人工血液第 16 巻 第 2 号 2008 年 10 月

目次
第15回年次大会プログラム
The 6th Current Issues on Blood Substitute Researchプログラム ……

38

ARTIFICIAL BLOOD
Vol. 16 No. 2 October, 2008

Contents
The 15th Annual Meeting Program
The 6th Current Issues on Blood Substitute Research Program ……

38

第15回日本血液代替物学会年次大会
The 15th Annual Meeting of the Society of Blood Substitutes, Japan
大会長： 堀之内 宏久（慶應義塾大学医学部呼吸器外科 准教授）
『血液代替物の開発戦略』
会期： 平成20年10月23日（木）会場： 慶應義塾大学信濃町キャンパス

北里講堂

東京都新宿区信濃町35

The 6th Current Issues on Blood Substitute Research
会長： 小林 紘一（慶應義塾大学 名誉教授）
Koichi Kobayashi（Keio University）

会期： 平成20年10月24日（金）・25日（土）October 24−25，2008

会場： 慶應義塾大学信濃町キャンパス 大会議室・中会議室（新棟11階）
東京都新宿区信濃町35
Keio University Shinanomachi Campus
35 Shinanomachi, Shinjuku-ku, Tokyo Japan

大会事務局〒160-8582 東京都新宿区信濃町35
慶應義塾大学医学部呼吸器外科
TEL: 03-5363-3806
FAX: 03-5363-3499
E-mail: amorjsbs@sc.itc.keio.ac.jp

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人工血液 Vol. 16 , No.2, 2008

年次大会 大会長 挨拶
このたびは第15回を迎えました日本血液代替物学会の年次大会をお世話することとなりました。今回の学会は小林紘一血液代替物学会会長が、２年に一度開催される、Current Issues on Blood SubutituteResearch（CIBSR）を主宰されるのに合わせて合同学会として開催させていただくこととなりました。当
初すべて英語でとも考えたのですが、日本語で自由にDisucussionができる時間もあったほうがよいとの意
見もありましたので、23日を日本の血液代替物学会年次大会として、24、25日を第６回のCIBSRとして行うこととしました。
年次大会では輸血代替を目的とした研究に関する演題が多く集まりましたので、シンポジウムのセッシ
ョンを「輸血代替としての人工酸素運搬体」として討論してゆきたいと思います。また、教育講演、招請
講演はCIBSRへ移して、小林弘祐先生に「組織への酸素運搬について」およびZapol先生に修飾ヘモグロビ
ンによる血圧上昇に対するNO吸入の効果について御講演いただくこととしました。

人工酸素運搬体は臨床応用が間近といわれながら実現への道筋が遠いようで、研究サイドで新たな知見を発表し、臨床応用への後押しができればと考えています。

一般会員のみならず、厚労省、日赤、企業の方のご参加もいただき、官産学にわたる交流ができればと期待しています。

多数のご参加を心からお待ちしております。

第15回日本血液代替物学会年次大会
大会長 堀之内 宏久（慶應義塾大学医学部呼吸器外科）

ARTIFICIAL BLOOD Vol. 16 , No.2, 200839

お知らせとお願い■会員・参加者の方へ＜開場及び受付開始＞
第15回年次大会およびThe 6th Current Issues on Blood Substitute Research（6th CIBSR）の開場時間は，10月23日（木）午前９時〜，
24日（金）
・25日（土）午前８時〜です．
＜参加登録＞
第15回年次大会の受付は23日（木）北里講堂前のみです．なお，事前受付は行いません．参加登録費は下記の通りです．
会員

10,000円

非会員

20,000円

学生

5,000円（受付にて必ず学生証を提示してください．
）

23日に第15回年次大会の参加登録を行った方は，追加登録なしで24・25日の6th CIBSRにご参加いただけます．
＜抄録集＞
抄録集は会員全員に事前送付しています．また当日は受付にて１部1,500円で販売いたします．
＜新入会受付＞
日本血液代替物学会に未入会の方は受付で入会手続きをおとりください．
年会費は正会員10,000円，購読会員6,000円，学生会員5,000円です．
＜懇親会＞
参加者相互の親睦を図るため，10月23日（木）午後５時30分よりレストラン オアシス（慶應義塾大学病院 新棟11階）におきまして懇親会を開催いたします．（終了: 午後７時30分頃）参加費は無料ですので，是非ご参加ください．
■The 6th Current Issues on Blood Substitute Research（6th CIBSR）への参加について24日（金）からのご参加は下記参加登録費となりますのでご注意ください．
一般参加30,000円

同伴者

15,000円

学生

5,000円（受付にて必ず学生証を提示してください．
）■演題発表される方へ
１．本学会の発表はすべてPCプロジェクターで行いますのでご了承ください．
２．Windowsの場合（動画を含む場合を除く）はUSBフラッシュメモリーまたはCD-Rに保存してご提出ください．CD-Rについて
は，ハイブリッドフォーマットのみといたします．MacintoshおよびWindowsで動画を含むデータの場合は，ご自身のパソコンをお持ちください．なお，当日お持込になるPCまたはメディアのウイルスチェックは必ず事前に行ってください．スクリー
ンセーバー，ウイルスチェックならびに省電力設定はご発表前にあらかじめ解除しておいてください．静止画像はJPEG形式で作成されることをお勧めいたします．
３．事務局でご用意しておりますPC（Windows）には，OS: Windows VistaでPowerPoint 2003をインストールしており，
PowerPoint 2000以降のバージョンには対応可能です．この環境にて正常に作動するデータをご用意ください．ご発表データ
は，会期終了後，事務局で責任を持って消去いたします．
４．当日はUSBフラッシュメモリー，CD-R等のバックアップデータを必ずご用意ください．当日のデータ及びバックアップとして使用させていただきます．
５．ご発表の１時間前までに受付にいらっしゃって，試写を行ってください．
６．ご自身のPCでの発表を希望される場合は，D-sub15ピンによるモニター出力が必要です．事務局でD-sub15ピンの接続ケーブ
ルをご用意いたしますが，ご持参いただくPCからD-sub15ピンへの変換コネクタが必要な場合には，各自でご用意ください．
D-sub15ピン以外では接続できませんのでご了承ください．
７．電源ケーブルを必ずご持参ください．バッテリーでのご使用はトラブルの原因となります．

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人工血液 Vol. 16 , No.2, 2008

８．発表時間は討論を含め一題20分です．
■各種会議日程
理 事 会：10月23日（木）12時〜

慶應義塾大学医学部 北里記念図書館２階

第１会議室

評議員会：10月23日（木）12時30分〜

慶應義塾大学医学部 北里記念図書館２階

北里講堂

総

16時30分〜

慶應義塾大学医学部 北里記念図書館２階

北里講堂

会：10月23日（木）■大会事務局〒160-8582

東京都新宿区信濃町35

慶應義塾大学医学部呼吸器外科
TEL: 03-5363-3806
FAX: 03-5363-3499
E-mail: amorjsbs@sc.itc.keio.ac.jp

学会のホームページにて，最新のお知らせ，注意事項，プログラムなどを掲載していきます．併せてご確認，ご利用ください．
学会ホームページ

http://www.blood-sub.jp/

th

6 CIBSR ホームページ（英語） http://www.convention.co.jp/b-sub2008

ARTIFICIAL BLOOD Vol. 16 , No.2, 2008

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人工血液 Vol. 16 , No.2, 2008

19:00
19:30

19:30

19:30

Tokyo Dome Hotel

19:00

Banquet19:00（新棟11階 大会議室前）

18:30（慶應義塾大学病院 新棟11階）

18:30

17:30

17:00

16:30

16:00

15:00
15:15
15:30

14:30

14:00

18:30

17:30

17:10

PARTⅡ

Cutting Edge of Research of Artificial Oxygen Carriers

Symposium Ⅰ

Coffee BreakCutting Edge of Research of Artificial Oxygen Carriers

PARTⅠ

13:30

13:00

12:30

12:00

11:20

11:00

10:30

18:00

懇親会 レストランオアシス

閉会の辞

Hirosuke Kobayashi（Kitasato University）

Presidential Address Koichi Kobayashi（6th CIBSR）（Keio University）
Symposium Ⅰ

9:10

8:30

9:50
Marcos Intaglietta
10:00（Univ. of California, San Diego）

Makoto Suematsu（Keio University）

Lunch Time Symposium

New Material

Educational Lecture

Coffee BreakKeynote Lecture Ⅰ

Plenary Lecture Ⅰ

Opening Remarks

10月24日（金）October 24
新棟 New Hospital Wing（Shinto）11F

18:00

18:00

17:00
17:10
17:30

16:30

16:30

総会

16:00

16:00

「機能性医薬」

休憩

15:00
15:10
15:30

14:00

13:40

13:10

12:10

15:00
15:10
15:30

第6回CIBSR参加受付

堀之内宏久（慶應義塾大学）

評議員会 12:30〜北里講堂

理事会 12:00〜第一会議室

11:50

11:10

10:20
10:30

14:30

一般口演

会長講演（第15回年次大会）

昼休み

シンポジウム 「輸血代替としての利用法」

開会の辞

9:40

8:50
9:00

14:30

14:00

13:20

13:00

12:30

12:00

11:30

11:00

10:30

9:50
10:00

9:00

10月23日（木）October 23
北里講堂

第15回年次大会 及び 第6回CIBSR 日程表

A. Gerson Greenburg（Biopure Corporation）

Warren M. Zapol（Massachusetts General Hospital）

Jonathan S. Jahr
（Univ. of California, Los Angeles）

Closing Remarks

Gas Carrier and Gas Biology

Session

Lunch

Invited Lecture

Acceptable Vascular Reaction in HBOC

Symposium Ⅱ

Coffee BreakKeynote Lecture Ⅱ

Plenary Lecture Ⅱ

10月25日（土）October 25
新棟 New Hospital Wing（Shinto）11F

交通案内図■JR総武線 「信濃町」駅下車 徒歩約 5 分■都営地下鉄大江戸線「国立競技場」駅下車（A 1 番出口），徒歩約 5 分

ARTIFICIAL BLOOD Vol. 16 , No.2, 200843

10月23日（木）October 23（THU）

9：50〜10：00

開会の辞

10：00〜12：00

シンポジウム「輸血代替としての利用法」
座長：高折 益彦（東宝塚さとう病院）

1. Beagle犬を用いた40％脱血ショックにおけるHb小胞体の蘇生効果および中長期生存後の安全性の検討
池田 達彦（慶應義塾大学医学部呼吸器外科）
2. 腎傷害による制御不能出血モデルに対するヘモグロビン小胞体の蘇生効果
勢司 泰久（慶應義塾大学医学部呼吸器外科）
3. 血管損傷による制御不能出血性ショックの蘇生―人工酸素運搬体の効果について―
堀之内 宏久（慶應義塾大学医学部呼吸器外科）
4. マウス肺切除＋周術期出血モデルでのヘモグロビン小胞体投与の有効性の検討
河野 光智（慶應義塾大学医学部呼吸器外科）
5. 人工酸素運搬体,ヘモグロビン小胞体（HbV） の in vitro におけるヒト造血幹/前駆細胞への影響
藤原 満博（北海道赤十字血液センター）
6. カプセル型人工酸素運搬体の臨床試験実施に向けた課題
金田 伸一（テルモ株式会社研究開発センター）
12：00〜13：20

13：20〜14：00

昼食

12：00〜12：30

理事会

12：30〜13：00

評議員会

会長講演
座長：小林 紘一（慶應義塾大学）

ガスキャリアとしての人工酸素運搬体と肺循環
堀之内 宏久（慶應義塾大学医学部呼吸器外科）
14：00〜15：00

一般口演
座長：東

寛（北海道赤十字血液センター）

1. ラット摘出潅流心臓での虚血―再潅流障害に対するヘモグロビン小胞体の保護効果―第3報中島 淳（防衛医科大学校内科1）
2. 出血性ショックモデルラットにおける頻回投与時のヘモグロビン小胞体の体内動態特性評価
田口 和明（熊本大学大学院医学薬学研究部）
3. ヘモグロビン小胞体（HbV）が免疫系に及ぼす影響
高橋 大輔（北海道赤十字血液センター）
15：00〜15：10

休憩

15：10〜16：30 「機能性医薬」
座長：武岡 真司（早稲田大学大学院先進理工学研究科）
1. 酸素親和性の異なるカプセル型人工酸素運搬体の作用
金田 伸一（テルモ株式会社研究開発センター）
2. ヘモグロビン小胞体の表面荷電基の特徴
宗 慶太郎（早稲田大学理工学術院理工学研究所）

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人工血液 Vol. 16 , No.2, 2008

3. アルブミンーフラーレン錯体の光物性と細胞毒性
小松 晃之（早稲田大学理工学術院理工学研究所）
4. 血小板凝集をトリガーとしてADPを放出するリポソームの血小板代替物としての止血能評価
岡村 陽介（早稲田大学大学院先進理工学研究科生命医科学専攻）
16：30〜17：00

総会

17：00〜17：10

閉会の辞

17：30〜19：30

懇親会（新棟11階

ARTIFICIAL BLOOD Vol. 16 , No.2, 2008

レストランオアシス）

45

会長講演
ガスキャリアとしての人工酸素運搬体と肺循環
堀之内 宏久
慶應義塾大学医学部呼吸器外科

呼吸器外科領域では治療手段として通常，肺切除を行ってい
る．肺葉切除が現在の治療の主流であるが，状態が許せば一側肺全摘を行うこともある．肺切除後のガス交換，循環状態がどのように変化するかは治療に直結する重要な検討課題である．
肺切除が行われると肺血管床が減少し，肺動脈圧が上昇していることが予想される．肺切除の周術期に Swan-Ganz カテー
テルを肺動脈内に留置して肺切除後の肺循環の変化について検討を行うと，術後に心拍出量は増大するが，肺動脈圧に予想されたような上昇は認められなかった．以上のことから，肺循環における肺動脈圧を規定する因子は肺血管床のみではないことが明らかである．また，周術期に急性肺傷害を呈した患者では肺動脈圧の上昇が認められていた．血管床の減少のみではなく，肺循環のコンプライアンスが減少していることが示唆された．
このような肺循環を調節する因子を明らかにすることが肺切除を安全に行うために必要と考えられる．
肺動脈圧を調節する因子は，数多くあるが，生理活性ガスが調節因子として深く関わっていることが知られている．酸素はガス交換の主役であるが，肺動脈のトーヌスを調節する重要な役割を果たしている．低酸素環境下で肺動脈圧が上昇する現象
が，いわゆる Hypoxic pulmonary hypertension であり，現在
我々はこの性質を利用して，片側肺を無気肺とし，無気肺となった肺の Hypoxic vasoconstriction を利用して VQ 不均等分布を意図的に作成，ガス交換を安定させて安全に肺切除を行っている．この反応は可逆的である．また，高濃度の酸素を長期に使用すると，Hyperoxia induced lung injuryを引き起こし，不可逆性の間質性変化を惹起することが知られている．Rat のHyperoxia lung injuryモデルを用いてサイトカインの影響を調べたところ，高濃度酸素暴露により，TNFα，PDGF が組織内で活性化していることが明らかとなり，酸素そのものがガス
交換局所で高い生理活性をもち，この生理活性により肺循環に変動が起こることが証明されている．もう一つの重要な生理活性ガスが，一酸化窒素 NO である．NO は血中で半減期が短く，局所の血流を調節する役割を担っている．肺循環には吸入により肺動脈の拡張が認められ，肺傷害に陥った肺の肺動脈圧を低下させる薬剤として使用されている．
一方人工酸素運搬体は体内で酸素を運搬するために開発されたが，修飾ヘモグロビンを用いた人工酸素運搬体では，投与により血圧，肺動脈圧が上昇することが明らかとなった．これは，人工酸素運搬体が NO を吸着するためであると信じられてきた．しかし，NO を吸入させることで，血圧の上昇を制御でき46

る可能性が示唆され，血管収縮には O2 ,N2 以外の要素があることが考えられる．
我々が開発中のヘモグロビン小胞体では，Beagle を用いた
50 ％脱血蘇生試験で 5 分間にわたる一過性の肺動脈の有意な上昇が観察された．投与直後の可逆性の変化は微粒子系であり，血管外への漏出がほとんどないためと考えられた．また，この一過性の肺動脈の上昇はインドメサシンの前投与では抑制できなかった．Hb 小胞体の持つ一過性の肺動脈上昇効果は Hb の持
つ本来の一酸化窒素消去現象である可能性がある．
一酸化炭素 CO も生体内では非常に強力な生理活性ガスであり，ヘムオキシゲナーゼにより産生されるが，現在まで定量的な投与法がなかったために，吸入による毒性のみが強調されてきた．人工酸素運搬体に CO を吸着させて投与することにより，定量的な投与が可能となり，種々の病態の治療に応用できる可能性がある．酒井らは血管収縮を制御して，臓器灌流を確保しながらショックの蘇生を行う蘇生液として，また，CO を放出
した後は酸素運搬体として臓器の酸素代謝を維持する機能を発揮して臓器機能を保全して蘇生効率を上げる酸素運搬体として機能することを発表している．
今後，人工酸素運搬体の研究を通してガス運搬体の機能解明が進むと，赤血球代替としてのみでなく，新たな酸素治療，生理活性ガス治療が開発されることになると考えられ，人工酸素運搬体開発の大きな一つの柱となるものと思われる．

人工血液 Vol. 16 , No.2, 2008

シンポジウム-1

シンポジウム-2

Beagle 犬を用いた 40 ％脱血ショックにおける腎傷害による制御不能出血モデルに対するヘモHb 小胞体の蘇生効果および中長期生存後の安グロビン小胞体の蘇生効果

全性の検討池田 達彦 1，堀之内 宏久 1，井澤 菜緒子 1，河野 光智 1，
泉 陽太郎 1，渡辺 真純 1，川村 雅文 1，宗 慶太郎 2，
酒井 宏水 2，土田 英俊 2，小林 紘一 1
1 慶應義塾大学医学部呼吸器外科
2 早稲田大学理工学術院理工学研究所＜目的＞ Hb 小胞体（HbV）の臨床応用を考えた場合，その安全性の評価は必須である．これまでに生体投与に関する有効性と安全性について様々な角度より検討がなされてきた．以前よりビーグル犬を用いて 50 ％出血ショックモデルを作成し，HbV はアルブミン生食，脱血血液と同様の蘇生効果があることが示された．今回は中長期の安全性の評価のため，40 ％出血モデルを作成し，HbV，または脱血血液を用いて蘇生し，その後生存させることとした．
＜方法＞月齢 6 ヶ月のビーグル犬 16 頭を使用した．全身麻酔を行った後に，右大腿動脈に動脈圧モニター用，および脱血用のカテーテルを挿入した．左前肢に静脈ラインをとり，蘇生液の投与経路とした．計測のための機器を装着し，状態が安定化した後に体重から求めた循環血液量の 40 ％相当量を脱血した．
脱血に従い血圧の低下を認め，60 分間にわたり平均血圧が 50mmHg を上回らないように維持した．その後，脱血血液（Autologous shed blood ASB 群），Hb 小胞体分散液（HbV を
5 ％アルブミン生食に分散した液体．Hb 濃度は 8.6 g/dl ： HbV群）を用いて蘇生を行った．蘇生液投与後 4 時間，全身麻酔下に循環動態を確認した後に半覚醒の状態とし，自室へ戻した．
以後 1 日目，3 日目，7 日目，14 日目，28 日目，56 日目，84 日目，168 日目，365 日目に体重，CBC，血液生化学を測定した．
また 28 日目，168 日目または 365 日目に犠牲死させ病理組織学的所見を検討した．
＜結果と考察＞ ASB 群で 7 頭，HbV 群で 9 頭使用した．28 日，
168 日，365 日で ASB 群は 2 頭，2 頭，3 頭犠牲死させ，HbV 群
は 3 頭ずつ犠牲死させた．全例とも生存し，HbV 群は ASB 群と同様の蘇生効果を示した．体重変化は 2 群とも同様に増加し，365 日後には 12kg に達した．Hct 値は HbV 群で蘇生後低下したが 14 日で ASB 群と同等な値に上昇した．WBC，PLT は 2 群ともにほぼ同様の変化を示した．AST，ALT，CPK が 1 日目に両群とも上昇したが 3 日目には baseline に復した．ショックの影響と考えられた．T-Chol は 3，7 日目に HbV 群で上昇したが14 日目以後は 2 群とも同様な経過を示した．Lipase は 7，14 日
目に HbV 群で低下したが 28 日目以後は 2 群とも同様な経過を示した．病理組織学的評価では HbV 群で 28 日目に犠牲死させた 3 頭で肝臓，脾臓において少量の褐色色素沈着を認めた．
HbV の代謝過程での変化と考えられた．その他，心臓，肺，腎臓，膵臓，小腸，大腸，食道，精巣，胸腺，副腎において明
らかな異常を認めなかった．
＜結論＞中動物での脱血ショック蘇生モデルを用いた中長期生存の実験での蘇生効果および安全性を確認した．

ARTIFICIAL BLOOD Vol. 16 , No.2, 2008

勢司 泰久 1，堀之内 宏久 1，相川 直樹 2，酒井 宏水 3，
土田 英俊 3，小林 紘一 1
1 慶應義塾大学医学部呼吸器外科
2 慶應義塾大学医学部救急医学
3 早稲田大学理工学術院理工学研究所【目的】大量出血患者の搬送に際して欧米では積極的な輸液蘇生が行われており，血液希釈を生じる．一方，本邦では輸液は通常行われておらず，血液循環量維持の点で不利である．そこ
で直径約 250nm の細胞型人工酸素運搬体であるヘモグロビン
小胞体（HbV）を輸液に用いることで，酸素供給と循環量維持を両立し，生存性向上が図れると考え，実験を行った．
【方法】体重約 350g の Wistar 系雄性ラットをセボフルラン吸入
麻酔下で維持し，血圧測定，輸液及び採血用にカテーテルを挿管した．また心電図，末梢組織に酸素電極及びレーザードップ
ラー血流計を設置した．更に HES を用いて血液交換し，ヘマ
トクリット値を約 1/3 に低下させた後，左腎臓へ経皮的に針金を刺入して引き絞り，臓器裂傷を作製した．その後，平均動脈圧（mAP）が 30mmHg に低下した時点から速度 56mL/kg/hrで輸液した．輸液中に mAP が 40mmHg 以上に上昇した場合は輸液を停止し，35mmHg 以下に低下した場合は再開して血圧を制御した．群構成は HbV を 5 ％リコンビナントアルブミン（rHSA）に分散した被験薬群（HbV 群）及び媒体である 5 ％rHSA の代用血漿群，無輸液の無蘇生群の計 3 群とした．観察は mAP=30mmHg 時点から最大 60min まで実施し，経時的に心拍数，mAP，呼吸数，酸素分圧，血流量，血中乳酸値及びpH を測定した．更に生存時間から 1hr 生存率，総出血量及び総輸液量から見かけの循環血液量変化率を算出した．
【結果】HbV 群は生存性が最も高く，死亡を認めなかったが，他群では途中止死亡例が出現した．見かけの循環血液量は各群とも減少を示したが，HbV 群は比較的小幅であった．更にHbV 群は，心拍数が序盤で低下したものの 10min 時点から安定，mAP が序盤で上昇した後，他群よりも高値，呼吸数が序
盤で低下したものの 10min 時点から安定に推移した．末梢酸素分圧は低下傾向を示したものの 10min 時点からは平均値で約15mmHg に安定し，末梢血流は輸液中にほとんど変動しなかった．血中乳酸値は序盤で上昇し，以後は極僅な上昇傾向に転じたが，他群に比較して抑制傾向であった．pH は出血直前より極僅な低下傾向に推移した．これらの結果は他群に比較して同等以上の良好な成績であった．
【考察】制御不能出血に対して人工酸素運搬体を用いた蘇生を試みたところ，無蘇生や代用血漿輸液と比較して，生存時間の延長など生存性向上効果が認められた．一方，末梢組織での酸素分圧は期待される程の上昇ではなく末梢血流上昇も充分ではなかった．しかし乳酸値及び pH の推移から，HbV を用いた蘇生は酸素代謝の改善に寄与したことが示されており，赤血球よりも粒子径の小さい HbV が低血流量下の末梢でも酸素を運搬すると推察された．

47

シンポジウム-3

シンポジウム-4

血管損傷による制御不能出血性ショックの蘇生

マウス肺切除＋周術期出血モデルでのヘモグロ―人工酸素運搬体の効果について―

ビン小胞体投与の有効性の検討

堀之内 宏久 1，勢司 泰久 1，河野 光智 1，泉 陽太郎 1，
酒井 宏水 2，小林 紘一 1，土田 英俊 2
1 慶應義塾大学医学部呼吸器外科
2 早稲田大学理工学術院理工学研究所

河野 光智 1，竹田 貴 1，堤 恭介 1，池田 達彦 1，
泉 陽太郎 1，渡辺 真純 1，堀之内 宏久 1，小林 紘一 1，
酒井 宏水 2，土田 英俊 2
1 慶應義塾大学医学部呼吸器外科
2 早稲田大学理工学術院理工学研究所

出血性ショックの治療は出血を制御し，減少した循環血液量を補てんし，臓器機能を回復させることにある．受傷現場で出血のコントロールがつかない場合に治療施設まで，輸液を行いながら搬送することが通常行われている．輸液製剤と輸液速度については現在まで，いろいろな検討が行われてきた．今回，血管損傷による制御不能出血性ショックモデルにおけるヘモグ
ロビン小胞体（HbV）の蘇生効果を検討した．
【方法】雄性 Wistar ラット（350g）を用い，開腹し，腹部大動
脈を剥離露出，プラスチック装具にて腸を圧排し，腹背方向に24G 針を貫通させ，出血を惹起した．１分以内に平均血圧が20mmHg 以下となった動物で実験を継続した．20mmHg 以下となった時点で輸液を開始し，規定量の輸液が終了した後 2 時間にわたって MAP，HR，呼吸数，Ht，白血球数，Hb，乳酸，
ピルビン酸を測定した．無蘇生群，および蘇生液として出血量の 3 倍量の生食，等量の同種血液，アルブミン生食，HbV を用いて検討した．
【結果】出血量は無蘇生群 8.0 ± 0.9ml，生食群 14.3 ± 2.9ml，同種血輸血群 12.6 ± 4.1ml，5 ％ rHSA 群 11.8 ± 4.0ml，HbV 群
9.8 ± 3.7ml であった．生食群では出血量も多く，血圧を上昇させて維持することはできなかったが，他の群では生食群に比して出血率の減少を認めた．HbV 群では出血量の減少，生存時間の延長，乳酸・ピルビン酸比の変化率の減少を認めた．
【考察】制御不能の出血性ショックの状態に対する蘇生では，大量の晶質液のみでは生存時間を延長することはできず，膠質液が生存時間の延長に有効であると思われた．臓器での嫌気解糖の程度を検討する目的で乳酸・ピルビン酸比を測定したが，血液による蘇生では，乳酸ピルビン酸比の上昇を認めず，HbVによる蘇生では，乳酸ピルビン酸比は上昇を認めたが，出血量，生存時間の延長を認めた．HbV による蘇生は生存を延長し，臓器の酸素代謝を改善する可能性があると考えられた．

輸血の代用として開発されたヘモグロビン小胞体は，ヘモグ
ロビンを期限切れのヒト赤血球から精製濃縮し，リン脂質小胞体に内包した小球粒子（直径： 250nm）である．血液型がなく，ウィルスなどの感染源を排除，室温で 2 年以上の備蓄が可能であり，これまでに酸素運搬体として機能することが確認され，出血性ショック蘇生液としての効果と安全性が動物実験において実証されている．臨床応用を視野に入れた多くの研究が進行中であるが，本研究では輸血の頻度が高い待機手術への応用を
動物モデルにおいて検討した．マウスに左肺切除と循環血液量の 25%，或いは 40 ％の交換輸液を行う，周術期出血を想定した動物モデルを新たに作成した．C57BL/6 マウス，雄性，8 〜
10 週齢（20 〜 22g）を用いて人工呼吸器管理下に左肺全摘術を行った．25 ％交換輸液は尾端を切断して出血を起こさせた後，尾静脈から異なる輸液を行った．40%交換輸液は頚動脈にカテーテルを挿入し，0.1ml の脱血と同量の試料溶液投与を 6 回繰り返して循環血液量の 40 ％を交換した．このモデルを用い，乳酸リンゲル液，5%アルブミン生食液，マウス保存血液，そしてヘモグロビン小胞体分散液を輸液し，生存率，体重減少，
摂食量，自発運動量の変化を観察した．回復の過程を比較する
ことで，ヘモグロビン小胞体の有効性と安全性を検討した．
25%の交換輸液実験では，左肺全摘術後，いずれの群においてもマウスは長期生存可能であった．ヘモグロビン小胞体の投与により，術後早期の体重減少は乳酸リンゲル液投与と比較して抑制される傾向にあった．また，摂食量はいづれの群においても術直後に低下するが，7 日目まで群間で有意差を認めず，回復した．術後に低下した自発運動量も群間に優位差を認めずに回復した．また，40 ％の交換輸液実験では，肺全摘術後，ヘモグロビン小胞体投与によりマウス保存血輸血と同等の生存率が得られた．術後の体重減少および回復，食事摂取量の回復もヘモグロビン小胞体投与後とマウス保存血投与後とでは有意差は認めなかった．左肺全摘による呼吸機能低下，酸素化能低下
状態でヘモグロビン小胞体が有効に機能したと考えられた．体重変化と摂食量による今回の検討では，外科的侵襲からの回復
過程に深刻な影響を与えなかった．これらの結果から，ヘモグ
ロビン小胞体は，外科手術による出血に対しても安全に使用できる可能性が示唆された．

48

人工血液 Vol. 16 , No.2, 2008

シンポジウム-5

シンポジウム-6

人工酸素運搬体，ヘモグロビン小胞体（HbV）

カプセル型人工酸素運搬体の臨床試験実施に向の in vitro におけるヒト造血幹/前駆細胞への

けた課題

影響
藤原 満博 1，東 寛 1，山口 美樹 1，高橋 大輔 1，
酒井 宏水 2，土田 英俊 2，池田 久實 1
1 北海道赤十字血液センター
2 早稲田大学理工学研究所【目的】HbV は，動物モデルにおいてその有効性が示されている．非臨床試験がおこなわれるうえで，HbV のヒト血液細胞への適合性を検討する必要がある．HbV は投与後，主に肝臓，脾臓のほか，骨髄にも分布することから，骨髄における造血機能に対し少なからず影響するのではないかと考えられた．そこ
でヒト造血前駆細胞の増殖・分化および造血幹/前駆細胞の増幅に対する影響について，in vitro の培養系において評価をおこなった．
【方法】（1）造血前駆細胞の増殖・分化の評価：ヒト臍帯血単核球より純化した CD34 陽性細胞を，異なる濃度（3 ％（v/v）
まで）の HbV を添加したイスコフ改変ダルベッコ培地（+ 30 ％FBS, IL-3, SCF）中で，10 日間の液体培養をおこなった．赤芽
球系の細胞（CD235a 陽性細胞）へ分化させるためには
erythropoietin を，顆粒系の細胞（CD15 陽性細胞）へ分化させるためには GM-CSF + G-CSF をそれぞれ上記培地に添加した．
HbV とのインキュベーション期間の影響をみる場合は，HbV
添加の上記培地にて CD34 陽性細胞を 20 時間および 3 日間培養
し，HbV を除去後，10 日目まで培養した．
（2）CD34 陽性細胞の増幅の評価： confluent にしたヒト骨髄ストローマ細胞株に
CD34 陽性細胞を浮遊させ，HbV を添加した無血清培地 XVIVO 10（+ 造血支持サイトカイン）にて共培養した．1 週間
後に等量の培地を加え，2 週間後に全細胞数および CD34 陽性細胞をカウントした． HbV とのインキュベーション期間の影
響を見る場合は，HbV 存在下で CD34 陽性細胞とストローマ細胞株を 3 日間または７日間共培養後，HbV を除去し，2 週間目まで共培養をおこなった．
【結果】（1）CD34 陽性細胞の液体培養にて，HbV が 10 日間継続してある場合は，HbV の濃度に依存して CD235a 陽性細胞および CD15 陽性細胞の増殖抑制がみられた．また，分化した赤芽球系の細胞（CD235a+CD45-）および顆粒球系の細胞（CD15+CD33-）の割合も減少した．一方，20 時間および 3 日間の HbV とのインキュベーションの場合には，CD235a 陽性細胞および CD15 陽性細胞の増殖抑制はみられなかった．
（2）ヒト骨髄ストローマ細胞株との共培養系において，HbV が継続してある場合には，HbV の濃度に依存して全細胞数およびCD34 陽性細胞の増幅の抑制がみられた． HbV に 7 日間曝した
場合は 14 日間と同様な抑制が見られたが，3 日間曝した場合の全細胞数および CD34 陽性細胞増幅の抑制は軽微であった．
【考察】HbV に曝される期間に依存して，ヒト造血前駆細胞の増殖・分化の抑制ならびにヒト造血幹/前駆細胞の増幅の抑制がみられた．In vivo では，HbV は骨髄マクロファージ内に投
与後 1-3 日に蓄積し，7 日では激減する．さらに HbV とヒト造血幹/前駆細胞と直接相互作用することは少ないと考えられる．
よって，HbV とヒト造血幹/前駆細胞が直接相互作用する invitro の閉鎖系において，3 日間ならば抑制効果が少ないという
結果は，in vivo において HbV の造血への影響は少ないことを示唆すると考えられる．

ARTIFICIAL BLOOD Vol. 16 , No.2, 2008

金田 伸一
テルモ株式会社研究開発センター

輸血用血液製剤の代替物，あるいは酸素治療用製剤として，
人工酸素運搬体の医療への応用が待たれて久しいが，未だ実用化に向けた開発の途上にある．最近，FDA 主催のワークショ
ップにてヘモグロビン（Hb）利用非カプセル型人工酸素運搬体について，その問題点が改めて指摘され，人工酸素運搬体の開発に大きな課題が突きつけられたと言える．一方，カプセル型人工酸素運搬体においても，これまで以上に，Hb をカプセ
ル化することの有用性，リポソーム製剤としての特性，安全性について，多くの情報を求められることが予測される．我々は，
ヒト赤血球由来の Hb をリポソーム内に封入したカプセル型人工酸素運搬体（LEH）の開発を行って来たが，本発表では，前述の課題への対応も含め，LEH の開発の進め方について議論
したい．
品質保証の観点からは，LEH ではリポソーム製剤の特性から，最終滅菌に課題が残り，無菌操作製剤として治験薬 GMPに準拠した製造が可能な工程を設定している．また，Hb はヒト血液に由来するものであり，LEH では，医薬品であるリポソーム製剤と，生物由来製品に求められる管理が共に必要とされると考えており，夫々に，対応すべき規制事項も踏まえた，試験評価，バリデーション等を行っていく必要がある．
また，人工酸素運搬体の酸素運搬能力については，理論的には，Hb 濃度，酸素親和性と動脈，末梢組織間での酸素分圧格
差に依存するが，必要とされる Hb 濃度，酸素運搬量や酸素親和性の最適域は，臨床適用の領域，使用条件により変る可能性もあり，議論の余地があると考えている．また，リポソーム製剤については，近年の DDS 製剤開発の過程で，その有用性と課題に関する情報が蓄積されてきている．LEH 開発に際しては，前述の Hb をカプセル化する意義の検証に加え，これらの情報も活用し，有効性や安全性の評価を進める必要がある．特に，リポソーム製剤に対する生体反応には種差が存在することを経験しており，複数の動物種を用いた評価による，臨床的なリスクの検討を進めている．さらに，血液代替物の場合，他のリポソーム製剤とはけた違いの大量投与や，高度の出血状態等，生体の恒常性が大きく損なわれた条件下での投与が想定され，また，酸素治療用製剤としても，虚血病変への影響等，非臨床試験に関しては，本製剤の使用条件の特殊性を考慮した試験設計に留意することが必要と考える．
また，臨床応用に向けては，各種の臨床検査指標に対し，
LEH は，その製剤の特性故に影響を与えることが確認されており，その対策を検討すると共に，特に血液代替物としては，臨床試験における代替エンドポイントの設定が重要な課題であると認識している．
以上のように，LEH の臨床応用に向けては，多くの課題があるが，従来に無い，新しい特徴を持った医薬品であることから，今後，早い段階から，規制当局との対話を持ちつつ，必要な要件に関し対応を行うことで，臨床試験開始に向け進んでいきたいと考えている．

49

一般口演-1

一般口演-2

ラット摘出潅流心臓での虚血-再潅流障害に対

出血性ショックモデルラットにおける頻回投与するヘモグロビン小胞体の保護効果−第 3 報時のヘモグロビン小胞体の体内動態特性評価

中島 淳，別所 基明，浜 御幸，山岸 正，柳田 茂樹，
楠原 正俊，大鈴 文孝
防衛医科大学校内科 1【目的】我々は，ラットの摘出心臓をランゲンドルフ潅流する
方法を用いて，ヘモグロビン小胞体（HbV）が虚血-再潅流時の心機能を有意に回復させることを明らかにしてきた．本研究
では，Hb 小胞体の心機能回復効果のメカニズムを解明する一環として，この効果が mitochondria KATP-channel 活性を経由する否かを検討するため，この channel 活性を選択的に抑制する
5-hydroxydecanoate（5-HD）を用いて以下の実験を行った．虚血-再潅流後の心機能を回復させる比較対照として，
mitochondria K ATP-channel opener の diazoxide（Dia）とischemic preconditioning（IPC）を用いた．
【方法】9-12 週齢の Wistar 系雄性ラットにヘパリン 1000 U を腹腔内投与し，nembutal 60 mg/Kg を腹腔内投与して麻酔した．
心臓を取り出し，Krebs-Henseleit buffer（KH-buffer）を用いて
静水圧 100 cmH2O，37 ℃でランゲンドルフ潅流した．左心室にラテックスバルーンを挿入し，圧トランスデューサーを介して，心機能を連続的に記録した．HbV は，Hb 濃度が 0.33 g/dLになるように KH-buffer で希釈・懸濁し，Dia と 5-HD はどちら
も 100 μM で KH-buffer に溶解させ，37 ℃に加温して 95% O2 +5% CO2 混合ガスを通気した．約 20 分間 KH-buffer で control 潅
流を行った後，対照群，HbV 群，5-HD + HbV 群，Dia 群，5HD + Dia 群，IPC 群，5-HD + IPC 群の 7 群（各群 n = 3）に分けて以下の実験を行った．対照群では，control 潅流の後，虚血 25 分-再潅流 30 分の処置を施した．HbV 群では，虚血 25 分再潅流 30 分処置の前に，その希釈・懸濁液を 10 分間潅流し，Dia 群ではその溶液を 15 分間潅流した．IPC 群では，虚血 25
分-再潅流 30 分処置の前に，虚血 5 分-再潅流 5 分を 3 回繰り返し
た．5-HD 溶液は，HbV 懸濁液あるいは Dia 溶液の潅流および
IPC 処置の 10-15 分前から潅流を開始し，虚血 25 分-再潅流 30 分
処置の前まで継続した．
【結果】① 対照群では，全例で再潅流後の左室発生圧の回復は認められなかった．② Dia 群と IPC 群では，再潅流開始後には全例で左室発生圧の回復が見られ，その程度は虚血開始前値の60%を越えた．③ 5-HD 100 μM を潅流することで，Dia と IPCの心機能回復効果は完全に抑制された．④ HbV 群でも，全例で再潅流後の左室発生圧の回復が認められたが，その効果は弱かった．⑤ HbV による心機能回復効果は，5-HD で抑制されなかった．
【考察】これらの実験結果は，HbV の虚血-再潅流後の心機能回復効果が mitochondria KATP-channel 活性を介さない可能性があることを示唆した．

50

田口 和明 1，丸山 徹 1，2，甲斐 俊哉 1，3，酒井 宏水 4，
土田 英俊 4，小林 絋一 5，小田切 優樹 1
1 熊本大学大学院医学薬学研究部
2 熊本大学薬学部付属育薬フロンティアセンター
3 ニプロ
（株）
医薬品研究所
4 早稲田大学理工学研究所
5 慶応義塾大学医学部外科【緒言】
ヘモグロビン小胞体（HbV）はリン脂質二重膜にヒト赤血球由来ヘモグロビン（Hb）を高濃度に封入した細胞型人工酸
素運搬体である．近年，同一動物にポリエチレングリコール（PEG）修飾リポソーム製剤を，1 回目投与後にある投与間隔で 2 回目投与を行うと 2 回目投与されたリポソームの血中滞留性が 1 回目投与時の血中滞留性より著しく低下する
Accelerated Blood Clearance 現象（ABC 現象）が報告されている．HbV の臨床適用は手術時や大量出血時であり，頻回投与が予想される．そのため，PEG 修飾リポソーム製剤である
HbV においても，ABC 現象が誘導される可能性は十分に考えられ，HbV の頻回投与時の体内動態の把握は HbV を上市する上で重要である．このような背景の下，本研究は，HbV の内部 Hb をヨウ素（125 I）標識した 125 I-HbV を用い，40 ％出血性ショックモデルラットにおける頻回投与時と単回投与時の体内動態を比較検討した．
【方法】
実験動物は SD 系雄性ラット（6 週齢，180 〜 200g）を用いた．
出血性ショックモデルラットの作製は血圧を 30 分間，40mmHg 以下に保ちながら，全血液量の 40 ％を脱血すること
で出血性ショックモデルを作製した．HbV 投与液は，IODOGEN を用い 125 I-HbV を調製した．体内動態の検討は，出血性ショックモデルラットの作成を行い，非標識 HbV 投与液（1400mg/kg）で蘇生し，1 時間後に 125 I-HbV（1400mg/kg）を再び投与し，経時的に開腹，採血及び肝臓・脾臓を摘出した．
血液及び各臓器の放射活性はオートウェルガンマーカウンターにより測定した．各速度論パラメータは MULTI を用い算出した．また，HbV 特異的 IgG 及び IgM の検出は ELISA 法により評価した．
【結果及び考察】
出血性ショックモデルラットにおける 2 回目投与時の消失半減期は単回投与時に比べ延長しており，その値は健常時と同レベルまで回復した．加えて，肝臓・脾臓への分布は，投与初期では単回投与時に比べて分布量が少ないものの，投与後 12 時
間を過ぎると初回投与に比べ臓器分布の増大と排泄の遅延が認められた．以上，本研究の条件では出血性ショック時においてHbV を頻回投与しても ABC 現象は誘導されないことが確認され，HbV の臨床使用に向けた有用な基礎データを提示することができた．

人工血液 Vol. 16 , No.2, 2008

一般口演-3

機能性医薬-1

ヘモグロビン小胞体（HbV）が免疫系に及ぼ

酸素親和性の異なるカプセル型人工酸素運搬体

す影響

の作用

高橋 大輔 1，藤原 満博 1，東 寛 1，宗 慶太郎 2
酒井 宏水 2，堀之内 宏久 3，小林 紘一 3，土田 英俊 2，
池田 久實１
1 北海道赤十字血液センター
2 早稲田大学理工学研究所
3 慶応義塾大学医学部呼吸器外科【目的】ヘモグロビン小胞体（HbV）は，高純度濃厚ヒトヘモ
グロビン溶液を脂質二分子膜で被覆し，表面をポリエチレング
リコールで修飾した人工酸素運搬体である．我々はこれまで，
HbV 投与後のラット脾細胞中に，正常ラット脾細胞の ConA 刺激による細胞増殖を一過性に抑制させる細胞が含まれることを見出してきた．今回は，細胞増殖の抑制に関与する細胞の特性を明らかにする目的で MHC classII 抗原の発現に着目して検討を行った．
【方法】WKAH ラットに 20%相当の HbV，対照として生理食塩
水をエーテル麻酔下，尾静脈より輸注し，16 時間後に脾臓の摘出を行った．摘出した脾臓から脾細胞浮遊液を調整し，ラット MHC classII 抗体（OX6）結合磁性ビーズを用い，MHC
classII 陽性および陰性画分を分画した．それぞれの画分についてサイトスピンを作成し，May-Grunwald Giemsa 染色を行い，顕微鏡下で HbV 貪食細胞の陽性率を算出した．また，それぞれの画分の増殖能に与える影響について評価を行うために，未処理ラット脾細胞に HbV 輸注ラットから分画したMHC classII
陽性および陰性画分を 1:1, 1:0.2 の割合となるように混合し，低濃度 ConA 刺激（0.3 μ g/mL）による正常ラット脾細胞の増殖能を測定した．さらにリポソーム貪食細胞の細胞表面マーカーを調べるため，FITC 標識空リポソームを輸注し，同様に
MHC classII 陽性および陰性画分を分画し，フローサイトメー
ターを用いてそれぞれの画分における FITC 陽性細胞，すなわ
ちリポソーム貪食細胞の細胞表面マーカーを調べた．
【結果・考察】HbV 輸注ラット脾細胞中の classII 陽性および陰性細胞画分どちらにも HbV 貪食細胞が含まれていた．両者の陽性細胞率は classII 陽性画分で 4 ％，陰性画分で 14 ％であり，HbV 貪食細胞は classII 陰性画分に有意に多く存在していた（p<0.01）．また，細胞増殖への影響を確認した結果，未処理脾
細胞： classII 陽性（陰性）脾細胞の混合比 1:1 の条件ではclassII 陽性，陰性いずれの画分でも増殖抑制を認めたが，1:0.2の条件では ClassII 陰性画分でのみ増殖抑制を認めた．貪食細胞の細胞表面マーカーはいずれの画分も CD3-,CD4mid,CD8α-,
CD11b bimodal, CD103-, CD43-であった．以上のことから HbV
輸注後のラット脾細胞に含まれる細胞増殖抑制に関与する細胞集団は MHC classII 分子の発現の有無により，2 種類のサブセ
ット分けられると考えられた．

ARTIFICIAL BLOOD Vol. 16 , No.2, 2008

金田 伸一，本山 慎二，石塚 隆伸，小原井 佳苗，
田辺 拓也，木村 哲寛，小島 潤也，筒井 洋治，上田 努，
後藤 博，粕川 博明
テルモ株式会社研究開発センター【緒言】
カプセル型人工酸素運搬体は，ヒト赤血球由来のヘモグロビン（Hb）をリポソーム内に封入した製剤である．カプセル型
人工酸素運搬体製剤の特徴として，内水相のアロステリック因子の量や，pH 等の条件により酸素親和性を比較的容易に調節可能な点が上げられる．本発表では，酸素親和性の異なる製剤の作用について，有効性，安全性の観点から評価した結果を報
告する．
【方法】
人工酸素運搬体製剤：高親和性製剤 H-LHb（P50 ： 9-12torr），
低親和性製剤 TRM-645（P50 ： 48-54torr）を用いた．
出血モデル：雄性 SD ラットにおいて，HespanderTM を用いた 40mL/kg の脱血，同時投与による血液希釈を行い，更に20mL/kg の脱血後，同量の HespanderTM を補液し，次いで 5，
10，20mL/kg の製剤を投与し，投与後，30 分，1 時間後の血漿中乳酸値を測定した．
脳梗塞モデル：雄性 SD ラットにおいて，中大脳動脈をナイ
ロン糸製栓子により 2 時間閉塞させ，一過性の脳虚血を招来した後，0.2，1，5mL/kg の製剤を虚血開始後 30 分に投与した．
血流再開 22 時間後に，神経症状を観察し，脳を摘出して脳切片を作製，TTC 染色し，画像解析にて計測した梗塞部面積より梗塞巣体積を算出した．
機能的 Hb 濃度：雄性 SD ラットに 20mL/kg の用量にて製剤を投与し，投与後，5 分，6，24 時間後に採血を行い，血中ヒト Hb 濃度を，抗ヒト Hb 抗体を用いた ELISA により，Hb メト
化率をシアンメト Hb 法により測定した．
血管収縮作用：雄性 SD ラット頚動脈を摘出，リング状標本を作製し，Krebs-Henseleit 液中に懸垂し，95%O2+5%CO2 の混合ガス通気下で，phenylephrine 添加時血管収縮の Ach による弛緩反応に対する製剤添加の影響を評価した．
【結果】
ラット出血モデルにおける血漿中乳酸値上昇に対し，高親和性，低親和性の何れの製剤もほぼ同等の投与用量依存的な抑制効果を示した．一方，一過性脳虚血モデルでも，何れの製剤も，投与用量依存的な梗塞体積の抑制効果を示したが，高親和性製剤の方が低用量で有意な効果を示した．また，神経症状スコアでは何れの製剤でも投与用量に依存し低減される傾向が見られたが，統計学的な差は認めなかった．血中での Hb の動態，メト化の進行速度には製剤間での統計学的な差は見られなかった．さらに，摘出血管の収縮性についても，製剤間で顕著な差は見られなかった．
【結論】
酸素親和性の異なる製剤間の比較において，局所の虚血に関しては高親和性の製剤がより効果的である可能性が示された
が，有効性，安全性の何れの点からも，著明な差は認められな
かった．

51

機能性医薬-2

機能性医薬-3

ヘモグロビン小胞体の表面荷電基の特徴

アルブミン−フラーレン錯体の光物性と細胞毒
性

宗 慶太郎，酒井 宏水，土田 英俊
早稲田大学理工学研究所【緒言】ヘモグロビン（Hb）小胞体に関するこれまでの動物投与試験やヒト血液を対象とするin vitro 混合試験では，補体系を含む免疫系の顕著な活性化を認めないことが確認されている．
Hb 小胞体の特徴的な表面物性の一つとして負電荷成分による電荷が挙げられる．本研究では，Hb 小胞体に使用している負電荷成分による表面電荷の特徴を明らかにすると共に，血液適合性における表面荷電基の構造特性について知見を得ることを目的とした．
【方法】小胞体の電気泳動移動度をレーザードップラー法により測定し，ゼータ電位を算出した．また，小胞体表面の負電荷が媒介する静電相互作用のモデルとして，Ca2+，カチオン性オ
リゴマー，カチオン性ポリマーを所定濃度で小胞体分散液に添加し，ゼータ電位の変動を測定した．雄性 Wistar ラット（体重 250 ± 20 g）に対し，エーテル自発呼吸麻酔下，尾静脈に挿入した留置針を介して小胞体試料を投与した（投与量： 5.6
mL/kg、投与速度： 1mL/min）．投与後覚醒させ，1 ないし 24
時間後に血液を採取し，遠心分離（1 × 10 3 g, 10 min）により血球成分を除去．続いて超遠心分離（3 × 10 5 g, 30 min）により小胞体を除去した．得られた試料について血清補体価を測定した．
【結果・考察】Hb 小胞体に使用している負電荷脂質は中性付近
では小胞体表面に比較的強い負電荷を与え,この電荷により Hbの内包効率が向上する．負電荷脂質含量の増大に伴いゼータ電位が負に増大して内包効率が向上するものの，-30mV 以下でこの効果はほぼ一定となった．Hb 小胞体の脂質組成では-40mV程度のゼータ電位を有していることから，負電荷脂質の含量が適正範囲に設定されていることを確認した．従来，負電荷リン脂質を含有する小胞体では，投与後の補体活性化や血小板減少などが報告され，表面電荷が補体活性化の主要因と考えられている．本検討では，Hb 小胞体に使用している負電荷脂質は負電荷リン脂質と同程度の電荷を小胞体表面に与え，この電荷が関与する静電的相互作用も同等であることが示された．一方，投与後の血清補体価の低下や血小板減少は負電荷リン脂質を含有する小胞体のみで認められた．これらの結果は，小胞体表面の負電荷基の構造が生体反応における重要な因子であることを
示唆する．

52

小松 晃之 1，屈 雪 1，土田 英俊 1，堀之内 宏久 2，
小林 紘一 2
1 早稲田大学理工学術院総合研究所理工学研究所
2 慶応義塾大学医学部呼吸器外科

ヒト血清アルブミン（HSA）は血漿蛋白質の約 60wt ％を占める補欠分子族を持たない単純蛋白質（分子量 66,500）であり，血流中では膠質浸透圧維持のほか，疎水性薬物・代謝産物の運搬／貯蔵の役割を担っている．メトヘモグロビンから解離した
鉄（III）プロトポルフィリン IX（ヘミン）も血中では HSA に捕捉され肝臓へと運ばれる．我々は HSA-ヘミン錯体の X 線結晶構造解析に成功し，ヘミンが HSA のサブドメイン IB にチロ
シン 161 との軸配位により結合していることを見出した（1）．
そこでヘモグロビン（Hb）のヘムポケット構造にならい，遺伝子組換え技術により HSA のサブドメイン IB 内へヒスチジン（近位軸塩）を導入してみると，驚いたことに得られた組換えHSA-鉄（II）ポルフィリン IX（ヘム）錯体は Hb のように酸素を吸脱着できるようになった（2, 3）．さらに配位酸素近傍のアミノ酸へ変異を加えると，rHSA-ヘム錯体の酸素親和性が調節できることもわかった（4）．他方，ヘムの中心金属を鉄から亜鉛に変換してみると，得られた HSA-Zn（II）ポルフィリン錯体は可視光照射により安定な励起三重項状態を形成することが可能で，白金コロイドを用いた水の還元反応（水素発生反応）の有効な光増感剤として作用することが明らかとなった（5）．
このように我々は，血漿蛋白質である HSA の多分子結合能を利用して，その内部に様々な機能性分子を結合させることにより，自然界には存在しないユニークな機能蛋白質を創製してきている．
今回は HSA にフラーレン誘導体を結合させた HSA-フラーレ
ン錯体を合成し，その構造，光物性，一重項酸素生成能，さらには可視光照射下にける細胞毒性について検討した．トリス（ジカルボキシメチレン）［60］フラーレン（CF）は HSA に 1:1（モル/モル）で結合し，安定な HSA-CF 錯体を形成する．窒素下におけるレーザーフラッシュ照射後の励起三重項寿命は46μs で酸素濃度の増大に伴い減少したことから，酸素分子へのエネルギー移動により一重項酸素が生成しているものと考えられる（6）．Stern-Volmerプロットから決定した速度定数（kq）
は 2.2 × 10 8 （M - 1 s - 1 ），量子収率は 0.46 であった．腫瘍細胞（LY80）に HSA-CF 錯体を添加し可視光照射すると，対照群（HSA-CF 非添加群）に比べ顕著な細胞数の低下が認められた．
HSA-CF 錯体は光線力学治療（PDT）の新しい光増感剤として期待される．
1. P. Zunszain, T. Komatsu, et al., BMC Struct. Biol. 3, 6（2003）.
2. T. Komatsu, et al. J. Am. Chem. Soc., 126, 14304（2004）.
3. T. Komatsu, et al. J. Am. Chem. Soc., 127, 15933（2005）.
4. T. Komatsu, et al. J. Am. Chem. Soc., 129, 11286（2007）.
5. T. Komatsu, et al. J. Am. Chem. Soc., 128, 16297（2006）.
6. Z. Qu, T. Komatsu, et al. Bioconjugate Chem., 19, 1556（2008）.

人工血液 Vol. 16 , No.2, 2008

機能性医薬-4
血小板凝集をトリガーとして ADP を放出する
リポソームの血小板代替物としての止血能評価
岡村 陽介 1，2，江藤 薫子 1，半田 誠 2，池田 康夫 3，
武岡 真司 1
1 早稲田大学大学院先進理工学研究科生命医科学専攻
2 慶應義塾大学輸血･細胞療法部
3 慶應義塾大学内科【目的】フィブリノーゲンγ鎖 C 末端ドデカペプチド（H12）を表面に結合させたリポソームは，活性化血小板間を架橋して血小板凝集を促進させる機能を持ち，血小板減少ラットに静脈投与すると出血時間を短縮させることを既に報告してきた．本報では，血小板凝集惹起剤（ADP）を内包させた H12 結合リポソーム（H12（ADP）リポソーム）を調製し，血小板代替物としての止血能増幅を評価した．
【方法】H12 をリポソームに担持し，内水相に ADP 水溶液（1mM, 血小板凝集試験では［8- 14 C］ADP を使用）を内包した．
H12（ADP）リポソームをPRP に分散させ（［PLT］= 2.0 x 10 5/μL，［小胞体］= f.c. 0.1 mg/mL），ADP 刺激（［ADP］= f.c. 3μM）にて血小板凝集を惹起させ，液体シンチレーションカウ
ンターにて ADP 放出率を測定した．血小板減少症ウサギ（11週齢，2.5 kg）に H12-（ADP）リポソームを投与し，投与 9 時間までの出血時間を経時的に測定した．
【結果･考察】H12-（ADP）リポソーム［1］と（ADP）リポソ
ーム［2］の血小板凝集能の比較では，［1］の透過度が約 10 ％増大した．そこで，RI 標識体を用いて同条件にて試験したところ，［1］は血小板凝集塊に有意に取込まれ，ADP の放出率
は 55.8 + 2.1 ％と算出されたのに対し，［2］は全く血小板凝集
塊に取込まれなかった．これは，［1］が H12 を介して血小板と多点結合して血小板凝集に巻込まれ，その際に受ける物理的な刺激によって内包 ADP が放出されたためと考えられる．
血小板減少症ウサギに H12-（ADP）リポソームをそれぞれ 10，20 mg/kg で投与したところ，出血時間はそれぞれ 881 ± 303，
442+45 秒であり，生理食塩水群（1696 ± 197 秒）あるいは（ADP）リポソーム群（10，20 mg/kg で 1503+481，1375+663秒）と比較して，投与量依存的に有意に出血時間を短縮させ，血小板輸血群のそれに匹敵した．この効果は投与 6 時間後まで持続でき，H12-（ADP）リポソームの有効血中濃度は 100 μg/
mL 以上であると算出された．従って，ADP 内包によって H12リポソームの止血能が顕著に向上できることを明らかにした．
更に，H12（ADP）リポソームの安全性に関する結果も併せて報告する．

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人工血液 Vol. 16 , No.2, 2008

The 6th Current Issues on Blood Substitute Research
October 24−25
2008

Program & Abstracts

President
Koichi Kobayashi（Keio University）

Congress Venue
Keio University Shinanomachi Campus
35 Shinanomachi, Shinjuku-ku,
Tokyo JapanARTIFICIAL BLOOD Vol. 16 , No.2, 200855

Welcome to the 6 th CIBSR
Koichi Kobayashi
Professor Emeritus, Keio University, School of Medicine

It is our great pleasure to offer an honorable opportunity to serve as a president of the 6th Current
Issues on Blood substitute Research. This meeting is held in conjunction with the 15th Annual
Meeting of the Society of Blood Substitutes Japan for which Dr. H. Horinouchi, Associate
Professor of School of medicine, Keio University, is the president to give dense scientific sessions topropel the research and development of this field.
Since artificial oxygen carriers should be injected into a blood stream, they cannot be free fromadverse effects or unexpected events. The history of artificial oxygen carrier is the history of
struggle against unsatisfactory reactions. Gold standard of oxygen therapeutics is allogeneic bloodtransfusion. Decades ago, it seemed that the benefit of transfusion has been considered superior to
the artificial oxygen carrier, although blood transfusion will have a considerable complications. To
minimize the undesirable effect of artificial oxygen carrier, either cell-free or cellular, a lot of
innovation has been made. It has been always a big question to what extent we can permit theundesirable effect when artificial oxygen carrier is brought in clinical use.
Meanwhile, understanding of vascular reaction to several gases has been deepened since gas
mediators were discovered in 90'. These gases seemed transferred to tissue by either specific gas
carriers or plasma. Oxygen compounds such as O2 , NO, CO are carried by heme-based gas
carriers: namely hemoglobin, myoglobin, and cytoplasmic hemeprotein. Research in the field of gas
biology will give us new insights and ways to develop artificial oxygen carriers.
We hope each of you enjoy the meeting and also take the opportunity to enjoy beautiful autumndays in Japan.

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人工血液 Vol. 16 , No.2, 2008

6th

CIBSR Schedule
October 25（SAT）
New Hospital Wing（Shinto）11F

October 24（FRI）
New Hospital Wing（Shinto）11F
8:30
8:50
9:00

Plenary Lecture I
9:40
Keynote Lecture I
10:20
10:30

Plenary Lecture II

Opening Remarks
Makoto Suematsu（Keio University）

10:30

Educational Lecture Hirosuke Kobayashi（Kitasato University）

11:00

New Material

11:20
Jonathan S. Jahr
（Univ. of California, Los Angeles）

12:30

Lunch

13:00
Oral Session

Presidential Address Koichi Kobayashi（6th CIBSR）（Keio University）

13:30

Symposium I

14:00

PART I

Cutting Edge of Research of Artificial Oxygen Carriers

15:00
15:10

Coffee Break

15:30

Symposium I

16:00

Acceptable Vascular Reaction in HBOC

12:00
Lunch Time Symposium

14:30

Coffee BreakInvited Lecture

12:10

14:00

Keynote Lecture II A. Gerson Greenburg（Biopure Corporation）

Symposium II

Coffee Break

11:50

13:40

9:10

9:50
Marcos Intaglietta
10:00（Univ. of California, San Diego）

11:10

13:10

Warren M. Zapol（Massachusetts General Hospital）

14:30
15:00
15:15

PART II

Cutting Edge of Research of Artificial Oxygen Carriers

Gas Carrier and Gas Biology

Closing Remarks

15:30
16:00
16:30

16:30

17:00

17:10
17:30

17:30

18:00

18:00
18:30

18:30
BanquetTokyo Dome Hotel

19:00

19:00

19:30

19:30

20:00

20:00

ARTIFICIAL BLOOD Vol. 16 , No.2, 2008

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人工血液 Vol. 16 , No.2, 2008

10月24日（金）8：50〜9：00

Opening Remarks

9：00〜9：40

Plenary Lecture I

October 24（FRI）

Chairperson： Koichi Kobayashi（Keio University）
Biomedical Application of Metabolome Analysis: Systematic Molecular Mining for Gaseous Signal Transducers
Makoto Suematsui（Keio University）
9：40〜10：20

Keynote Lecture I
Chairperson： Hiromi Sakai（Waseda University）

Microvascular Reactions in Designing Oxygen Carrying Blood Substitutes
Marcos Intaglietta（University of California, San Diego）
10：20〜10：30

Coffee Break

10：30〜11：10

Educational Lecture
Chairperson： Hirohisa Horinouchi（Keio University）

Physiological Aspects of Oxygen Transport to Tissue
Hirosuke Kobayashi（Kitasato University）
11：10〜11：50

New Material
Chairperson： Shinji Takeoka（Waseda University）

NM−1

Hemostatic Effects of Liposome Carrying Fibrinogenγ-Chain
Dodecapeptide on the Surface and Adenosine 5'-Diphosphate Inside as a Platelet Substitute
Makoto Handa（Keio University）

NM−2

O2 Binding Properties of Recombinant Albumin-heme Complexes Having Arginine at the Entrance of the Heme
PocketAkito Nakagawa（Waseda University）

12：10〜13：10

Lunch Time Symposium
Chairperson： Koichi Kobayashi（Keio University）

LS−1

Hb-vesicle, a Cellular Hb-based Oxygen Carrier, Fulfills the Physiological Roles of the RBC Structure
Hiromi Sakai（Waseda University）

LS−2

The Effect of Hemoglobin Vesicle Administration on Ventilator Induced Lung Injury
Yotaro Izumi（Keio University）

LS−3

Effect of Artificial Oxygen Carrier Hb Vesicle on Cerebral Blood Flow During and After Hemodiluted
Cardiopulmonary Bypass in Rat
Ryo Aeba（Keio University）

13：10〜13：40

Presidential Address
Chairperson： Eishun Tsuchida（Waseda University）

Will Our Dream "Clinically Applicable Artificial Oxygen Carrier" Come True in Foreseeable Future?
Koichi Kobayashi（Keio University）

ARTIFICIAL BLOOD Vol. 16 , No.2, 200859

13：40〜15：00
PART ISY−Ⅰ−1SY−Ⅰ−2

Symposium I

Cutting edge of research of artificial oxygen carriers（PART 1）

Chairperson： Yotaro Izumi（Keio University）

Second Generation PEGylated Hbs with a Range of O2 Affinities to Improve Tissue Oxygenation without
Activating Autoregulation Induced Vasoconstriction
Seetharama A. Acharya（Albert Einstein College of Medicine of Yeshiva University）
Effect of the Initially Injected Hemoglobin Vesicles（HbV）on the Pharmacokinetics of Second Injection of HbV in
Mice
Kazuaki Taguchi（Kumamoto University）

SY−Ⅰ−3

Small Volume Resuscitation from Hemorrhagic Shock with Polymerized Bovine Hemoglobin
Pedro Cabrales（La Jolla Bioengineering Institute）

SY−Ⅰ−4

Liposome-encapsulated Hemoglobin Transfusion Rescues Rats Undergoing Progressive Hemodilution from Lethal
Organ Hypoxia without Scavenging Nitric Oxide
Yashiro Nogami（National Defense Medical College）

15：00〜15：10

Coffee Break

15：10〜17：10

Symposium I

PART II
SY−Ⅰ−5

Cutting Edge of Research of Artificial Oxygen Carriers（PART 2）

Chairperson： Amy Tsai（University of California, San Diego）

Pegylation Destabilizes the Haemoglobin Tetramer
Luca Ronda（University of Parma）

SY−Ⅰ−6

Vanished Oxygen Affinity of Myoglobin by Pyridine-containing Heme
Saburo Neya（Chiba University）

SY−Ⅰ−7

Hemospan○R : Rational Design, Preclinical Effects, and Clinical Development of PEG-conjugated Human Hemoglobin
Mark A. Young（Sangart, Inc.）

SY−Ⅰ−8Transient Induction of Immune-suppressor Cells in Rat Spleen by Massive Injection of Hemoglobin-vesicle（HbV）
Hiroshi Azuma（Hokkaido Red Cross Blood Centre）

SY−Ⅰ−9Potential Tumor Oxygenation by Systemic Administration of Hemoglobin Vesicle in a Mouse Lewis Lung
Carcinoma Model
Yotaro Izumi（Keio University）

SY−Ⅰ−10

Polymerized Placenta Hemoglobin Improves Cardiac Functional Recovery and Reduces Infarction Size of Isolated
Rat Heart
Qian Yang（Chengdu Medical College）

18：30〜60

Banquet at Tokyo Dome Hotel

人工血液 Vol. 16 , No.2, 2008

10月25日（土）8：30〜9：10

October 25（SAT）

Plenary Lecture II
Chairperson： Koichi Kobayashi（Keio University）

Prevention of the Pulmonary Vasoconstrictor Effects of HBOC-201 in Awake Lambs by Continuously Breathing
Nitric Oxide
Warren M. Zapol（Massachusetts General Hospital）
9：10〜9：50

Keynote Lecture II
Chairperson： Masuhiko Takaori（Higashitakarazuka Sato Hospital）

HBOCs and Vasoactivity: An Alternate Perspective
A. Gerson Greenburg（Biopure Corporation and Brown University）
9：50〜10：00

Coffee Break

10：00〜11：20

Symposium II

Acceptable Vascular Reaction in HBOC

Chairperson： Hae Kim（Brown University）
SY−Ⅱ−1

Hemoglobin-Based Oxygen Carrier Mediated Vasoactivity : Proposed Mechanisms and Potential Remedies
Hae W. Kim（Brown University）

SY−Ⅱ−2

Does Lowering Oxygen Affinity of Polyethylene Glycol Conjugated Hemoglobins Cause Arteriolar Vasoconstriction?
Amy G. Tsai（University of Carifornia, San Diego）

SY−Ⅱ−3Mechanism to Avoid Vasoconstriction and Maintain Perfusion of Pegylated Hemoglobins
Pedro Cabrales（La Jolla Bioengineering Institute）

SY−Ⅱ−4

Effect of Artificial RBCs on Murine Hemorrhagic Shock Model
Yutaka Tomita（Keio University）

11：20〜12：00

Invited Lecture
Chairperson：Hiroshi Morisaki（Keio University）

Clinical and Translational Advances in HBOCs: One Investigator's Perspective
Jonathan S. Jahr（University of California, Los Angeles）
12：00〜13：00

LUNCH

13：00〜15：00

Oral Session

Gas Carrier and Gas Biology

Chairperson： Teruyuki Komatsu（Waseda University）
S−1

Design and Evaluation of S-nitrosylated Human Serum Albumin as a Novel Anticancer Drug
Toru Maruyama（Kumamoto University）

S−2

Hemoglobin-vesicles as O2 - and CO-carriers
Hiromi Sakai（Waseda University）

S−3

Injectable O-15 System for Oxygen Metabolism Studies Using Hemoblobin-vesicle（HbV）: Automatic Labeling andApplication in Rats Using PET
Vijay Narayan Tiwari（University of Fukui）

ARTIFICIAL BLOOD Vol. 16 , No.2, 2008

61

S−4

Comparison of Nitric Oxide-induced Oxidation of Recombinant Oxyhemoglobin Subunits Using a Competition
Experiment
Yen-Lin Lin（National Chung Cheng University）

S−5

Polymerized Human Placenta Hemoglobin Decreased the Pathological Nitric Oxide Production in Cold Stored Rat
Heart
Tao Li（Sichuan University）

S−6

Polymerized Human Placenta Hemoglobin Ameliorates Oxidative Stress and Energy Deficiency in Cold Stored
Rat Heart
Tao Li（Sichuan University）

15：00〜15：15

Closing Remarks

PosterPO−1

Assessment of Inflammation-altered Intestinal Permeability Using Arterially Perfused Murine Jejunal Loop
Premysl Bercik（McMaster University）

PO−2

Hemoglobin Encapsulation in Vesicles Retards the Reaction with NO by Intracellular Diffusion Barrier
Hiromi Sakai（Waseda University）

PO−3

Effect of HbV as a Resuscitation Fluid in Uncontrolled Hemorrhage-shock Model: Blunt Kidney Injury Model
Yasuhisa Seishi（Keio University）

PO−4

Resuscitation of Hemorrhagic Shock due to Uncontrolled Hemorrhage-effect of Hemoglobin-vesicle in Vascular
Injury Model
Hirohisa Horinouchi（Keio University）

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人工血液 Vol. 16 , No.2, 2008

Presidential Address
Presidential Address

Will Our Dream "Clinically Applicable Artificial Oxygen Carrier"Come True in Foreseeable Future?
Koichi Kobayashi, President of the Society of Blood Substitutes
Japan
Dept. General Thoracic Surgery, School of Medicine, Keio University, Japan
E-mail: kobayash@sc.itc.keio.ac.jp

In 20th Century, blood transfusion was one of the major therapeutic achievements, but still we havebeen facing difficult task to keep transfusion medicine safe and reliable. Looking at the newly
emerged blood borne infectious diseases and shortage of donor, developing artificial blood
component will promise the progress of medical science in 21st Century. Clinical trials of various
kinds of cell-free HBOCs are underway especially in North America. Some were suspended andsome are now in the final stage. However, the Natanson's paper recently published in JAMA wasvery surprising. Meta-analysis of cell-free HBOCs clinical data showed that they increase the risk of
myocardial infarction and death. The Food and Drug Administration of the United States held apublic workshop entitled "Safety of Hemoglobin-Based Oxygen Carriers (HBOCs)" in the end of
April 2008. Recent results of clinical trials, safety issues of clinical and preclinical studies, remedies
to the side effects, and future directions were discussed. In response to the Natanson's paper, someargument arose. Whatever, it has to be emphasized that HBOCs are necessary for unmet medical
needs; a situation where safe RBC transfusion is not available.
Research and development of Hb-vesicle (HbV) and synthetic hemes have been performed by close
collaboration of Waseda and Keio for more than 20 years. HbV is a cellular HBOC and it isexpected that toxic effects of cell-free HBOCs would be shielded by the cellular structure. Even
though we have not come to a clinical stage yet, the preclinical results of our safety and efficacy
evaluations of HbV make us confident in advancing to further development of HbV for eventual
realization.
Whether cell-free, or cellular HBOCs, I hope a clinically available oxygen carrier to save life andalleviate human suffering will appear in foreseeable future.

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人工血液 Vol. 16 , No.2, 2008

Keynote LectureⅠ
Keynote LectureⅠ Microvascular Reactions in Designing Oxygen Carrying Blood
Substitutes

Marcos Intaglietta, Amy G. Tsai, Pedro Cabrales,
Beatriz Y. Salazar Vazquez
Department of Bioengineering, University of California San Diego, CA, USA

The effective restitution of blood volume and oxygen carrying capacity to the circulation requiresthat the microcirculation, where blood exerts its primary role in gas and material exchange remains
functional in the presence of changes of the composition of blood. This requires normal capillary
perfusion (functional capillary density, FCD, capillaries with the passage of red blood cells), a
parameter that must be above a critical threshold to insure tissue survival. The key factor inmaintaining FCD is capillary pressure which is directly and linearly related to FCD. Capillarypressure is the resultant of central blood pressure and how this is transmitted from the major bloodvessels through the arteriolar microcirculation, which depends on microvessel vasoactivity andblood viscosity, factors directly affected by the local interaction of blood and the endothelium.
Maintenance or increased plasma viscosity is beneficial in resuscitation when the reduction ofhematocrit lowers blood viscosity, maintaining and/or increasing shear stress on the endothelium
and the production of nitric oxide (NO) and prostacyclin. Addition of oxygen carrying capacitywith hemoglobin based oxygen carriers must insure that the natural tendency of hemoglobin toscavenge NO is balanced by its production via other mechanisms. Increased shear stress andnitrate reductase activity provide such a balance. Over-oxygenation of the arteriolar walls due tofacilitated diffusion and low affinity oxygen carriers also constitute vasoconstrictive mechanismsthat impair microvascular function. Optimal microvascular function is determined by a varying
combination of oxygen transport properties and mechanical factors and not solely on blood's
intrinsic oxygen carrying capacity. This leads to effective resuscitation via mechanism thatemphasize either restoration of oxygen carrying capacity or improvement of microvascularfunction, a situation that in many instances diminishes the need of restituting intrinsic oxygen
carrying capacity, if microvascular functionality can be augmented, as evidenced by retransfusion
procedures that promote vasodilatation.

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Keynote LectureⅡ
Keynote LectureⅡ HBOCs and Vasoactivity: An Alternate Perspective
A Gerson Greenburg
VP Medical Affairs, Biopure Corporation; Professor of Surgery Emeritus, Brown
University, RI, USA

The observation of vasoactivity, often referred to as "hypertension" in the literature ― in reality anelevation of blood pressure ― has plagued the development of hemoglobin based oxygen carriers,
HBOCs, for over 20 years. Regulatory agencies have considered it the underlying basis for the
emergence of many of the significant and durable serious adverse events (SAE), e.g. myocardial
infarction, stroke, renal failure and death. Licensing and even approval for clinical trials has been
withheld with vasoactivity expressed as the reason.
HBOC-201, Hemopure®, was evaluated in regulatory agency specified preclinical protocols oforgan flow, organ oxygenation and microcirculation and in specific studies of coronary flow andmyocardial function in both animals and humans. The objective of the studies was to determine
whether there is vasoactivity and to what extent it can be localized to organs of concern. In
exchange models skeletal muscle was the only bed showing vasoconstriction, an observation
substantiated by the tissue oxygen and microcirculation studies. Top loading in patients withcoronary artery disease did not induce coronary vasoconstriction (direct observation of coronary
function) and perfusion of an occluded coronary artery for 3 minutes with oxygenated HBOC-201
preserved function fully without evidence of ischemia. These, and other studies, demonstrate thatvasoactivity is not the operative agent in the emergence of SAEs in a pivotal Phase 3 orthopedic
surgery trial. By properly done subset analysis of the 350 patients treated in this trial, the safety
profile of HBOC-201 was equal to that of blood when 3 or fewer units of red cells were used. In
this trial elevations in blood pressure were observed but only a few at levels that required treatment.
SAEs were not associated with the blood pressure changes.
In a series of 54 patients treated with HBOC-201 for various "anemia" indications, there were a few
instances of elevated blood pressure, investigators made aware of the possibility beforehand, andthese were successfully treated with nitrite donors, bets-blockers or calcium channel blocker withgood results. In some instances, an elevation in blood pressure may be helpful to insure tissue
perfusion. There is evidence in models of traumatic brain injury that the elevation in pressure isbeneficial in an otherwise lethal model. Moreover, there is evidence in the treatment of humanstroke that survival and improved recovery are achieved with elevated blood pressure. A new
definition of "hypotension" has been proposed with systolic below 110 considered detrimental for
hypovolemic shock. Have we been generally under-resuscitating patients?
Vasoactivity is a known side effect of this class of agents. Evidence is accumulating to indicate it
may not be the basis for the emergence of SAEs. Optional explanations will be presented.

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Plenary LectureⅠ
Plenary LectureⅠ Biomedical Application of Metabolome Analysis: Systematic
Molecular Mining for Gaseous Signal Transducers
Makoto Suematsu
Department of Biochemistry & Integrative Medical Biology, School of Medicine,
Keio University, Japan
E-mail: gasbiology@z6.keio.jp
Gaseous metabolites such as NO, CO and H2S are too small to handle for mining their receptors
through nanobeads technology. We have recently applied metabolome analysis based on CE-MS
that allowed us to collect systematic information on small molecular metabolites responding todisease conditions. Using differential metabolomics analyses, we compared alterations in themetabolites, the molecular size of which are less than 1000, between normal and theacetaminophen (AAP)-induced liver injury model using mice where CO was overproduced via
heme oxygenase (HO) through cytochrome P450 degradation. Results indicate increases inmethionine and S-adenosylmethionine in the liver and hyperhomocystinemia, and decreases
cystathionine, cysteine and GSH. Although the decreases in transsulfuration metabolites appeared
to result from consumption of thiols for the xenobiotic detoxification, mechanisms for the increases
in remethylation metabolites remain to be solved. Since the increases in the remethylation
metabolites were inhibited by blocking HO through Zn-protoporphyrin-IX, we hypothesized that
CO generated by HO might inhibit the heme-containing cystathionine β-synthase (CBS), the ratelimiting enzyme for transsulfuration pathway. As expected, the recombinant enzyme wasinhibitable by CO but not by NO. These results suggest that the CO-CBS system serves as aprotective mechanism against xenobiotic detoxification. Moreover, heterozygous CBS-knockout
mice lacked CO responsiveness for regulating transsulfuration pathway. To note is that the
inhibitory action of CO on CBS is stabilized by H2S, an end product of the enzyme, suggesting a
product inhibition mechanism operated by multiple gases. Metabolome analysis appears a
powerful strategy for mining gas-responsive signal transducers in vivo. A variety of biomedical
application of this technology for Gas Biology will be shown in the lecture.

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Plenary LectureⅡ
Plenary LectureⅡ Prevention of the Pulmonary Vasoconstrictor Effects of HBOC201 in Awake Lambs by Continuously Breathing Nitric Oxide
Binglan Yu, Gian Paolo Volpato, Kenneth D. Bloch,
Warren M. Zapol
Anesthesia Center for Critical Care Research, Department of Anesthesia andCritical Care, Harvard Medical School at Massachusetts General Hospital, MA,
USA
Background:
Hemoglobin (Hb)-based oxygen-carrying solutions (HBOC) provide emergency alternatives toblood transfusion to carry oxygen to tissues without the risks of disease transmission or transfusion
reaction. Two primary concerns hampering the clinical acceptance of acellular HBOC are theoccurrence of systemic and pulmonary vasoconstriction and the maintenance of the heme-iron in
the reduced state (Fe+2). We recently demonstrated that pretreatment with inhaled nitric oxide(NO) prevents the systemic hypertension induced by HBOC-201 (polymerized bovine Hb)
infusion in awake mice and sheep without causing methemoglobinemia. In this study, weinvestigated the pulmonary and systemic hemodynamic effects of breathing NO both before andafter the administration of HBOC-201 in awake lambs.
Methods and Results:
Intravenous administration of HBOC-201 (12 ml/kg; Biopure Corporation, Cambridge, MA) in
healthy, awake lambs induced prolonged systemic and pulmonary vasoconstriction. Pretreatment
with inhaled NO (80 parts per million (ppm) for 1 h) prevented the HBOC-201-induced increase
in mean arterial pressure, but not the increase of pulmonary arterial pressure, systemic vascular
resistance, or pulmonary vascular resistance. Pretreatment with inhaled NO (80 ppm, 1 h) followed
by breathing a lower concentration of NO (5 ppm) during and after HBOC-201 infusion prevented
systemic and pulmonary vasoconstriction without increasing methemoglobin levels.
Conclusions:
These findings demonstrate that pretreatment with inhaled NO followed by breathing a low
concentration of NO during and after administration of HBOC-201 may enable administration ofan acellular Hb substitute without vasoconstriction while preserving its oxygen-carrying capacity.

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Invited Lecture
Invited Lecture

Clinical and Translational Advances in HBOCs: One Investigator's
Perspective
Jonathan S. Jahr
Professor of Clinical Anesthesiology
David Geffen School of Medicine at UCLA, CA, USA

Extensive work from my laboratories over the past twelve years in translational and clinical models
has resulted in significant publications [1 - 5, 7 - 19]. Clinical Phase I, II, and III trials with
Biopure's Hemopure, Hemosol's Hemolink, and Alliance's Perflubron have produced useful data,
although both Hemosol and Alliance have not pursued these products, probably due to poor study
designs in cardiac surgery models, and lack of cooperation with independent investigators (refusal
to publish existing data, etc) (1,2). This has been reviewed (3,4). Northfield's PolyHeme andSangart's Hemospan are moving forward with Phase II and III trials, but have been reluctant to let
independent investigators test product critically (5,6). A recent meta-analysis has comparedmortality and morbidityof the current products (Hemopure, PolyHeme, and Hemospan and older
discontinued products, Hemolink and HemeAssist) not to primary or secondary endpoints, but tomortality, all derived from post-hoc subset data analysis (6, 6a)In our translational laboratories, large and small animal experiments have been important todelineate product efficacy of oxygen delivery and safety in four HBOCs from three manufacturers
independently (7 - 19).
In a canine hypovolemia resuscitation model, Oxyglobin reconstituted splanchnic perfusion andoxidatative metabolism in spite of pronounced systemic vasoconstriction and insufficient
restoration of cardiac output and DO 2 ; it may improve diffusive oxygen transport in the
microvasculature by virtue of hemodilution an its high efficiency in the uptake and release ofoxygen (7)In the same model as reference 7, a study confirmed that real-time (in vivo) microvascular studies,
which were conducted only in small rodent models in the past, can be performed simultaneouslywith systemic function, hemodynamic, and oxygenation studies in a large animal model for relevant
data correlation (8).
In a similar canine model that included different resuscitation strategies after hemorrhagic shock,Oxyglobin infusion may not improve oxygen delivery more than hetastarch, likely due tohemodilution caused by its high colloid oncotic pressure, but may facilitate diffusive oxygen
transport to tissues (9)In a more advanced canine model of hemorrhagic shock with pre-hospital and hospital resuscitation
modes, including use of vasopressin, we concluded that low-volume crystalloid or Oxyglobin
resuscitation posthemorrhage may improve (but not restore) macro- and microvascular functions
and tissue oxygenation, while arginine vasopressin infusion may only improve blood pressures andresult in lower overall systemic perfusion compared with low-volume saline or hemoglobin
glutamer-200 treatment and worsening of anaerobic conditions in skeletal muscle (10).

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In an attempt to use HBOC's to estimate circulating blood volumes, we concluded that circulatingplasma volume and circulating blood volume may be accurately estimated by using plasma
hemoglobin concentration measurements after HBOC infusion (11).
In a rabbit model using magnetic resonance imagining, noninvasive in vivo magnetic resonancemeasurement of oxygen saturation is valid for whole blood mixed with stroma-free hemoglobin.
Therefore, magnetic resonance oximetry may be clinically useful for assessing oxygenation status inpatients resuscitated with an HBOC (12).
OxyVita, a new generation HBOC, and Oxyglobin, HBOCs with different molecular weights, had
similar effects on coagulation as measured by thrombelastography. The impairment of coagulation
by HBOCs and hetastarch occurred at doses corresponding to 12 mL/kg or a blood volume
replacement of 17%. The use of HBOCs at doses corresponding to 23 mL/kg or a blood volume
replacement of 33% significantly decreased coagulation to levels associated with increased clinical
bleeding in this preliminary study. Minimal coagulopathic effects are expected with use of OxyVita
at the manufacturer's anticipated effective dose of 10 g or 2 to 3 mL/kg (13).
In a clinical laboratory, effects of Hemopure on coagulation testing were evaluated; mechanicaldetection methods (fibrometer, STA, CS-190) and the MDA-180 methods were less affected byincreasing levels of Hemopure than optical detection devices for all test parameters (14)
In measuring hemoglobin, using a point of care device the HemoCue Plasma/Low Hemoglobin
System is a reliable instrument for detecting and measuring small concentrations of three different
HBOCs in plasma (15). The B-hemoglobin photometer accurately determined the concentrationof three HBOCs solutions dissolved in canine plasma.
The validity of arterial and mixed venous oxygen saturation measurements in a canine hemorrhage
model after resuscitation with varying concentrations of hemoglobin-based oxygen carrier wasdetermined (16). We concluded that the administration of this oxygen therapeutic may interferewith the values of some monitors.
Lactate measurements with three HBOCs were evaluated, and based on the samples tested in thisstudy, results indicate true lactate levels in the presence of HBOC-200 may be consistently
underestimated when measured in a spectrophotometry-based lactate analyzer, especially at higher
lactate concentrations. The clinical implications of this study are that with increasing levels of an
HBOC in human plasma, lactate interpretation may become inaccurate when measured by aspectrophotometric lactate analyzer, causing underestimation of "true" lactate values and possible
under-treatment of the patient. Therefore, caution must be exercised when interpreting lactate
results from the Synchron LX® 20 when a HBOC is present in plasma (17).
Methemoglobin concentration increases in patients receiving HBOCs. When interpreting lactate
concentrations from a patient with an HBOCpresent in plasma, underestimation of true lactate
levels may occur unrelated to methemoglobin concentrations (18).
High methemoglobin concentrations in Oxyglobin cause additive coagulation impairment that likely
results from the effects of oxidative substances on platelet function and coagulation proteins.
Oxidative products adversely react with coagulation factors and modify redoxsensitive sites in theplatelets. Therefore, if methemoglobinemia occurs as a result of HBOC administration and if thelevels are significantly elevated (greater than 10%), impairment of coagulation is possible (19) .

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In summary, work on HBOCs is presented, providing independent validation of safety and efficacy,
crucial for FDA and other regulatory registration for any product, and increased acceptance.
Independent investigation, regardless of outcome, is crucial for good science to prevail.
REFERENCES:
1.
Levy JH, Goodnough LT, Greilich PE, Parr GVS, Stewart RW, Gratz I, Wahr J, Williams J, Comunale ME, Doblar D,
Silvay G, Cohen M, Jahr JS, Vlahakes GJ. Polymerized bovine hemoglobin solution as a replacement for allogeneic red
blood cell transfusion after cardiac surgery: Results of a randomized, double-blind trial. The Journal of Thoracic andCardiovascular Surgery 2002, 124:35-42.
2.
Jahr JS, Mackenzie C, Pearce B, Pitman A, Greenburg AG. HBOC-201 as an alternative to blood transfusion: efficacy
and safety evaluation in a multicenter phase III trial in elective orthopedic surgery. Journal of Trauma-Injury Infection& Critical Care 2008; 64(6):1484-1497.
3.
Jahr JS, Walker V, Manoochehri K. Blood Substitutes as Pharmacotherapies in Clinical Practice. Current Opinion inAnaesthesiology 2007, 20:325-330.
4.
Jahr JS, Moallempour M, Lim JC. HBOC-201, HEMOGLOBIN GLUTAMER 250 (BOVINE), HEMOPURE®
(Biopure Corporation, Cambridge, MA). Expert Opinion in Biological Therapy 2008; 8(9): 1425-1433.
5.
Jahr JS, Varma N. PolyHeme. IDrugs 2004; 7(5):478-482.
6.
Natanson C, Kern SJ, Lurie P, et al. Cell-free hemoglobin-based blood substitutes and risk of myocardial infarction anddeath: a meta-analysis. JAMA 2008; 299(19):2304-2312.
6a.
Greenburg AG. Biopure refutes the JAMA meta-analysis - June 6, 2008, www.biopure.com/, accessed 9/17/2008.
7.
Driessen B, Jahr JS, Lurie F, Griffey SM, Gunther RA. Effects of hemoglobin-based oxygen carrier Hemoglobin
glutamer-200 (bovine) on intestinal perfusion and oxygenation in a canine hypovolemia model. British Journal of
Anaesthesia 2001, 86(5);683-692.
8.
Cheung ATW, Jahr JS, Driessen B, Duong PL, Chew JT, Chan MS, Lurie F, Golkaryeh MS, Kullar RK, Gunther RA.
Effects of a hemoglobin-based oxygen carrier (HBOC, Hemoglobin glutamer-200 [bovine]) on the microcirculation in acanine hypovolemia model: a non-invasive, computer-assisted intravital microscopy study. Anesthesia and Analgesia
2001; 93:832-838.
9.
Driessen B, Jahr JS, Lurie F, Golkaryeh M, Gunther RA. Arterial oxygenation and oxygen delivery after hemoglobinbased oxygen carrier infusion in canine hypovolemic shock: A dose-response study. Critical Care Medicine 2003;
31:1771-1779.
10.
Driessen B, Zarucco L, Gunther RA, Burns PM, Lamb SV, Vincent S, Boston RA, Jahr JS, Cheung ATW. Effects oflow volume Hemoglobin glutamer-200 (bovine) versus normal saline and arginine vasopressin resuscitation on systemic
and skeletal muscle blood flow and oxygenation in a canine hemorrhagic shock model. Critical Care Medicine 2007;
35(9):2101-2109.
11.
Jahr JS, Lurie F, Xi S, Golkaryeh MS, Kuznetsova OA, Kullar RK, Driessen B. A novel approach to measuring
circulating blood volume: use of a hemoglobin-based oxygen carrier (HBOC) in a rabbit model. Anesthesia andAnalgesia 2001;92:609-614.
12.
Chan FP, Jahr JS, Driessen B, Daunt D, Li KCP. Validation of MR in vivo measurement of oxygen saturation after
resuscitation with a polymerized hemoglobin-based oxygen carrier in a rabbit model. Academic Radiology 2001; 8:583590.
13.
Jahr JS, Weeks DL, Desai P, Lim JC, Butch AW, Gunther R, Driessen B. Does OxyVita, a new generation hemoglobinbased oxygen carrier, or oxyglobin acutely interfere with coagulation compared with normal saline or 6% hetastarch? An
ex vivo thrombelastography study. Journal of Cardiothoracic and Vascular Anesthesia 2008; 22(1):34-39.
14.
Jahr JS, Lurie F, Gosselin R, Lin JS, Wong L, Larkin E. Effects of a Hemoglobin-based oxygen carrier (HBOC-201) on
coagulation testing. American Journal of Therapeutics 2002; 9: 431-436.
15.
Lurie F, Jahr JS, Driessen, B. The novel HemoCue® Plasma/Low Hemoglobin System accurately measures low
concentrations of three different hemoglobin-based oxygen carriers (HBOC) in plasma: Hemoglobin glutamer-200(bovine), (Oxyglobin®), Hemoglobin glutamer-250 (bovine), (Hemopure®) and Hemoglobin-raffimer (HemolinkTM).
Anesthesia and Analgesia 2002; 95:870-873.
16.
Lurie F, Driessen B, Jahr JS, Reynoso R, Gunther RA. Validity of arterial and mixed venous oxygen saturation
measurements in a canine hemorrhage model after resuscitation with varying concentrations of hemoglobin-based
oxygen carrier. Anesthesia and Analgesia, 2003; 96:46-50.
17.
Jahr JS, Osgood S, Rothenberg SJ, Li Q-L, Butch AW, Gunther RA, Cheung AT, Driessen B. Lactate interference byhemoglobin-based oxygen carriers (Oxyglobin®, Hemopure® and HemolinkTM). Anesthesia and Analgesia 2005;
100:431-6.
18.
Osgood SL, Jahr JS, Desai P, Tsukamoto J, Driessen B. Does methemoglobin from oxidized HBOC (Hemoglobin
glutamer-200) interfere with lactate measurement (YSI 2700 Biochemistry Analyzer)? Anesthesia and Analgesia 2005;
100:437-9.
19.
Moallempour M, Jahr JS, Lim JC, Weeks D, Butch A, Driessen B. Methemoglobin effects on coagulation: a dose
response study with HBOC-200 (Oxyglobin®) in an ex vivo thrombelastogram Model. Journal of Cardiovascular andThoracic Anesthesia, 2008.

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Educational Lecture
Educational Lecture

Physiological Aspects of Oxygen Transport to Tissue
Hirosuke Kobayashi
Kitasato University School of Allied Health Sciences, Department of Medical
Engineering and Technology, JapanThe oxygen metabolism at mitochondria is similar, but there is a considerable diversity in theoxygen transport system among living things. Gill was developed in fish and has a fine countercurrent gas exchange system. Lungs were developed from esophagus in amphibians, reptiles, birds
and mammals. Bird's lung has a cross-current efficient gas exchange system, while mammals havepool lungs with large gas exchange area. Current topic is that dinosaurs had bird-type lungs andsome of them evolved into birds. The oxygen transport in the mammalian lungs is determined by
two limiting factors: perfusion limitation and diffusion limitation. In healthy humans, perfusion
limitation is the main limiting factor, except at high altitude on exercise, while the diffusion
limitation is the main limiting factor in patients with low diffusing capacity, particularly during
exercise. Circulation system can also be considered as a part of respiratory system, where oxygen
bound to oxygen carrier molecules is transported to microvessels. Arterial vessels and venous
vessels are running parallel, and gas exchange occurs between arterial and venous vessel. The Bohreffect is considered to contribute to oxygen transport in the lungs and tissues. It is also possible thatperipheral oxygen pressure is stabilized through counter-current gas exchange between
microvessels in the presence of the Bohr effect.

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SymposiumⅠ
SY−Ⅰ−1

Second Generation PEGylated Hbs with a Range of O2 Affinities
to Improve Tissue Oxygenation without Activating Autoregulation
Induced Vasoconstriction
Seetharama A. Acharya1, Fantao Meng2, Hongying Liang2,
Marcos Intaglietta3
1

Department of Physiology and Biophysics & Department of Medicine, Albert
Einstein College of Medicine, NY, USA
2
Departments of Physiology and Biophysics, Albert Einstein College of Medicine,NY, USA
3
Department of Bioengineering, University of California San Diego, CA, USA
HexaPEGylated Hb, (SP-PEG5K)6-Hb, and its prototype MP4 are vasoinactive. The taming of
the vasoactivity of Hb on PEGylation has been attributed to (i) the plasma expander like propertiesof hexaPEGylated Hb and (ii) high oxygen affinity. The high oxygen affinity minimizes theoversupply of oxygen to vascular walls on the arterial side, thereby limiting the autoregulation
mediated vasoconstriction. This high oxygen affinity of hexaPEGylated Hbs, however, makes them
function predominantly as plasma expanders. To facilitate better tissue oxygenation, it is necessary
to delineate the correlation between the oxygen affinity of PEGylated Hb and tissue oxygenation
without activating the vasoconstrictive activity. We have now designed PEGylated Hbs with a range
α-fumaryl Hb (P50 ~30 mm Hg) as the primary substrate forof O2 affinities using low O2 affinityα
PEGylation and acylation chemistry mediated PEGylation to engineer optimal plasma expander
like properties. The oxygen affinity of αα-fumaryl Hb has been modulated either by NEM
modification of Cys-93(β) (P50 ~12 mmHg) or by carboxymethylation of the α-amino group of
Val-1(β) (P 50 ~63 mm Hg). HexaPEGylation of αα-fumar yl Hb with site specificcarboxymethylation on Val-1(β), α
α-fumaryl Hb and NEM modified αα-fumaryl Hb using SPAPEG5K generates products with a P50 around 42, 20 and 10 mmHg, respectively. All the threehexaPEGylatedαα-Hb display a molecular radius of about 5.8 nm, and a COP and viscosity at 4%
around 53 mmHg and 1.9 cP, respectively. DecaPEGylation of αα-fumaryl Hb with site specificcarboxymethylation on Val-1(β) with SPA-PEG3K generates a product with a P50 ~ 30 mmHg.
The oxygen affinity of these four PEGylated products is expected to facilitate the identification of
the optimum oxygen affinity and the concentration of Hb needed in the plasma to optimize thetissues oxygenation with minimal impact on the autoregulation mediated vasoactivity.

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SymposiumⅠ
SY−Ⅰ−2

Effect of the Initially Injected Hemoglobin Vesicles (HbV) on the
Pharmacokinetics of Second Injection of HbV in Mice
Kazuaki Taguchi1, Yukino Urata1, Makoto Anraku1,
Toru Maruyama1, Toshiya Kai1, Daisuke Kadowaki1,
Hiromi Sakai2, Koichi Kobayashi3, Eishun Tsuchida2,
Masaki Otagiri1
1

Graduate School of Pharmaceutical Sciences, Kumamoto University, Japan
Research Institute for Science and Engineering, Waseda University, Japan3
Department of Surgery, School of Medicine, Keio University, Japan
2

Objective: Hemoglobin-vesicle (HbV) is an artificial oxygen carrier and has liposome structurewith polyethylene glycol (PEG). Recently, it reported that PEG-liposome lose their long-circulating
characteristic when they are administrated twice in the same animals (referred to as "accelerated
blood clearance (ABC) phenomenon" ), and anti-PEG IgM, which elicits a response by the spleen,
plays an essential role in induction of the ABC phenomenon. Therefore, we investigated whether
the first dose of HbV at a dose of 0.1mg/kg or 1400mg/kg have an effect on the pharmacokinetic
behavior of the second dose in mice. Additionally, we also studied the induction of IgG or IgM
against HbV. Method: Seven days after the first injection of non-labeled HbV (0.1 or 1400 mg
Hb/kg body weight), the male ddY mice received the 125I-HbV, which labeled the internal Hb of
HbV with iodine (the volume was identical to the volume of first injection). The IgG and IgMagainst HbV were detected by ELISA. Result and Discussion: The second dose was cleared
rapidly from the circulation and was dramatically accumulated in liver, as compared to first
injection, when mice were received first dose at a dose of 0.1mg/kg, but not 1400mg/kg. However,
the amounts of IgM against HbV, which was promoted ABC phenomenon, were produced byadministration at a dose of both 0.1mg/kg and 1400mg/kg, while that of IgG against HbV was notdetected. These results show that IgM against HbV is elicited by even administration of HbV, andthe suppression of ABC phenomenon at a dose of 1400mg/kg would be saturation of phagocytic
processing. Therefore, it would need to consider the induction of ABC phenomenon in repeatedadministration of HbV.

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SymposiumⅠ
SY−Ⅰ−3

Small Volume Resuscitation from Hemorrhagic Shock with
Polymerized Bovine Hemoglobin
Pedro Cabrales1, Amy Tsai2, Marcos Intaglietta2
Bioengineering, La Jolla Bioengineering Institute, CA, USA
Department of Bioengineering, University of California San Diego, CA, USA

1
2

Systemic and microvascular hemodynamic responses to hemorrhagic shock volume resuscitation
with hypertonic saline (HTS) followed by infusion of polymerized bovine Hb (PBH) at different
concentrations were studied in the hamster window chamber model, to determine role of plasma
oxygen carrying capacity and vasoactivity during resuscitation. Moderate hemorrhagic shock was
induced by arterial controlled bleeding of 50% of blood volume (BV), and a hypovolemic state wasmaintained for one hour. Volume was restituted by infusion of HTS (7.5% NaCl) 3.5% of BV offollowed by 10% of BV of PBH at two different concentrations. Resuscitation was followed for 90
minutes and was carried out using 13gPBH/dl [PBH13], PBH diluted to 4gPBH/dl in albumin
solution at matching colloidal osmotic pressure (COP) [PBH4], and an albumin only solution atmatching COP [PBH0]. Systemic parameters, microvascular hemodynamics and functional
capillar y density (FCD) were determined during hemorrhage, hypovolemic shock andresuscitation. PBH13 caused higher arterial pressure without reverting vasoconstriction andhypoperfusion. PBH4 and PBH0 had lower MAP and partially reverted vasoconstriction. Only
treatment with PBH4 restored perfusion and FCD when compared to PBH13 and PBH0. Bloodgas parameters and acid-base balance recovered proportionally to microvascular perfusion. Tissue
pO2 was significantly improved in the PBH4 group showing that limited restoration of oxygen
carrying capacity is beneficial and compensates for the effects of vasoactivity characteristic ofmolecular hemoglobin solutions proposed as blood substitutes. Supported by NIH BRP R24 HL64395, PPG HL71064, grants R01-HL62354, R01-HL76182 and A RMY PR023075.

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SymposiumⅠ
SY−Ⅰ−4

Liposome-encapsulated Hemoglobin Transfusion Rescues Rats
Undergoing Progressive Hemodilution from Lethal Organ
Hypoxia without Scavenging Nitric Oxide
Yashiro Nogami1, Manabu Kinoshita2, Bonpei Takase3,
Satoshi Syono4, Shinichi Kaneda5, Daizou Saitoh6,
Makoto Kikuchi7, Masayuki Ishihara8, Tadaaki Maehara1
1

Department of Surgery,National Defense Medical College,Japan
Department of Immunology and Microbiology
3
Department of Intensive Care Medicine
4
Department of Defense Medicine
5
Terumo Research Institute6
Division of Traumatology,National Defense Medical College Research Institute7
Department of Medical Engineering
8
Division of Biomedical Engineering, National Defense Medical College ResearchInstitute2

Objective: To investigate the efficacy of liposome-encapsulated hemoglobin (LHb) transfusion inrats undergoing lethal progressive hemodilution. Summary Background Data: Unlike otheracellular hemoglobin-based oxygen carriers, LHb has lipid bilayer membranes, similar to
mammalian red blood cells (RBCs), which prevent hemoglobin from direct contact with blood
components and the endothelium. Acellular hemoglobin has high affinity to nitric oxide (NO), andreportedly, acellular hemoglobin-based oxygen carriers have pressor effects on peripheral vessels,
because they behave as NO scavengers. The vasoconstrictor effects of acellular hemoglobin may
cause decreased peripheral perfusion in massive hemorrhage, thereby diminishing oxygen delivery.
Methods: Rats were subjected to blood withdrawal (0.2 mL/min) and simultaneously resuscitatedby isovolemic fluid transfusion with either LHb, 5% albumin, or washed rat RBCs for 150 min(n=15 in each group). Results: All rats transfused with LHb or RBCs were rescued from lethal
progressive hemodilution, whereas none of albumin-transfused rats survived. LHb did not affectthe plasma NO metabolite levels, suggesting it was not a potent NO scavenger. LHb also improved
hemodilution-induced metabolic acidosis, and reduced exaggerated neuroendocrine responses andinjuries to the heart, liver, and kidney. It suppressed expression of hypoxia-inducible factor-1alphain the liver and kidney, suggesting improvement of hypoxia at molecular response levels. However,neither transfused LHb nor RBCs improved the acute lung injury seen after progressive
hemodilution. Conclusion: LHb transfusion is very effective in rescuing rats undergoingprogressive hemodilution from lethal organ hypoxia without scavenging NO. (Ann Surg 2008;248:
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SymposiumⅠ
SY−Ⅰ−5Pegylation Destabilizes the Haemoglobin Tetramer
Luca Ronda1, Dario Caccia2, Raffaella Frassi2, Michele Perrella2,
Elena Del Favero3, Stefano Bruno1, Barbara Pioselli1,
Andrea Mozzarelli1
1Department of Biochemistry and Molecular Biology, University of Parma, Italy
Department of Biomedical Sciences and Technologies, University of Milan, Italy
3
Department of Chemistry, Biochemistry and Biotechnology for Medicine,
University of Milan, Italy
2

Haemoglobin conjugated with 6-7 molecules of 5 kDa polyethylene glycol (PEG) exhibits a
prolonged life-time in the plasma circulation. This effect is due to the increase in the molecularweight and hydrodynamic volume, which reduces renal filtration and endothelium permeation.
However, haemoglobin PEGylated under aerobic conditions shows high oxygen affinity, low
modulation of allosteric effectors and almost no cooperativity. To avoid these changes with respect
to HbA the PEG conjugation was carried out under anaerobic conditions. The product exhibits an
oxygen affinity, response to allosteric modulators, and cooperativity that are closer to HbA than
PEGylated oxy-haemoglobin. The alteration of functional properties is partly explained by anincreased dissociation of the PEGylated haemoglobin molecules into dimers as suggested by theconcentration dependence of the oxygen affinity and the elution profiles in size exclusion
chromatography. The chromatographic pattern is consistent with an altered tetramer-dimer
equilibrium due to a decreased rate of re-association of the dissociated subunits into tetramericspecies. We demonstrated by SDS-PAGE and MALDI-TOF analyses that PEG conjugation yields
a reproducible product with an average of 6.7 ± 1 PEG molecules per Hb tetramer and a predictedbroad PEG/haemoglobin ratio. This heterogeneity, possibly common to other PEGylated
haemoglobins, needs to be considered to evaluate in vivo physico-chemical and physiologicalparameters in the perspective of their use as safe blood substitutes.

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SY−Ⅰ−6

Vanished Oxygen Affinity of Myoglobin by Pyridine-containing
Heme
Saburo Neya1, Masaaki Suzuki1, Tyuji Hoshino1,
Teruyuki Komatsu2,
1

Department of Physical Chemistry, Chiba University, Japan
Waseda University, Japan2

The iron complex of oxypyriporphyrin, a porphyrinoid containing a keto-substituted pyridine, wascoupled with apomyoglobin. The reconstituted ferric myoglobin was found to be five-coordinate
without iron-bound water molecule. The anionic ligands such as CN and N 3 bound the
myoglobin with high affinities while neutral imidazole did not. The IR observation indicated thatazide complex was pure high-spin although the corresponding native protein was in the spin-state
equilibrium. The reduced myoglobin was five-coordinate, but exhibited no measurable affinity forO2. The affinity for CO was lowered down to 1/2400 as compared with native myoglobin. These
anomalies were ascribed to the deformation in the iron coordination core by a relatively large
pyridine unit. The ligand binding analyses for the ferric and ferrous myoglobin suggests that theproximal histidine pulls up the iron atom from the deformed core to reduce the interaction between
the iron and exogenous ligands. Similarity of the reconstituted myoglobin with guanylate cyclase, a
NO-responsive signaling hemoprotein, was pointed out.

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SY−Ⅰ−7

Hemospan®: Rational Design, Preclinical Effects, and ClinicalDevelopment of PEG-conjugated Human Hemoglobin
Mark A. Young1, Peter Keipert1, Kim D. Vandegriff 1,
Robert M. Winslow1,2
1Sangart, Inc., San Diego, CA, USA
University of California San Diego, CA, USA

2

The effectiveness of hemoglobin-based oxygen carriers (HBOCs) to deliver oxygen is dependent
on the profile of oxygen affinity, hemoglobin concentration and oxygen content, and cardiovascularactions. The ideal molecular and solution properties for HBOCs and the associated actions inanimals and humans remain a subject of intense investigation. Hemospan® is polyethylene-glycol(PEG)-conjugated human hemoglobin tetramer with a large molecular radius and high oxygen
affinity (P50 =5 mmHg), formulated at low hemoglobin concentration (4.3 g/dL) and high colloid
osmotic pressure (70 mmHg). We have demonstrated that these characteristics limit facilitateddiffusion of oxygen from cell free hemoglobin and, as a result, avoid autoregulator yvasoconstriction associated with oversupply of oxygen to the arteriolar smooth muscle. In anumber of experimental animal models, inspiration of 100% oxygen induces hypertension and adecrease in cardiac output, effects which are mimicked by αα crosslinked Hb or unmodified
stroma-free Hb (SFH). The consequence of vasoconstriction withααHb or SFH is to compromise
oxygen delivery, acid base status and hemodynamic stability after hemodilution or hemorrhage. In
contrast, administration of Hemospan is not associated with vasoconstriction and, instead,maintains oxygen delivery, cardiac output, and acid-base status following hemodilution or severe
hemorrhage. Thus Hemospan preserves perfusion and oxygen delivery compared with other
HBOCs exhibiting peripheral vasoconstriction. Measurement of perivascular nitric oxide afteradministration of HBOCs suggests that the lack of vasoconstriction with Hemospan is not due todifferential NO scavenging. In addition to the critical lack of vasoconstriction, plasma volume
expansion with Hemospan is also important in maintenance of peripheral perfusion and oxygen
delivery. Hemospan expands the intravascular volume by approximately 1.6-fold the infused
volume, similar to that of 10% pentastarch, but significantly greater than that ofααHb or the newgeneration hetastarch, Voluven. Our studies have demonstrated that the combined effects ofoxygen carrying capacity and volume expansion improve hemodynamic stability, acid-base status
and survival after hemodilution and hemorrhage relative to other HBOCs or matched colloids.
The clinical development program for Hemospan has strived to capitalize on the hemodynamic
stability and oxygen delivery observed in preclinical models. Phase II studies in orthopedic surgery
patients demonstrated significantly improved hemodynamic stability and maintenance of arterialpressure during spinal anesthesia. Two Phase III studies were deliberately designed to establish asafety database in the controlled clinical setting of orthopedic surgery, with the avoidance of
surgical hypotension as the primary objective. Secondary endpoints were designed to capitalize on
the demonstrated hemodynamic stability to determine benefit in composite indices for organ
morbidity and ischemia. These trials have recently completed enrollment of approximately 850
patients and data analysis is currently underway.

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SymposiumⅠ
SY−Ⅰ−8Transient Induction of Immune-suppressor Cells in Rat Spleen by
Massive Injection of Hemoglobin-vesicle(HbV)
Hiroshi Azuma1, Hideki Abe1, Daisuke Takahashi1,
Mitsuhiro Fujihara1, Hiromi Sakai2, Keitarou Sou2,
Hirohisa Horinouchi3, Koichi Kobayashi3, Eishun Tsuchida2,
Hisami Ikeda1
1

Research Division,Hokkaido Red Cross Blood Centre,Japan
Advanced Research Institute for Science and Enginnering, Waseda University,Japan
3
Department of Lung Surgery, Keio University School of Medicine, Japan
2[Objective] HbV, a liposomal oxygen carrier containing human hemoglobin is one of the candidates
for artificial red blood cells. The safety and efficacy of HbV has been evaluated in various aspects.
However, its effects on immune system have not been fully estimated. [Materials and Methods]
HbV or empty vesicle(EV) was intravenously infused into rats with or without pre-immunization by
Keyhole Limpet Hemocyanin (KLH). Then, splenic T cells (or lymph node T cells) derived fromthe rats at various interval were cultured with Concanavalin A (Con A) and/or KLH. The cellproliferation was evaluated by 3H-thymidine incorporation into DNA. Expression of high affinity
IL-2 receptor (CD25) on T cell and IL-2 secretion from T cell were also evaluated by flow
cytometry. In some experiment, assay was done in the presence of inducible nitric oxide synthase(iNOS) inhibitor. [Results] Con A(0.3μg/ml)-induced proliferation of splenic T cells derived fromrat loaded with HbV and/or EV was significantly suppressed compared with that from saline-loaded
rat. The proliferation of HbV-loaded lymph node T cells was also suppressed but the suppression
was not comparable to that of splenic T cells. Secondary response to KLH was also suppressed.
When splenocytes were taken 7days after HbV injection, no suppression was observed at all. HbVor EV- loaded splenocytes suppressed Con A-induced proliferation of saline-loaded splenic T cells.
No significant reduction of high affinity IL2 receptor expression and IL2 secretion was observed in
HbV-loaded splenic T cells. In addition, iNOS inhibitor was shown to restore T cell proliferation toa certain extent. [Conclusion] Massive liposome injection induced immune-suppressor cells in rat
spleen, which lead to transient suppression of splenic T cell proliferative response to Con A and
KLH. Nitric oxide appeared to be involved in the suppression.

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SymposiumⅠ
SY−Ⅰ−9Potential Tumor Oxygenation by Systemic Administration of
Hemoglobin Vesicle in a Mouse Lewis Lung Carcinoma Model
Yotaro Izumi1, Manabu Yamamoto1, Ken Takeuchi1,
Masazumi Watanabe1, Hirohisa Horinouchi1, Yuji Teramura2,
Hiromi Sakai2, Shinji Takeoka2, Eishun Tsuchida2,
Koichi Kobayashi1
1Division of General Thoracic Surgery, Keio University School of Medicine, Japan
Advanced Research Institute for Sci and Eng, Waseda University, Japan

2

Background: Hemoglobin Vesicle (HbV) is currently being developed as an artificial oxygen
carrier. It contains concentrated hemoglobin solution within a phospholipid vesicle. Due to its
unique oxygen transporting capabilities in comparison to red blood cells, we hypothesized that it
may augment tumor tissue oxygen status. Methods: Lewis lung carcinoma fragments were
implanted in the right hindlegs of C57 mice. Once the tumors reached approximately 8 mm indiameter, the animals were randomly assigned to receive systemic infusion of 0.9ml/kg of HbV(HbV group) or saline (saline group). Tumor tissue oxygen tension was measured using
phosphorescence decay by Oxyspot before infusion, and up to 40 minutes after infusion. Also, in a
separate group of animals, the tumors were irradiated (20 Gy) after the completion of infusion.
Results: In the HbV group, tumor tissue oxygen tension increased significantly at approximately 10
to 20 minutes following infusion, after which it retuned to preinfusion range. This increase was notobserved in the saline group. Additionally, tumor tissue HIF1alpha level was decreased after HbV
administration. There was also a significant tumor growth delay after irradiation in the HbV groupcompared to the saline group. Discussion: The present study suggests that systemic administrationof HbV transiently increases tumor tissue oxygen tension. The effect of irradiation given during
this period may be augmented, at least in part, by this effect.

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SY−Ⅰ−10

Polymerized Placenta Hemoglobin Improves Cardiac Functional
Recovery and Reduces Infarction Size of Isolated Rat Heart
Qian Yang1, Tao Li2, Jin Liu2, Wei Wu2, Mingliang Yi2,
Chengmin Yang3
1

Department of Medicinal Chemistry, School of Pharmacy, Chengdu Medical
College, People's Republic of China
2
Laboratory of Anesthesiology and Critical Care Medicine, West China Hospital,
Sichuan University, China
3
Institute of Tianjin Union Biotechnology Development, China
Objective: Polymerized human placenta hemoglobin (PolyPHb) allowing transporting more O2 tothe hypoxia tissues owing to its higher O2 affinity than adult peripheral blood hemoglobin, as wellas having a lower viscosity and smaller mean diameter than human red blood cells, suggest
PolyPHb may be helpful to microcirculation perfusion and thereby alleviate myocardialischemia/reperfusion injury. This study was therefore designed to investigate its protective effect toisolated rat heart. Methods and Results: Isolated rat hearts were perfused with Langendorffmodel, after 30 minutes of baseline, the hearts were arrested and stored by St.Thomas' solution(STS) without (STS group) or with 0.5 gHb/dL PolyPHb (PolyPHb group) at 4 °C for 8 hours,
then reperfused for 2 hours. Compared with STS group, PolyPHb in STS greatly improved therecovery of left ventricular developed pressure (LVDP, P<0.01), maximum LVDP increase anddecrease rate (± dp/dt, P<0.01), coronary flow rate (CF, P<0.01). Also, both the cardiac enzyme
release, including creatine kinase (CK) and lactate dehydrogenase (LDH), and myocardial
infarction size were significantly reduced in PolyPHb group. Conclusion: our study provided
direct evidence that PolyPHb was beneficial to improving cardiac functional recovery and reducing
myocardial infarction of 8-hour hypothermic stored rat heart.

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Hemoglobin-based Oxygen Carrier Mediated Vasoactivity :Proposed Mechanisms and Potential Remedies
Hae Won Kim, Chi-Ming Hai
Brown University Medical School, Providence, RI, USA

A key impediment to regulatory approval of HBOCs appears to be an undesirable BP elevation(hypertension) often observed following IV administration in preclinical and clinical studies. The
HBOC-mediated hypertension, as it occurs without increased cardiac output, appears to be
primarily mediated by systemic vasoconstriction. In fact, a recent statement by FDA indicates thatall HBOC products reviewed are vasoactive at the doses proposed for clinical use. Because
vasoconstriction can cause subnormal blood flow to affected organs, a crucial question is whether
HBOC administration could lead to critical organ dysfunction. However, there has not been any
published report that definitively links HBOC to observed serious organ dysfunction. Whilepotential benefits of moderate hypertension in certain patient groups (e.g., hemorrhagic shock) is
being debated, an acute BP elevation could be detrimental in vulnerable patients. Acceptablevasoactivity is difficult to define generally since it depends on indications and individual patient's
condition. Vascular response to HBOCs may also be influenced by such factors as underlying
pathologies, concurrent medications, and infusion protocol. The etiology of HBOC-mediatedvasoactivity appears to be complex and may involve multiple pathways. Currently proposedmechanisms include HBOC interference with endothelial NO, vascular autoregulator ymechanisms, adrenergic receptors and activation of endothelin and other vasoactive agents.
Regardless of mechanisms, the HBOC-mediated hypertension could generally be modulated bymany conventional anti-hypertensive drugs including nitrovasodilators, Ca++ channel blockers,
ACE inhibitors. It may also be possible to prevent the effect by pre- or co-administration of proper
vasodilators such as inhaled NO and nitrites. However, metHb formation (non-oxygen carrier)and difficulty of proper dosing in hemodynamically unstable patients would pose significant
challenges. This presentation will primarily focus on mechanistic aspect of HBOC-mediatedvasoactivity and potential biochemical/pharmacologic means of modulation.

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Does Lowering Oxygen Affinity of Polyethylene Glycol ConjugatedHemoglobins Cause Arteriolar Vasoconstriction?
Amy G. Tsai1, Pedro Cabrales2, Seetharama Acharya3,
Joel M. Friedman3, Marcos Intaglietta1
1

Department of Bioengineering, University of California San Diego, CA, USA
La Jolla Bioengineering Institute, CA, USA
3
Department of Biophysics and Physiology and of Medicine, Albert Einstein Collegeof Medicine, NY, USA
2

Microvascular autoregulatory mechanisms prevent exposure of the tissue to extreme levels ofoxygen. The site of this control is the arterioles which vasodilate and vasocontrict thereby allow
and restrict oxygen delivery to tissue respectively. Sensitivity of these vessels to hyperoxia led to thedesign of high oxygen affinity acellular hemoglobins. Their aim was to protect the arterioles fromexposure to high oxygen levels and to target oxygen delivery to anoxic tissue regions. Polyethylene
glycol conjugated hemoglobins (PEG-Hb) are ideally suitable since their P50 is very low(approximately 4 - 6 mmHg). However in some clinical situations, it maybe advantageous todeliver oxygen to tissue before it becomes nearly anoxic. The objective of these studies was todetermine if an increase in oxygen affinity of a PEG-Hb would result in vasoconstriction as well asdelivery oxygen to tissue. Microvascular studies were performed with the awake hamster window
model. Vasoactivity was studied in a topload experiment using PEGylated alpha-alpha Hbmolecule which has a much higher P50 of 30 mmHg compared to first generation PEG-Hbs.
Results show that arteriolar vasoconstriction obtained when the microcirculation is exposed toapproximately 0.5 g/dl alpha-alpha Hb (P50 = 23 mmHg) is eliminated when the same molecule is
PEGylated. Mean arterial pressure was slightly elevated without vasoconstriction. Extreme
exchange transfusion experiments with the PEGylated alpha-alpha Hb were also performed todetermine if tissue oxygenation is increased by this PEGylated alpha-alpha Hb.
Research supported in part by NIH HL064395, NIH HL071064 and HL062318.

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SymposiumⅡSY−Ⅱ−3Mechanism to Avoid Vasoconstriction and Maintain Perfusion of
Pegylated Hemoglobins
Pedro Cabrales1, Amy G Tsai2, Joel M Friedman3,
Marcos Intaglietta 2
1

Bioengineering, La Jolla Bioengineering Institute, CA, USA
Department of Bioengineering, University of California San Diego, CA, USA
3
Department of Biophysics and Medicine, Albert Einstein College of Medicine, NY,USA
2

Despite the advances in oxygen carrier blood substitutes, the development of materials that
effectively maintain blood volume, perfusion and oxygen delivery remain the priority for
emergency care and trauma. There is increasing evidence that blood transfusions per se, might not
be the optimal blood replacement fluid. Vasoconstriction due to nitric oxide (NO) scavenging andoxygen autoregulation are the problem of small hemodynamic radius hemoglobin (Hb) based
oxygen carriers (HBOC). Conversely, Hb based materials with large hydrodynamics radius due to
Hb encapsulation and Hb surface conjugations of polyethylene glycol (PEG) are vasoinactive.
PEGylated Hb (PEG-Hb) traps water creating a shielding around the Hb, eliminating hypertension
and vasoconstriction and even inducing vasodilation. These effects may be related to PEG-Hb's
NO management, namely: i) Shear stress related production of NO by mechanotransduction due
to increased plasma viscosity and direct molecular interaction; ii) NO transport in an allostericallycontrolled reversible chemical reaction, and, iii) Nitrite reductase activity of the PEG-Hb.
Additionally to increase mechanical interaction with the vascular endothelium, PEG-Hb novel
biochemical properties produces intermediates that extract an electron from nitrite, leading to theformation of reaction residues which generate nitrosothiols. The concerns with these mechanisms
are: i) How do these reactions generate bioactive NO? ii) How does free NO arrive to the site ofaction without reacting or being sequestered?; and, iii) If free NO does not form nitrosothiols, how
are these being generated? These NO generating mechanisms attributed to PEG-Hb/NO
interaction are the pathways for PEGylated Hbs to remain vasoinactive, and may in someconditions, cause them to act as vasodilators, even though PEG-Hb is an effective NO scavenger.
Supported by NIH BRP R24-HL64395, PPG HL71064, grants R01-HL62354, R01-HL76182
and ARMY PR023075.

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Effect of Artificial RBCs on Murine Hemorrhagic Shock Model
Yutaka Tomita1, 2, Haruki Toriumi2, Jemal Tatarishvili2,
Minoru Tomita2, Miyuki Unekawa2, Hiromi Sakai3,
Eishun Tsuchida3, Hirohisa Horinouchi4, Koichi Kobayashi4,
Norihiro Suzuki2
1

Department of Preventive Medicine for Cerebrovascular Disease, School of
Medicine, Keio University, Japan2
Department of Neurology, Keio University, Japan
3
Research Institute for Science and Engineering, Waseda University, Japan
4
Division of General Thoracic Surgery, Department of Surgery, Keio University,
Japan
Purpose: Effect of artificial RBCs1) injections on brain oxygen tension after withdrawal of bloodfrom the mice was examined. Methods: Under isoflurane anesthesia, a cranial window was opened
above the left parieto-temporal cortex of the head of C57BL/6J mice (n=8). An oxygen electrode(Eikohkagaku) was placed in an avascular area near the branch of left MCA of the brain surface. A
femoral artery and a tail vein were catheterized. After withdrawal of 0.3-0.5 ml blood, the sameamount of saline (n=3) or artificial RBCs (n=5) was systemically injected. During injection, thechanges of brain microvasculature, blood pressure (Muromachi) and oxygen tension (PO2) wereobserved. The movement of FITC-labeled RBCs running through single capillaries in the ROI of
the brain parenchyma (50μm depth) was monitored continuously with video camera (30 frames/s)or high speed laser scanning confocal fluorescent microscope (500 frames/s) 2). Results: The control
values of blood pressure and PO2 at the brain surface were 80.5 ± 15.5 mmHg and 32.9 ± 4.1
mmHg respectively. After withdrawal of blood, these values decreased to 25.6 ± 5.6 mmHg and22.9 ± 2.9 mmHg (statistically significant; P ＜ 0.05). Stoppage or decrease of velocities of RBCs in
the MCA branch was observed. In the group of saline injection, PO2 at the brain surface elevatedtransiently then decreased a few minutes later. On the other hand, in the group of artificial RBCs
injection, PO2 at the brain surface slightly elevated and showed a transient unstable change, thenre-elevated to the basal control level and sustained. Velocities of RBCs in the microvessels
recovered. Conclusion: It was suggested that artificial RBCs injections during hemorrhagic shock
were effective to prevent profound microcirculatory injury of the brain. References:1) Artif Cells,
Blood Substitutes, Biotechnol. 35: 81-91, 2007. 2) Microcirculation. 15: 163-174, 2008.

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Oral Session
S−1

Design and Evaluation of S-nitrosylated Human Serum Albumin
as a Novel Anticancer Drug
Toru Maruyama1, Naohisa Katayama2, Keisuke Nakajou2,
Yu Ishima1, Hisakazu Komori1, Kunitoshi Uchida2,
Jun-ichi Yokoe2, Norikiyo Yasui2, Hisashi Yamamoto2,
Toshiya Kai2, Makoto Sato2, Masaki Otagiri1
1

Department of Biopharmaceutics, Kumamoto University, Japan
Pharmaceutical Research Center, Nipro Corporation, Japan

2

Objective: In recent studies, the cytotoxic activity of NO has been investigated for its potential use
in anticancer therapies. Nitrosated human serum albumin (NO-HSA) may act as a reservoir of NO
in vivo. However, there are no published reports regarding the effects of NO-HSA on cancer.
Therefore, the present study was undertaken to investigate the antitumor activity of NO-HSA.
Method: NO-HSA was prepared by incubating HSA, which had been sulfhydrylated using
iminothiolane, with isopentyl nitrite (6.64 mol NO/mol HSA). Antitumor activity was examined invitro using murine colon 26 carcinoma (C26) cells and in vivo using C26 tumor-bearing mice.
Result and Discussion: Exposure to NO-HSA increased the production of reactive oxygen
species in C26 cells. Flow cytometric analysis using rhodamine 123 showed that NO-HSA caused
mitochondrial depolarization. Activation of caspase-3 and DNA fragmentation were observed in
C26 cells after incubation with 100 μM NO-HSA for 24 h, and NO-HSA inhibited the growth of
C26 cells in a concentration-dependent manner. The growth of C26 tumors in mice wassignificantly inhibited by administration of NO-HSA compared with saline and HSA treatment.
Immunohistochemical analysis of tumor tissues demonstrated an increase in terminal
deoxynucleotidyl transferase dUTP nickend labeling-positive cells in NO-HSA-treated mice,suggesting that inhibition of tumor growth by NO-HSA was mediated through induction of
apoptosis. Biochemical parameters (such as serum creatinine, blood urea nitrogen, aspartate
aminotransferase, and alanine aminotransferase) showed no significant differences among the threetreatment groups, indicating that NO-HSA did not cause hepatic or renal damage. Conclusion:
These results suggest that NO-HSA has the potential for chemopreventive and/or
chemotherapeutic activity with few side effects.

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S−2

Hemoglobin-vesicles as O2- and CO-carriers
Hiromi Sakai1, Hirohisa Horinouchi2, Koichi Kobayashi2,
Eishun Tsuchida1
Research Institute for Science and Engineering, Waseda University, Japan
Department of Surgery, School of Medicine, Keio University, Japan1
2

E-mail: hiromi@waseda.jp
Hb-vesicles (HbVs, 250 nm) are artificial O2-carriers that encapsulate concentrated Hb solution inphospholipid vesicles. We confirmed that the resuscitative effect of the HbV fluid was comparablewith that of RBC transfusion, though both O2-carrying fluids inevitably showed some oxidative
damage to the tissues (Crit Care Med 2004; Shock, in press). Recent reports on cytoprotective
effects of exogenous CO, by CO-inhalation and injection of CO-releasing molecules, urged us totest infusion of CO-bound HbV (CO-HbV) and RBC (CO-RBC) to improve tissue viability overthat of O2-HbV and O2-RBC. Male Wistar rats were anesthetized with 1.5%-sevoflurane inhalation(FiO2=21%) while spontaneous breathing was maintained. Shock was induced by 50% blood
withdrawal from femoral artery. Fifteen min later, they received CO-HbV, CO-RBC, O2-HbV, O2RBC, or empty vesicles (EV) suspended in 5% rHSA. All groups showed prompt recovery of bloodpressure and blood gas parameters just after resuscitation, and survived for 6 h of observation
period. However, only EV group showed significant hypotension at 3 and 6 h. Plasma enzyme
levels were elevated at 6 h, especially in the O2-HbV, O 2-RBC, and EV groups. They weresignificantly lower in the CO-HbV and CO-RBC groups than in the O2-bound fluids groups.
Immunohistochemical staining of 3-nitrotyrosine exhibited less oxidative damage in the liver andlung for CO-HbV and CO-RBC groups. Blood HbCO levels (26-39% immediately after infusion)
decreased to less than 3% at 6 h while CO was exhaled through the lung. Both HbV and RBC
gradually gained the O2 transport function. Collectively, both CO-HbV and CO-RBC reduced
oxidative damage to organs in comparison to O2-HbV and O2-RBC. Adverse and poisonous effects
of CO gas were not evident in this experimental model.

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S−3

Injectable O-15 System for Oxygen Metabolism Studies Using
Hemoblobin-vesicle (HbV) : Automatic Labeling and Applicationin Rats Using PET.
Vijay Narayan Tiwari, Hidehiko Okazawa, Masato Kobayashi,
Tetsuya Mori, Shingo Kasamatsu, Yasuhisa Fujibayashi
Biomedical Imaging Research Center, University of Fukui, Japan

Abstract: Artificial red blood cells have emerged as one of the most exciting and promisingdevelopments to substitute normal blood at least for a transient period of time. Evidence fromexperimental and clinical studies suggests that they are stable enough in vivo and hence can be usedas oxygen carrier for diagnostic purposes. Intravenously injectable O-15 preparations could be
more efficient in terms of utilization and safety than the established O-15 inhalation method formeasuring hemodynamic parameters like cerebral blood flow (CBF), oxygen extraction fraction(OEF) and cerebral metabolic rate of oxygen (CMRO2) in brain using positron emission
tomography (PET). This study examined the possibility of developing an injectable O-15 systemusing Hemoglobin vesicles (HbV) a type of artificial red blood cell and later to measure brain
oxygen consumption in rats using O-15 labeled HbV. Though primarily HbV were labeled with O15 gas using direct bubbling, various other strategies were employed to enhance the labelingefficiency of HbV including the use of a vacuum pump to eliminate helium fraction from target gas,
bubbling combined with vortexing and addition of L-Cysteine as reductant to protect hemoglobin
molecule from oxidation. L-Cysteine was found to play a significant role in increasing the bindingof O-15 with HbV. A maximum radioactivity of 204 MBq/ml of HbV was obtained under optimal
conditions. CMRO2 values of 6.8 ± 1.4 mL.min-1.100g-1 (n=5) were obtained in rats using asmall animal PET scanner after the injection of O-15 labeled HbV. O-15 labeled HbV can be
successfully utilized to measure brain oxygen consumption in rats using PET which indicates thatHbV is capable of carrying oxygen in vivo and subsequently in delivering it to cerebral tissue.
Keywords: Hemoglobin-vesicle (HbV), Injectable O-15, L-Cysteine, Labeling efficiency, Positronemission tomography (PET).

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Oral Session
S−4

Comparison of Nitric Oxide-induced Oxidation of Recombinant
Oxyhemoglobin Subunits Using a Competition Experiment
Yen-Lin Lin, Kuang-Tse Huang
Department of Chemical Engineering, National Chung Cheng University, Taiwan

A low reaction rate with nitric oxide (NO) is one of the important characteristics of hemoglobin(Hb)-based oxygen carriers. The reaction rate between oxyHb and NO is usually measured bystopped-flow spectrophotometry. However, the reported rates vary due to the difficulty ofaccurately determining the NO concentration and the limit of the instrument dead time. To
circumvent these problems, we developed an experiment using oxymyoglobin (oxyMb) to competewith oxyHb for NO that is released from an NO donor. Determination of the rate constants in thecompetition experiment no longer depends on accurate measurement of time or NO concentration,
since this approach instead measures the ratio of rate constants for the reaction of oxyHb andoxyMb with NO. For recombinant mutant Hb α(L29F)β, the rates for α(L29F) and β areapproximately 150- and 1.7-fold smaller than for wild-type Hb. In conclusion, the competition
experiment provides an alternative method for determination of relative reaction rates ofrecombinant Hb subunits with NO.

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S−5

Polymerized Human Placenta Hemoglobin Decreased the
Pathological Nitric Oxide Production in Cold Stored Rat Heart
Tao Li, Pu Zhang, Jin Liu, Ronghua Zhou, Guohua Li
Department of Anesthesiology, West China Hospital, Sichuan University, People's
Republic of China

Ischemia, inflammation and heart failure result in induced nitric oxide synthase (iNOS) expression
in heart, and the subsequent depression of contractile activity and oxygen consumption has been
attributed to nitric oxide (NO) inhibition of mitochondrial respiration, which caused damage tomyocardium. Considering hemoglobin was the largest reservoir for oxygen and NO in the body, we
speculated that polymerized human hemoglobin (PolyPHb), the polymer of hemoglobin, might
have potential influence on the NO produced in ischemia/reperfusion (I/R) injury. Isolated
Sprague-Dawley rat hearts were divided into 3 groups: sham group, St.Thomas' solution (STS)group and STS+PolyPHb group, and perfused with Langendorff apparatus. After basal perfusedfor 30 minutes, the last 2 group hearts were arrested and stored with STS and STS plus PolyPHb(0.5g/dL), respectively, at 4 °C for 8 hours, then reperfused for 2 hours. The sham group hearts
were perfused for 2.5 hours without cold storage. PolyPHb added in STS greatly improved the left
ventricle contractile performance during reperfusion, including left ventricular developed pressure(LVDP), maximum LVDP increase (+dp/dt) and decrease rate (-dp/dt). In 3 group hearts, nodifferences in eNOS expression and eNOS phosphorylation were observed, whereas the iNOS
expression in STS group was greatly increased. PolyPHb in STS inhibited the iNOS expression
and significantly decreased the NO formation in I/R heart, these results were further proved by theless formation of peroxynitrite (ONOO ) in STS+PolyPHb group. In conclusion, PolyPHb in STS
is beneficial to the cardiac contractive function recovery and this effect was partially associated with
reduced iNOS-derived pathological NO production.

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Oral Session
S−6

Polymerized Human Placenta Hemoglobin Ameliorates Oxidative
Stress and Energy Deficiency in Cold Stored Rat Heart
Tao Li, Pu Zhang, Jin Liu, Ronghua Zhou, Guohua Li
Department of Anesthesiology, West China Hospital, Sichuan University, People's
Republic of China

Growing evidence indicated that augmented superoxide, nitric oxide (NO) and other free radicals
from ischemia/reperfusion (I/R) injury caused the oxidative stress and further contributed toenergy deficiency. Excessive NO during I/R injury was implicated to an early reversible inhibitionof cytochrome-c oxydase in complex IV, then followed by a persistent inhibition of complex I,
which decreased the oxygen availability and ATP synthesis in I/R heart. Our previous studies havedemonstrated the cardioprotective effect of polymerized human placenta hemoglobin (PolyPHb) in
isolated rat heart, this study therefore was designed to investigated the underlying mechanism of
this protective effect. Using Langendorff model, isolated Sprague-Dawley rat hearts were divided
into 3 groups: sham group, St.Thomas' solution (STS) group and STS+PolyPHb group. After
perfused for 30 minutes as baseline, the last 2 group hearts were arrested and stored with STS andSTS plus PolyPHb (0.5g/dL), respectively, at 4 °C for 8 hours, then reperfused for 2 hours. The
sham group hearts were perfused for 2.5 hours without cold storage. Compared with sham group,
STS group exhibited significant increase in NO production, companied by increased oxidative
stress in left ventricular tissue, PolyPHb in STS greatly decreased the NO production andameliorated the oxidative stress, evidenced by elevated superoxide dismutase (SOD) activity andinhibited malondialdehyde (MDA) formation. Consistent with these results, the mitochondria
hydrogen peroxide (H2O2) content and cytochrome-c release in STS+PolyPHb group were greatly
reduced, as compared to STS group. Further, the mitochondria ATP synthesis in STS+PolyPHb
group was also significantly higher than that in STS group, which conjointly proved themitochondria respiratory chain was well preserved by PolyPHb after 8-hour cold storage and 2hour reperfusion. Therefore, our study demonstrated that the cardioprotective effect of PolyPHb
might be associated with attenuation of oxidative stress and restoration of energy synthesis in cold
stored rat heart.

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New Material
NM−1

Hemostatic Effects of Liposomes Carrying Fibrinogen γ-Chain
Dodecapeptide on the Surface and Adenosine 5'-Diphosphate
Inside as a Platelet Substitute
Makoto Handa1, Yosuke Okamura1, 2, Shinji Takeoka2, Yasuo Ikeda3
1

Department of Transfusion Medicine & Cell Therapy, School of Medicine, Keio
University, Japan2
Department of Life Science and Medical Bio Science, Graduate School of Advanced
Science and Engineering, Waseda University, Japan3
Department of Internal Medicine, School of Medicine, Keio University, Japan
E-mail: mhanda@sc.itc.keio.ac.jp
We studied platelet substitutes for treatment of bleeding by focusing on a dodecapeptide;
HHLGGAKQAGDV (H12), which corresponded to the fibrinogen γ-chain carboxy-terminal
sequence (γ400-411), a specific binding module of the ligand for platelet GPIIb/IIIa complex. In
this study, we conjugated H12 to the surface of a liposome into which adenosine 5'-diphosphate(ADP) was included to prepare H12-(ADP)liposome, and evaluated their in vitro and in vivo
characteristics related to platelet hemostatic function.
The effect of H12-(ADP)liposome (φ 250 ± 80 nm) on agonist-induced platelet aggregation was
evaluated on an aggregometer (Hema Tracer T-638, Nico Bioscience, Tokyo). Thrombocytopenic
animals (rat or rabbit) were made by busulphan infusion, and a template-guided incision(QuikheelTM) was made 1 cm from the tip of tail or ear. The tail or ear was immersed in a 50 mL
cylinder of saline and the time taken to stop bleeding was measured. Using H12-liposomes having acontrast dye, iopamidol inside, computed tomography (CT) analysis (eXplore LocusTM, GE
Healthcare UK, Buckinghamshire, England) was performed to detect the accumulation of theiopamidol-incorporated liposomes to the sites of vascular injury in normal rats.
As compared with the H12-liposomes, the H12-(ADP)liposomes significantly enhanced thecollagen-induced platelet aggregation, suggesting that release of ADP from the H12(ADP)liposome was triggered by platelet aggregation. In vivo hemostatic effect was studied bymeasuring the tail bleeding time of thrombocytopenic rats at 5 min after the intravenous
administration of the H12-(ADP)liposomes. The bleeding times of the normal ([platelet] = 8.1 ±0.9x10 5 /μL) and thrombocytopenic rats ([platelet] = 2.0 ± 0.3x10 5 /μL) were 178 ± 56 and682 ± 198 s, respectively. The H12(ADP)liposomes at a dose of 10 mg/kg shortened the tailbleeding time to 349 ± 49 s, whereas the H12-liposomes at the same dose were without effects(572 ± 127 s). The similar hemostatic ability of the H12(ADP)liposomes was also seen inthrombocytopenic rabbits. It was thus suggested that the H12-(ADP)liposomes would amplify thein vivo hemostatic ability by releasing ADP. Indeed, with CT analysis, the H12-liposomes wereshown to specifically accumulate at the sites of vascular injury. These results indicated that theH12-liposomes would be a promising candidate as an ideal synthetic platelet substitute that is
specifically recruited to and exerts its hemostatic ability at sites of vascular injury.

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New Material
NM−2

O2 Binding Properties of Recombinant Albumin-heme Complexes
Having Arginine at the Entrance of the Heme Pocket
Akito Nakagawa1, Teruyuki Komatsu1,2, Stephen Curry 3,
Eishun Tsuchida1
1Research Institute for Science and Engineering, Waseda University, Japan
PRESTO, Japan Science and Technology Agency.
3
Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial
College London, South Kensington Campus, UK
2E-mail: akito@aoni.waseda.jp
In our blood stream, iron(III)-protoporphyrin IX (hemin) released from methemoglobin is boundwithin a D-shaped hydrophobic cavity in subdomain IB of human serum albumin (HSA) with anaxial coordination of Tyr-161 and salt bridges between the porphyrin propionates and a triad ofbasic amino acid residues (Arg-114, His-146 and Lys-190). We found that a pair of site-specificmutations into the subdomain IB of HSA allows the heme to bind O 2 ; (i) introduction of aproximal His at the Leu-185 position and (ii) substitution of Tyr-161 with non-coordinating Leu(rHSA(HK)). The rHSA-heme could be an artificial O2 carrier acting not only as a red blood cell(RBC) substitute but also as an O2-providing therapeutic reagent. However, the rHSA(HK)-heme
showed two O2-binding affinities which are probably due to two different orientations of the heme
in rHSA. In this paper, we report new rHSA having Arg at entrance of the heme pocket (146
and/or 190 position) and its effect on O2-binding properties. The introduction of Arg yielded aC); the value of which was almost the same
single O2-binding affinity (P50: 6-9 Torr, pH 7.0 at 22 °as that of RBC (8 Torr). Resonance Raman and infrared spectra of this rHSA-heme revealed thatthe strained coordination disappeared by the introduction of the Arg at 146 and 190 position. We
can conclude that introduction of Arg into the entrance of the heme pocket of rHSA-heme iseffective at excluding the low O2-binding affinity conformer.

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Lunch Time Symposium
LS−1

Hb-vesicle, a Cellular Hb-based Oxygen Carrier, Fulfills the
Physiological Roles of the RBC Structure
Hiromi Sakai1, Keitaro Sou1, Yotaro Izumi2,
Hirohisa Horinouchi2, Koichi Kobayashi2, Eishun Tsuchida1
1Research Institute for Science and Engineering, Waseda University, Japan
Department of Surgery, School of Medicine, Keio University, Japan2

E-mail: hiromi@waseda.jp
Biconcave RBCs deform to a parachute-like configuration to flow through a narrower capillary.
This profile is believed to be effective to facilitate the gas exchange. On the other hand,
physicochemical analyses have revealed that O2-unloading and binding of Hb are significantly
retarded by compartmentalization in RBC. Why nature has selected such an inefficient cellular
structure for gas transport? Interestingly, some of the answers to this question have been revised by
the research of blood substitutes.
Hb-vesicles (HbV) are artificial oxygen carriers encapsulating concentrated Hb solution (35 g/dL)
with a phospholipid bilayer membrane. Concentration of the HbV suspension is extremely high([Hb] = 10 g/dL, volume fraction, ca. 40 vol%) and it has an oxygen carrying capacity that iscomparable to that of blood. HbV is much smaller than RBC (250 vs. 8000 nm), but it recreates
the functions of RBCs; (i) The O2 unloading of HbV is slower than that of a cell-free Hb solution;(ii) COP is zero. For a massive dosage, HbV has to be co-injected with or suspended in a plasma
substitute such as albumin; (iii) The viscosity of HbV is adjustable to that of blood; (iv) HbV isfinally captured by RES, and then degraded and excreted promptly; (v) Co-encapsulation of anallosteric effector regulates O2 affinity; (vi) Hemolysis is minimal during circulation and the lipid
bilayer membrane prevents a direct contact of Hb and vasculature; (vii) Reaction of NO is retardedby an intracellular diffusion barrier, and HbV does not induce vasoconstriction.
We admit HbV has only a few days of functional half-life after intravenous injection. On the other
hand, the obvious advantages are that it is pathogen-free and blood-type-antigen-free; moreover, it
can withstand long-term storage for stockpiling. HbV has a variety of potential applications not
only as a transfusion alternative but also as an O2 or CO therapeutic fluid that cannot be attained by
the present RBC transfusion.

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Lunch Time Symposium
LS−2

The Effect of Hemoglobin Vesicle Administration on Ventilator
Induced Lung Injury
Yotaro Izumi1, Hiromasa Nagata2, Takashige Yamada2,
Hiroshi Morisaki2, Hiromi Sakai3, Hirohisa Horinouchi1,
Junzo Takeda2, Eishun Tsuchida3, Koichi Kobayashi1
1Division of General Thoracic Surgery, Keio University School of Medicine, Japan
Department of Anesthesia, Keio University School of Medicine, Japan
3
Research Institute for Sci and Eng, Waseda University, Japan
2

Hemoglobin Vesicle (HbV) is currently being actively developed as an artificial oxygen carrier. It
contains concentrated hemoglobin solution within a phospholipid vesicle. As a resuscitative fluid, it
will potentially be administered to patients receiving care in the intensive care unit, which includes
mechanical ventilation. Mechanical ventilation differs in a number of ways from physiologicalbreathing. Lung injury may be affected by positive pressure ventilation, known as ventilator
induced lung injury (VILI). In the present study, we studied the effects of HbV administration in a
rabbit VILI model. HbV did not affect lung function when administered under spontaneous
breathing. In this study, we administered HbV when the lung was mechanically ventilated. Rabbits
were mechanically ventilated (tidal volume, 30ml/kg), and 30%, or 60% exchange transfusion was
done with HbV, saline, or 5% albumin. Hemodynamics, and blood gas parameters were monitored
for 4 hours. The lung was then resected for histology. Wet to dry ratio was also measured. In the
30% exchange groups, there was no apparent change in systemic blood pressure in any of thegroups. Arterial oxygen tension tended to decrease gradually, but there was no significant
difference between groups. Slight lung edema was observed. There was no significant difference inthe wet to dry ration between the groups. The moderate decrease in arterial oxygen tension
suggested the presence of ventilation induced lung injury, but there was nothing to indicate that it
was aggravated by HbV administration. In the 60% exchange groups, HbV administration tended
to improve arterial oxygen tension. (Supported by MHLW of Japan)

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Lunch Time Symposium
LS−3

Effect of Artificial Oxygen Carrier Hb Vesicle on Cerebral Blood
Flow During and After Hemodiluted Cardiopulmonary Bypass in
Rat.
Ryo Aeba1, Yasuhisa Seishi2, Hirohisa Horinouchi2,
Masataka Yamazaki1, Eishun Tsuchida3, Hiromi Sakai3,
Koichi Kobayashi2
1

Division of Cardiovascular Surgery, Keio University School of Medicine, Japan
Division of General Thoracic Surgery, Keio University School of Medicine, Japan
3
Research Institute of Science and Engineering, Faculty of Science and Engineering,
Waseda University, Japan
2

Background
Donor blood continues to be priming for clinical cardiopulmonary bypass (CPB) in neonate andinfant to avoid unacceptable level of hemodilution. We have previously demonstrated that
hemoglobin vesicle (HbV) has contributed preservation of neurocognitive function after
hemodiluted CPB in rat. The mechanism of the effect is to be clarified.
MethodNine rats subjected CPB (90minutes and 200ml/kg/min of bypass flow), without the use of donor
blood priming. Hb V was added in the priming of CPB (Hb V group, n=4), whereas the other 5
animals served as control group. Brain tissue oxygen tension and regional cerebral blood flow(rCBF) were monitored during and after CPB.
ResultIn both the groups hematocrit decreased to 13.3+/- 0.5, 12.0+/-1.4 during CPB, but maintained
brain tissue oxygen tension during and after CPB. rCBF increased in both the groups during andafter CPB, although Hb V group showed less increase during CPB and more increase after CPB(p=0.489) compared with the control group.
Discussion
In hemodiluted CPB Hb V may contribute in oxygen delivery and better maintain cerebral oxygen
metabolism after CPB. Thus the use of Hb V for clinical CPB prime in neonates and infants should
be warranted.

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Poster
PO−1

Assessment of Inflammation-altered Intestinal Permeability Using
Arterially Perfused Murine Jejunal Loop
Premysl Bercik1, Jun Lu1, Elena F Verdu1, Hiromi Sakai2,
Eishun Tsuchida2, Stephen M Collins1
1

Department of Medicine, McMaster University, Canada
Advanced Research Institute for Science and Engineering, Waseda University,
Japan

2

We have shown previously that arterial perfusion with hemoglobin vesicles (HbV) is superior tooxygenated Krebs solution and maintains structural and functional integrity of the isolated jejunal
loop for up to 2 hours. In this study we investigated whether our model is able to detect changes inintestinal permeability induced by chronic inflammation. Balb/c mice (n=30), healthy or infectedwith Helicobacter pylori (Hp), received antibiotics or placebo for 2 weeks. Intestinal permeability
was measured 2 months later. 4-5 cm segment of jejunum was selected, the terminal branch of themesenteric artery was cannulated. The loop was perfused arterially with HbV dispensed in a
modified Krebs solution with 5% albumin. Macromolecular markers 51Cr-EDTA and 14C-mannitol
were administered luminally. Their concentration in the venous outflow was measured using
scintillation counter and expressed as a percentage of the luminal content. Tissue samples weretaken and examined for Hp, inflammation and structural damage. Data are presented as means ±SE. The perfusion with HbV resulted in a normal histology with preserved epithelium in 83.3% ofexperiments while in 16.7% segments we found moderate/severe epithelial damage, mostly result of
prolonged surgical manipulation. Hp infection induced chronic gastritis but did not affect jejunum.
Paracellular permeability for 51Cr-EDTA was higher in Hp infected mice compared to uninfected
controls (0.57 ± 0.08 vs 0.27 ± 0.07, p<0.05) and tended to improve after eradication treatment.
Membrane permeability for 14C-mannitol was also higher in Hp infected mice compared to controls(1.10 ± 0.10 vs 0.63 ± 0.15, p<0.05) however this did not improve after eradication treatment(1.15 ± 0.16, p<0.05 vs control). In conclusion, Hp infection in mice impairs intestinal epithelial
barrier. Hp eradication partially improves paracellular permeability but does not affect membrane
permeability. HbV-perfused intestinal loop is a valuable tool for assessing gut function in both
health and disease.

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Poster
PO−2

Hemoglobin Encapsulation in Vesicles Retards the Reaction with
NO by Intracellular Diffusion Barrier
Hiromi Sakai1, Atsushi Sato2, Kaoru Masuda3, Shinji Takeoka2,
Eishun Tsuchida1
1Research Institute for Science and Engineering, Waseda University, Japan
Graduate School of Science and Engineering, Waseda University, Japan
3
Kobelco Research Institute, Japan2

The reaction of NO, an endothelium-derived vasorelaxation factor, and Hb is restricted byencapsulation in RBCs to prevent vasoconstriction. This essence is applied to an artificial O2
carrier, Hb-vesicles (HbV), that mimic the cellular structure of RBCs, and actually we confirmedthat HbV becomes vaso-inactive in blood circulation. However, the mechanism of the NO-binding
restriction is controversial; i) an extracellular diffusion barrier surrounding the RBC; ii) a RBC
cytoskeletal submembrane constituting a diffusion barrier; and iii) retardation of NO-diffusion due
to the viscous Hb in RBCs. To clarify the mechanism, we analyzed HbV with different intracellular
Hb concentrations, [Hb]in , and different particle sizes using stopped-flow spectrophotometry [1].
The apparent NO-binding rate constant, k'NO , of deoxygenated HbV at [Hb]in = 1 g/dL was almost
equal to kNO of molecular Hb, indicating that the lipid bilayer membrane cannot be a barrier. With
increasing [Hb]in to 35 g/dL, k'NO decreased by 1/3, which was further decreased by 1/5 withenlarging particle diameter from 250 to 450 nm. For carbon monoxide (CO)-binding, which is
intrinsically much slower than NO-binding, k'NO did not change greatly with [Hb]in and the particle
diameter. Results obtained using diffusion simulations coupled with elementary reactions concurwith these tendencies and clarify that NO is trapped rapidly by Hb from the interior surface region
to the core of HbV at a higher [Hb]in , retarding NO-diffusion toward the core of HbV. In contrast,
slow CO-binding allows time for further CO-diffusion to the core. Simulations extrapolated to alarger particle (8 μm) showing retardation even for CO-binding. The obtained k'NO and k'CO yield
similar values to those reported for RBCs.
In summary, the intracellular diffusion barrier is predominant owing to the intrinsically rapid NObinding that induces a sink of NO from the interior surface to the core, retarding further NOdiffusion and binding. A high [Hb]in , 35 g/dL, and a larger particle size are the key factors toregulate the NO-binding and to optimize artificial O2 carriers comprised of phospholipid vesicles.
Refs: [1] Sakai et al, JBC 2008;283:1508-17; BBA 2008;1784;1441-7.

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Poster
PO−3

Effect of HbV as a Resuscitation Fluid in Uncontrolled Hemorrhageshock Model: Blunt Kidney Injury Model
Yasuhisa Seishi1, Hirohisa Horinouchi1, Naoki Aikawa2,
Hiromi Sakai3, Eishun Tsuchida3, Koichi Kobayashi1
1Department of Surgery, Keio University, School of Medicine, Japan
Department of Emergency and Critical Care Medicine, Keio University, School of
Medicine, Japan
3
Advanced Research Institute of Science and Engineering, Waseda University,
Japan
2【Background】Blunt trauma is often accompanied by massive hemorrhage from solid organs'injury such as liver, kidney, pancreas. A large amount of blood can be flow out into the peritoneal
space. Therefore fluid resuscitation to restore the circulation volume as well as maintenance oforgan perfusion is important for these patients. Artificial oxygen carrier with colloid solution may
act as volume resuscitation and oxygen infusion to tissues.
【Method】Male Wistar rats weighing 350g were used. After general anesthesia using sevofluren,
67% hemodilution was done by serial withdrawal and infusion with HES. Then left kidney wasdestroyed by cutaneous manipulation. Macroscopic effect of this procedure revealed to resemble
blunt rupture of the kidney. After injury, MAP decreased and Fluid resuscitation was begun when
MAP reached 30mmHg. Animals were randomly divided into four groups, non-resuscitationgroup, 5%HSA group, HbV group.
【Results】There are no deaths in HbV group and length of survival was good. Estimatedcirculation volume decreased in each group but in HbV group, decrease of circulation volume wassmaller than other groups. MAP was maintained after initial drop in HbV group. Subcutaneous
blood flow decreased as well as PtO2 even after resuscitation in 5%HSA group and nonresuscitation group, but in HbV group, Pt O2 was maintained at the level of 15 Torr. Increase of
Lactate level was inhibited in HbV group after resuscitation.
【Discussion】Resuscitation with HbV could improve survival and circulatory indices after
traumatic abdominal uncontrolled hemorrhagic injury. Stable PtO2 and inhibition of Lactate
increase indicated HbV can carry enough oxygen at low blood pressure, low flow condition in
peripheral tissue.

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Poster
PO−4

Resuscitation of Hemorrhagic Shock due to Uncontrolled
Hemorrhage-effect of Hemoglobin-vesicle in Vascular Injury
Model
Hirohisa Horinouchi1, Yasuhisa Seishi1, Mitsutomo Kohno1,
Yotaro Izumi1, Hiromi Sakai2, Koichi Kobayashi1,
Eishun Tsuchida2
1

Department of Surgery, Keio University, School of Medicine, Japan
Advanced Research Institute of Science and Engineering, Waseda University,
Japan

2

Uncontrolled hemorrhage is usually fatal if fluid resuscitation and adequate hemostasis cannot be
achieved. Low blood pressure causes sparse perfusion and hypoxia in peripheral tissue and organfunction is deteriorating in a short period. Artificial oxygen carrier may improve oxygen
metabolism of organs and elongate survival.
Method: Male Wistar rats were used. After general anesthesia with sevofluren, lapalotomy was
done. Uncontrolled hemorrhage was created by penetrating the aorta with 24 G needle. MAP
decreased swiftly and when MAP decreased lower than 20 mmHg within a minute, resuscitation
was initiated. Several resuscitation fluid such as large volume of saline, same volume of allogenic
blood, same volume of 5% albumin, HbV resolved in 5%HSA was administered. MAP, HR, WBC,
Ht, Hb, Lactate, Piruvate were measured. When animal died, time was recorded.
Results: Bleeding volume was 8.0+/-0.9ml in no resuscitation group, 14.3+/-2.9ml in high volume
saline group, 12.6+/-4.1ml in allogeneic transfusion group, 11.8+/-4.0ml in 5%HSA group, and9.8+/-3.7ml. In high volume saline group, constant decrease of MAP was observed even large
resuscitation fluid was administered. There are significant difference in blood loss between salinegroup and other groups. In HbV group, blood loss was significantly smaller in amount. Change of
Lactate/pyruvate ratio was smaller in allogeneic blood group and HbV group.
Discussion: In uncontrollable hemorrhage model, MAP could not be maintained by large volumeof crystalloid fluid. Colloid containing fluid seemed effective for elongation of survival. Oxygen
carrying capacity inhibited the increase of lactate/pyruvate ratio. HbV was thought to ameliorate
organ oxygen metabolism and increase the survival time.

ARTIFICIAL BLOOD Vol. 16 , No.2, 2008137

Call for Papers
Artificial Blood, the official bilingual journal of The Societyof Blood Substitutes, Japan, welcomes papers and otherarticles contributing to the research and development ofblood substitutes.
If you wish to submit an article for publication, please email
it to the following address after first confirming the
instructions for authors.
kita@kenkousya.jp

Instructions for Authors（draft last revised Sept. 30, 2008）
The Journal's purpose is to publish research and related articles
contributing to research on the development of blood
substitutes, information on Society proceedings, regulations, andother matters of interest to the Society members, and it
welcomes original articles from a range of contributors
regardless of format. Although contributors should ideally bemembers of the Society, this is not a requirement. Decisions on
acceptance of manuscripts are made by the Editorial Board
based on the results of peer review. Original articles will not be
accepted if they have been previously published or are being
considered for publication in another journal.
If an article is coauthored, the consent of all coauthors is
required before submission. As copyright to articles must betransferred to the Society, the representative of the author（s）
must sign and seal a copy of the Copyright Transfer Agreementfound in the Journal or downloadable from the Society's website（http://www.blood-sub.jp/home/index.html）, and submit it to
the Editorial Board by post, fax, or by email as a PDF fileattachment.
Manuscripts should, as a rule, be prepared by word-processor.
However, handwritten manuscripts may be accepted.
1）Articles should be categorized into one of the followings:
original articles, review articles, conference reports, topicalpieces, and opinion pieces. The category into which amanuscript falls should be clearly indicated at the top right-handcorner of the first page. Manuscripts that do not fall into any ofthese categories may also be accepted, and manuscripts may
also be re-categorized depending on the opinion of the138

reviewers. Submit your manuscripts to the Editor-in-Chief byeither of the following methods with a covering letter（of any
format）:
i）Submission by email of electronic files of the text and figures（indicate the software used）. Text and tables should be in DOC
or TXT formats, and figures should be in PPT, JPG, or TIFF
formats.
ii）Submission by post of four sets of hardcopies.
2）Manuscripts are reviewed by researchers in the field ofartificial blood selected by the Editor-in-Chief, and revisions may
be required depending on the opinion of the reviewers. Revisedmanuscripts should be submitted with a "Response to
Reviewers" to the covering letter that responds to each of thepoints made by the reviewers, indicating any revisions made to
the manuscript.
3）Once informed of the decision to accept for publication, theauthor should send by post files containing the text and figuresof the accepted paper saved in electronic media to the address
specified（indicate the software used）. Text and tables should bein DOC or TXT format, and figures should be in PPT, JPG, or
TIFF format.
4）Manuscripts should be typed on A4 or letter size paper. The
title page should include the title, names of authors, institutions
to which all the authors belong, and the address of thecorresponding author. Handwritten manuscript should be
written consisting of 20 lines to 1 page.

人工血液 Vol. 16 , No.2, 20085）Original articles, review articles, topical pieces, and opinion
pieces should include an abstract and about 6 keywords on thesecond or subsequent pages.
6）Research conducted with the aid of an official grant must be
acknowledged, and any conflict of interests（for example, if theauthor has an interest in a company distributing the drug
described in the manuscript: being an employee or consultant tothat company, receiving research funding, owning shares orpatents, and so on）must be described in a footnote on the firstpage or in acknowledgment section.
7）If a manuscript describes the results of research on humans
or animals, it should be indicated that such research was
performed in accordance with the guidelines of the instituteconcerned in the methods or other appropriate sections of themanuscript.
8）Abbreviations should be spelled out on their first appearance.
The names of drugs, medical drugs, laboratory equipment, andso on should be given. The type, distributor（manufacturer）andthe address should also be indicated.
Example: Rhodamine B（Sigma-Aldrich, St. Louis, USA）
Polygraph system（LEG-1000; Nihon Kohden Corporation,
Tokyo）.
9）The English fonts should be Times, Helvetica, Courier, orSymbol. Text should be typed in lower-case one byte characters.
However, sentences and proper nouns should begin with an
upper-case letter.
10）Figures should be expressed in Arabic numerals. Weights
and measurements should be expressed in units such as thefollowings: m, cm, mm, μm, L, mL, μL, mol, g, mg, μg, ng, pg,
fg, N/10.
11）Figures and tables should be numbered in order of citation,and it should be clearly indicated where they are to appear in
the main text. The title, legends and description in tables andfigures should be written in English. Figures will be printed bydirect offset printing. Tables will be inputted by the Editorials
as originals.
12）References should be cited numerically in order of appearance
in the text using superscript letters as follows: 2）, 3-5）, 1, 4-6）, etc.
References should be listed using the Vancouver style as follows:
Names of all authors. Title of paper. Title of journal. Year of
publication; volume number: inclusive page numbers.
Abbreviations of journal names should be in accordance with
Index Medicus. References to books should be given as follows:
Names of all authors. Title of paper. Name of editor（s）. Booktitle. Place of publication: Publisher, year; inclusive page
numbers.
References to electronic sources should be given as follows:

ARTIFICIAL BLOOD Vol. 16 , No.2, 2008

Name of website.
Address on new line（month and year of last access）.
Examples:
１. Wong NS, Chang TM. Polyhemoglobin-fibrinogen: a novel
oxygen carrier with platelet-like properties in a hemodiluted
setting. Artif Cells Blood Substit Immobil Biotechnol
2007;35:481-489.
２. Natanson C, Kern SJ, Lurie P, Banks SM, Wolfe SM. Cell-freehemoglobin-based blood substitutes and risk of myocardial
infarction and death: a meta-analysis. J Am Med Assoc
2008;299:2304-2312.
３. Sakai H, Sou K, Takeoka S, Kobayashi K, Tsuchida E. In:
Winslow RM, ed. Blood Substitutes. London: Academic Press（Elsevier）, 2006;514-522.
４. Oxygen Infusion Project, Waseda University, Japan.
http://www.waseda.jp/prj-artifblood/index-ja.html（ lastaccessed Sept 2008）13）In the case of citation or reproduction of previously
published figures or tables and other content, the permission of
the copyright holder（s）must first be obtained. Copyright in the
published papers shall belong to the Society.
14）Regarding secondary use and copyright in works publishedin the Journal, secondary use may be made of the Journal, in
whole or in part, via media such as CD-ROM or the Internet.
Reproduction rights, translation rights, film rights, dominion, andpublic transmission rights（including the right to make theworks transmittable）are transferred to the Society by theauthor's submission of the aforementioned Copyright Transfer
Agreement. This clause shall not restrict reuse by the author
himself/herself, but the Editor-in-Chief must be informed in theevent of reuse.
15）No publication fee is charged for publication in the Journal,
and the author（s）shall receive as a gift 30 offprints of theircontributions. Authors will be charged for copies in excess of
this number（approximately 100 yen per copy）. Authors wanting
prints of color photos or on art paper, etc. must pay the actual
cost of such prints.
16）Address for manuscripts to be sent:
Attn: Hiromi Sakai（Editor-in-Chief）
Artificial Blood Editorial Office
The Society of Blood Substitutes, Japan
Oxygen Infusion Project
Bldg. ＃55S-Rm. 703
Research Institute for Science and Engineering
Waseda University
3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan
Tel: +81-3-5286-3122 Fax: +81-3-3205-4740
E-mail: kita@kenkousya.jp139投稿規定（平成20年9月30日改訂）
本誌は，血液を構成するあらゆる成分について，その代替物を開発する研究に貢献する論文，関連する情報，学会会員のための会報，学会諸規定等を掲載するが，形式にはこだわらず創意ある投稿を広く集める．
本誌への投稿者は本学会会員であることが望ましいが，投稿を希望する者は誰でも投稿することが出来る．原稿掲載の採否は，査読結果に従って編集委員会が決定する．
原著論文について，他誌に既発表あるいは投稿中の論文は掲載しない．
共著者がいる場合には，共著者全員の承諾を得てから投稿する．論文の版権は本学会に譲渡しなければならない．このため，
著者の代表者は，本誌に添付の著作権譲渡同意書（Copyright Transfer Agreement）
或 は ，本 会 の ホ ー ム ペ ー ジ サ イ ト（http://www.blood-sub.jp/home/index.
html）からダウンロードしたものに署名捺
印の上，郵送，Fax，またはpdfファイルとしてE-mailにて編集委員会宛に提出する．
ワープロを用いて作製した原稿の投稿を原則とする．ただし，手書き原稿による投稿でも受け付ける．欧文による投稿を歓迎する．

こと．
６）投稿論文に記載の研究が公的助成を受けて実施された場合には，謝辞にその旨を記載すること．また，Conflict of Interests（例えば，論文に記載された薬品を販売する企業と著者との利害関係: 雇用，コンサルタ
ント，研究助成，株式，特許など）があれ
ば，これを第１頁の脚注，謝辞などに記載すること．
７）ヒトを対象とした研究結果，および動物実験の結果を掲載する場合には，各研究機関のガイドラインに従って実施したことを方法等に明記すること．
８）論文中の略語は初出の際に省略しない
こと．薬品，医薬品，測定装置等は，外国語名の場合は言語のまま用い，日本語化しているものはカタカナとする．型式，販売（製造）元とその所在地も記入すること．
（例） Rhodamine B（Sigma-Aldrich, St.
Louis, USA）, ポリグラフシステム（LEG1000; 日本光電工業, 東京）
９）句読点はコンマ（，）ピリオド（．）とする．

１）原稿の種類は，「原著論文」，「総説」，「学会報告」，「トピックス」，「オピニオン」，「海外文献紹介」から選び，これを第１頁の右肩上に明記すること．これらに該当しない原稿も受け付ける．査読意見によっては種類が変更される場合がある．次のいずれかの方法により，送付状（任意のフォーマット）を添えて編集委員長宛に投稿する．

10）文中の英語に使用するフォントは，Times,
Helvetica, Courier, Symbolを原則とし，英文半角小文字とする．ただし，文頭および固有名詞は大文字で書きはじめること．

i ）文章と図表の電子ファイルをE-メールで送付する（使用したソフトを明記すること）．文章・表のファイル形式
は，doc, txtが好ましい．図は，ppt,
jpg, tiffが好ましい．

12）FigureとTable：引用順にそれぞれ番号を付けること．表題，説明，図表中文字は，全て英文とすることが好ましい．本文
中に挿入箇所を明記すること．Figureは直接オフセット印刷とする．Tableは編集部にて入力し原図とする．

11）数字はアラビア数字を使い，度量衡の単位はm, cm, mm, μm, L, mL, μL, mol, g,
mg, μg, ng, pg, fg, N／10などを用いる．

ii）ハードコピー4部を郵送する．
２）投稿論文の査読は，編集委員長が選んだ人工血液分野の研究者に依頼する．査読
意見によっては，原稿の修正を求める場合がある．修正論文（Revised Manuscript）の投稿に際しては，送付状に「査読意見に対する回答」を添え，意見に対して一つ一つ回答をするとともに，修正箇所がある場
合にはこれを明記する．
３）掲載決定通知の後，著者は採択論文の文章・図表のファイルを電子媒体として，指定する宛先に送付すること（使用したソ
フトを明記すること）．文章・表のファイル
形式は，doc, txtが好ましい．図は，ppt,
jpg, tiffが好ましい．
４）原稿はA4版の大きさとし，第1頁には
表題，英文表題，著者名，全著者所属，英文著者名，英文著者所属，次いで連絡の取れる著者（corresponding author）の住所，
英文住所を記入する．手書き原稿の場合は
Ｂ5版，1行20字，20行とする．
５）「原著論文」，「総説」，「トピックス」，「オピニオン」については，第2頁以降に和文抄録，Keywords（英文で6個程度）を付
け，最終頁または別紙に英文抄録を付ける

13）文献：本文に引用した順序に番号を付け，文中では2），3-5），1，4-6）などとする．文献の記載法はthe Vancouver styleに従う．全
著者名．論文題名．誌名 西暦発行年；巻数：頁〜頁．とし，誌名の省略は医学中央雑誌またはIndex Medicus に準拠する．単行本の場合は全著者名．題名．編集者名．
書名．発行地：発行書店，年号；頁〜頁．
の順とする．電子文献の場合は，ホームページ名．改行してアドレス（引用した西暦
年月）とする．
（例）
１. 高折益彦. 人工酸素運搬体:その将来への期待. 人工血液 2007;15:90-98.
２. 橋本正晴. 単回投与毒性試験. 野村 護,
堀井郁夫, 吉田武美 編. 非臨床試験マニュアル. 東京: エルアイシー, 2001;37-48.
３. Wong NS, Chang TM. Polyhemoglobinfibrinogen: a novel oxygen carrier
with platelet-like properties in ahemodiluted setting. Artif Cells Blood
Substit Immobil Biotechnol 2007;35:
481-489.
４. Natanson C, Kern SJ, Lurie P, Banks
SM, Wolfe SM. Cell-free hemoglobinbased blood substitutes and risk of
myocardial infarction and death: ameta-analysis. J Am Med Assoc

2008;299:2304-2312.
４. Sakai H, Sou K, Takeoka S, Kobayashi
K, Tsuchida E. In: Winslow RM, ed.
Blood Substitutes. London: Academic
Press（Elsevier）, 2006;514-522.
５. 早稲田大学酸素輸液プロジェクト．
http://www.waseda.jp/prj-artifblood/index-ja.html（2008年9月現在）
14）既発表の図表，その他を引用，転載する場合には，あらかじめ版権所有者の許可を得ること．また，掲載論文の著作権は本学会に帰属する．
15）二次掲載について．本誌は，他の言語
ですでに掲載された論文を和文で二次掲載することは二重投稿ではなく正当な掲載と認めるが，著者は以下の事項を遵守する．
ａ）すでに掲載された論文であること．
ｂ）著者は両方の雑誌の編集者より許可を得ていること．二次掲載する編
集者に最初に掲載されたもののコ
ピー，別刷，もしくは原稿のいず
れかを添付すること．
ｃ）論旨を変えないこと．執筆者は同一（順不同）であること．
ｄ）二次掲載版のタイトル・ページに掲載される脚注には，その論文の全体もしくは一部分がすでに掲載されている旨を明記し，更に初出文
献も示すこと．適切な脚注の例を
以 下 に 示 す ．「 This article isbased on a study first reported inthe［...雑誌タイトル（完全な典拠情報を添えたもの）...］（訳：この論文記事は，［...］に最初に報告された研究に基づくものである）」．
これらの要件を満たしている場合は，その旨を明記して，総説または論文記事（二次掲載）として投稿する．
16）本誌掲載著作物の二次利用および著作権について．本誌の一部，もしくは全部を
CD-ROM，インターネットなどのメディアに二次利用する場合がある．本誌に掲載する著作物の複製権・翻訳権・上映権・譲渡権・公衆送信権（送信可能化権を含む）は，著者が上述の著作権譲渡同意書を提出することにより，本学会に譲渡される．本項は，著作者自身の再利用を拘束するものでは無いが，再利用する場合は，編集委員長に通知をすること．
17）掲載料．掲載料は無料とし，論説，総説，原著，報告等については別刷り30部を贈呈する．それを越える分についての費用は著者の負担とする（およそ1部100円）．カ
ラー写真掲載・アート紙希望などの場合は，著者の実費負担とする.
18）原稿の送付先〒169-8555 東京都新宿区大久保3-4-1
早稲田大学理工学研究所 55号館S棟703室
酸素輸液プロジェクト内
日本血液代替物学会 会誌「人工血液」編集部
酒井 宏水 宛
電話: 03-5286-3122, FAX: 03-3205-4740,
E-mail: kita@kenkousya.jp

編集委員会●酒井宏水（委員長），東 寛，大谷 渡，武岡真司，寺嶋克幸，堀之内宏久，村田 満，渡辺真純●日本血液代替物学会 会誌■発行

日本血液代替物学会■編集・制作「人工血液」編集委員会

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vol.16（2） 2008年10月14日発行〒160−8582 東京都新宿区信濃町35
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