared to normal values (AST: 64 ± 13 U / L; ALT: 32 ± 8 U / L). In general, AST is present in a high concentration in the myocardium, liver, and brain, and then abundantly contained in skeletal muscle, kidney, and the like. ALT is most often contained in the liver, and the following is the order of kidney, heart muscle, and skeletal muscle. Clinically, an increase in AST and ALT is seen mainly during liver disease. Therefore, judging from the fact that the AST value and the ALT value were markedly increased, it means that ischemia-reperfusion injury occurred in the liver as a result of ischemia due to hemorrhagic shock and reperfusion due to artificial red blood cell administration. Also, the LDH-1 values of the HbV group, SAB group, and RBC group are 308 ± 111, 287 ± 157 and 207 ± 129 U / L, respectively, which are extremely high compared to the normal value (32 ± 17 U / L). Became value. In general, LDH-1 is abundant in the myocardium and clinically becomes high when there is a disorder such as myocardial infarction. Therefore, judging from the fact that LDH-1 significantly increased, it means that ischemia-reperfusion injury occurred in the myocardium as a result of ischemia due to hemorrhagic shock and reperfusion due to artificial red blood cell administration.
実施例１
比較例１で調製し、脱酸素状態でガラス容器に封入保存されていたＨｂ小胞体の分散液（容量１０ｍＬ）について、ブチルゴム製の蓋に金属製の導入針を刺し、ＣＯガスをバブルし、Ｈｂの配位子を全てＣＯとし、つまり１００％ＨｂＣＯとした。この溶液８．６ｍＬに対し、２５％濃度のアルブミン溶液を１．４ｍＬ添加し、膠質浸透圧を約２０ｍｍＨｇに調節した。結果としてＨｂ濃度は８．６グラム／ｄＬになった。これを蘇生液（ＨｂＶ−ＣＯ／ＨＳＡ）とした。
Example 1
For the Hb vesicle dispersion (capacity 10 mL) prepared in Comparative Example 1 and stored in a glass container in a deoxygenated state, a metal introduction needle was inserted into a butyl rubber lid, and CO gas was bubbled. All the ligands of Hb were CO, that is, 100% HbCO. 1.4 mL of 25% strength albumin solution was added to 8.6 mL of this solution, and the colloid osmotic pressure was adjusted to about 20 mmHg. As a result, the Hb concentration was 8.6 grams / dL. This was used as a resuscitation liquid (HbV-CO / HSA).
比較例１に記載の方法と同じ方法により、ウィスター系ラット（雄、体重２５０〜３００ｇ）を１．５％セボフルレンの吸入で麻酔状態とし、自発呼吸を維持し、頸動脈と頸静脈にカテーテルを留置した。頸動脈より循環血液量の５０％相当の血液を１ｍＬ／分の速度で抜いた。１５分間放置した後、脱血量と同量のＣＯを結合したＨｂ小胞体分散液を投与し、６時間に亘り血行動態、血液ガス組成、をモニタリングした。また、採血液中のＨｂＣＯの濃度を可視吸収スペクトルから計測した。脱血前の血圧約９６ｍｍＨｇは、脱血後に約２９ｍｍＨｇまで低下し、投与後に約８９ｍｍＨｇにまで回復した。これは比較例１記載のＨｂＶ／ＨＳＡ群よりも僅かに低い値であった。ＨｂＣＯの比率は、投与直後２５±３％であったが、３０分後に１４±４％、１時間後に６±２％、３時間後には１±２％にまで低下しており、ＨｂＶの殆どが酸素運搬体として機能していた。６時間経過後の血液生化学検査の結果、ＡＳＴ値は１８５±８２Ｕ／Ｌ、ＡＬＴ値は５０±１６Ｕ／Ｌ、ＬＤＨ−１値は９７±３８Ｕ／Ｌであり、比較例１に記載の場合に比較して顕著に低下していたことから、肝機能障害および心筋障害（虚血再灌流障害）が低減されていることが明らかになった。 Using the same method as described in Comparative Example 1, Wistar rats (male, body weight 250-300 g) were anesthetized by inhalation of 1.5% sevoflurane, maintained spontaneous breathing, and catheters were placed in the carotid artery and jugular vein. Detained. Blood equivalent to 50% of the circulating blood volume was drawn from the carotid artery at a rate of 1 mL / min. After leaving for 15 minutes, an Hb endoplasmic reticulum dispersion combined with the same amount of CO as the blood removal amount was administered, and hemodynamics and blood gas composition were monitored for 6 hours. Further, the concentration of HbCO in the collected blood was measured from the visible absorption spectrum. The blood pressure of about 96 mmHg before blood removal decreased to about 29 mmHg after blood removal and recovered to about 89 mmHg after administration. This was slightly lower than the HbV / HSA group described in Comparative Example 1. The ratio of HbCO was 25 ± 3% immediately after administration, but decreased to 14 ± 4% after 30 minutes, 6 ± 2% after 1 hour, and 1 ± 2% after 3 hours, and most of HbV Was functioning as an oxygen carrier. As a result of the blood biochemical test after 6 hours, the AST value is 185 ± 82 U / L, the ALT value is 50 ± 16 U / L, and the LDH-1 value is 97 ± 38 U / L. It was revealed that liver dysfunction and myocardial injury (ischemic reperfusion injury) were reduced.
実施例２
ウィスター系ラットの赤血球を得るため、ラット（雄、体重２５０〜３００ｇ）をエーテル麻酔下、開腹し、下大静脈より採血した。これを遠心分離（２０００ｇ、１５分）して赤血球層を得て、さらに５％アルブミン溶液を添加して遠心分離、再分散する操作を２回繰り返した。この洗浄赤血球分散液１０ｍＬを５０ｍＬガラス容器に入れ、ブチルゴム製の蓋で封入した。金属製の導入針を刺し、ＣＯガスを通気し、Ｈｂの配位子を全てＣＯとし、つまり１００％ＨｂＣＯとした。通気の操作で溶血が認められたので、再度遠心分離して上澄みを除去し、再度５％アルブミン溶液を添加し、最終的にＨｂ濃度を８．６ｇ／ｄＬに調節した。これを蘇生液（ｗＲＢＣ−ＣＯ／ＨＳＡ）とした。採血から投与試験に用いるまでの所要時間は、３時間以内とした。
Example 2
In order to obtain erythrocytes of Wistar rats, rats (male, body weight 250-300 g) were opened under ether anesthesia, and blood was collected from the inferior vena cava. This was centrifuged (2000 g, 15 minutes) to obtain an erythrocyte layer, and the operation of adding a 5% albumin solution, centrifuging and redispersing was repeated twice. 10 mL of this washed erythrocyte dispersion was placed in a 50 mL glass container and sealed with a butyl rubber lid. A metal introduction needle was stabbed, CO gas was bubbled, and all the ligands of Hb were changed to CO, that is, 100% HbCO. Since hemolysis was observed by aeration, the supernatant was removed by centrifugation again, 5% albumin solution was added again, and the Hb concentration was finally adjusted to 8.6 g / dL. This was used as a resuscitation liquid (wRBC-CO / HSA). The required time from blood collection to administration test was within 3 hours.
比較例１および実施例１に記載の方法と同じ方法により、ウィスター系ラット（雄、体重２５０〜３００ｇ）の出血性ショックモデルに対し、ｗＲＢＣ−ＣＯ／ＨＳＡを投与した。脱血前の血圧約１００ｍｍＨｇは、脱血後に約３１ｍｍＨｇまで低下し、投与後に約８９ｍｍＨｇにまで回復し、そのまま６時間推移した。ＨｂＣＯの比率を血液ガス組成測定装置（ラジオメータ社製、ＡＢＬ−７００）で測定したところ、投与直後３９±２％であったが、３０分後に２５±２％、１時間後に１４±２％、３時間後には４±１％、６時間後に漸く１％程度にまで低下しており、ｗＲＢＣの殆どが酸素運搬機能を回復してとしていた。赤血球のＣＯ放出速度は明らかにＨｂ小胞体よりも遅かった。６時間経過後の血液生化学検査の結果、ＡＳＴ値は１２６±５４Ｕ／Ｌ、ＡＬＴ値は３３±９Ｕ／Ｌ、ＬＤＨ−１値は７２±４６Ｕ／Ｌであり、比較例１に記載の場合に比較して顕著に低下していたことから、肝機能障害および心筋障害（虚血再灌流障害）が低減されていることが明らかになった。 By the same method as described in Comparative Example 1 and Example 1, wRBC-CO / HSA was administered to a hemorrhagic shock model of Wistar rats (male, body weight 250 to 300 g). The blood pressure before blood removal, about 100 mmHg, dropped to about 31 mmHg after blood removal, recovered to about 89 mmHg after administration, and remained unchanged for 6 hours. The HbCO ratio was measured with a blood gas composition measuring device (ABL-700, manufactured by Radiometer Co., Ltd.), which was 39 ± 2% immediately after administration, 25 ± 2% after 30 minutes, and 14 ± 2% after 1 hour. It decreased to 4 ± 1% after 3 hours and gradually decreased to about 1% after 6 hours, and most of wRBC had recovered the oxygen carrying function. The red blood cell CO release rate was clearly slower than the Hb endoplasmic reticulum. As a result of the blood biochemical test after 6 hours, the AST value is 126 ± 54 U / L, the ALT value is 33 ± 9 U / L, and the LDH-1 value is 72 ± 46 U / L. It was revealed that liver dysfunction and myocardial injury (ischemic reperfusion injury) were reduced.
実施例３
修飾Ｈｂとして、ウシヘモグロビンをグルタルアルデヒドで重合したポリＨｂ溶液について、Ｈｂ小胞体とＣＯの放出速度の相違を検討した。先ずポリＨｂおよびＨｂ小胞体分散液（［Ｈｂ］＝１０ｇ／ｄＬ）に一酸化炭素ガスを通気し、一酸化炭素を結合させた溶液を調製した。ラットの血液から洗浄赤血球を調製し、Ｈｂ濃度を１０ｇ／ｄＬにした。酸素を結合したラット赤血球４．５ｍＬに対し、一酸化炭素が結合したポリＨｂまたはＨｂ小胞体を０．５ｍＬ添加し、速やかにボルテックスミキサーで混合した。ポリＨｂまたはＨｂ小胞体に結合した一酸化炭素が徐々に遊離して赤血球に結合するので、混合後３０秒、１分、３分後に、０．５ｍＬを採取し、遠心分離（２０００ｇ、３０秒）して上澄みのポリＨｂまたはＨｂ小胞体の分画について、カルボニルヘモグロビン（ＨｂＣＯ）の割合を測定した。亜ニチオン酸ナトリウム粉末を添加して酸素を除去したときの可視吸光度スペクトルにおけるデオキシＨｂ（４３０ｎｍ）とＨｂＣＯ（４１９ｎｍ）のピーク比からＨｂＣＯの割合を算出した。ポリＨｂ溶液の場合、ＨｂＣＯの割合は混合後３５％（３０秒）、１５％（１分）、９％（３分）のように低下したのに対し、Ｈｂ小胞体の場合は、４２％（３０秒）、２３％（１分）、１１％（３分）となり、Ｈｂ小胞体の方がポリＨｂよりもＣＯ放出速度が遅く、ＣＯ徐放性に優れていた。
Example 3
As a modified Hb, the difference in the release rate of Hb endoplasmic reticulum and CO was examined for a poly-Hb solution obtained by polymerizing bovine hemoglobin with glutaraldehyde. First, carbon monoxide gas was passed through a poly Hb and Hb endoplasmic reticulum dispersion ([Hb] = 10 g / dL) to prepare a solution in which carbon monoxide was bonded. Washed erythrocytes were prepared from rat blood and the Hb concentration was 10 g / dL. 0.5 mL of poly Hb or Hb vesicle bound with carbon monoxide was added to 4.5 mL of rat erythrocytes bound with oxygen, and immediately mixed with a vortex mixer. Since carbon monoxide bound to poly Hb or Hb endoplasmic reticulum is gradually released and binds to erythrocytes, 0.5 mL is collected 30 seconds, 1 minute and 3 minutes after mixing and centrifuged (2000 g, 30 seconds). The fraction of carbonyl hemoglobin (HbCO) was measured for the fraction of the supernatant poly Hb or Hb endoplasmic reticulum. The ratio of HbCO was calculated from the peak ratio of deoxy Hb (430 nm) and HbCO (419 nm) in the visible absorbance spectrum when oxygen was removed by adding sodium nithionite powder. In the case of the poly Hb solution, the HbCO ratio decreased to 35% (30 seconds), 15% (1 minute), and 9% (3 minutes) after mixing, whereas in the case of the Hb endoplasmic reticulum, 42% (30 seconds), 23% (1 minute), and 11% (3 minutes). The Hb vesicle had a slower CO release rate than poly Hb, and was excellent in CO sustained release.
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