IBC’s 5th Annual Conference on Blood Substitutes

ShorrIBC’s 5th Annual Conference on Blood Substitutes IBC’s 5th Annual Conference on Blood

Substitutes

November 20-21, 1997, Boston, MA, USA

Robert GL Shorr

Enzon, Inc., 40 Kingsbridge Road, Piscataway, NJ 08854, USA

IB C’s 5th An nual Con fer ence on Blood Sub sti tutes was held on No vem ber20-21, 1997. Ap proxi mately 100 medi cal re search ers, aca demic sci en tists,blood sub sti tute com pany speak ers and rep re sen ta tives from ma jor phar -ma ceu ti cal com pa nies with an in ter est or part ner in the field at tended. Thepa pers pre sented fo cused on the prog ress in clini cal tri als for those com -pounds in hu man study, on pre clini cal mod els for pre dict ing ef fi cacy in hu -mans, and on novel ap proaches and agents for the de liv ery of oxy gen. Both per fluoro car bons (PFC) and haemoglobin- based oxy gen car ri ers were de -scribed. Sev eral lec tures ad dressed the his tory of the field and fu ture di rec -tions for labo ra tory and clini cal in ves ti ga tion. The fol low ing is a sum maryof some of the pres en ta tions. This sum mary is di vided into 3 sec tions. Thefirst sec tion pro vides a his tori cal over view and dis cusses the changes in theper ceived need for a blood sub sti tute. Sec tion 2 com prises an up date ofcom pany ac tivi ties. The fi nal sec tion fo cuses on likely fu ture di rec tions forlabo ra tory and hu man clini cal study.

Keywords: blood substitutes, haemoglobin, nitric oxide, perfluorocarbons,polyethylene glycol, shock, transfusions, trauma

Exp. Opin. Invest. Drugs (1998) 7(2):283-291

1. Blood substitutes: historical overview and changes in theperceived need for a blood substitute

The safety of the Ameri can do nor blood sup ply was ques tioned with thedis cov ery that HIV- associated AIDS could be trans mit ted through bloodtrans fu sion. Pub lic alarm over AIDS, hepa ti tis and as yet to be dis cov eredad ven ti tious agents fu elled re search into po ten tial blood sub sti tutes.

The rec og ni tion that a blood sub sti tute could be used for mili tary and ci vil -ian trauma medi cine and elec tive sur gery as so ci ated trans fu sion also stimu -lated medi cal and phar ma ceu ti cal com mu ni ties, as well as in ves tors, topar tici pate in blood sub sti tute re search. Both start- up and de vel op mentstage com pa nies with blood sub sti tute pro grammes emerged with ‘warchests’ and de vised strate gies to meet the ar ti fi cial blood chal lenge. The aim was not only to elimi nate the need for typ ing and cross match ing, but wasalso to de velop a safe, ef fi ca cious and read ily avail able al ter na tive to redblood cells.

Dr R Winslow (Uni ver sity of Cali for nia & Vet er ans Af fairs Hos pi tal, San Di -ego, USA) sum ma rised 12 years of col lec tive ef forts in the field, com par ingre cent clini cal prog ress (see sec tion 2) with ear lier at tempts us ing moretoxic ma te ri als. He stated that con sid er able ad vances had been made with

2831998 © Ash ley Pub li ca tions Ltd. ISSN 1354- 3784

Meeting Highlight

1. Blood substitutes: historicaloverview and changes in theperceived need for a bloodsubstitute

2. Company updates

2.1 Enzon

2.2 Alliance

2.3 Apex

2.4 Biopure

2.5 Hemosol

2.6 Northfield Laboratories

2.7 Baxter Healthcare

2.8 Other oxygen therapeutics

3. Future directions for bloodsubstitute research anddevelopment

Bibliography

http://www.ashley- pub.com

Ex pert Opin ion on Investigational Drugs

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the pu ri fi ca tion of hae mo glo bin from cell stroma andother toxic con tami nants. Im prove ments in the sta bi li -sa tion of hae mo glo bin mole cules by chemi cal and ge -netic means, the un der stand ing of tox ic itymecha nisms and un ex pected physio logi cal re sultswere also high lighted. With re gard to the lat ter, DrWinslow dis cussed the im proved un der stand ing ofthe role of ni tric ox ide (NO) and other va soac tivecom pounds, and the regu la tion of blood flow andoxy gen de liv ery at the level of the mi cro cir cu la tion [1]. Dif fer ences in oxy gen de liv ery be tween red bloodcell- encased hae mo glo bin and cell free so lu tionswere also ad dressed rela tive to the regu la tion of blood flow and pres sure.

Dr Winslow also de scribed the dif fer ing roles andcon tri bu tions of the mili tary, aca demic sci en tists,phar ma ceu ti cal com pa nies and the FDA. Two main is -sues were ad dressed: the slower than pre dicted prog -ress made with blood sub sti tutes and the im provedsafety of the US blood sup ply. To day the US bloodsup ply is con sid ered safe and the prob abil ity of trans -mis sion of AIDS or hepa ti tis via trans fu sion re mote.FDA guide lines for more tightly con trolled blood col -lec tion and proc ess ing were cred ited in part for thisim prove ment [2]. Other im prove ments in clude rou -tine so phis ti cated test ing for com mon as well as rarecon tami nat ing in fec tive agents. The ex clu sion fromdo na tion of do nors with high- risk life style pro files,autolo gous blood do na tion pro grammes, the use oferythro poietin to en hance the rate of red blood cellpro duc tion, the sal vage of shed blood and des ig nateddo nor pro grams have also been in sti tuted. A re defi ni -tion of the ‘tran sf usion trig ger’ by sur geons and an aes -the si olo gists for pa tients re quir ing elec tive sur gery oremer gency care has also been called for [3-5]. In short, a ‘co mpet itive’ analy sis for blood looks con sid era blybet ter to day than a dec ade ago.

Dr Winslow also cred ited in ter na tional meet ings, aca -demic sym po sia, and NIH spon sored dia logue un derFDA guid ance as con trib ut ing to the de vel op ment ofblood sub sti tutes. It was quickly rec og nised that‘blood sub sti tutes’ was a mis no mer and hae mo glo binor per fluoro car bon (PFC)-based oxy gen car ri ers is amore de scrip tive term. It also emerged that whilst allcould judge the safety of vari ous prod ucts and ap -proaches di rectly, ‘e ff icacy’ re mained to be de fined.Against the back drop of a ques tion able blood sup ply,many be lieved that a prod uct that could be used inplace of red blood cells, i.e., ‘spa ring’ the use of thisre source, was ef fi ca cious.

To day, how ever, there is no doubt that the risk ofpara site,vi ral or bac te rial dis ease trans mis sion viablood trans fu sion in the USA has dra mati cally de -clined. The US blood sup ply is now con sid ered safe.Fur ther more, while pe ri odic and lo cal blood short -ages may oc cur, an ade quate sup ply of do nor blood isavail able. In deed, the use of blood has ac tu allyplateued or de clined since 1992 [6,7]. Dr Winslow al -luded to the re cent US Army Gulf War ex pe ri ence.This war es tab lished a new para digm of short pe ri odsof in tense con flict rather than pro tracted com bat. De -spite the US Army’s sub stan tial in vest ment in bloodsub sti tute re search (es ti mated at over $20 m), the de -sired char ac ter is tics and ap pro pri ate ness of a bloodsub sti tute for the mod ern bat tle field have been calledinto ques tion [8].

With blood con sid ered safe [9-11], some have ques -tioned the mar ket need for a red blood cell sub sti tutein the de vel oped world. The FDA is es pe cially cau -tious re gard ing blood sub sti tutes. Dr Fratan tonti,former Head of Hae ma tol ogy for the FDA, stated thata ‘d ecrease in the use of a thera peu tic agent as so ci ated with mor bid ity, such as al lo ge neic trans fu sion, is apos si ble end- point, but there must be a re al is tic as -sess ment of risk/bene fit. The pub lic and me dia as sess -ments of the dan gers of al lo ge neic trans fu sion are notcon sis tent with avail able data’ [12]. Chang ing per cep -tions of the need for blood sub sti tutes could in ducethe re- definition of ef fi cacy stud ies and end- points.The Bax ter ex pe ri ence (see sec tion 2) could prove tobe a har bin ger for fu ture regu la tory agency re sponseto red blood cell spar ing tri als. The re shuf fling of ef fi -cacy study pri ori ties would, how ever, ig nore the de -vel op ing world where the in fra struc ture for the safecol lec tion, proc ess ing, and dis tri bu tion of blood islack ing and where there is lit tle ‘safe’ blood to com -pete against [13].

Few com pa nies with blood sub sti tute pro grammespos sess the re sources to un der take a world- wide strat -egy for the de vel op ment, ap proval and dis tri bu tion ofblood. Some ‘su bst itute’ com pa nies have looked to in -her ent phar ma col ogi cal prop er ties of their prepa ra -tions to guide more com peti tive in di ca tion se lec tion.For ex am ple, So ma to gen in ves ti gated the hae ma to -poie tic ac tiv ity as so ci ated with cell free hae mo glo binand anae mia, and Apex stud ied the changes in or ganblood flow and blood pres sure due to NO scav engeand treat ment of shock. Other re search ers havelooked for clini cal in di ca tions where the de liv ery ofoxy gen is not ade quately achieved and can not beachieved by red blood cells, but where oxy gen

© Ash ley Pub li ca tions Ltd. All rights re served. Exp. Opin. Invest. Drugs (1998) 7(2)

284 IBC’s 5th Annual Conference on Blood Substitutes

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de liv ery it self would be bene fi cial. En zon is the fur -thest ad vanced in this re gard, ex plor ing the sen si ti sa -tion of hy poxic solid tu mours to ra dia tion andche mo ther apy [14,15]. Clini cal tri als dem on strat ing ef -fi cacy in ar eas where medi cal de mand has not beenmet are the most likely to ad vance rap idly and suc -ceed in the mar ket place of the de vel oped world.

Thus, it would seem that ‘blood sub sti tute’ niches inthe USA and Europe are now likely to be seen as com -pet ing with an avail able blood sup ply that is both safeand ef fi ca cious, thereby ‘rai sing the bar’ for cell freeso lu tions. How ever, in the de vel op ing world manycoun tries con sider the avail abil ity of a safe and ef fi ca -cious blood sub sti tute a na tional pri or ity. Re cently,the Bra zil ian gov ern ment has es tab lished a Net workPro gramme for Ar ti fi cial Blood with the ob jec tive ofpro vid ing Bra zil ian medi cine with us able blood sub -sti tutes. In es tab lish ing strate gies for the de vel op mentand ap proval of blood sub sti tutes, it will be nec es saryfor com pa nies to rec og nise both the global dif fer -ences, as well as the shifts in com peti tive as sump tionsand mar ket needs.

2. Company updates

2.1 Enzon

En zon has fo cused its at ten tion on poly eth yl ene gly -col con ju gated bo vine hae mo glo bin (PEG Hb) as anoxy gen car rier. The com pany has adopted a clini caltrial strat egy based upon the dem on stra tion of thesen si ti sa tion of hy poxic tu mours to ra dia tion can certher apy. Solid tu mour hy poxia is be lieved to be a fac -tor in ra dia tion re sis tance and poor prog no sis formany can cers [16-18]. The com pany is con fi dent thatthis ap proach will re sult in ac cel er ated ap proval dueto the un met medi cal need by red blood cells in thisarea. While the com pany also be lieves its prod uct tohave util ity as a red blood cell sub sti tute for traumaand trans fu sion medi cine, these in di ca tions will bepur sued later or in col labo ra tion with a cor po ratepart ner.

PEG Hb is for mu lated as a 6 g/dl so lu tion and storedfro zen, hav ing a shelf life of nearly 2 years. Con ju ga -tion is based upon the at tach ment of monoac ti vatedpoly eth yl ene gly col strands (MW = 5000 Da) to thesur face amino groups of hae mo glo bin. No crosslink -ing or pyri doxy la tion of the pro tein is per formed [19].

The com pany has com pleted Phase Ia es ca lat ing dosesafety stud ies in healthy vol un teers and is pres ently

con duct ing Phase Ib es ca lat ing mul ti ple dose safetystud ies in can cer pa tients un der go ing ra dia tion ther -apy. The com pany plans to com plete these stud iesand be gin ef fi cacy stud ies in 1998.

In hu mans a pro longed cir cu lat ing life time with ahalf- life of > 48 h has been ob served. The com panybe lieves this fea ture to be bene fi cial to can cer pa tients re ceiv ing frac tion ated ra dia tion ther apy on a daily ba -sis for sev eral weeks, as re duced, more con ven ientdos ing is made pos si ble.

Pre clini cal ro dent stud ies have shown that hy poxicsolid tu mours can be oxy gen ated with PEG Hb andthat this oxy gena tion leads to an in crease in sen si tiv ity to ra dia tion treat ment. De pend ing upon the mod elsse lected, com plete tu mour re gres sion or sub stan tialgrowth de lay can be achieved [14]. In stud ies withdogs pre sent ing spon ta ne ous na sal car ci noma, thecom pany has shown that the ad mini stra tion of PEGHb in com bi na tion with ra dia tion ther apy re sults inmarked tu mour re gres sion. Na sal car ci no mas in dogsare thought to be hy poxic and typi cally re spondpoorly to ra dia tion treat ment.

Dr G Ara et al. (Har vard Medi cal School Dana- FarberCan cer In sti tute, Bos ton, USA) have in ves ti gated thecom bi na tion of solid tu mour oxy gena tion via PEG Hbwith che mo ther apy [15]. Dr Ara ex plained that tu -mours are het ero ge ne ous and of ten con tain re gions of hy poxia. Many an ti neo plas tic agents have been dem -on strated to de pend on cel lu lar oxy gena tion for op ti -mal ef fects. Mecha nisms in clud ing the meta bolic stateof cells and sus cep ti bil ity to cy to toxic agents, as wellas the re quire ment by some agents of mo lecu lar oxy -gen for drug ac tiv ity, have been sug gested. Dr Arashowed that PEG Hb has a pro longed cir cu lat inghalf- life in rats (> 20 h) and oxy gen ates tu mours in thepres ence of room air, 28% oxy gen or car bo gen (95%oxy gen, 5% car bon di ox ide). In the pres ence of 28%oxy gen or car bo gen, this ef fect con tin ued for morethan 48 h and was found to be dose- dependent. Op ti -mal re sults were ob tained for 4 and 6 ml/kg doses. Us -ing a mi croe lec trode in ser tion tech nique, Dr Arashowed that the per cent age of rat mam mary tu mourcells (13762) which are hy poxic (PO2 < 5 mmHg)could be de creased from 55 - 46%, 45 - 26%, and 36 -19% for room air, 28% oxy gen and car bo gen, re spec -tively. When com bined with ra dia tion (2, 3, or 4 Grayper frac tion daily for 5 days) a dose of 6 ml/kg PEG Hb was found to sub stan tially en hance growth de lay over ra dia tion plus air, 28% oxy gen or car bo gen onlycon trols.


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Dr Ara de scribed the re sults ob tained with an ti neo -plas tic agents, such as pa cli taxel, cy clo phos pha mide,adria my cin, fluor ou ra cil and BCNU. In these stud ies,mice bear ing mam mary EMT-6 tu mours re ceived PEGHb (6 ml/kg), the che mo thera peu tic se lected at afixed dose and sched ule pat terned af ter hu man use,and breathed room air, 28% oxy gen or car bo gen. A 2-to 4- fold in crease in growth de lay was ob served along with a de crease in the number of lung me tas ta ses forthose ani mals re ceiv ing PEG Hb over those which re -ceived the che mo thera peu tic and air, 28% oxy gen orcar bo gen only. Im por tantly, Dr Ara noted that this in -crease in sen si tiv ity of tu mours to well known an ti -neo plas tic agents was not ac com pa nied by a markedin crease in bone mar row tox ic ity [15].

Drs Acha rya and Fried man (Al bert Ein stein Col lege ofMedi cine, Bronx, NY, USA), not as so ci ated with En -zon, con tin ued stud ies with hae mo glo bin PE Gy la tion. They pre sented their re sults with hu man hae mo glo -bin and Bis Mal PEG 2000, a sulf hy dryl di rectedcrosslink ing agent. Con ju ga tion was oxy con for ma -tion spe cific, had very lit tle in flu ence on oxy gen af fin -ity and could be achieved un der mild con di tions.

2.2 Alliance

Al li ance in con junc tion with its part ners Or tho Bio -tech and John son & John son has fo cused on the de -vel op ment of PFC emul sions as a blood sub sti tute [20]. Un like hae mo glo bin so lu tions, the com pa ny’s leadprod uct, OXYGENT, is non- proteinaceous PFC (per -fluoro oc tyl bro mide) emul si fied with egg yolk phos -pholipid [21]. As the ma te rial is en tirely syn thetic,there is no risk of dis ease trans mis sion from hu man orani mal blood prod ucts.

Dr P Keipert (Al li ance, San Di ego, CA, USA) de scribed the sub mi cron (~ 0.17 µm) PFC par ti cles as float ing inthe plasma and eas ily per me at ing the micro vas cu la -ture to pre vent or al le vi ate tis sue hy poxia. He re -ported that in clini cal stud ies a short du ra tion feb rilere sponse 4 - 6 h post- dosing and a tran sient de creasein plate let count were ob served. Com pared to ear lierPFC for mu la tions the in ci dence, du ra tion and mag ni -tude of fe ver were greatly re duced (1 - 2 °C in 5/32 pa -tients re ceiv ing OXYGENT). Over 200 healthyvol un teers have now re ceived per flub ron emul sion. A sup ple men tal study in 48 healthy vol un teers (24 male, 24 fe male), in ves ti gat ing the ef fects on plate let ho -meo sta sis and im mune func tion pa rame ters, showeda de crease in plate let count (less than 20% at 3 dayspost dos ing) in the high- dose groups (1.8 g/kg). No

ef fect on any in di ca tors of plate let func tion tested (exvivo ag gre ga tion, tem plate bleed ing times, co agu la -tion pa rame ters, PT, PTT and fi brino gen), com ple -ment ac ti va tion im mu no genic or evi dence ofsup pressed hu mo ral or cel lu lar im mune re spon sive -ness were ob served. No liver, pul mo nary, re nal orclini cally mean ing ful ef fects on blood chem is try werefound and no hae mo dy namic changes or evi dence ofvaso con stric tion was seen.

The com pany be lieves that the PFC emul sion par ti cles are cleared from the plasma by phago cytic up takethrough the Kupf fer cells of the liver and macro phagein the spleen (RES). Phar ma coki netic analy sis shows a dose- dependent cir cu lat ing half- life of 6.1 ± 1.9 h for a 1.2 g/kg dose, and 9.4 ± 2.2 h for a 1.8 g/kg dose.Clini cal stud ies in elec tive sur gery pa tients are ex am -in ing doses up to 2.7 g/kg.

Al li ance is in ves ti gat ing the en hance ment of autolo -gous red blood cell use [20,21]. Dr L Steh ling elabo -rated fur ther on the util ity of ad juncts to autolo goustrans fu sion tech niques, stat ing that tem po rary oxy gen car ri ers could en able sub stan tially greater vol umes ofblood re moval dur ing autolo gous do na tion, sig nifi -cantly de lay the need for blood re in fu sion and pro -vide a greater safety mar gin at lower hae ma to crit.

Two mul ti cen tre, ran dom ised, con trolled, single- blind Phase IIb stud ies with per flub ron emul sion have been com pleted. A to tal of 256 (147 or tho pae dic, 109urologi cal or gy nae co logi cal) sur gi cal pa tients inEurope or the USA were evalu ated with per flub ronuse as a tem po rary oxy gen car rier. Study ob jec tiveswere to dem on strate the util ity of per flub ron in re -vers ing trans fu sion trig gers (physio logi cal pa rame tersthat in di cate a need for blood), in com pari son toautolo gous blood. Ex am ples of pre de fined trans fu -sion trig ger pa rame ters meas ured were: in creases incar diac out put, heart rate, and de creased blood pres -sure. As in healthy vol un teers no clini cally sig nifi cantside- effects were re ported and Dr Kiepert stated thatthe de sired pri mary study end- points were met withper flub ron emul sions, sig nifi cantly de lay ing the needfor sub se quent blood trans fu sion. Full re sults fromthese stud ies will be pre sented at the 1998 Euro peanSo ci ety of An aes the si ol ogy Meet ing (Bar ce lona,Spain) and the 1998 In ter na tional An aes the sia Re -search So ci ety Meet ing (Or lando, USA). Piv otal tri alsto dem on strate the ef fi cacy of per flub ron as a tem po -rary oxy gen car rier for trans fu sion trig ger re ver sal areex pected to be gin soon.



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Two Phase II stud ies us ing per flub ron emul sion, look -ing for im proved per form ance over red blood cells incar diac sur gery were also de scribed. Ap proxi mately60 pa tients re ceiv ing car dio pul mon ary by- pass wereen rolled. One study fol lowed cere bral and sys temicoxy gena tion and post by- pass neu ro be hav iouralfunc tion. The sec ond study was de signed to de ter -mine the low est hae ma to crit that could be safelyreached dur ing by- pass fol low ing autolo gous bloodhar vest. A third study in this pa tient group to de ter -mine dose- related phar ma coki net ics is in prog ress.

Post by- pass neu ro be hav ioural defi cit of vari ous de -grees and du ra tion is as so ci ated with this sur gi cal pro -ce dure. De creased brain oxy gena tion due to bloodloss/di lu tion and by- pass pump me di ated pul sa tileflow may be in volved. In creased brain oxy gena tionand the dem on stra tion of im proved neu ro be hav ioural out come would be an im por tant and valu able medi cal ad vance in the area of cere bral is chae mia and reper fu -sion [22].

Likely fu ture in di ca tions to be ex plored for per flub ron emul sions are neu ro pro tec tion dur ing ca rotid end- arterectomy or in trac ra nial vas cu lar sur gery, myo car -dial pro tec tion dur ing by- pass, and limb and kid neypro tec tion dur ing vas cu lar or re nal sur gi cal pro ce -dures where per fu sion is im paired.

2.3 Apex

Apex has fo cused its at ten tion on the use of pyri doxy -lated polyoxyeth yl ene (POE) con ju gated hu man hae -mo glo bin (PHP) for the treat ment of NO- inducedshock [23]. NO- induced shock is char ac ter ised by apre cipi tous de cline in mean ar te rial pres sure. Thecom pa ny’s poly mer con ju ga tion strat egy util ises a bi -func tional POE unit of MW = 3000 Da to pro duceHb-(OCH2CH2)-COO- along with a mix ture ofcrosslinked tet ra mer, oc tamer and 12- mer in de finedra tios.

Dr D Tra ber (Uni ver sity of Texas, Galve ston, USA) de -scribed NO as the most ac cepted di la tor for sep sis. Ofthe three forms of ni tric ox ide syn thetase (NOS), thein duci ble form (iNOS) is in duced by en do toxin andcy to ki nes as so ci ated with sep tic shock. NO- mediatedshock may oc cur in as many as 800,000 cases per yearwith the pri mary ini tial treat ment ob jec tive be ing asta bi li sa tion of hae mo dy nam ics and pre ven tion ofoxy gen defi cit due to NO- mediated in creases in lo cal -ised vas cu lar bed vaso di la tion. The pres ent treat mentre lies on the use of cate cho la mines to coun ter actvaso di la tion in the larger ves sels. Dr J DeAn gelo

(Apex, North Caro lina, USA) sug gested that the NOsen si tive ves sels are the micro vas cu la ture and that this would be the most im por tant site of ac tion for NO- directed thera pies.

Dr Tra ber de scribed the con ver sion of NO to ni tritesand ni trates on in ter ac tion with hae mo glo bin. Stud iesof PHP il lus trat ing the im pact of PHP ad mini stra tion in ovine mod els of sep tic shock were dis cussed [24,25].A con tinu ous in fu sion of 20 mg/kg could re turn meanar te rial pres sure to base line lev els. On re moval of thedrug, pres sure again fell to lev els as so ci ated withshock. Dr DeAn gelo sug gested that PHP is able toscav enge NO as i t ex tra vasates from themi cro cir cu la tion.

In Phase II clini cal tri als in pa tients suf fer ing shock the com pany is ad min is ter ing 25, 50, and 100 mg/kgdoses as 200 ml bo lus in fu sions over 30 min. The com -pany re ports the drug to be well- tolerated and ca pa -ble of in creas ing mean ar te rial pres sure at the 50mg/kg and 100 mg/kg doses.

2.4 Biopure

Bio pure is de vel op ing two for mu la tions of bo vinehae mo glo bin po lym er ised with glu taral de hyde:HBOC-301 (OXYGLOBIN) for vet eri nary ap pli ca tionsand HBOC-201 (HEMOPURE) for hu man thera peu ticuse [26]. The safety and ef fi cacy of HBOC-301 hasbeen re cently ap proved by the FDA Cen ter for Vet eri -nary Medi cine for use in anae mic dogs. Po lym eri sa -tion re sults in a prod uct mix ture com pris ing a range of mo lecu lar weights. HBOC-301 has an av er age mo -lecu lar weight of 200,000 Da and HBOC-201 has anav er age mo lecu lar weight of 250,000 Da. HBOC-201con tains smaller amounts of lower mo lecu lar weighthae mo glo bin spe cies. Both prod ucts are sta ble atroom tem pera ture for at least two years and are for -mu lated as 13 g/dl so lu tions with a P50 of 38 and an in -tra vas cu lar half- life of 24 - 36 h.

Dr M Gaw ryl (Bio pure, Cam bridge, MA, USA) statedthat Bio pure’s hae mo glo bin pu ri fi ca tion and po lym -eri sa tion pro cess was well con trolled and highly re -pro duci ble. She also de scribed Bio pure’s auto matedplant, re cently pre- approval in spected with out ci ta -tion by the FDA.

Dr Gaw ryl dis cussed the prob lem of Mad Cow Dis -ease, i.e., Bo vine Spongi form En cepha li tis (BSE), andthe po ten tial for con tami na tion of raw ma te ri als withpri ons or vi rus, pro pos ing the mini mi sa tion of risk byherd man age ment. In par ticu lar, re strict ing the use of


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ani mals to those ob tained from BSE free re gions of the world, who are less than 30 months of age and withdocu men ta tion from birth of good health. The com pa -ny’s pu ri fi ca tion pro ce dure as well as po lym eri sa tionchem is try was also shown in vali da tion stud ies con -ducted by MA Bioserv ices to re move and/or in ac ti vate model vi rus and BSE- type causa tive agents.

Dr Gaw ryl also ad dressed the po ten tial im mune sys -tem re ac tions to bo vine hae mo glo bin, point ing outthe greater than 92% amino acid se quence ho mol ogybe tween bo vine and hu man hae mo glo bin and high -light ing the ex pe ri ence with 350 pa tients who re -ceived sin gle or mul ti ple doses of HBOC-201 with outim mune re sponse.

Dr V Rentko (Bio pure) pre sented the re sults of clini cal stud ies ex am in ing HBOC-301 for the treat ment ofanae mia in dogs. Anae mic dogs re quir ing trans fu sionwere ran domly as signed to two groups: a treat mentgroup that im me di ately re ceived treat ment withHBOC-301 and a con trol group that re ceived no treat -ment un less their con di tion de te rio rated. If dogs in ei -ther group re quired ad di tional oxy gen car ry ingso lu tion in fu sion within 24 h they were iden ti fied astreat ment fail ures and were ad min is tered HBOC-301(treat ment group) or packed red blood cells (no treat -ment group). Plasma hae mo glo bin, at ti tude, ac tiv ityand heart rate of these dogs were evalu ated for 24 hand dogs moni tored for a to tal of 72 h. HBOC- 301- treated ani mals showed sta tis ti cally sig nifi cant im -prove ments com pared to con trols in fail ure rate, timeto fail ure and gen eral physi cal con di tion and at ti tude.Drug- related ef fects were mild tran sient mu cousmem brane dis col ou ra tion, hae mo globin uria, in -creased blood pres sure, ele vated as par tate ami no -trans fe rase ac tiv ity and gas tro in tes ti nal (GI) ef fects.

Dr W Hoff man (The Cleve land Clinic Foun da tion,USA) de scribed hu man clini cal stud ies withHBOC-201 (see also [27,28]) and sum ma rised the re -sults of a Phase I/II sur gi cal study in volv ing 6 US cen -tres. The pur pose of the study was to evalu ate a sin glein traop era tive dose of HBOC-201 (0.6, 0.9, 1.2, 1.5,2.0, and 2.5 gHb/kg) com pared to pla cebo Lac tatedRin ger’s So lu tion con trols. Pa tients were ex pected tore quire at least 1 unit of blood, had no sig nifi cant co- morbidity and gave in formed con sent. The sin gleHBOC-201 dose was given af ter ap proxi mately 500 ml of blood loss. In this study, 64 adult, ran dom ised pa -tients re ceived HBOC-201 (42 pa tients, to tal dose 35.4- 205 g) or Lac tated Rin ger’s So lu tion (22 pa tients).HBOC-201 was well- tolerated. One case study

in volv ing an ab domi nal aor tic an eu rysm re pair washigh lighted. This pa tient re ceived a sin gle 118 g doseof HBOC-201 which was cred ited with achiev ing in -traop era tive sta bil ity with out tox ic ity or re quire mentfor ad di tional in traop era tive red blood cell trans fu -sion. No hae mo globin uria or re nal dys func tion wasob served. The com pany is con tinu ing this study along with oth ers de signed to dem on strate red blood cellspar ing in the peri- operative set ting and both car diacand non- cardiac sur gery arena.

2.5 Hemosol

He mo sol has fo cused its at ten tion on the de vel op -ment of o- raffinose po lym er ised hae mo glo bin(HEMOLINK) as a red blood cell sub sti tute [29]. Dr AMag nin (He mo sol, Eto bi coke, On tario, Can ada) de -scribed the com pa ny’s his tory and tech nol ogy plat -form for hae mo glo bin pu ri f i ca t ion andpo lym eri sa tion. In ad di tion to bio chemi cal char ac teri -sa tion, Dr Mag nin also re ported on vi rus re moval andin ac ti va tion vali da tion stud ies. Both pas teuri sa tionand mem brane fil tra tion stud ies were shown to in ac ti -vate or re move model test vi rus, re spec tively.

Dr G Biro (He mo sol) de scribed a se ries of ani malstud ies dem on strat ing safety and ef fi cacy in a va ri etyof mod els. Phase I hu man clini cal tri als ex am in ingsafety and phar ma coki net ics in healthy vol un teerswere also de scribed. An es ca lat ing dose (0.025 - 0.6g/kg), ran dom ised, placebo- controlled blind studyfol lowed 42 sub jects (33 of whom re ceivedHEMOLINK) on the pre- infusion day, 72 h post- infusion and at 1 and 6 weeks follow- up. Hae mo dy -nam ics, pul mo nary func tion, clini cal chem is try, hae -ma tol ogy, GI exam and phar ma coki net ics weremoni tored. The plasma cir cu lat ing half- life was 12 - 17 h. A mild bra dy car dia and dose- dependent in crease in blood pres sure were ob served along with com plaintsof GI up set, gas, pain and dysphagia. Clini cal evalua -tion look ing at HEMOLINK util ity and red blood cellspar ing in or tho pae dic sur gery is in prog ress.

2.6 Northfield Laboratories

Northfield Labo ra to ries is de vel op ing hu man hae mo -glo bin, pyri doxy lated and po lym er ised with glu taral -de hyde (POLYHEME), as a blood sub sti tute for largevol ume use [30]. The av er age mo lecu lar weight forPOLYHEME is 150,000 Da with lit tle or no sin gle tet ra -mer or na tive hae mo glo bin pres ent. For mu la tion is at10 g/dl. The P50 value is 28 - 30 and the cir cu lat inghalf- life in hu mans is 24 h. In Phase II hu man clini caltest ing in trauma pa tients, 51 pa tients re ceived 1 - 2



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units (500 ml/unit POLYHEME), 20 pa tients re ceived 3 - 4 units, 7 pa tients re ceived 21 units and 10 pa tientsre ceived 10 units. No feb rile re sponse, ele va tions inblood pres sure, changes in heart rate or in su lin clear -ance were ob served [31-33]. In ad di tional Phase IIstud ies the com pany evalu ated POLYHEME bloodlev els. The re sults sug gested that POLYHEME can cor -re late to whole blood on a unit to unit ba sis [30]. A 240pa tient Phase III mul ti ple site clini cal trial ex am in ingthe elimi na tion of al lo ge neic blood use in elec tive sur -gery is ex pected to re quire 12 months for com ple tion.

2.7 Baxter Healthcare

Bax ter Health care is en gaged in de vel op ing Di as pi rincrosslinked hu man hae mo glo bin (DCLHb), alsoknown by its trade name He mAs sist [34]. DCLHb hasbeen evalu ated in 12 com pleted Phase I/II clini cal tri -als and was the first modi fied hae mo glo bin for mu la -tion to be evalu ated in ran dom ised con trolled piv otalclini cal stud ies. The mo lecu lar weight of DCLHb is64,000 Da and the prod uct is pack aged as a 10 g/dl250 ml unit.

In piv otal tri als at 8 hos pi tals in France, Bel gium, andthe UK, 209 car diac sur gery pa tients re ceiv ing up to750 ml DCLHb were evalu ated post by- pass sur gery.Fifty- nine per cent of pa tients for whom blood trans fu -sion was in di cated did not re quire trans fu sion on theday of sur gery. Af ter 7 days, 19% of pa tients did not re -quire trans fu sion. In these stud ies, DCLHb in fu sionwas well- tolerated with tran sient skin dis col ou ra tion,hae ma turia, ele vated blood pres sure and en zyme ele -va tions ob served in some pa tients.

In April 1997, a mar ket ing authori sa tion ap pli ca tionwas filed in Europe. Sur pris ingly, this ap pli ca tion wasde layed and ad di tional clini cal trial data re quested. Dr T Estep (Bax ter Health care, Deer field, IL, USA) statedthat ad di tional data would come from one or more ofthe three Phase III clini cal tri als cur rently un der way inEurope and the US. These tri als are ex am in ing DCLHb in pa tients suf fer ing from haem or rhagic shock. Thepri mary end- point in this popu la tion, with an ex -pected mor tal ity of 40%, will be mor tal ity at 28 days.The sec on dary end- point will be the evalua tion ofmul ti ple or gan mor bid ity. Dr Estep stated that, as sum -ing the suc cess ful com ple tion of one or more of thesePhase III tri als, mar ket ing authori sa tion is ex pected inlate 1999 or early 2000.

2.8 Other oxygen therapeutics

Dr R Spears (Wayne State Uni ver sity, USA) and PaulZale sky (Therox, USA) in tro duced the con cept of theuse of satu rated aque ous oxy gen in fu sates for oxy gende liv ery. The core tech nol ogy is based upon the abil -ity to dis solve large amounts of oxy gen in fluid in or -der to cre ate a sta ble su per satu rated oxy gen so lu tion.To de liver this so lu tion bub ble free, an ex tra cor po realcir cuit is used to con trol re gional ar te rial hy per ox ae -mia. A pump is used to main tain flow through a stan -dard guide cathe ter to achieve re gional de liv ery. DrSpears used a rab bit model for hy pox ae mia and lowflow myo car dial is chae mia to il lus trate the safety andef fi cacy of this tech nique. In a ca nine model for myo -car dial is chae mia com pared to autoreper fu sion con -trols, aque ous oxy gen de liv ery showed a markedim prove ment of left ven tricu lar func tion both glob ally and re gion ally, with a re duc tion in ex tra sys tole fre -quency and an im prove ment of micro vas cu lar bloodflow. Nor mox ae mic reper fu sion was as so ci ated withno im prove ment in left ven tricu lar func tion and an in -crease in ex tra sys tole fre quency.

3. Future directions for blood substituteresearch and development

Since the prog ress of ex ist ing blood sub sti tute can di -date mole cules into the clinic, ques tions and an swersre gard ing trans fu sion medi cine prac tice, the col lec -tion and dis tri bu tion of blood and the safety of thesup ply have also arisen.

At the most ba sic level, the is sue of how low hae ma -to crit can go be fore a trans fu sion is cur rently de bated.Com pa nies, such as Bio time, are claim ing that plasmaex pander so lu tions, com bined with cryo sur gi cal pro -ce dures, can re duce the need for blood. Blood sal -vage ma chines and erythro poietin are now also be ingused.

In re ac tion to AIDS and hepa ti tis con cerns, both FDAand blood col lec tion/dis tri bu tion or gani sa tions havees tab lished and fol lowed guide lines for so phis ti catedtest ing and qual ity con trols.

Thus, the cur rent situa tion ap pears to be one wherethe use of blood is chang ing and where the risk fordis ease trans mis sion by con tami na tion (while real) isper ceived as small. For the de vel oped world, there -fore, pres sure to de fine niches for blood sub sti tuteswhere higher per form ance bene fits over blood can be shown is likely to in crease.


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The idea of red blood cell ‘spa ring’ as the jus ti fi ca tionfor the in tro duc tion of blood sub sti tutes onto theUS/Euro pean mar ket is dif fi cult to main tain with there ali sa tion that the blood sup ply is ‘safe’. This hasbeen il lus trated by the Bax ter case. Bax ter was re -quested for a greater dem on stra tion of ef fi cacy af ter amar ket ing ap pli ca tion was filed in Europe for DCLHbto be used as a red blood cell ‘spa ring’ agent.

How ever, clini cal trial re sults with PFC emul sions andhaemoglobin- based oxy gen car ri ers are yield ing im -pres sive re sults. Fur ther more, as pointed out by Dr HKlein (War ren G Mag nu son Clini cal Cen ter, Na tionalIn sti tutes of Health, USA), the po ten tial for hu man er -ror ex ists and the threat of as yet un known vi ruses orin fec tious agents re mains. As il lus trated by Dr S Gould (Northfield Labo ra tory, USA), the time taken for typ -ing and cross match ing of blood may be criti cal formany pa tients suf fer ing from large vol ume blood lossand trauma. Clearly, the avail abil ity of a so lu tion freeof this re quire ment would add enor mous flexi bil ity tothe res cue pro cess.

For the de vel op ing world, where a blood col lec tionand dis tri bu tion in fra struc ture is lack ing or pro hibi -tively ex pen sive, a sub sti tute for blood across di verseuses is not a mat ter of in cre mental im prove ment orcon ven ience, but a na tional pri or ity.

Pos si ble fu ture strate gies for blood sub sti tute re search in clude the con tinu ing dem on stra tion of the abil ity to‘spare blood’ in spe cific niches, and, more im por -tantly, the abil ity to ‘truly’ sub sti tute for red blood cells in more di verse ap pli ca tions. This lat ter ca pa bil itymay re quire the modi fi ca tion of ex ist ing mole cules, or the in ven tion of new prod ucts with longer cir cu lat inglife times, and per haps other unique fea tures. Thebene fits of long cir cu lat ing life so lu tions ca pa ble ofde liv er ing oxy gen, but yet pos sess ing a high col loidos motic pres sure, are be ing seen in a va ri ety of ani mal mod els for re sus ci ta tion. These so lu tions, most no ta -bly PEG Hb, may pos ses both plasma ex pan sion ca pa -bili ties, ri val ling ear lier or in ves ti ga tive starchso lu tions, while main tain ing an abil ity to de liver oxy -gen [35].

For haemoglobin- based oxy gen car ri ers, side- effectshave been de scribed as GI up set, dysphagia, ele vatedblood pres sure, gas, oe so phag eal spasm and pain.With most hae mo glo bin so lu tions both in ten sity anddu ra tion have var ied. Some com pounds have beengen er ally given to pa tients un der an aes the sia wheresymp toms would not be re ported, other drugs weread min is tered to healthy vol un teers, or pa tients whom

were con scious, only some of whom were mildly pre-or post- medicated with well char ac ter ised smoothmus cle re lax ants.

For those com pa nies de vel op ing hae mo glo bin prod -ucts with the more se vere ef fects, re search and de vel -op ment of more side- effect free ‘ge ner ations’ is likely.Al ready, So ma to gen has re ported the de vel op ment ofhae mo glo bin mu tants with de creased re ac tivi ties to -wards NO. Ex plo ra tion of clini cal in di ca tions whereblood sub sti tutes can be uniquely ef fec tive over redblood cells is not only likely to con tinue, but also toex pand as the phar ma col ogi cal prop er ties of the dif -fer ent ma te ri als be come bet ter un der stood.

All would agree that the dem on stra tion of ef fi cacy inar eas where medi cal need is not fully met by cur rentmedi cal prac tice will be most likely to re ceive ac cel er -ated ap proval and rapid mar ket pene tra tion. The suc -cess of blood sub sti tutes will not be based on theen hance ment or sub sti tu tion of red blood cells, butthe abil ity to ‘out -perform’ blood it self.

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2. Guide line for qual ity as sur ance in blood es tab lish -ments 7/11/95. Rockville MD Cen ter for Bi ol ogics Re searchand Re view/FDA (1995).

3. STEH LING L, SI MON TL: The red blood cell trans fu siontrig ger: physi ol ogy and clini cal stud ies. Arch. Pa thol.Lab. Med. (1994) 118:429- 434.

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7. SI MON TL: The quan ti ta tive analy sis of trans fu sionmedi cine. (1995) 35:799- 800

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26. LIGHT WR, JA COB EE, RENTKO VT, GAW RYL MS: Use ofHBOC- 201 as an oxy gen thera peu tic in the pre clini caland clini cal set ting. In: Red Blood Cell Sub sti tutes. Ru -dolph AS, Ra bi no vici R, Fuer stein GZ (Eds.), Mar cellDekker, USA (1998):421- 436.

27. OR RIN GER EP, GON ZALEZ PM, HACK NEY AC et al.: Aphase I study of po lym er ized bo vine hae mo glo bin(PNH) in adult pa tients with sickle cell dis ease not incri sis at time of study. In: Sickle Cell Dis ease and Tha las -semias. Beuzard Y, Lu bin B, Rosa J (Eds.), John Lib bey Eu ro -text, Mon trose, USA (1995) 234:301- 302.

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32. GOULD S, MOORE E, MOORE F et al.: The clini cal util ityof hu man po lym er ized hae mo glo bin as a blood sub sti -tute fol low ing trauma and emer gent sur gery. J. Trauma(1995) 39:157.

33. GOULD S: Hu man po lym er ized hae mo glo bin firstthera peu tic use as a blood sub sti tute in trauma andsur gery. Ameri can col lege of Sur geons 81st Clini cal Con -gress. New Or leans, LA, USA (1995) Oct 22- 27.

34. NEL SON DJ: He mAs sist: de vel op ment and clini cal pro -file. In: Red Blood Cell Sub sti tutes . Ru dolph AS, Ra bi no viciR, Feuer stein GZ (Eds.), Mar cell Dekker, USA (1998):353- 400.

35. VAN DE GRIFF KD, ROHLFS RJ, WINSLOW RM: Col loid os -motic ef fects of hae mo glo bin based oxy gen car ri ers.In: Ad vances in Blood Sub sti tutes: In dus trial Op por tu ni tiesand Medi cal Chal lenges. Winslow RM, Van de griff KD,Intali etta M (Eds.), Birk hauser, Bos ton, MA, USA(1997):207- 223.

Robert GL Shorr Enzon, Inc., 40 Kingsbridge Road, Piscataway, NJ 08854, USA


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y.

Documents

IBC’s 5th Annual Conference on Blood Substitutes