JP2010215517A - Hemoglobin-containing liposome suspension having controlled oxygen affinity to medium oxygen affinity - Google Patents
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本発明は、出血治療又は梗塞治療などに用いるヘモグロビン含有リポソーム懸濁液に関する。 The present invention relates to a hemoglobin-containing liposome suspension used for treatment of bleeding or infarction.
出血治療又は梗塞治療などに用いる人工酸素運搬体として、ヘモグロビン含有リポソーム懸濁液が様々に検討されて来た。ヘモグロビンベースの人工酸素運搬体はアロスリック因子を用いて、酸素親和性を制御する事が可能である。通常の血液循環における肺の酸素分圧100mmHgと組織末端の酸素分圧40mmHgの間の酸素運搬量を多くする為、酸素親和性を制御する方法が検討されて来た（特公平4-66456、特開2008-120760）。出血ショック時又は梗塞時は血流不全の為、組織末端は酸素不足に陥っており、通常の組織末端の酸素分圧40mmHgより更に低くなっている。この場合、酸素分圧100mmHgと酸素分圧40mmHgの間の酸素供給だけでなく、酸素分圧40mmHg以下の部位への酸素供給を十分に考慮する必要がある。特に梗塞部位等への適用では酸素分圧40mmHg以下への酸素供給が重要である。酸素親和性を制御する主な因子はヘモグロビンに対するアロステリック因子添加量である。また、アロステリック因子はヘモグロビン収率（仕込みヘモグロビン量に対するリポソーム化されたヘモグロビン量の割合（％））にも関与する事を、本発明者等は見出しており（特願2008-79396）、この観点からの検討は従来十分ではなかった。酸素親和性を制御する因子はアロステリック因子だけでなく、リポソーム内水相のpHも関与し、リポソーム内水相のpHには、リポソーム化前のヘモグロビンのpHだけでなくリポソーム膜形成脂質組成も影響を与える。そして、リポソーム膜形成脂質組成はリポソーム内のヘモグロビン保持機能等にも関与する。これらの因子を総合的に検討し、アロステリック因子を含有したヘモグロビン溶液を内水相とするリポソーム懸濁液において、酸素親和性を適切に制御し、且つ、ヘモグロビン収率を向上させる検討は、従来、行なわれていなかった。
ヘモグロビンベースの人工酸素運搬体において、ヒト血液を原料とする場合、赤血球からヘモグロビンを取り出す工程で、ヒト赤血球に元々存在するアロステリック因子の2,3-DPG（酸素放出能を高める燐酸化合物）が失われる。その結果として、酸素解離曲線（詳細は0010に記述）は左にシフトし、酸素運搬効率（詳細は0010に記述）は低くなってしまう問題があった。この問題を解決する為、ヘモグロビン溶液型の人工酸素運搬体では、ヘモグロビンにアロステリック因子を化学結合させたり、ヘモグロビン含有リポソーム型の人工酸素運搬体では、予めヘモグロビン溶液にアロステリック因子を添加する技術を用いている。これらは何れも通常の血液循環における肺の酸素分圧100mmHgと通常の組織末端の酸素分圧40mmHgの間の酸素運搬量を多くする為に、酸素放出能を制御する方法であった（特公平4-66456、特開2008-120760）。本発明では、このタイプを低酸素親和性〔高酸素親和性（後述）と比較して、通常の血液循環における酸素分圧領域（100mmHg〜40mmHg）において酸素を放し易い〕と呼び、天然赤血球と比較して、酸素解離曲線が右にシフトしている状態を示す。
また、人工酸素運搬体としてのヘモグロビン含有リポソームの平均粒子径は0.2μm前後である。天然赤血球（7〜8μm）と比較すると、非常に小さいので、血栓、栓塞等で血流が阻害されて、虚血により低酸素状態に陥った部位（酸素分圧が、通常の組織末端の酸素分圧40mmHgより更に低い）に対して、狭窄部位（天然赤血球では通過困難な）を通過して、或いは側副血行路や周囲の毛細血管を介して、酸素を供給する事も可能である（Circulation, 2004;100(Suppl-Ｉ)：483、CirculationJ.,2004:68(Suppl-Ｉ)：Ｉ-133）。また、癌組織においては、時として癌細胞の増殖に見合った血管新生を伴わないので、その血管網は無秩序で且つ、脆弱で有り、癌組織の血流は不安定で、一時的な血流遮断を繰り返している。この為、癌組織内部の細胞は低酸素状態にさらされている。この低酸素状態により、癌治療の為の放射線照射に対して抵抗性を示すが、人工酸素運搬体としてのヘモグロビン含有リポソーム懸濁液は、天然赤血球では到達不可能な癌組織内低酸素部位までに、酸素を供給出来る可能性がある（ASAIOJ.,2004:50:164）。この為、ヘモグロビン含有リポソーム懸濁液を人工酸素運搬体として、梗塞部位、ガン部位など、通常の組織末端（酸素分圧：40mmHg）より、更に低酸素状態の部位に、酸素を供給し易くする事を目的として、酸素分圧40mmHgと0mmHgの間の酸素運搬量を多くする為に、酸素放出能を制御したヘモグロビン含有リポソーム懸濁液を、本発明者等は鋭意検討して来た（特願2006-308816）。本発明では、このタイプを高酸素親和性〔低酸素親和性（前述）と比較して、通常の血液循環における酸素分圧領域（100mmHg〜40mmHg）において酸素を放し難い〕と呼び、天然赤血球と比較して、酸素解離曲線が左にシフトしている状態を示す。
In a hemoglobin-based artificial oxygen carrier, when human blood is used as a raw material, 2,3-DPG (a phosphate compound that increases oxygen release capacity), an allosteric factor originally present in human erythrocytes, is lost in the process of removing hemoglobin from erythrocytes. Is called. As a result, there is a problem that the oxygen dissociation curve (details are described in 0010) shifts to the left, and the oxygen carrying efficiency (details is described in 0010) is lowered. In order to solve this problem, a hemoglobin solution-type artificial oxygen carrier uses a technology that chemically bonds an allosteric factor to hemoglobin, or a hemoglobin-containing liposome-type artificial oxygen carrier that adds an allosteric factor to the hemoglobin solution in advance. ing. These were methods for controlling the oxygen release capacity in order to increase the amount of oxygen transported between the normal oxygen circulation pressure of 100 mmHg and the normal oxygen pressure at the end of the tissue of 40 mmHg. 4-66456, JP 2008-120760). In the present invention, this type is called low oxygen affinity (compared to high oxygen affinity (described later), oxygen is easily released in the oxygen partial pressure region (100 mmHg to 40 mmHg) in normal blood circulation) In comparison, the oxygen dissociation curve is shifted to the right.
Moreover, the average particle diameter of the hemoglobin-containing liposome as the artificial oxygen carrier is around 0.2 μm. Compared with natural erythrocytes (7-8μm), it is very small, so blood flow is blocked by thrombus, embolism, etc., resulting in hypoxia due to ischemia (oxygen partial pressure is oxygen at the end of normal tissue) It is also possible to supply oxygen through a stenotic site (which is difficult to pass with natural erythrocytes) or via collateral blood vessels and surrounding capillaries (partial pressure lower than 40 mmHg) ( Circulation, 2004; 100 (Suppl-I): 483, Circulation J., 2004: 68 (Suppl-I): I-133). In addition, in cancer tissue, there is sometimes no angiogenesis commensurate with the growth of cancer cells, so that the vascular network is disordered and fragile, the blood flow of the cancer tissue is unstable, and the temporary blood flow Repeated blocking. For this reason, the cells inside the cancer tissue are exposed to hypoxia. This hypoxic state is resistant to radiation for cancer treatment, but the hemoglobin-containing liposome suspension as an artificial oxygen carrier can reach hypoxic sites in cancer tissues that cannot be reached with natural erythrocytes. In addition, there is a possibility that oxygen can be supplied (ASAIOJ., 2004: 50: 164). For this reason, the hemoglobin-containing liposome suspension is used as an artificial oxygen carrier to make it easier to supply oxygen to sites that are hypoxic than normal tissue terminals (oxygen partial pressure: 40 mmHg), such as infarcted sites and cancer sites. In order to increase the oxygen transport amount between oxygen partial pressures of 40 mmHg and 0 mmHg, the present inventors have intensively studied hemoglobin-containing liposome suspensions with controlled oxygen release capacity. (Application 2006-308816). In the present invention, this type is referred to as high oxygen affinity (compared to low oxygen affinity (described above), it is difficult to release oxygen in the oxygen partial pressure region (100 mmHg to 40 mmHg) in normal blood circulation). In comparison, the oxygen dissociation curve is shifted to the left.
更に、出血ショックにおいては、血液が失われ、血流不全となる為、組織末端は酸素不足に陥っているので、通常の酸素分圧40mmHgより低い状態にある。従って、出血ショック治療初期においては、酸素分圧40mmHg以下の部位への酸素供給が重要であり、人工酸素運搬体が投与され、低酸素分圧領域への酸素供給及び血管内循環量が確保された後は、今度は、通常の血液循環における酸素分圧100mmHg〜40mmHgの間での酸素供給が重要となる。この点に着目し、低酸素親和性の人工酸素運搬体と高酸素親和性の人工酸素運搬体を併用して用いる事を特徴とする人工酸素運搬体も検討して来た。（特願2008-219207）。しかし、この方法は、低酸素親和性と高酸素親和性の２つの製剤が必要となる問題がある。 Furthermore, in the hemorrhagic shock, blood is lost and blood flow is insufficiency, so that the end of the tissue is deficient in oxygen, so that the oxygen partial pressure is lower than 40 mmHg. Therefore, in the early stage of hemorrhagic shock treatment, it is important to supply oxygen to sites with an oxygen partial pressure of 40 mmHg or less, and an artificial oxygen carrier is administered to ensure oxygen supply to the low oxygen partial pressure region and the amount of intravascular circulation. After that, oxygen supply at an oxygen partial pressure of 100 mmHg to 40 mmHg in normal blood circulation becomes important. Focusing on this point, an artificial oxygen carrier characterized by using a low oxygen affinity artificial oxygen carrier in combination with a high oxygen affinity artificial oxygen carrier has been studied. (Japanese Patent Application No. 2008-219207). However, this method has a problem that two preparations having low oxygen affinity and high oxygen affinity are required.
本発明では、出血治療時だけでなく、梗塞部位、ガン部位などの治療時にも、適切な酸素運搬が可能で有り、しかも、１つの製剤で、その目的を達する為、酸素分圧100mmHg〜40mmHgの酸素運搬と酸素分圧40mmHg〜0mmHgの酸素運搬がバランスよく出来る中酸素親和性の人工酸素運搬体を検討した。本発明では中酸素親和性とは、酸素解離曲線が、低酸素親和性と比較して左にシフトし、且つ、高酸素親和性と比較して右にシフトしている状態を示す。中酸素親和性は低酸素親和性と比較した場合、より低酸素領域（酸素分圧40mmHg〜0mmHg）への酸素供給を増加させ、出血ショック治療初期、梗塞部位、ガン部位への酸素供給に有利となる。高酸素親和性と比較した場合は、より通常の酸素分圧領域（酸素分圧100mmHg〜40mmHg）への酸素供給を増加させる事が出来、高酸素親和性と比較し、酸素分圧100mmHg〜40mmHgにおける酸素供給及び酸素分圧40mmHg~0mmHgにおける酸素供給のバランスが良くなる。また、酸素親和性を制御する主な因子はヘモグロビンに対するアロステリック因子量であるが、アロステリック因子添加量はヘモグロビン収率にも影響を与える事を本発明者等は見出しており、アロステリック因子添加量を減少させると、ヘモグロビン収率が増加する傾向にある。そして、酸素親和性を制御する因子はアロステリック因子だけでなく、リポソーム内水相のpHも関与している。リポソーム内水相のpHはリポソーム化前のヘモグロビンのpHだけでなくリポソーム膜形成脂質組成中のステアリン酸の影響も受ける（特開2008-120760、特願2006-308816）。また、ステアリン酸はヘモグロビン収率、内水相ヘモグロビンの保持機能、ヘモグロビンメト化率へも影響を与える（詳細は0011に記述）。今回の発明においては、これらの因子を総合的に検討し、中酸素親和性に酸素運搬能を制御し、出血治療時だけでなく、梗塞部位、ガン部位などの治療時にも適切な酸素運搬が、１つの製剤で可能であり、且つ、ヘモグロビン収率を向上させたアロステリック因子を含有したヘモグロビン溶液を内水相とするリポソーム懸濁液を提供する。 In the present invention, appropriate oxygen transport is possible not only at the time of treatment of bleeding but also at the time of treatment of infarcted sites, cancer sites, and the like, and in order to achieve the purpose with one preparation, oxygen partial pressure of 100 mmHg to 40 mmHg We investigated a medium oxygen affinity artificial oxygen carrier capable of well-balanced oxygen transportation and oxygen partial pressure of 40mmHg to 0mmHg. In the present invention, the medium oxygen affinity indicates a state in which the oxygen dissociation curve is shifted to the left as compared with the low oxygen affinity and is shifted to the right as compared with the high oxygen affinity. Compared with low oxygen affinity, medium oxygen affinity increases oxygen supply to the lower oxygen region (oxygen partial pressure 40mmHg to 0mmHg), which is advantageous for the early stage of hemorrhagic shock treatment, oxygen supply to infarcted area and cancer area It becomes. Compared with high oxygen affinity, oxygen supply to more normal oxygen partial pressure region (oxygen partial pressure 100mmHg ~ 40mmHg) can be increased. Compared with high oxygen affinity, oxygen partial pressure 100mmHg ~ 40mmHg The balance between oxygen supply and oxygen supply at an oxygen partial pressure of 40 mmHg to 0 mmHg is improved. The main factor that controls oxygen affinity is the amount of allosteric factor relative to hemoglobin, but the present inventors have found that the amount of allosteric factor added also affects the hemoglobin yield. When it is decreased, the hemoglobin yield tends to increase. The factor controlling oxygen affinity is not only the allosteric factor but also the pH of the aqueous phase in the liposome. The pH of the aqueous phase in the liposome is influenced not only by the pH of hemoglobin prior to liposome formation but also by the stearic acid in the liposome membrane-forming lipid composition (Japanese Patent Application No. 2008-120760, Japanese Patent Application No. 2006-308816). In addition, stearic acid also affects the hemoglobin yield, the retention function of the inner aqueous phase hemoglobin, and the hemoglobin metration rate (details are described in 0011). In the present invention, these factors are comprehensively examined, and the oxygen carrying capacity is controlled to the medium oxygen affinity, so that appropriate oxygen carrying is possible not only at the time of bleeding treatment but also at the treatment of infarcted sites, cancer sites, etc. Provided is a liposome suspension in which a hemoglobin solution containing an allosteric factor, which is possible with one preparation and has an improved hemoglobin yield, is used as an inner aqueous phase.
ヘモグロビン含有リポソーム懸濁液において、（１）ヘモグロビン濃度（２）アロステリック因子濃度（３）リポソーム膜構成脂質濃度（４）ステアリン酸濃度を適切に設定する事により、中酸素親和性に酸素親和性を制御し、出血治療時だけでなく、梗塞部位、ガン部位などの治療時にも適切な酸素運搬が、１つの製剤で可能であり、且つ、ヘモグロビン収率を向上させたアロステリック因子を含有したヘモグロビン溶液を内水相とするリポソーム懸濁液が下記のごとく提供される。 In the hemoglobin-containing liposome suspension, (1) hemoglobin concentration, (2) allosteric factor concentration, (3) liposome membrane constituent lipid concentration, and (4) stearic acid concentration are set appropriately to make oxygen affinity to medium oxygen affinity. A hemoglobin solution containing an allosteric factor that is controlled and can carry oxygen appropriately not only for bleeding treatment but also for treatment of infarcted sites, cancer sites, etc., with a single formulation and improved hemoglobin yield Is provided as follows.
(1)アロステリック因子を含有したヘモグロビン溶液を内水相とするリポソーム懸濁液であって、前記リポソーム膜形成脂質がステアリン酸を含み、前記リポソーム懸濁液中のヘモグロビン濃度が5.6〜6.7w/v%であり、アロステリック因子濃度が0.033〜0.045w/v%であり、リポソーム膜形成脂質濃度が3.05〜5.10w/v%であり、ステアリン酸濃度が0.41〜0.77w/v%である事を特徴とする前記リポソーム懸濁液。
(2)前記アロステリック因子がフィチン酸12ナトリウムである事を特徴とする(1)に記載のリポソーム懸濁液。
(3)前記リポソーム懸濁液中のヘモグロビンメト化率が10%以下である事を特徴とする(1)に記載のリポソーム懸濁液。
(1) A liposome suspension having a hemoglobin solution containing an allosteric factor as an inner aqueous phase, wherein the liposome membrane-forming lipid contains stearic acid, and the hemoglobin concentration in the liposome suspension is 5.6 to 6.7 w / v%, allosteric factor concentration is 0.033 to 0.045 w / v%, liposome membrane-forming lipid concentration is 3.05 to 5.10 w / v%, and stearic acid concentration is 0.41 to 0.77 w / v%. The said liposome suspension characterized.
(2) The liposome suspension according to (1), wherein the allosteric factor is 12 sodium phytate.
(3) The liposome suspension according to (1), wherein a hemoglobin metation rate in the liposome suspension is 10% or less.
以上、詳述した様に、本発明はアロステリック因子を含有したヘモグロビン溶液を内水相とするリポソーム懸濁液において、(1)ヘモグロビン濃度(2)アロステリック因子濃度(3)リポソーム膜構成脂質濃度(4)ステアリン酸濃度について、特に酸素親和性に直接的に影響するアロステリック因子濃度及び間接的に影響を与えるステアリン酸濃度を適切に設定し、出血治療時だけでなく、梗塞部位、ガン部位などの治療時にも適切な酸素運搬が、１つの製剤で可能であり、且つ、ヘモグロビン収率を向上させたアロステリック因子を含有したヘモグロビン溶液を内水相とするヘモグロビン含有リポソーム懸濁液が提供される。 As described above in detail, the present invention is a liposome suspension having a hemoglobin solution containing allosteric factor as an inner aqueous phase, (1) hemoglobin concentration (2) allosteric factor concentration (3) liposome membrane constituent lipid concentration ( 4) Regarding the stearic acid concentration, especially the allosteric factor concentration that directly affects oxygen affinity and the stearic acid concentration that indirectly affects stearic acid concentration are set appropriately, not only at the time of bleeding treatment, but also in the infarct site, cancer site, etc. A hemoglobin-containing liposome suspension having a hemoglobin solution containing an allosteric factor having an improved hemoglobin yield as an inner aqueous phase, which is capable of carrying oxygen appropriately at the time of treatment, is provided.
以下、本発明を具体的に説明する。
＜リポソーム膜構成脂質＞
本発明におけるリポソーム膜形成脂質は天然又は合成の脂質が使用可能である。特にリン脂質が好適に使用される。これらを常法に従って水素添加したものがあげられる。更にリポソーム膜形成脂質には所望によりステロール等の膜強化剤や荷電物質として高級飽和脂肪酸を添加しても良い。リン脂質としては水素添加大豆リン脂質、膜強化剤としてコレステロール、荷電物質としてステアリン酸等が好適に使用される。
The present invention will be specifically described below.
<Liposome membrane constituent lipid>
The liposome membrane-forming lipid in the present invention can be a natural or synthetic lipid. In particular, phospholipids are preferably used. These may be hydrogenated according to a conventional method. Further, higher lipophilic fatty acids may be added to the liposome membrane-forming lipid as desired, such as a membrane reinforcing agent such as sterol or a charged substance. As the phospholipid, hydrogenated soybean phospholipid, cholesterol as the film strengthening agent, stearic acid or the like as the charged substance are preferably used.
＜リポソーム内水相に含有されるヘモグロビン＞
本発明のリポソーム内水相に含有されるヘモグロビンは、公知の方法によりヒト期限切れ濃厚赤血球製剤より白血球、血小板、血漿及び赤血球膜を除去した後、濃縮したヒト由来濃厚ヘモグロビンが用いられる。
<Hemoglobin contained in liposome aqueous phase>
As the hemoglobin contained in the aqueous phase of liposome of the present invention, concentrated human-derived hemoglobin is used after removing leukocytes, platelets, plasma and erythrocyte membrane from a human expired concentrated erythrocyte preparation by a known method.
＜リポソーム凝集抑制剤＞
リポソーム表面への蛋白吸着抑制剤又はリポソーム凝集抑制剤として、公知の方法 ( 特開2008-024646など ) により、一端に疎水性を有し、且つ、他端に親水性高分子を有する化合物が用いられる。ポリエチレングリコールとリン脂質が共有結合したポリエチリングリコール結合リン脂質が好適に用いられる。
<Liposome aggregation inhibitor>
As a protein adsorption inhibitor or a liposome aggregation inhibitor on the liposome surface, a compound having a hydrophobic property at one end and a hydrophilic polymer at the other end is used by a known method (JP 2008-024646, etc.). It is done. Polyethylene glycol-linked phospholipid in which polyethylene glycol and phospholipid are covalently bonded is preferably used.
＜アロステリック因子による酸素解離曲線の制御＞
本発明に記載されるアロステリック因子とは、酸素解離曲線（ヘモグロビンの酸素飽和度と酸素分圧との関係を示す曲線。ヒト天然赤血球の酸素解離曲線は図1参照）に影響を与える因子である。アロステリック因子としては、特開昭57-26621号に記載のものも使用出来るが、安全性、保存安定性、価格、入手のし易さ、効果の点でフィチン酸が好ましく、フィチン酸12ナトリウムがより好ましい。アロステリック因子は酸素解離曲線を右にシフトさせ、その結果として酸素運搬効率を高くする。一般的には、天然赤血球の酸素運搬効率とは、通常の血液循環における肺の酸素分圧である100mmHgと酸素供給先である組織末端の酸素分圧40mmHgとの間のヘモグロビンの酸素飽和度の差を示す。図1が示す様に、ヒト天然赤血球では肺（酸素分圧100mmHg）で、酸素飽和度は約100%であり、組織末端（酸素分圧40mmHg）では酸素飽和度は約75%なので、通常の血液循環において、肺と組織末端との間で、酸素飽和量の約25%を組織に供給する。人工酸素運搬体としてのヘモグロビン含有リポソーム懸濁液において、ヒト血液を原料とする場合、赤血球からヘモグロビンを取り出す工程において、ヒト赤血球に元々存在するアロステリック因子の2,3-DPG（酸素放出能を高める燐酸化合物）が失われる。その結果として、酸素解離曲線は左にシフトし、天然赤血球で得られた100mmHgと40mmHgの間での酸素運搬効率が低下してしまう問題が有った。本発明者らは、予めヘモグロビン溶液にアロステリック因子を添加し、これをリポソーム化する事により、この問題を解決する方法を鋭意検討して来た（特公平4-66456、特開2008-120760）。このタイプを本発明では低酸素親和性と呼んでいる（0003に既述）。
しかし、梗塞部位、ガン部位などの治療時における酸素供給では（特願2008-79396）、末梢循環不全となり、組織末端では酸素不足に陥っており、組織末端の酸素分圧は通常の組織末端の酸素分圧40mmHgよりも更に低くなっている。酸素分圧40mmHgよりも更に低い酸素分圧の部位に酸素を供給する為には、酸素分圧40mmHgと酸素分圧0mmHgとの間での酸素運搬効率が重要であり、酸素解離曲線を適切な範囲で左にシフトさせれば、天然赤血球と比較して、酸素分圧100mmHg〜40mmHgの間では、酸素を離し難く、酸素分圧40mmHg以下の部位で酸素を離し易くなる。梗塞部位又はガン部位などの低酸素領域に運搬可能な酸素運搬量を増加させる為には、酸素解離曲線を右にシフトさせる作用のあるアロステリック因子については添加量を少なくするか、或いは添加しない技術を用いる高酸素親和性の人工酸素運搬体が有利である（特願2008-79396）。
<Control of oxygen dissociation curve by allosteric factor>
The allosteric factor described in the present invention is a factor that affects the oxygen dissociation curve (a curve showing the relationship between the oxygen saturation of hemoglobin and the partial pressure of oxygen. See FIG. 1 for the oxygen dissociation curve of human natural erythrocytes). . As the allosteric factor, those described in JP-A-57-26621 can be used, but phytic acid is preferable in terms of safety, storage stability, price, availability, and effects, and 12 sodium phytate is used. More preferred. Allosteric factors shift the oxygen dissociation curve to the right, resulting in higher oxygen carrying efficiency. In general, the oxygen transport efficiency of natural erythrocytes is the oxygen saturation of hemoglobin between 100 mmHg, the partial pressure of oxygen in the lungs in normal blood circulation, and 40 mmHg, the partial pressure of oxygen at the end of the tissue to which oxygen is supplied. Indicates the difference. As shown in Figure 1, human natural erythrocytes have a lung (oxygen partial pressure of 100 mmHg), oxygen saturation is about 100%, and at the end of tissue (oxygen partial pressure of 40 mmHg), oxygen saturation is about 75%. In the blood circulation, approximately 25% of the oxygen saturation is supplied to the tissue between the lung and the end of the tissue. In a hemoglobin-containing liposome suspension as an artificial oxygen carrier, when human blood is used as a raw material, in the process of taking out hemoglobin from red blood cells, 2,3-DPG (which enhances the oxygen release capacity) of the allosteric factor originally present in human red blood cells Phosphoric acid compound) is lost. As a result, the oxygen dissociation curve shifted to the left, and there was a problem that the oxygen carrying efficiency between 100 mmHg and 40 mmHg obtained with natural erythrocytes was reduced. The present inventors have intensively studied a method for solving this problem by adding an allosteric factor to a hemoglobin solution in advance and converting it into a liposome (Japanese Patent Publication No. 4-66456, JP 2008-120760). . This type is called low oxygen affinity in the present invention (as described in 0003).
However, oxygen supply during treatment of infarcted sites, cancer sites, etc. (Japanese Patent Application No. 2008-79396) resulted in peripheral circulatory insufficiency and oxygen deficiency at the end of the tissue. The oxygen partial pressure is even lower than 40 mmHg. In order to supply oxygen to a part with an oxygen partial pressure lower than the oxygen partial pressure of 40 mmHg, the oxygen transport efficiency between the oxygen partial pressure of 40 mmHg and the oxygen partial pressure of 0 mmHg is important, If the range is shifted to the left, it is difficult to release oxygen at an oxygen partial pressure of 100 mmHg to 40 mmHg compared to natural red blood cells, and oxygen is easily released at a site where the oxygen partial pressure is 40 mmHg or less. Technology to reduce or not add allosteric factors that act to shift the oxygen dissociation curve to the right in order to increase the amount of oxygen transportable to hypoxic regions such as infarctions or cancer sites An artificial oxygen carrier having a high oxygen affinity using benzene is advantageous (Japanese Patent Application No. 2008-79396).
出血ショック時の治療初期においては、血液が失われ、血流不全となる為、組織末端は酸素不足に陥っているので、高酸素親和性のヘモグロビン含有リポソーム懸濁液（アロステリック因子の添加量が少ないか或いは添加しない）を投与し、酸素分圧40mmHg以下の低酸素部位への効率の良い酸素供給を行なう。その後では、今度は通常の肺の酸素分圧100mmHgと組織末端の酸素分圧40mmHgとの間での酸素供給が必要となるので、低酸素親和性の人工酸素運搬体が有利となる。そこで、高酸素親和性の人工酸素運搬体投与の後で、低酸素親和性の人工酸素運搬体を投与する事を特徴とする人工酸素運搬体も考えられる（特願2008-219207）。しかし、この方法の短所としては、低酸素親和性と高酸素親和性の２つ
の製剤を必要とする事がある。
In the early stage of treatment at the time of hemorrhagic shock, blood is lost and blood flow becomes insufficiency, so the tissue ends are in a state of oxygen deficiency. Therefore, hemoglobin-containing liposome suspension with high oxygen affinity (the amount of allosteric factor added is Less oxygen or not added) to provide an efficient oxygen supply to hypoxic sites with an oxygen partial pressure of 40 mmHg or less. Thereafter, oxygen supply between the normal oxygen partial pressure of 100 mmHg of the lung and the oxygen partial pressure of 40 mmHg at the end of the tissue is required, so that an artificial oxygen carrier having a low oxygen affinity is advantageous. Therefore, an artificial oxygen carrier characterized by administering a low oxygen affinity artificial oxygen carrier after administration of a high oxygen affinity artificial oxygen carrier is also considered (Japanese Patent Application No. 2008-219207). However, the disadvantage of this method is that it requires two preparations of low oxygen affinity and high oxygen affinity.
本発明においては、出血治療、梗塞治療、ガン治療において、１つの製剤で、適切な酸素運搬が可能となる様に、低酸素親和性と比較した場合は、より低酸素領域（酸素分圧40mmHg〜0mmHg）への酸素供給を増加させ、且つ、高酸素親和性と比較した場合は、より通常の酸素分圧領域（酸素分圧100mmHg〜40mmHg）への酸素供給を増加させた中酸素親和性のヘモグロビン含有リポソーム懸濁液を鋭意検討した。中酸素親和性では、低酸素親和性と比較して、酸素解離曲線が左にシフトしており、ヘモグロビンに対するアロステリック因子添加量は少なくて済み、その結果、ヘモグロビン収率は向上する。ヘモグロビン収率が向上するだけでなく、低酸素親和性と比較して、低酸素領域（酸素分圧40mmHg〜0mmHg）への酸素供給が増加するので、出血ショック治療初期又は梗塞部位、ガン部位などの治療に有利となる。以上により、本発明において、適切に設定した前記リポソーム懸濁液中のヘモグロビン濃度（詳細は0013に記述）、及びステアリン酸濃度（詳細は0011に記述）に対する、前記リポソーム懸濁液中のアロステリック因子であるフィチン酸12ナトリウム濃度は0.033〜0.045w/v%であり、より好ましくは0.035〜0.043w/v%である。 In the present invention, in the case of bleeding treatment, infarction treatment, and cancer treatment, when compared with low oxygen affinity so that appropriate oxygen transport can be achieved with one preparation, a lower oxygen region (oxygen partial pressure of 40 mmHg) can be obtained. Increased oxygen supply to ~ 0mmHg) and increased oxygen supply to more normal oxygen partial pressure region (oxygen partial pressure 100mmHg ~ 40mmHg) when compared with high oxygen affinity The hemoglobin-containing liposome suspension was intensively studied. In the medium oxygen affinity, the oxygen dissociation curve is shifted to the left as compared with the low oxygen affinity, and the amount of allosteric factor added to hemoglobin is small, and as a result, the hemoglobin yield is improved. Not only is the hemoglobin yield improved, but oxygen supply to the hypoxic region (oxygen partial pressure 40mmHg to 0mmHg) is increased compared to low oxygen affinity, so the initial stage of bleeding shock treatment or infarcted area, cancer area, etc. It is advantageous for treatment. As described above, in the present invention, the allosteric factor in the liposome suspension with respect to the hemoglobin concentration (described in detail in 0013) and the stearic acid concentration (detailed in 0011) in the liposome suspension set appropriately in the present invention. The concentration of 12 sodium phytic acid is 0.033 to 0.045 w / v%, more preferably 0.035 to 0.043 w / v%.
＜ステアリン酸組成比が内水相pH及びヘモグロビン保持機能などに及ぼす影響＞
酸素解離曲線に影響を与える因子はアロステリック因子だけでなく、内水相ヘモグロビンのpHも影響を与える。内水相ヘモグロビンのpHには、リポソーム化前の赤血球膜除去濃厚ヘモグロビンのpHだけでなく、リポソーム膜構成脂質中のステアリン酸組成比も影響を与える。つまり、ステアリン酸組成比が高いと内水相pHは低下する傾向に有り、酸素解離曲線は右にシフトし、ステアリン酸組成比が低いと、ステアリン酸組成比が高い場合と比較し、内水相pHは上昇する傾向に有り、酸素解離曲線は左にシフトする。
ステアリン酸組成比は内水相pHを介して、酸素解離曲線に影響を与えるばかりでなく、内水相ヘモグロビンの保持機能にも関与する。つまり、ステアリン酸組成比が高いとヘモグロビン保持機能が低くなり、ステアリン酸組成比が低いとヘモグロビン保持機能が向上する傾向に有る。ヘモグロビン保持機能が低い場合、ヘモグロビン含有リポソーム懸濁液が生体に投与された時、血漿中にリポソーム内部のヘモグロビンが漏れ出す懸念がある。血漿中に漏れ出したヘモグロビンは、血漿中に存在するハプトグロビンにより、肝臓に運ばれ、分解、再利用されるが、一定量を越えると、ハプトグロビンによる処理能力を超えるので、可能な限り、ヘモグロビン保持機能を向上させておく必要が有る。他方、ステアリン酸組成比はヘモグロビン収率にも影響を与え、ステアリン酸組成比が高いとヘモグロビン収率が高くなり、ステアリン酸組成比が低いとヘモグロビン収率が低くなる。これらを総合的に検討する必要が有り、今回、これらの因子を総合的に考慮し、本発明におけるヘモグロビン含有リポソーム懸濁液中のステアリン酸濃度を0.41〜0.77w/v%、より好ましくは0.47〜0.71w/v%に設定した。
<Effect of composition ratio of stearic acid on inner aqueous phase pH and hemoglobin retention function>
Factors affecting the oxygen dissociation curve are not only allosteric factors, but also the pH of the internal aqueous phase hemoglobin. The pH of the inner aqueous phase hemoglobin affects not only the pH of the erythrocyte membrane-removed concentrated hemoglobin prior to liposome formation, but also the stearic acid composition ratio in the liposome membrane-constituting lipid. That is, when the stearic acid composition ratio is high, the pH of the inner aqueous phase tends to decrease, the oxygen dissociation curve shifts to the right, and when the stearic acid composition ratio is low, the internal water phase is compared with the case where the stearic acid composition ratio is high. The phase pH tends to increase and the oxygen dissociation curve shifts to the left.
The stearic acid composition ratio not only affects the oxygen dissociation curve via the inner aqueous phase pH, but also contributes to the retention function of the inner aqueous phase hemoglobin. That is, when the stearic acid composition ratio is high, the hemoglobin retention function is low, and when the stearic acid composition ratio is low, the hemoglobin retention function tends to be improved. When the hemoglobin retention function is low, when the hemoglobin-containing liposome suspension is administered to a living body, there is a concern that hemoglobin inside the liposome leaks into the plasma. Hemoglobin leaked into the plasma is transported to the liver by haptoglobin present in the plasma, where it is degraded and reused. However, if it exceeds a certain amount, it will exceed the processing capacity of haptoglobin, so that hemoglobin is retained as much as possible. It is necessary to improve the function. On the other hand, the stearic acid composition ratio also affects the hemoglobin yield. When the stearic acid composition ratio is high, the hemoglobin yield increases, and when the stearic acid composition ratio is low, the hemoglobin yield decreases. It is necessary to consider these comprehensively, and this time, considering these factors comprehensively, the stearic acid concentration in the hemoglobin-containing liposome suspension in the present invention is 0.41 to 0.77 w / v%, more preferably 0.47. Set to ~ 0.71w / v%.
＜ヘモグロビン含有リポソーム懸濁液の酸素運搬量設定＞
本発明における、ヘモグロビンを内水相とするリポソーム懸濁液1mLが酸素分圧100mmHgと酸素分圧40mmHgの間で運搬可能な酸素運搬量（通常の血液循環における酸素供給を想定）又は、酸素分圧40mmHgと0mmHgの間で運搬可能な酸素運搬量（梗塞部位、ガン部位への酸素供給を想定）は、本発明において、以下の3項目により理論的に算出する。（１）前記リポソーム懸濁液中のヘモグロビン濃度（ヘモグロビンが酸素運搬の主役である）（２）前記リポソーム懸濁液中のヘモグロビンメト化率（ヘモグロビンが酸化されて、メトヘモグロビンとなると酸素運搬能を失う）（３）前記リポソーム懸濁液の酸素運搬効率。本発明の酸素運搬効率は通常の血液循環における酸素供給の為には、酸素分圧10mmHgと40mmHgの間で設定される（酸素解離曲線において酸素分圧100mmHgと40mmHgの間の酸素飽和度の差）。また、梗塞部位又はガン部位への酸素供給の為には、酸素分圧40mmHgと0mmHgの間で設定される（酸素解離曲線において酸素分圧40mmHgと0mmHgの間の酸素飽和度の差）。
前記リポソーム懸濁液中のヘモグロビン濃度：Aw/v%、前記リポソーム懸濁液中のヘモグロビンメト化率：Ｂ％、前記リポソーム懸濁液の酸素運搬効率（「通常の血液循環における酸素供給を想定」又は「梗塞部位又はガン部位への酸素供給を想定」）：C%とすると、前記リポソーム懸濁液1mLが、酸素分圧100mmHgと40mmHgの間で、又は、酸素分圧40mmHgと0mmHgの間で運搬可能な酸素量DmL（37℃、1気圧）は、以下の様に理論的に計算される。
リポソーム懸濁液1mL中のヘモグロビンに結合可能な酸素分子数（moL）は、ヘモグロビンに結合可能な酸素分子が4つである事から、
｛A (1−B / 100) × 4 / 64500｝/ 100．．．．．(1)となる。
更に、酸素運搬効率がC%である事から、リポソーム懸濁液1mLが放出する酸素分子数(moL)は、
（１）×（C / 100）．．．．(2)となる。
また、気体の状態方程式PV＝nRT、R(atm・1 / K・moL)＝0.082より、
D(mL)＝（2）×0.082×（37＋273）×1000．．．．(3)となる。
以上により、前記ヘモグロビン含有リポソーム懸濁液において、（1）前記リポソーム懸濁液中のヘモグロビン濃度(2)前記リポソーム懸濁液中の酸素運搬効率(3)前記リポソーム懸濁液中のヘモグロビンメト化率を適切に制御し、設定する事により、適切な酸素運搬量の設定が可能となる。
<Oxygen carrying amount setting of hemoglobin-containing liposome suspension>
In the present invention, 1 mL of liposome suspension having hemoglobin as an inner aqueous phase can carry oxygen between oxygen partial pressure of 100 mmHg and oxygen partial pressure of 40 mmHg (assuming oxygen supply in normal blood circulation) or oxygen content The oxygen carrying amount that can be carried between pressures of 40 mmHg and 0 mmHg (assuming oxygen supply to the infarcted site and cancer site) is theoretically calculated according to the following three items in the present invention. (1) Hemoglobin concentration in the liposome suspension (hemoglobin is the main role of oxygen transport) (2) hemoglobin metration rate in the liposome suspension (when hemoglobin is oxidized to methemoglobin, oxygen transport capacity (3) Oxygen carrying efficiency of the liposome suspension. The oxygen carrying efficiency of the present invention is set between oxygen partial pressures of 10 mmHg and 40 mmHg for oxygen supply in normal blood circulation (difference in oxygen saturation between oxygen partial pressures of 100 mmHg and 40 mmHg in the oxygen dissociation curve). ). In addition, the oxygen partial pressure is set between 40 mmHg and 0 mmHg for oxygen supply to the infarcted site or cancer site (difference in oxygen saturation between the oxygen partial pressures of 40 mmHg and 0 mmHg in the oxygen dissociation curve).
Hemoglobin concentration in the liposome suspension: Aw / v%, hemoglobin metration rate in the liposome suspension: B%, oxygen transport efficiency of the liposome suspension (“assuming oxygen supply in normal blood circulation” ”Or“ assuming oxygen supply to the infarct or cancer site ”): Assuming C%, 1 mL of the liposome suspension is between oxygen partial pressures of 100 mmHg and 40 mmHg, or between oxygen partial pressures of 40 mmHg and 0 mmHg. The amount of oxygen DmL (37 ° C, 1 atm) that can be transported is calculated theoretically as follows.
The number of oxygen molecules that can bind to hemoglobin (moL) in 1 mL of liposome suspension is four oxygen molecules that can bind to hemoglobin.
{A (1-B / 100) × 4/64500} / 100. . . . . (1)
Furthermore, since the oxygen carrying efficiency is C%, the number of oxygen molecules (moL) released by 1 mL of liposome suspension is
(1) x (C / 100). . . . (2)
Also, from the equation of state of gas PV = nRT, R (atm · 1 / K · moL) = 0.082,
D (mL) = (2) × 0.082 × (37 + 273) × 1000. . . . (3)
As described above, in the hemoglobin-containing liposome suspension, (1) hemoglobin concentration in the liposome suspension (2) oxygen transport efficiency in the liposome suspension (3) hemoglobin methation in the liposome suspension By appropriately controlling and setting the rate, an appropriate oxygen carrying amount can be set.
＜リポソーム懸濁液中のヘモグロビン濃度＞
本発明における人工酸素運搬体としてのヘモグロビン含有リポソーム懸濁液の酸素運搬効率の主役はヘモグロビンである。前記リポソーム懸濁液中のヘモグロビン濃度が高過ぎると、ヘモグロビンをリポソーム化する為のリポソーム膜形成脂質の濃度が必然的に高くなり、生体に投与される総脂質濃度が高くなって、安全性の面で懸念がある。また、前記リポソーム懸濁液中のヘモグロビン濃度が低過ぎると、酸素運搬の主役であるヘモグロビンの絶対量が不足して、酸素運搬量設定に不利となる。従って、前記リポソーム懸濁液中の適切なヘモグロビン濃度は5.6〜6.7w/v%であり、より好ましくは5.7〜6.6w/v%である。
<Hemoglobin concentration in liposome suspension>
The main role of the oxygen carrying efficiency of the liposome suspension containing hemoglobin as the artificial oxygen carrier in the present invention is hemoglobin. If the hemoglobin concentration in the liposome suspension is too high, the concentration of liposome membrane-forming lipid for liposomal hemoglobin will inevitably increase, and the total lipid concentration administered to the living body will increase, resulting in safety. There is concern in terms. On the other hand, if the hemoglobin concentration in the liposome suspension is too low, the absolute amount of hemoglobin, which is the main oxygen transporter, is insufficient, which is disadvantageous in setting the oxygen transport amount. Therefore, the appropriate hemoglobin concentration in the liposome suspension is 5.6 to 6.7 w / v%, more preferably 5.7 to 6.6 w / v%.
＜リポソーム懸濁液中のヘモグロビンメト化率＞
ヘモグロビンは酸化されて、メトヘモグロビンとなると、酸素運搬能を失うので、人工酸素運搬体としてのヘモグロビン含有リポソームにおいては、ヘモグロビンの酸化防止（ヘモグロビンメト化防止）は、重要課題の１つである。ヘモグロビンのpHが過度に低下すると、ヘモグロビンの酸化が促進するので、製造工程を低温に保つと同時に、製造工程ではヘモグロビンのpH制御を行い、公知の方法（特開2006-104069）により、還元剤使用による脱酸素化及び脱酸素化状態のまま、製造バッグに無菌充填した後、脱酸素化状態を維持出来る様に外包装を行う。前記リポソーム懸濁液製造直後及び有効期間中のヘモグロビンメト化率は10%以下である。ヘモグロビンメト化率がこれより高くなると、前記リポソーム懸濁液の酸素運搬量が低下し、酸素運搬体として不利となる。
<Hemoglobin methaization rate in liposome suspension>
When hemoglobin is oxidized to methemoglobin, the oxygen carrying ability is lost. Therefore, in hemoglobin-containing liposome as an artificial oxygen carrier, antioxidation of hemoglobin (prevention of hemoglobin methation) is one of important issues. When the pH of hemoglobin is excessively lowered, the oxidation of hemoglobin is promoted, so that the manufacturing process is kept at a low temperature and at the same time, the pH of hemoglobin is controlled in the manufacturing process, and a reducing agent is obtained by a known method (Japanese Patent Laid-Open No. 2006-104069). After aseptically filling the production bag in the deoxygenated and deoxygenated state after use, outer packaging is performed so that the deoxygenated state can be maintained. Immediately after the production of the liposome suspension and during the effective period, the hemoglobin metation rate is 10% or less. If the hemoglobin metrification rate is higher than this, the amount of oxygen transported in the liposome suspension is lowered, which is disadvantageous as an oxygen transporter.
次に本発明の実施例により具体的に説明するが、本発明は、これらの実施例に限定されるものではない。なお、前記リポソーム懸濁液の製造工程は無菌環境下での操作とした。 EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. In addition, the manufacturing process of the said liposome suspension was made into operation in aseptic environment.
＜中酸素親和性のヘモグロビン含有リポソーム懸濁液の製造＞
アロステリック因子添加量により、酸素解離曲線を低酸素親和性より左に且つ、高酸素親和性より右にシフトさせる。
水素添加ホスファチジルコリン192g、コレステロール94g、ステアリン酸49gからなる均一混合脂質に水335gを加えて、85℃で30分間加熱して水和膨潤均一混合資質を調整した。期限切れ濃厚赤血球製剤からヘモグロビンを精製、濃縮し、アロステリック因子として、フィチン酸12ナトリウムをヘモグロビンに対して、0.5モル添加したヘモグロビン濃度42.6w/v%の濃厚ヘモグロビン溶液を調製した。前記水和膨潤均一混合脂質670gに前記濃厚ヘモグロビン溶液2393gを添加し、均一に攪拌し前乳化を行なった。前記前乳化後に更に強力な攪拌により、本乳化を行なった。前記本乳化後の混合液を生理食塩水により希釈して、0.45μｍ膜を用いて、循環濾過により粒子径の制御を行なった。次に10mg/mL濃度の亜硫酸ナトリウム生理食塩水を使用し、亜硫酸ナトリウムによる脱酸素化を行なった後、分画分子量30万の限外濾過膜を用いて、0.5mg/mL濃度の亜硫酸ナトリウム生理食塩水による加水濃縮で、リポソーム化されなかったヘモグロビン及びフィチン酸12ナトリウムを除去し、ヒト由来濃厚ヘモグロビン及びアロステリック因子含有リポソーム懸濁液を作成した。前記リポソーム懸濁液に、PEG結合リン脂質として、DSPE-PE5000（日本油脂製）を生理食塩水に溶解させたPEG結合リン脂質水溶液を添加した。前記リポソーム及びPEG結合リン脂質を含有した前記リポソーム懸濁液中のリポソーム膜構成脂質濃度が4.48w/v%であり、PEG結合リン脂質が0.41w/v％である様に調製した後、37℃、24時間処理し、PEG結合リン脂質をリポソーム表面に固定化した前記リポソーム懸濁液を5181mL得た。
前記リポソーム懸濁液中のヘモグロビン濃度は6.1w/v%であり、アロステリック因子であるフィチン酸12ナトリウム濃度は0.0360w/v%であり、ステアリン酸濃度は0.59w/v%であり、ヘモグロビン収率は31%であった。製造直後の前記リポソーム懸濁液中のヘモグロビンメト化率は4.3％であった。
<Manufacture of Liposome Suspension Containing Hemoglobin with Medium Oxygen Affinity>
The oxygen dissociation curve is shifted to the left from the low oxygen affinity and to the right from the high oxygen affinity by the amount of allosteric factor added.
335 g of water was added to a uniformly mixed lipid consisting of 192 g of hydrogenated phosphatidylcholine, 94 g of cholesterol, and 49 g of stearic acid, and heated at 85 ° C. for 30 minutes to prepare a hydrated and swollen homogeneous mixed quality. Hemoglobin was purified and concentrated from the expired concentrated erythrocyte preparation, and a concentrated hemoglobin solution having a hemoglobin concentration of 42.6 w / v%, in which 0.5 mol of 12 sodium phytate was added to hemoglobin as an allosteric factor, was prepared. 2393 g of the concentrated hemoglobin solution was added to 670 g of the hydrated and swollen uniformly mixed lipid, and the mixture was stirred uniformly and pre-emulsified. After the pre-emulsification, the emulsification was carried out by a stronger stirring. The mixed solution after the main emulsification was diluted with physiological saline, and the particle size was controlled by circulating filtration using a 0.45 μm membrane. Next, after deoxygenation with sodium sulfite using 10 mg / mL sodium sulfite physiological saline, 0.5 mg / mL sodium sulfite physiological The hemoglobin and 12 sodium phytate that were not made into liposomes were removed by hydroconcentration with saline, and a liposome suspension containing human-derived concentrated hemoglobin and allosteric factor was prepared. To the liposome suspension, an aqueous PEG-linked phospholipid solution in which DSPE-PE5000 (manufactured by NOF Corporation) was dissolved in physiological saline was added as a PEG-linked phospholipid. The liposome membrane-constituting lipid concentration in the liposome suspension containing the liposome and the PEG-bound phospholipid was 4.48 w / v%, and the PEG-bound phospholipid was 0.41 w / v%. 5181 mL of the above-described liposome suspension treated with PEG-conjugated phospholipids on the liposome surface was obtained at 24 ° C. for 24 hours.
The hemoglobin concentration in the liposome suspension is 6.1 w / v%, the concentration of 12 sodium phytate, an allosteric factor, is 0.0360 w / v%, the stearic acid concentration is 0.59 w / v%, and the hemoglobin concentration is The rate was 31%. The hemoglobin metation rate in the liposome suspension immediately after production was 4.3%.
＜酸素運搬量の算出＞
前記リポソーム懸濁液の酸素解離曲線（37℃）から求めた通常の酸素分圧（通常の血液循環における）である酸素分圧100mmHgと酸素分圧40mmHgの間の酸素運搬効率は25％であり、低酸素領域（出血ショック治療初期又は梗塞部位、ガン部位など）である酸素分圧40mmHgと酸素分圧0mmHgの間の酸素運搬効率は61％であった。前記リポソーム懸濁液中のヘモグロビン濃度6.1w/v%、ヘモグロビンメト化率4.3w/v%、前記リポソーム懸濁液の酸素運搬効率（通常の酸素分圧設定又は低酸素領域酸素分圧設定）を前述0011に記載の（３）式に当てはめると、前記リポソーム懸濁液1mLが、通常の酸素分圧（通常の血液循環における）である酸素分圧100mmHgと酸素分圧40mmHgの間で運び得る酸素運搬量（37℃、1気圧）は0.023mLと算出された。一方、低酸素領域（出血ショック治療初期又は梗塞部位、ガン部位など）の酸素分圧40mmHgと酸素分圧0mmHgの間で運び得る酸素運搬量（37℃、1気圧）は0.056ｍＬと算出された。
<Calculation of oxygen transport amount>
The oxygen transport efficiency between the oxygen partial pressure of 100 mmHg and the oxygen partial pressure of 40 mmHg, which is the normal oxygen partial pressure (in normal blood circulation) determined from the oxygen dissociation curve (37 ° C) of the liposome suspension, is 25% The oxygen carrying efficiency between the oxygen partial pressure of 40 mmHg and the oxygen partial pressure of 0 mmHg in the hypoxic region (early hemorrhagic shock treatment or infarcted site, cancer site, etc.) was 61%. Hemoglobin concentration in the liposome suspension 6.1 w / v%, hemoglobin metation rate 4.3 w / v%, oxygen transport efficiency of the liposome suspension (normal oxygen partial pressure setting or low oxygen region oxygen partial pressure setting) Is applied to the formula (3) described in 0011 above, 1 mL of the liposome suspension can be carried between an oxygen partial pressure of 100 mmHg and an oxygen partial pressure of 40 mmHg, which are normal oxygen partial pressures (in normal blood circulation). The oxygen carrying amount (37 ° C, 1 atm) was calculated to be 0.023 mL. On the other hand, the oxygen carrying amount (37 ° C, 1 atm) that can be carried between oxygen partial pressure 40mmHg and oxygen partial pressure 0mmHg in the hypoxic region (early bleeding shock treatment or infarcted area, cancer site, etc.) was calculated to be 0.056mL. .
＜中酸素親和性のヘモグロビン含有リポソーム懸濁液の製造＞
アロステリック因子添加量により、酸素解離曲線を低酸素親和性より左に且つ、高酸素親和性より右にシフトさせる。
水素添加ホスファチジルコリン3149g、コレステロール1543g、ステアリン酸809gから成る均一混合脂質に水5501gを加えて、85℃で30分間加熱して水和膨潤均一混合脂質を調整した。期限切れ濃厚赤血球製剤からヘモグロビンを精製、濃縮し、アロステリック因子として、フィチン酸12ナトリウムをヘモグロビンに対して、0.5モル添加したヘモグロビン濃度42.6w/v%の濃厚ヘモグロビン溶液を調整した。前記水和膨潤均一混合脂質11002gに前記濃厚ヘモグロビン溶液39295gを添加し、均一に攪拌し前乳化を行なった。前記前乳化後に更に強力な攪拌により、本乳化を行なった。前記本乳化後の混合液を生理食塩水により希釈して、0.45μm膜を用いて、循環濾過により粒子径の制御を行なった。次に10mg / mL濃度の亜硫酸ナトリウム生理食塩水を使用し、亜硫酸ナトリウムによる脱酸素化を行なった後、分画分子量30万の限外濾過膜を用いて、0.5mg mL濃度の亜硫酸ナトリウム生理食塩水による加水濃縮で、リポソーム化されなかったヘモグロビン及びフィチン酸12ナトリウムを除去し、ヒト由来濃厚ヘモグロビン及びアロステリック因子含有リポソーム懸濁液を作成した。前記リポソーム懸濁液にPEG結合リン脂質として、DSPE-PE5000（日本油脂製）を生理食塩水に溶解させたPEG結合リン脂質水溶液を添加した。前記リポソーム及びPEG結合リン脂質を含有した前記リポソーム懸濁液中のリポソーム膜構成脂質濃度が4.09w/v%であり、PEG結合リン脂質が0.43w/v%である様に調整した後、37℃、24時間処理し、PEG結合リン脂質をリポソーム表面に固定化した前記リポソーム懸濁液を87683mL得た。
前記リポソーム懸濁液中のヘモグロビン濃度は6.3w/v%であり、アロステリック因子であるフィチン12ナトリウム濃度は0.040w/v%であり、ステアリン酸濃度は0.62w/v%であり、ヘモグロビン収率は33%であった。製造直後の前記リポソーム懸濁液のヘモグロビンメト化率は5.0%であった。
<Manufacture of Liposome Suspension Containing Hemoglobin with Medium Oxygen Affinity>
The oxygen dissociation curve is shifted to the left from the low oxygen affinity and to the right from the high oxygen affinity by the amount of allosteric factor added.
5501 g of water was added to a uniformly mixed lipid composed of 3149 g of hydrogenated phosphatidylcholine, 1543 g of cholesterol and 809 g of stearic acid, and heated at 85 ° C. for 30 minutes to prepare a hydrated and swollen uniformly mixed lipid. The hemoglobin was purified and concentrated from the expired concentrated erythrocyte preparation, and a concentrated hemoglobin solution having a hemoglobin concentration of 42.6 w / v% added with 0.5 mol of 12 sodium phytate as an allosteric factor to hemoglobin was prepared. 39295 g of the concentrated hemoglobin solution was added to 11002 g of the hydrated and swollen uniformly mixed lipid, and the mixture was uniformly stirred and pre-emulsified. After the pre-emulsification, the emulsification was carried out by a stronger stirring. The mixture after the main emulsification was diluted with physiological saline, and the particle size was controlled by circulation filtration using a 0.45 μm membrane. Next, after deoxygenation with sodium sulfite using 10 mg / mL sodium sulfite physiological saline, 0.5 mg mL sodium sulfite physiological saline using an ultrafiltration membrane with a fractional molecular weight of 300,000 Hydrophobic concentration with water removed hemoglobin and 12 sodium phytate, which were not made into liposomes, to prepare a liposome suspension containing human-derived concentrated hemoglobin and allosteric factor. A PEG-conjugated phospholipid aqueous solution in which DSPE-PE5000 (manufactured by NOF Corporation) was dissolved in physiological saline was added to the liposome suspension as PEG-conjugated phospholipid. After adjusting the liposome membrane-constituting lipid concentration in the liposome suspension containing the liposome and PEG-bound phospholipid to 4.09 w / v% and adjusting the PEG-bound phospholipid to 0.43 w / v%, 37 After treating at 24 ° C. for 24 hours, 87683 mL of the above-mentioned liposome suspension in which the PEG-linked phospholipid was immobilized on the liposome surface was obtained.
The hemoglobin concentration in the liposome suspension is 6.3 w / v%, the phytine 12 sodium concentration as an allosteric factor is 0.040 w / v%, the stearic acid concentration is 0.62 w / v%, and the hemoglobin yield Was 33%. The hemoglobin metration rate of the liposome suspension immediately after production was 5.0%.
＜酸素運搬量の算出＞
前記リポソーム懸濁液の酸素解離曲線（37℃）から求めた通常の酸素分圧（通常の血液循環における）である酸素分圧100mmHgと酸素分圧40mmHgの間の酸素運搬効率は23%であり、低酸素領域（出血ショック治療初期又は梗塞部位、ガン部位など）である酸素分圧40mmHgと酸素分圧0mmHgの間の酸素運搬効率は63%であった。前記リポソーム懸濁液のヘモグロビン濃度6.3w/v%、ヘモグロビンメト化率5.0%、前記リポソーム懸濁液の酸素運搬効率（通常の酸素分圧設定又は低酸素領域酸素分圧設定）を前述0012に記載の（３）式に当てはめると、前記リポソーム懸濁液1mLが、通常の酸素分圧（通常の血液循環における）である酸素分圧100mmHgと酸素分圧40mmHgの間で運び得る酸素運搬量（37℃、1気圧）は0.022mLと算出された。一方、低酸素領域（出血ショック治療初期又は梗塞部位、ガン部位など）の酸素分圧40mmHgと酸素分圧0mmHgの間で運び得る酸素運搬量（37℃、1気圧）は0.059mLと算出された。
<Calculation of oxygen transport amount>
The oxygen transport efficiency between the oxygen partial pressure of 100 mmHg and the oxygen partial pressure of 40 mmHg, which is the normal oxygen partial pressure (in normal blood circulation) determined from the oxygen dissociation curve (37 ° C) of the liposome suspension, is 23% The oxygen transport efficiency between the oxygen partial pressure of 40 mmHg and the oxygen partial pressure of 0 mmHg in the hypoxic region (early hemorrhagic shock treatment or infarcted site, cancer site, etc.) was 63%. The liposome suspension has a hemoglobin concentration of 6.3 w / v%, a hemoglobin metation rate of 5.0%, and the oxygen suspension efficiency of the liposome suspension (ordinary oxygen partial pressure setting or low oxygen region oxygen partial pressure setting) When applied to the equation (3) described above, the amount of oxygen that can be carried between 1 mL of the liposome suspension between an oxygen partial pressure of 100 mmHg, which is a normal oxygen partial pressure (in normal blood circulation), and an oxygen partial pressure of 40 mmHg ( 37 ° C, 1 atm) was calculated to be 0.022 mL. On the other hand, the oxygen transport amount (37 ° C, 1 atm) that can be transported between oxygen partial pressure 40mmHg and oxygen partial pressure 0mmHg in the hypoxic region (early bleeding shock treatment or infarcted area, cancer site, etc.) was calculated to be 0.059mL. .
＜低酸素親和性及び高酸素親和性のヘモグロビン含有リポソーム懸濁液との比較＞
低酸素親和性のヘモグロビン含有リポソーム懸濁液（後述、比較例1）、中酸素親和性のヘモグロビン含有リポソーム懸濁液（前述、実施例1、実施例2）、高酸素親和性のヘモグロビン含有リポソーム懸濁液（後述、比較例2）の各々が、通常の血液循環における酸素分圧100mmHg〜40mmHgの間で運び得る酸素運搬量（37℃、1気圧）及び低酸素分圧領域（出血ショック治療初期又は梗塞部位、ガン部位など）40mmHg〜0mmHgの間で運び得る酸素運搬量（37℃、1気圧）を表1に纏める。実施例1の中酸素親和性のヘモグロビン含有リポソーム懸濁液は比較例1の低酸素親和性のヘモグロビン含有リポソーム懸濁液と比較すると、低酸素分圧領域（40mmHg〜0mmHg）へ運び得る酸素量が1.47倍に増加し、出血ショック治療初期、梗塞部位、ガン部位への酸素供給に有利となる。比較例2の高酸素親和性のヘモグロビン含有リポソーム懸濁液と比較すると、通常の血液循環における酸素分圧領域（100mmHg〜40mmHg）へ運び得る酸素量が8.21倍に増加している事が確認された。高酸素親和性では低酸素領域での酸素運搬に極端に特化しており、これに対して中酸素親和性では高酸素親和性と比較して、低酸素領域への酸素運搬と、通常の血液循環における酸素分圧での酸素運搬のバランスが取れており、且つ、低酸素親和性と比較し、低酸素領域への酸素運搬量が増加している事が分かる。実施例2も比較例1及び比較例2と比較して同様の傾向が認められた。また、実施例1、実施例2と同じくアロステリックエフェクターを用いて酸素運搬を制御する比較例1を比較すると、実施例1、実施例2の中酸素親和性ではヘモグロビン収率が各々2.52倍、2.58倍に増加した。
<Comparison with low oxygen affinity and high oxygen affinity hemoglobin-containing liposome suspension>
Low oxygen affinity hemoglobin-containing liposome suspension (described later, Comparative Example 1), medium oxygen affinity hemoglobin-containing liposome suspension (previously described in Example 1 and Example 2), high oxygen affinity hemoglobin-containing liposome Each of the suspensions (described later, Comparative Example 2) can carry an oxygen partial pressure of 100 mmHg to 40 mmHg in normal blood circulation (37 ° C, 1 atm) and a low oxygen partial pressure region (bleeding shock treatment) Table 1 summarizes the amount of oxygen transport (37 ° C, 1 atm) that can be carried between 40 mmHg and 0 mmHg. Compared with the low oxygen affinity hemoglobin-containing liposome suspension of Comparative Example 1, the amount of oxygen that the medium oxygen affinity hemoglobin-containing liposome suspension of Example 1 can carry to the low oxygen partial pressure region (40 mmHg to 0 mmHg) Increases 1.47 times, which is advantageous for oxygen supply to the initial stage of hemorrhagic shock treatment, infarcted area and cancer area. Compared with the high oxygen affinity hemoglobin-containing liposome suspension of Comparative Example 2, it was confirmed that the amount of oxygen that can be carried to the oxygen partial pressure region (100 mmHg to 40 mmHg) in normal blood circulation increased 8.21 times. It was. High oxygen affinity is extremely specialized in oxygen transport in the low oxygen region, whereas medium oxygen affinity is compared to high oxygen affinity in terms of oxygen transport to the low oxygen region and normal blood. It can be seen that the oxygen transport at the oxygen partial pressure in the circulation is balanced and the oxygen transport amount to the low oxygen region is increased as compared with the low oxygen affinity. The same tendency was observed in Example 2 as compared with Comparative Example 1 and Comparative Example 2. Further, when comparing Comparative Example 1 in which oxygen transport is controlled using an allosteric effector in the same manner as in Example 1 and Example 2, the hemoglobin yield is 2.52 times and 2.58 in the medium oxygen affinity of Example 1 and Example 2, respectively. Doubled.
（比較例1）
(Comparative Example 1)
＜低酸素親和性のヘモグロビン含有リポソーム懸濁液の製造＞
アロステリック因子を添加して、酸素解離曲線を天然赤血球と比較して、より右にシフトさせる。
水素添加大豆ホスファチジルコリン182g、コレステロール89g、ステアリン酸46gからなる均一混合脂質に水317gを加えて、85℃で30分加熱して水和膨潤均一混合脂質を調整した。期限切れ濃厚赤血球製剤からヘモグロビンを精製、濃縮し、アロステリック因子として、フィチン酸12ナトリウムをヘモグロビンに対して等モル添加したヘモグロビン濃度42.6w/v%の濃厚ヘモグロビンを調整した。前記水和膨潤均一混合脂質634gに前記フィチン酸12ナトリウム添加濃厚ヘモグロビン溶液2264gを添加し、水和膨潤均一混合脂質中のステアリン酸を中和する量の水酸化ナトリウムを添加しつつ、均一に攪拌し、前乳化を行なった。前記前乳化後に更に強力な攪拌により、本乳化を行なった。前記本乳化後の混合液を生理食塩水により希釈して、0.45μm膜を用いて、循環濾過により粒子径の制御を行なった。次に10mg/ml濃度の亜硫酸ナトリウム生理食塩水を使用し、亜硫酸ナトリウムによる脱酸素化を行なった後、分画分子量30万の限外濾過膜を用いて、0.5mg/ml濃度の亜硫酸ナトリウム生理食塩水による加水濾過濃縮で、リポソーム化されなかったヘモグロビン及びフィチン酸12ナトリウムを除去し、ヒト由来濃厚ヘモグロビン及びアロステリック因子含有リポソーム懸濁液を作成した。前記リポソーム懸濁液に、PEG結合リン脂質として、DSPE-PEG5000（日本油脂製）を生理食塩水に溶解させたPEG結合リン脂質水溶液を添加した。前記リポソーム及びPEG結合リン脂質を含有した前記リポソーム懸濁液中のリポソーム膜構成脂質濃度が4.04w/v%であり、PEG結合リン脂質濃度が0.33w/v%である様に調整した後、37℃、24時間処理し、PEG結合リン脂質をリポソーム表面に固定化した前記リポソーム懸濁液1991mLを得た。
前記リポソーム懸濁液中のヘモグロビン濃度は6.2w/v%であり、アロステリック因子であるフィチン酸12ナトリウム濃度は0.077w/v%であり、ステアリン酸濃度は0.60w/v%であり、ヘモグロビン収率は12.8%であった。製造直後の前記リポソーム懸濁液中のヘモグロビンメト化率は4.5%であつた。
<Production of low oxygen affinity hemoglobin-containing liposome suspension>
Allosteric factor is added to shift the oxygen dissociation curve more to the right compared to natural red blood cells.
317 g of water was added to a uniformly mixed lipid consisting of 182 g of hydrogenated soybean phosphatidylcholine, 89 g of cholesterol and 46 g of stearic acid, and heated at 85 ° C. for 30 minutes to prepare a hydrated and swollen uniformly mixed lipid. Hemoglobin was purified and concentrated from the expired concentrated erythrocyte preparation, and concentrated hemoglobin having a hemoglobin concentration of 42.6 w / v% was prepared by adding equimolar amounts of 12 sodium phytate to hemoglobin as an allosteric factor. Add 2264 g of the concentrated hemoglobin solution with 12 sodium phytate to 634 g of the hydrated and swollen uniformly mixed lipid, and stir uniformly while adding sodium hydroxide in an amount to neutralize the stearic acid in the hydrated and swollen uniformly mixed lipid. And pre-emulsified. After the pre-emulsification, the emulsification was carried out by a stronger stirring. The mixture after the main emulsification was diluted with physiological saline, and the particle size was controlled by circulation filtration using a 0.45 μm membrane. Next, 10 mg / ml sodium sulfite physiological saline was used, and after deoxygenation with sodium sulfite, a 0.5 mg / ml sodium sulfite physiological solution was used using an ultrafiltration membrane with a molecular weight cut off of 300,000. The hemoglobin and 12 sodium phytate which were not made into liposomes were removed by hydrofiltration concentration with saline solution, and a human-derived concentrated hemoglobin and allosteric factor-containing liposome suspension was prepared. To the liposome suspension, an aqueous PEG-conjugated phospholipid solution in which DSPE-PEG5000 (manufactured by NOF Corporation) was dissolved in physiological saline was added as a PEG-conjugated phospholipid. After adjusting the liposome membrane-constituting lipid concentration in the liposome suspension containing the liposome and PEG-bound phospholipid to 4.04 w / v% and adjusting the PEG-bound phospholipid concentration to 0.33 w / v%, By treating at 37 ° C. for 24 hours, 1991 mL of the above-mentioned liposome suspension in which the PEG-linked phospholipid was immobilized on the liposome surface was obtained.
The hemoglobin concentration in the liposome suspension was 6.2 w / v%, the 12 sodium phytate concentration, which is an allosteric factor, was 0.077 w / v%, the stearic acid concentration was 0.60 w / v%, and the hemoglobin concentration was The rate was 12.8%. The hemoglobin metation rate in the liposome suspension immediately after production was 4.5%.
＜酸素運搬量の算出＞
前記リポソーム懸濁液の酸素解離曲線（37℃）から求めた酸素運搬効率（高酸素親和性設定：酸素分圧40mmHgと酸素分圧0mmHgの間の酸素飽和度の差）は41%であった。前記リポソーム懸濁液中のヘモグロビン濃度6.2%、ヘモグロビンメト化率4.5%、前記リポソーム懸濁液の酸素運搬効率（高酸素親和性設定）41%を前述0013に記載の(3)式に当てはめると、前記リポソーム懸濁液1mLが酸素分圧40mmHg〜0mmHgの間で運び得る酸素運搬量（37℃、1気圧）は0.038mLと算出された。一方、酸素分圧100mmHgと40mmHgの間の運搬可能な酸素量（37℃、1気圧）は、酸素解離曲線が右にシフトしており、この場合の酸素運搬効率（低酸素親和性設定）が37%となるので、同じく前述0012に記載の(3)式に当てはめると0.035mLとなる。
（比較例2）
<Calculation of oxygen transport amount>
The oxygen carrying efficiency (high oxygen affinity setting: difference in oxygen saturation between oxygen partial pressure 40 mmHg and oxygen partial pressure 0 mmHg) determined from the oxygen dissociation curve (37 ° C) of the liposome suspension was 41% . When the hemoglobin concentration in the liposome suspension is 6.2%, the hemoglobin metation rate is 4.5%, and the oxygen carrying efficiency (high oxygen affinity setting) of 41% in the liposome suspension is applied to the equation (3) described in the above 0013. The amount of oxygen transport (37 ° C., 1 atm) that can be carried by 1 mL of the liposome suspension between an oxygen partial pressure of 40 mmHg to 0 mmHg was calculated to be 0.038 mL. On the other hand, the amount of oxygen that can be transported between oxygen partial pressures of 100 mmHg and 40 mmHg (37 ° C, 1 atm) has the oxygen dissociation curve shifted to the right. In this case, the oxygen transport efficiency (low oxygen affinity setting) Since it is 37%, it is 0.035 mL when applied to the equation (3) described in 0012.
(Comparative Example 2)
＜高酸素親和性のヘモグロビン含有リポソーム懸濁液の製造＞
アロステリック因子を添加せず、酸素解離曲線を天然赤血球と比較して、より左にシフトさせる。
水素添加大豆ホスファチジルコリン182g、コレステロール89g、ステアリン酸46gからなる均一混合脂質に水317gを加えて、85℃で30分間加熱して水和膨潤均一混合脂質を調整した。期限切れ濃厚赤血球際剤からヘモグロビンを精製、濃縮し、ヘモグロビン濃度42.0w/v%の濃厚ヘモグロビンを調整した。前記水和膨潤均一混合脂質634gに前記濃厚ヘモグロビン溶液2264gを添加し、均一に攪拌し前乳化を行なった。前記前乳化後に更に強力な攪拌により、本乳化を行なった。前記本乳化後の混合液を生理食塩水により希釈して、0.45μm膜を用いて、循環濾過により粒子径の制御を行なった。次に10mg/mL濃度の亜硫酸ナトリウム生理食塩水溶液を使用し、亜硫酸ナトリウムによる脱酸素化を行なった後、分画分子量30万の限外濾過膜を用いて、0.5mg/mL濃度の亜硫酸ナトリウム生理食塩水溶液による加水濃縮で、リポソーム化されなかったヘモグロビンを除去し、ヒト由来濃厚ヘモグロビン含有リポソーム懸濁液を作成した。前記リポソーム懸濁液に、PEG結合リン脂質として、DSPE-PEG5000（日本油脂製）を生理食塩水に溶解させたPEG結合リン脂質生理食塩水溶液を添加した。前記リポソーム及びPEG結合リン脂質を含有した前記リポソーム懸濁液中のリポソーム膜形成脂質濃度が4.05w/v%であり、PEG結合リン脂質濃度が0.31w/v%である様に調整した後、37℃、24時間処理し、PEG結合リン脂質をリポソーム表面に固定化した前記リポソーム懸濁液8000mLを得た。
前記リポソーム懸濁液中のヘモグロビン濃度は6.3w/v%であり、ステアリン酸濃度は0.58w/v%であり、ヘモグロビン収率は53.0%であった。製造直後の前記リポソーム懸濁液中のヘモグロビンメト化率は4.0%であった。
<Production of high oxygen affinity hemoglobin-containing liposome suspension>
No allosteric factor is added and the oxygen dissociation curve is shifted to the left compared to natural erythrocytes.
317 g of water was added to a uniformly mixed lipid composed of 182 g of hydrogenated soybean phosphatidylcholine, 89 g of cholesterol and 46 g of stearic acid, and heated at 85 ° C. for 30 minutes to prepare a hydrated and swollen uniformly mixed lipid. Hemoglobin was purified and concentrated from the expired concentrated red blood cell preparation to prepare concentrated hemoglobin having a hemoglobin concentration of 42.0 w / v%. 2634 g of the concentrated hemoglobin solution was added to 634 g of the hydrated and swollen uniformly mixed lipid, and the mixture was stirred uniformly and pre-emulsified. After the pre-emulsification, the emulsification was carried out by a stronger stirring. The mixture after the main emulsification was diluted with physiological saline, and the particle size was controlled by circulation filtration using a 0.45 μm membrane. Next, 10 mg / mL sodium sulfite physiological saline solution was used, and after deoxygenation with sodium sulfite, a 0.5 mg / mL sodium sulfite physiological solution was used using an ultrafiltration membrane with a molecular weight cut off of 300,000. The hemoglobin that had not been converted to liposomes was removed by hydroconcentration with a saline solution to prepare a human-derived concentrated hemoglobin-containing liposome suspension. A PEG-linked phospholipid physiological saline solution in which DSPE-PEG5000 (manufactured by NOF Corporation) was dissolved in physiological saline was added to the liposome suspension as a PEG-conjugated phospholipid. After adjusting the liposome membrane-forming lipid concentration in the liposome suspension containing the liposome and PEG-bound phospholipid to 4.05 w / v% and adjusting the PEG-bound phospholipid concentration to 0.31 w / v%, After treatment at 37 ° C. for 24 hours, 8000 mL of the above-described liposome suspension in which PEG-linked phospholipid was immobilized on the liposome surface was obtained.
The hemoglobin concentration in the liposome suspension was 6.3 w / v%, the stearic acid concentration was 0.58 w / v%, and the hemoglobin yield was 53.0%. The hemoglobin metation rate in the liposome suspension immediately after production was 4.0%.
＜酸素運搬量の算出＞
前記リポソーム懸濁液の酸素解離曲線（37℃）から求めた酸素運搬効率（高酸素親和性設定。酸素分圧40mmHgと酸素分圧0mmHgの間の酸素飽和度の差）は97%であった。前記リポソーム懸濁液中のヘモグロビン濃度：6.3w/v%、ヘモグロビンメト化率：4.0%、前記リポソーム懸濁液の酸素運搬効率（高酸素親和性設定）：97%を前述0015に記載の（３）式に当てはめると、前記リポソーム懸濁液1mLが酸素分圧40mmHg〜0mmHgの間で運び得る酸素運搬量（37℃、1気圧）は0.092mLと算出された。一方、酸素解離曲線が天然赤血球より左にシフトしているので、100mmHgと40mmHgの間の酸素運搬効率は僅か3%となり、100mmHgと40mmHgの間で運搬可能な酸素量は、前述0012に記載の（３）式に当てはめると0.0028mLとなる。
<Calculation of oxygen transport amount>
The oxygen carrying efficiency (high oxygen affinity setting. Difference in oxygen saturation between oxygen partial pressure 40 mmHg and oxygen partial pressure 0 mmHg) determined from the oxygen dissociation curve (37 ° C) of the liposome suspension was 97% . Hemoglobin concentration in the liposome suspension: 6.3 w / v%, hemoglobin metration rate: 4.0%, oxygen transportation efficiency (high oxygen affinity setting) of the liposome suspension: 97% 3) When applied to the equation, the oxygen carrying amount (37 ° C., 1 atm) that 1 mL of the liposome suspension can be carried between oxygen partial pressures of 40 mmHg to 0 mmHg was calculated to be 0.092 mL. On the other hand, since the oxygen dissociation curve is shifted to the left from the natural erythrocytes, the oxygen carrying efficiency between 100 mmHg and 40 mmHg is only 3%, and the amount of oxygen that can be carried between 100 mmHg and 40 mmHg is as described in 0012 above. When applied to the equation (3), it becomes 0.0028 mL.
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| WO2013047263A1 (en) * | 2011-09-28 | 2013-04-04 | テルモ株式会社 | Hemoglobin-containing liposome and method for producing same |
| WO2020178598A1 (en) * | 2019-03-06 | 2020-09-10 | Cytoseek Ltd | Antitumor cell comprising a charge modified globin |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013047263A1 (en) * | 2011-09-28 | 2013-04-04 | テルモ株式会社 | Hemoglobin-containing liposome and method for producing same |
| CN103796668A (en) * | 2011-09-28 | 2014-05-14 | 泰尔茂株式会社 | Hemoglobin-containing liposome and method for producing same |
| JPWO2013047263A1 (en) * | 2011-09-28 | 2015-03-26 | テルモ株式会社 | Hemoglobin-containing liposome and method for producing the same |
| CN103796668B (en) * | 2011-09-28 | 2016-12-28 | 泰尔茂株式会社 | Liposome containing hemoglobin and manufacture method thereof |
| WO2020178598A1 (en) * | 2019-03-06 | 2020-09-10 | Cytoseek Ltd | Antitumor cell comprising a charge modified globin |
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