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PFC study

PFC study

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tSBTScicn('('S~iesI2006J 1.

152-160

ORIGINAL PAPER

e 2006 Th! Authors.

Journ~1 compilation@ 2006 Blackwell Publiy,;ng

4PS-22-01

Overview of artificial 02 carriers

A. Pape. H. Kertsroo, J. Meier, B. Zwissler a o. Hubler Johann Wolfgang ~the Uni~'t'rsity, Frankfurt Am Moifl. Germany

Introduction

Artifrclal 02 earners aim at increasing arterial O2 content and 02 delivery to the tissues. thereby offering a promlslng alternative to the transfusion of allogeneic red blood cells IRBC).

Despite continuous advancement of Quality assurance regarding the production and transfusion of allogeneic blood products, the transfusion of bank cd blood is still 35S0- elated with relevant risks, the most serious of which being allergic reactions. transfuslon-rela led lung injul)' (1RALI), the accidental transfusion of incompatible blood ('clerical error' related to the mix-up of cross-milt chlng specimen or blood product) and the transmission of viral and bacterial infections (Hepatitis. HIV, eMY, EBV}. Moreover, a deliberate transfusion regimen (i.e. indication for transfusion at Hb ;. 10 gld]) seems to be associated with devilled perioperative morbidity and mortality [1].

Transfusion-related morbidity as well as continuously rising costs of blood products impose significant costs on the public health systems: althoughthe willingness to donate blood had temporarily increased after 1 J September 2001 in the USA, the Food and Drug Administration (FDA) realises a constantly decreasing rate of blood donation and therefore prognoses a shortage of 4 million units of banked blood by 2030. AcC'ol1ijng to public health Statistics, 43Clb of all donated blood products are transfused to patients aged 65 or older. Because this patient pcpulatlon is growing consistently with the common demographic development, the demand for banked blood will increase significantly within the next years. Due to the growing imbnlancc between decreasing availability and increasing demand, the costs of blood products are supposed to double until 20)0 [2,3].

11](: expected cost exploslon in transfusion medicine underlines the sodn-economic signifrcance of snfcly and effectively applicable altcmatives to the transfusion of allogeneic RDC. The Iargc-scalcproductlen and application of'synthetic 0.2 camel'S, however, requires the proof of cfTIcacy in situations normally requiring the trnnsfusi on ofRBC (i.e. extreme anaemia with critical limitation of 0.2 transport capacity). Moreover. the risk profile of artihcial 0.2 carriers should at least bearthe comparison with the excellent quality standard of banked blood.

152

Presently. 1\'10 types of artificial 0.2 carrieres are under experimental and clinical research:

1 Haemoglobin-based oxygen carriers (HBOC), Le, solutions based on isolated human or bovine haemoglobin (Hb).

2 Synthetically processed pcrtluorocarbons tpFC).

Haemoglobin-based oxygen carriers

Although first experimental injections of Hb have already been reported in 189B (4]. the three-dimensional structure of the terrameric haemoglobin molecule was frrst completely elucidatedby Perutzand Kendrew in 1969 [5}. The 02~ dissociation-kinetic of the Hb molecule is described by a sigmoid CUNe witha physiological p50 ll.e, 0.2 partial pressure at which 50% of Hb is saturated with 02) of 26·8 mmHg. Hb used for manufacturing of HBOCs originates from outdated human red cells. from bovine blood or is genetically engineered.

First-generation HBOC

The first approach to use an HBDe therapeutically was documented by Amberson in 1949. A 22-year-old female patient suffered a severe postpartum haemorrhage due to incomplete placental separation. After transfusion of all compatible blood products available in the blood bank haemorrhagic shock was still persisting, so that Amberson infused a total of 2 J of a self-made haemoglobin solution. Already after Infusion of 300 ml, shock-related hypotension reverted instantaneously. Desplte a haematocrit of only 5'5%. the patient was breathing spontaneously: haernodynamic stability as well as adequate Vigilance were maintained, both indicating sufficlent oxygenation of heart and brain. However, on day 9 post partum, the patient evolved severe pyrexia and finally died from acute renal failure [5].

Despitethe fatal outcome of thepatient, this case report provides principal evidence about the eflkacy of isolated haemoglobin regarding 0l transport and tissue oxygenation. In the following 30 years, a couple of unfavourable sideeffects of'fnst generation' HBOCs had been identified:

I Contamination of the Hb-solution with erythrocytestroma and endotoxlnes.

2 Spontaneous dissociation of extracellular Hb-tetramers into a~-dimers, (X- or Ii-monomers. Consequences: Increase of plasma-osmolarity, accelerated glomerular filtration, reduction of intravascular half-life.

3 Nephrotoxic effects related to erythrocyte stroma. endotoxlnes and to the glcmcrular filtration of mono- and dimmers with consecutive precipitation in the ascending part of the loop of Henle.

4 Markedly increased 02 affmity (decrease of p50-[0- 15 mmHg with consecutive left-shift or02 dissociation curve.l due to the absence of 2,J-DPG as allosteric regulator and the acidotic extracellular pH-milieu.

S Increased formation of Met-Hb due 10 oxidation and missing erythrecyticenzymes [superoxide-dismutasc, glutathionperoxidase, katalase, Met-Hb-redultase), Met-Hb does not transport 02 and favours the formation of toxic 02 species.

6 Vasoconstriction of fret Hb. Underlying mechanisms include scavenging of the endogenous vasodilator nitric oxide (NO-scavengingl, augmented release ofthc endogenous vasopressor cndothelln, stimulation of cndothelin-receptors and adreno-rcceptors. TIll,' extravasation ofHb molecules is discussed as a prerequisite for these vasopressor effects [71.

Second-generation HBOC

Further research and development activttles filmed at optimizing 02 transport propertiesof acellular Hb and to attenuate undesirable effects [8], Thls goal could be achieved by (I) the development of techniques 10 obtain ultra-purified Hb solutions and (2) by chemical modlfrcations of the Hb molerule, Innovative methods of hac moly sis, dialysis, crystallization and filtration reduced the contamlnntion with erythrocyte debris and endotoxlnes, which enabled the production of haemoglobin solutions according to 'good laboratory practice: To avoid nephrotoxic effects related to the breakdown of the Hb-tetramer in mono- and dlmers, n couple of chemical modifications of'the Hb-molecule had been developed (Fig. I).

Crosslink of a-subunits

Lysine residues (position 99) of botho-chalns arc crosslinked With J.4-dibromosalicyl-fumarate. In particular, this chemical modifu:ation has been implemented by Baxter Heahhcare (Diasphin Cross-linked Hemoglobin. Hcmasslst'"]. Aside from stablllsatiun of the molecule. the intramolecular crosslink modifres molecular confonnatlon, thereby decreasing 02 alTmity Ipso c 32 mmHg).

Polymerization oj Hb-tetramers

Hb originatlng from outdated human blood is polymerized by gllltaraldehyde-(PolyHl'meT'M, Nonhf1e1d Laboratory lnc., USA) or by o-Raffinose (Hemo\ink"rtl·, HClllDSOJ Inc .• Canada). Additional conjugation with dextrane or polycthylenglycol increases the diameter of the Hb-(maCTol-molccuh: by adsorption of

H20, thereby preventing glomerular frltration as well as extravasation through the endothelial barrier.

TIle 0l affmity ofPolyHeme"rtll is lower than in native blood (p50 = 26-32 mmllg]. This has been achieved by pyridoxylation of'Hb-molccules (pLP acting as an analogue to 2,J-DPGI.

Bovine fIb

BOVine Hb polymerized with glutaraldehyde is void of nephrotoxic effects, TIle risk of'bovine sponglform encephalopathy (BSE) seems to be extremely low, hut Is still a matter of concern [a]. Wlu~rcas the O~ ;lffmity of isolated human Hb is markedly elevated. the p50 of isolated bovine Hb is comparable With human Hb within the erythrocytic milieu, but is independent on the regulation by 2,3-DPG (91.

Due to the high incidence of infectious diseases among blood donors in South Africa. the bovine HBOC Hemopure'tM [Biopure Inc., Cambridge, USA) had been appmvedby the South African Ministry ofHcalth on Aprll6th, 2001. In 2002, Biopure filedthe approval by the FDA, the procedure is still pending.

Maleimide-actizlated polyr1Jr.v/(,IIC glycol modified Hb (MP4)

Targeting tissue oxygenation on the microcirculatory level, an innovative concept has been realized with the design of MP4 [malefmlde-actlvated polyethylene glycol modified haemoglobin Hemospan'", Sangart Corp., San Diego, CA). MP4is a low-dose HBOe (Bb content 4 girl!) with high O2 affmity (p50 5·9 mmHg) and high Viscosity (2·5 cP) (10).

These in the Iust view coumcrinrultive characteristics of MP4 (high 0l affmity) are based on the proposition that rapid 02 unloading - as intended by conventional HBOCs with low 02 affinity - induces tissue hyperoxia and arteriolar vasoconstriction. which impairs microvascular blood flow {11]. Contrasting this. the low 0l affinity ofMP4 entails that O2 release is preferentially directed to hypoxic tissues. thereby avoiding hyperoxia in normoxic areas. Due to its high viscosity. MP4 increases plasma viscosity. thereby arousing endothelial shear stress W1tll consecutive NO release and vasodilation. As a consequence, functional capillary density has been demonstrated to increase. indicating improved microvascular perfusion [12J.

The low Hb cement allows for an effective exploltatton of the raw material; one unit outdared RaCs yields about four units MP4, A similar concept is represented by HP3Hb, a lowdose preparation of ultrapurifted porcine Hb (') g/dl). Molecules arcjnined to PEG-conjugated hyperpolymers (MW 800 KDJ using glutaraldehyde. Bioeompntibility has been tested in volunteers [13), data in IlIC treatment of severe anaemia are not Jet available.

GellelicaUy engineered Hb

Using genetically engineered Hb (e.g. via bacteria and yeast cultures) offers two incentives: (I) Manufacturing could

e l006 The Authors.

Journal compilation © 2006 Blackwell Publishlng l.td./SBT Scienc{' Seriet: (2006} I. 152-160

154 A. Pape N al.

B

A

,

t

a

D

Fig. 1 Modjfiratjon~ of the lib molecule appli~d ill Selttral HBOCs. (al Intr.lmol~C11lar cr(l~link of a-subunits (o:-o:-Crosslink]: (b] insertion of pyrit!oxal-5'· phosphate acting as a 2,3·DGP-analogue; (e) colljllgation of polyt'thyltnc-!Itycolto the surface of the Hlrmoleculc; (d) polymerization of Hb-molecutes using glutaraldehyde or D-r.lffinoSl:; Ie) tntbeddin9 of native or modified Hb moteeules in phOSpholipid,-vesitits (iiposoml:S, non-n-partides, 'artificial red ctl1s1 with additiMal incorpollition of enzyme systems [Met-Hb-Red: Met·Hb-reducla~. SOD: superoode-dismutase, KAT:katalasc), adopted from Habler rr (It [B).

become independent on the raw material 'outdated banked blood' [21 Selective exchange of particular amino adds modifies the tertiary and quaternary structure of the HIlmolecule, thereby preventing the binding of NO [14]. and vasoconstriction related to NO-scavenging (15). The large scale production of genetically engineered HBOCs is however, at the present time point extremely cost-intensive.

Liposomr-cllcapsulated Hb

By embedding native or modified Hb-molecules into phospholipid-vesicles (llposomcsl unfavourable effects of free Hb cirrulating in the plasma can be avoided [16]. Together with Hb, enzymatic systems [superoxide-tlismutase, katalase,

Met-Hb-reductase-systcmsl and allosteric factors (2.3,-DPGanalogues like hexalnosltol-triphesphatel can be incorporated [17,IBl, PEG-modification of the liposome-surface increased plasma half life and diminished the aggregation of vesicles.

Aq uaso Illes

Another innovative Hb modification consists in the formation of Hb aquasomes, Bovine Hb molecules are adsorbed to spherical hydroxyapatite cores, A ]4% suspension of this HBOC was reported to possess almost physiological 02- transport properties (pSO 2B·4 mmllg], to be void of vasopressor effects and effective in tissue oxygenation [19).

e 2006 The AuUtors.

Journal compilation © 2006 Blackwell Publishing ud.lSBT Sdtntc Sfrit$ (2006) 1. 152-160

Anifl~ial O~ carriers 155

Table I l'hysito-dlemic"al char.arltri~tics and actual state of clinical ~alCh on HBOrs
SoulttofHb Conc. [g/d!] MW!Da] POO fmmHg} Indication Phase of dlniC<llltsting
PHP'" Human B 123-{)oo 23.6 Hemodynamic instability in septic shock 11/111
HemAssist'" Human 10 6&000 32 Reduction of perioperatfve transfusion rate tip to III stopped
r-Hb 1·1'" Rcrombina11 5-10 64-000 31·32 Rrduction of p~[)peratM tran~fusiOn tate f'II,stopp~
r.HbZ-O'" Recombinant 10 32!HlOO ll-J2 Reduction of ptripperativ~ transfusion rate 1/1I,stoppel!
Hemopure" lIo~i~e 13 2S1l-OOO 38 Reduction Df perioperative transrusion rate III
Polyheme'" Human 10 150000 26-32 Reductlon of perioperative transfusion ratt '"
Hemolink'" Human 10 120-18(}OOO 39 Reduction (If perioper.alive transfusion rate III
Hemospan"" Human 4 95000 6 Reduclilll1 of perioperauve transfusion rate II PHp"U _ pyrid[)l(ilated. potytthVlene-gl'1COl conjugattd Hb (Curacytt Htall" XI~I1Ces. Munith. Germanvl. H~mAssist'" - Diaspirin ctOSSlinktd Hb IDCLHb. Baxter Htalthcarc:,lIound lake, USA].

r-Bb 1-1 ~ Recombinant Hb. vtl'5ion l'lISDtlIal()gtn lnc, Boulder, U5t., Inter Baxter Heallhr:arc:). r-Hb 2·0 - R~ombinant Hb, version 2'() 18;Jxter Htalthearc:l.

HtmopurcT" .. Polvmerized bovine Hb [HBOC 201. Bropure Corp., Cambridge, USA).

Polyhemt'~ - Pyridoxylated. gtutaraldthyde-polymcri:sed Hb INorthlilcd taboratorv tnc .• EVanstDn, USA). HcmDlin~"" • Jib r3flimtr (Hemosollnc.., Toronto, Canada).

Hrmospan'" '" Maleimide-~ttiVllled polytthylene glyml-modified Hb IMP4. Sanqart INC, San Diego. USA).

Experimental and clinical application of HBOCs

Most of HBOCs arc hyperoncotic solutions characterized as 'oxygen-transporting plasma expanders: Due to these properties, HBOCs seem to be predestined for fluid resuscitation from haemerrhaglcshock, Hypovolemia can be treated effectively and decreased arterial O2 content resulting from dilutional anaemia is compensated by the excellent 0ltIansport capacity.

Indeed, in experimental shock studies, HBOes were superior 10 conventional fluid resuscitation with crystallcids and colloids, when haernorrhaglc shock was induced by withdrawal of> 50<11) of circulating blood volume. The infusion of the HEOC consistently effected a sustained stabilization of haemodynamics and tissue oxygenation and significantly decreased mortality [20-23].. Moreover, the post-ischaemlc interaction between leucoeytes and the endothelium could be attenuated by infusion ofHBOO; based 011 human [24.25] as well as on bovine Hb [26].

Surprisingly, the long-time favourite among the HBOes, DCLHb, was abandoned in 1998 after an interim analysis of a trauma study performed in the USA. After enrolment of \12 patients, the 24- and 48-h mortality was signifIcantly higher in patients treated with DClHb [27]. Although severe deficiencies regarding design and performance of the study (under-resuscitation and over-proportional enrolment of desperate cases in the DClHb-group}, the study has been terminated prematun:ly and was never restarted [28].

Thereupon. Baxter shilled research and development act IVHies towards gcnetkally engineered Haoes (r-Hb). The firstgeneration r-Hb was structurally an analogue to DCLffil and had identical side effects. However, the second-generation r-Hh [r-Hb 2'0) was a PEG-conjugated Hb-polymcr, which

featured 20-30-fold less NO-scavenging [8J. An expertmental shock-model in pigs yiekled promising results: regarding haemodynamic stabllhy and 6-daysurvival, r-Hb 2'0 Was as effective as whole blood and superior to conventional fluid resuscitation with crystalloid and colloids [29}.

Because the clinical application of r-Hb H yielded unfavourable effects comparable with the side-effects ofDCLHb li.e, hypertension, hyperbilirubinemia, hyperamylasernia, hyperllpasemia [3D}), Baxter stopped all research activities in this field. Results of a clinical phase-l study with r-Hb 2·0 have up to date not been published.

Contrasting this .. PolyHerne™ proved to be an effective resuscitation fluid, when 171 patients suffering massive haemorrhage were treated wIth the HBOC. Compared with a historical control group, 30-day mortality could be reduced signiftcantly 164'SIfu \'5. 25%) (31]. However. this report docs not comment on potentlal side-effects ofPolyHcme'IM. Shanty, Northfield has initiated another prehospltal phase 111 study aiming at the enrolment of720 patients at !6 US trauma centrcs.Pritnnry endpoint of this study is the 30 days mortality, secondary endpoints are the reduction of allogeneic blood transfusions and the incidence of multi- organ failure (32).

Aside from fluid resuscitation from haemorrhagtc shock, HBOCs arc also suitable for thc treatment of intraoperative blood loss. During isovolemlc replacement of lost blood. the 02-transport properties of lite HBOC allow for haemodlluttcn to a lower haematocrit than t10 crystalloid and colloid solutions. In the best case, the transfusion of allogeneic blood can be postponed until surgical bleeding is under control.

HBOO; have been tested in several clinical phase 111 studies fcf. Table I). including cardiac and non-cardiac (genernl. vascular; trauma) surgery (31.33-40). Frequently observed side-effects

@ 2006 The Authors.

JDum~1 compllation I!' 2006 Blackwell Publi$hing Ltd. ISBT Science Serics (2006) I, 152-160

155 A. l'aJl~ d al.

Table 2 PhVSiro·chrmical characteristic and actual state of clinical research on PFC

Origin Conc.lwM Indl~ali~" Php~e of clinical ttst'ng
O~Y9CnIT. Synthdit GO Reduction of penoperative transfusion rate Ill, pres<:ntly suspended
OxyFluorT~ Synthetic 40 Reduction or gas· embolism during cardiopulmonary bypass I. presently s~pendtd
Perforan'u Synthetic 10 Reduction of perioperative transfusion rate Approved since 1996 in Russia Oxygent'" • Per1luorOOl:lylbramid~ (Alliance Pharmaceutical Corp .• USA), Oxyfluor'" - Ptrfluorodichlorooctan IHcmaGtn lne., USA].

Ptrftor:;nT~ • Prrl1uorodecaline/- paramdllylcydohtxil piperidine (Perflar-n Corp •• Russia).

consisted in increased systemic and pulmonary arterial resistances. decrease of cardiac output. jaundice. increased activities of amy lase, lipase and hepatic transaminases [36- 38,40J. Whether the increased enzyme activities must be judged as signs of pancreatitis or may be related to interference with photometric laboratory tests has up to date not been fully elucidated 141].

However. it sustained reduction of allogeneic blood transfusion (up to postoperative day 7) attributable to the use of an HBOC has only been reported by two authors {38,42). bUI till' blood-sparing potential was limlted to only 260- 600 ml pRI3C. The clinical relevance of this fmding has been critically discussed by the authors. A reason for this may be the short Intravascular half life of HBOCs. TIll: short-term npplleation only postpones the time point of allogeneic blood transfusion. To achieve an effeclivl' reduction ofRBCtransfusions. HBOCs must be infused over a longer term. theoretically until Ole erythropoicsls can provide a sufficient quantity of autologous RDCs, Regarding the long-term usc of HBOCs, only case reports are presently available [43.44).

Perfluo roca rbon s(PFC)

PFC are simply constructed molecules (MW 450-500 D) derived from cyclic or straight-chain hydrocarbons with hydrogen atoms replaced by halogens (i.e. fluorine or bromide). PFC are chemically and biologically inert. insoluble in water and haw therefore to be emulsffied before tv. infusion. After intravenous administration. emulsion droplets (diameter: 0'1-0,3 ~m) are rapidly (within few hours) taken up by the reticulo-endothelial system (RES). This farst step of elimination determines the intravascular half life (5-9 h) in a dose-dependent manncrI45].ln a second elimination-phase (about 20 days). PFC are redlstributed to the blood, transported to the IU1Igs and finally excreted via exhalation (461. In the case of overdosing, potential toxic effects of PFC emulsions are ducto overload ofthe RES. resulting in hepatosplenomegaly. vacuolated macrophages and finally in compromised immunological function of the RES 147J.

PFC are characterized by a high gas-dissolving capacity (C02> O2 > N2) and a linear relationship between arterial 02

partial pressure and 02 content, which fundamentally contrasts with the sigmoidal 02 kinetics ofHb 145.481, Therefore, hyperoxic ventilation [i.e. ventilation with supra-physiologic Fi02, usually with pure oxygen) is necessary to achieve: high arterial 02 partial pressures and to maximize the amount of 02 transported by PFC (491.

A1tbough at high arterial 02 partial pressures the absolute 0l content is higher in whole blood than in prc, 02 release to the tissues from PPC is almost complete in the presence of a high pOl gradient between arterial blood and the tissues (cr. Fig. 2). Depending on the dosage and ccncentration of the PFC emulsion applied. the amount of 02 released can even exceed the amount OfOl released from Hb, e.g, 100 g ora 60% perfluorn-octobromyl-cmulsion release 15 ml 02 given that lhep02 gradient between arterial blood lind the tissues is 560mmHg [arterial pOl 600 nllnHg. tissue p02 40 mmHgJ. The same amount of 02 is provided by 450-rnl Whole blood with an Hb-concentration of 14 g/d). After intravenous infusion, PFC droplets arc exclusively distributed within the plasma compartment, thereby elcvatlng plasmn solubility for 02 and arterial °1 content. Regartling 02 delivery to the tissues. it is discussed that PFC also facilitates the release ofHbtransported 02 by lowering the diffusion barrier between the erythrocyte and the plasma ('facilitated diffusion') raj.

First-generation PFC

TIll.' fust PFC-emulsion cxperhnentally and clink-ally investigated in a larger scale was Fluosol-DNM (Green Cross Corp .. Osaka. Japan), a 201l'Q (welgtu/volume. w/v) emulsion consisting of perfluorodccaline, pcrfluorctrtpopylamln and the synthetic emulsifterpluronic F-68n.1.ln clinical studies performed from 1979 through 1988 in Japan, high dosages of FluosolDNM (20 and 50 mljkg) had been applied to about 650palicnts [50,51]. For the majority of'paticnts. the Investlgaiors did not report adverse events. although other authors reponed relcvant side-effects associated with the emulsifying agent (anaphylactic reactions, interference with neutrophil chemotaxis [521. activation of the complement system and interference with pulmonary surfactant causing pulmonary hyperinflation [53], However, Fluosol-DAHt failed to provide sufficient

e :WOIj TIle Authors.

Journal compilation © 2006 Blackwell PubHslting Ltd. /SEr Sckncc Scrit:5 (20061 I. 152-\60

Artificial OJ carriers 157

F F

f b F b f

F"b.~~~j~~~6~F

IFf 1

F/~"-..!~ "-..b~ I .... F

F I F I ~

F F

Perlluorodecalin (C1nF IS)

Perllucrolripropytamin ICsF2IN)

Perlluoroocylbromid (C~Fj7Br)

Blood

20
~ 15
E..
i:
~ 10
e
8
a 5
o
o 40 200 300 400 500
pOl (mmHg) PFC Emulsion 60% (w/v)

Fig. 2 Chtmit:al struc!lIrt of PFC Itop) and OJ-dissociation kinetics of native blood (sigmoidal) ~nd J 60% IwM O>tygcnl'" emulsion IIi near.llbottom} .. At a given li5SU~ pOi Of 40 mmHg. 01 exl r:tction trom pft h ~ in contrast to blood - almost complete (01 extraction rate 80-90% PFC V5. 25% blood). adopltd from Habler rl ollal.

O.Aranspon prepentesin severely anaemic patients denying the transfusion of allogeneic blood for religious reasons [54,55]. Although all clinical studies investigating tile i.v. infusion ofFluosol-DA Thrin anaemic patients had been suspended by the end of the 1980s, at least the short-term potential of Fluosol-DAThf to provide sufficient tissue oxygenation remained unchallenged. In 1989, the substance was therefore approved by the FDA for intcrvcntionnl cardiology as an additive 0l transporter during percutaneous transluminal coronary angioplasty [!'TeA). With this mode of application. myocardial hypoxia distal to the catheter balloon could be reduced slgniflcantly [56J. Due to low sale figures, the production ofFluosol-DN'" was fmally suspended in 1993.

In 1996 however, pcrflaoromethyl-cyclohcxylpiperidin (Pcmoran™ 10%, Perftoran Corp., Pushchino, Russia), a substance analogous to Fluousol-DN'" has been approved by the Russian Ministry of Health [cf. Table 2] [57). Clinical data are predominantly published in Russian and report signifmmt blood savings attributed to the infusion of Perftoran [58].

Second-generation PFC

By the end oftlte 1980s, an improved second-generation PFe with higher concentration (60% w/vl and superior O2 transpan properties had been developed by the Alliance Pharmaceulical Corp., San Diego, CA, USA (Oxygen rIM). In several rnulticentre Phase-l-studles performed in the USA and In Europe, 0,45-3 g/l;g Oxygent'lN was infused in 145 volunteers and in 30 surgical patients, No relevantadverseeffects regard ing haematology, coagulation or organ function (liver, kidney, lungs) had been observed after infusion ofOxygent'Pot [59]. Frequently reponed side-effects of mild to moderate severity included flu-like symptoms. primarily fever (1-2 ·C rise in temperature), chills, headache. nausea and myalgia.

These symptoms were related to the clearance process by cells of the RES and were sensitive 10 prophylaxis with cyclooxygcnase inhibitors and corticosterolds 146). Moreover, a transient decrease of plate lei count by approxlmately 15% had been reported 3 days post dosing, which completely retumcd to normal by day 7 145]. Bolh, the release of'cytokines and the sequestration of'tbrcmboeytes could be almost elimInated by further development of emulsifying substances.

Experimental and clinical application ofPFC

Due to potential adverse effects related to the over-dosage of PFC (compromised immunological function due to overload of the RES), the manufacturers recommend the application of rather low doses e.g. for 60% Ozygent1M the recommended maximum dosage is only 2·7 gJkg. i.e. 4·5 ml/kg. In contrast to HBOCs, .PFC therefore appears unsuitable as a primary resuscitation fluid for the treatment of hacmorrhaglc shock, However. the supplementation of conventional shock therapy [i.e, crystalloids and colloids) with PFe may be advantageous. In splenectomised dogs, Kemrnlng and coworkers demonstrated beneficial effects of this treatment modality (supplementation with 60% Oxygen""'. 4·5 ml/kg) on survival and tissue oxygenation (60).

In electtve surgery withanticipated severe blood loss, the application ofPFC as a part of a multlmodal treatment strategy provides a signlfrcant blood sparing potential: prior to surgczy. autologous blood is harvested by acute ncrmovolemic haernodilution (ANH). In the case of major bleeding with further decrease of Hb-content during acellular fluid replacement. adequate tissue oxygenation may be maintained by the combination of byperoxlc ventilation and repetitive

e 2006 The Authors.

Journal compilatlcn © 2006 Blackwell Puhlishing Ltd. JSDT Scimc» Series {2006} 1. 152-160

t 58 A. Pape ~I al.

co-administration of low-dose boluses of PFC. In the best case, the re-transfuslon of autologous blood can be postponed until surgical bleeding is under control, thereby avoiding that transfused RBCs are immediately spilled OUt via the ongoing surgical blood loss. The concept of bridging an extensive blood Joss by extreme haemodilution under the protection of hyperoxlc ventilation and application of PFC has been patented as 'augemented hacrnodilution' (A-ANHfM) by Alliance Pharmaceutical Corp [41l!.

lnsplencctomisr:d dogs, Habler and coworkers demonstrated that this concept allowed the extension of acute nOTmovolemic anaemia from Her 21% to Hct 8% without any signs of impaired tissue oxygenation or compromised myocardial contractility (49,61).

In patients undergoing cardiac surgery Frumento and coworkers found that the application of2'7 g/kg Oxygemllo' provided adequate gastrolntesdnal tlssue oxygenation at Hb to 6·6 ± 0·4 gldl [62}.1n non-cardiac surgical pattents [orthopaedit and general surgery), the low-dose bolus administration of ~ Oxygem™ (0-9, 1·8 ad 2·7 gld!) allowed to postpone the transfusion of'allogenelc blood by 80 min [63]. In it recent multleentre phase-Ill-study, the number ofpRBC-uniL<; transfused until postoperative day 3 was signifH:anOy lower in patients treated with PFC [64J. However, some methodological concerns arose about the latter STUdy regarding the comparability of the groups Ipreopcrative ANH and hyperoxlc ventilation have exclusively been performed in the PFCgroup) [65] •. lndeed, hyperoxlc vemllatlon alone has been demonstrated to significantly increase the tolerance of acute normovolernic anaemia by providing sufficient oxygenation of vital organs [49, 66-58}.

Aside from the above-mentioned side-effects ofPFC (flulike symptoms, primarily fever. chills, headache, nausea and myalgia). an increased incidence of postoperative ileus has been reponed {64]. Moreover. patient enrolment in a phase 1U study in cardiac surgery was suspended in 2001 due to increased rate of neurological complications [4a}. The manuIacturers seck to perform additional multlcentre studies in Europe and the USA before fIling for market approval.

Summary and conclusion

The growing imbalance between increasing demand and decreasing availability of allogeneic blood underlines the socio-economic significance of safe a nd effective artlficla I 0l tamers as an alternative to the transfusion of allogeneic RBC. The blood-sparing potential of both types of artificial O~ carriers currently under investigation (HBOes and PFCI has been proven In experimental as well as in cl lnlcal studies. As a Part of a multimodal treatment strategy including normovolemlc haemodllution and hypcroxia. the low-dose-administration of PFC effectively Increases intraoperative anaemin tolerance. Although the bovine HBOC HemopureTloI was approved

in South Africa for treatment of acutely anaemic surgical patients in 200\, the approval of a particular moc by the FDA is presently not foreseeable. Nevertheless, further development of safe and effectivc synthetic 0l carriers remains an issue of substantial interest.

References

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Bulletin UASVM, Veterinary Medicine 65(2)/200R pISSN ! R43-5270: cISSN 1843-5378

COMPARATIVE STUDY REGARDING THE INFLUENCE OF PERFTORAN-HSS ASSOCIATION ON BLOOD GASES PARAMETERS IN RAT MODEL OF HAEMORAGIC SHOCK

'Faculty of Veterinary Medicine Timisoara, Calea Aradului nr. 119, Timisoara, Romania, 2University of Medicine and Fannacy "Victor Babes", Timisoara, Romania vivian. vanatu@gmail.com

Keywords: blood substitutes, haemorrhagic shock, perftoran, perfluorocarbons, hypertonic saline solution, therapy.

Abstract: This study tested Perftoran blood substitute and a association of Pcrftoran and Hypertonic Saline Solution for the use in haemorrhagic shock in rats. Four groups were made (n=! 0): P (Perftoran), PSH (Perftoran HSS), SH (HSS) and M (Control). Haemorrhagic shock was induced by controlled exsanguination until MAP decreased below 45 mmHg. Resuscitation therapy was initiated after 30' of shock and was made with 0.9% NaCI solution in group M, 3.5% NaCI solution in group SH, Pcrftoran in group P and Perftoran associated with 2.9% NaCl solution in group PSH. Data analysis shows that both Pcrforan and PSH arc efficient ill oxygen delivering and volume resuscitation.

INTRODUCTION

Exsanguination and haemorrhagic shock are the most frequent causes of death after acute trauma. Haemorrhagic shock is a critical imbalance between oxygen and nutrients delivery to the cell and the consume of those by the cell. In hipovolemia oxygen delivery to the tissues decreases, producing the breakdown of the oxidative metabolism and the activation of anaerobic metabolism, Anaerobic metabolism is associated with the increase of indicators for ischemia (lactic acid, base deficit). Those indicators reflect cellular oxygen need, proven as valid predictors for the probability of death after haemorrhagic shock in animals [1].

Bleeding control and compensation of the deficit in circulating blood volume represent the emergency treatment of haemorrhagic shock. This is done mainly by the use of asanguinous solutions (crystalloid and colloid). Those solutions provide volume replacement but do not provide oxygen transport. Dilution anemia is induced by asanguinous solutions and, to a certain degree, is compensated by an increase in cardiac output and arterial oxygen extraction. However, when hematocrit falls below critical values, these compensatory mechanisms become exhausted. Tissue hypoxia occurs then, even in the presence of nonnovolemia, and transfusion of red blood cells becomes indispensable to restore the oxygen transport capacity of blood.

Blood transfusion may be a problem for veterinarians because of the large number of blood groups, lack of universal donors and absence of blood storage facilities for animals. In this cases, blood substitutes may be a useful treatment solution. Blood substitutes are oxygencarrying fluids that are designed to provide an alternative to the blood transfusion. Perfluorocarbons are a category of blood substitutes which rely on the ability of this substances to dissolve and transport oxygen. Perftoran is a perfluorocarbon emulsion made of

31

perfluorodecalin and perfluoromethylcyclohexylpiperidin that solves 7% vol. of oxygen [3, 4].

The purpose of the present study was to investigate the effects of primary fluid resuscitation with Perf toran-Hypertonic Saline Solution association as compared with single administration of Perf toran, Hypertonic Saline Solution 3.5% and physiologic NaCI solution 0.9% [5).

MATERIAL AND METHODS

Animals. Experiments were performed in 40 healthy adult Wistar rats, weighing between 280-360 g (mean 317.6 ± 17.8 g). All animals included in this study received care in compliance with "3R" principles of animal wellness.

Animal preparation. Anesthetic induction and maintain were made with Xylazine 2% (7,5-10 mg/kg) and Ketamine 10% (60- 90 mg/kg) by intramuscular injection. Following anesthesia, rats were intubated with an adapted intravenous catheter with the help of the intubation device described by Ordodi et al. Rats were mechanically ventilated with 100% oxygen at a rate of 70 ventilations/minute and positive end expiratory pressure of 3 em H20 using the rat respirator described by the same author. A warming pad (Labovolt Prolab) was used to maintain core body temperature >3rC (98.6°F) [6, 7].

The following probes and catheters were placed: ECG probes for recording of lead 11, rectal thermometer probe, 24G catheter (VasoFix Certo, Braun Medical) in extemal carotid artery for mean arterial pressure (MAP) monitoring and blood withdraw.

Carotid artery catheterization was made by sterno-mandibular incision of the skin, muscle retraction and isolation of the right carotid artery from the right artery-vein-nerve package. A 24G catheter was placed inside the artery. At 24G catheter a 3 way stopcock with arterial pressure monitoring line was attached (fig. 1).

ECG lead II and MAP values were recorded on a computer using Digidata 1200 acquisition system and Axoscope 10 acquisition software.

Blood volume. Total blood volume was calculated with the help ofthe formula described by Lee and Blaufox [2]:

Blood volume (ml) = 0.06 x Body Weight + 0.77

Blood samples. Gas analysis arterial blood samples were taken in 2 ml syringe with lithium heparin. Arterial blood gases were determined using a GEM Premium 3000 blood gas analyzer. Hemoleucogram samples were taken in 500 ~tl Capiject EDT A microtainers (Terumo) and determined by impedance and flow cytometry with a Cell-Dyn 3700 device.

Samples were collected before exsanguination (0 min, baseline), after haemorrhagic shock installation (30 min, shock) and 30 minutes after therapy administration (60 nun, therapy).

Fig. I: Catheterized rat connected to pressure transducer bv a 3 way stoncock.

32

Experimental protocol. After completion of surgical preparation, a 20 minutes stabilization period was allowed before samples of blood were collected for baseline values. During this period ECG and MAP were permanently recorded. Mean arterial pressure was then reduced within 10 minutes to 45 mmHg by repeated withdrawals of blood and maintained at this value for 30 minutes.

After 30 minutes of hipovolemia blood was collected for haemorrhagic shock values.

For therapy, the volume of blood removed during exsanguination period was replaced in a randomized order by three times volume of NaCI 0.9% solution (M or control group), 31ml!kg NaCI 3.5% hypertonic solution (HSS group) , 20 ml/kg Perftoran blood substitute (Perftoran or P group) or 32 ml/kg from a mixed solution made of Perftoran and NaCl 2.9% in equal parts (Perftoran-HSS group). Exsanguinations, blood sampling and therapy were made through arterial catheter.

Statistics. Statistical analysis was performed with Office Excel 2003 (Microsoft) and StatPlus 2005 (Analystxoft), Obtained data were expressed as mean ± standard error. Statistic analysis was made using Man-Whitney test for compare inside the group and between the groups. Values ofp<0.05 were considered statistically significant.

Experiments were funded from CNCSIS grant TD-431/2007, contract 484/2007.

Table I. Characteristics of Perftorau (after Maevsky and col.,
cited by Lowe) [1,2]
Characteristics Perftoran
PFCs Perfluorodecalin
Perfluoromethylcyclopiperidine
Emulsifiers (surfactants) Proxano1268
Egg yolk phospholipids
Total PFC content (% w/v) 20.0
A verage droplet diameter/urn 0.07
pH 7.3
Osmolarity/mOsm kg 300
Viscosity/cP 2.3 RESULTS AND DISCUSIONS

Decreasing MAP (fig. 2) below 45 mmHg required withdrawal of 1.95 ± 0.06 mLiIOOg in group M, 2.00 ± 0.05 mLiIOOg in group SH, 2.29 ± 0.15 mLiIOOg in group P and 2.1 0 ± 0.15 mLiIOOg in group PSH. Mean arterial pressure decreased after exanguination bellow 45 mmHg and was maintained at this level for 30 minutes.

Primary resuscitation consisted in intra-arterial infusion of 5.25 ± 0.23 mLilOOg NaCl 0.9% in group M, 2.75 ± 0.24 mLilOOg NaCI 3.5% in group SH, 3.33 ± 0.28 mLilOOg Perftoran in group P and 3.50 ± 0.35 mLilOOg Perftoran + NaCI 2.9% 111 group PSH. After resuscitation MAP increased to 59 mmHg in group M (0.9% NaCl solution), 60 mmHg in group SH (3.5% NaCl), 94 mmHg in Group P (Perftoran) and 83

Fig, 2; Mean Arterial Pressure (mmHg).

pH

Fig. 3; pH.

mmHg in group PSH (Perftoran - HSS association) [8].

PH values (fig. 3) decreased after exanguination and continued to decrease after therapy in all groups. At the end of haemorrhagic shock period, pH decreased with 2.91 % in group M, ] .33% in group SH, 2.5% in group P and 2.39% In group PSH. After therapy administration pH increased with 0.68% in group M and decreased with 1.57% in group SH, 0.43% in group P, 0.75% in group PSH. This values together with lactic acid and pe02 values show that decrease of pH occurred because of lactic acidosis.

Partial pressure of carbon dioxide from arterial blood dynamic (fig. 4) was similar to all groups. After hemorrhage decreased in all groups and after therapy administration pe02 increased in all groups. Animals were ventilated with 100% oxygen at a constant rate of 66-72 respirations/minute and variations did not occurred between stages of experiment. pe02 values bellow normal were recorded because of 100% oxygen ventilation and not because of hyperventilation. Thus, the decrease of pe02 values after hemorrhage was caused by a decrease in oxygen consume after decreased tissue perfusion. The increase of peol values after therapy was caused by the reestablish of tissue perfusion and oxygen consume. This also explains why pe02 values were higher in P and PSH groups compared to M and SH groups. The difference between P and PSH values is probably caused by the Perftoran dose, which was reduced to half in PSH group [9].

~~--------------------------------,

Partial O2 pressure (fig. 5) was in

normal range for animals breathing 100% oxygen. P02 slightly increased after hemorrhagic shock, but not significant. P02 values increased with 54.43 mmHg after Perftoran and with 62.75 mmHg after PSH administration. The P02 values also increased in M and SH groups but the increase is much lower (21 mmHg in group M, 32.8 mmHg in group).

Lactate values (fig. 6) increased after shock and decreased after therapy. In insufficient oxygen NADH cannot release the H+ and they build up in the celL To prevent the rise in acidity, pyruvic acid accepts H+ fanning lactic acid that then dissociates into lactate and H+ [9].

PC02

Fig. 4: CO2 Partial Pressure (mmHg)

Pa02

600.00 T~--~-'--------------

200.00

100.00

c. 00

.M "51-!

Fig. 5: O2 partial pressure (mml-lg)

Fig. 6: Lactate (rnmol/L)

34

Baseline values of base deficit (fig. 7) were 5,53± 1,04 mmollL in group M, O,35±O,61 mmollL in group SH, 1,07±O,84 mmoliL in group P and 2,75±O,72 mmol/L in group PSH. This values indicate a slightly acidosis in group M.

Base deficit increased at the end of

hemorrhagic shock phase to 15,7±2,72 mmollL in group M, 1O,52±I,06 mmollL in group SH, 12,90±0,81 mmoliL in group P and at J5,23±J,II mmollL in group PSH, indicating severe acidosis.

After therapy base deficit decreased to 12,70±1,69 mmollL in group M, increased to 1l,08±1,61mmolJL in group SH, decreased to 8,29±1,16 mmoIlL in group P and decreased to 12,lO±2,73 mmol/L in group PSH. Showed results indicate that base deficit decreases most after Perftoran administration [9].

Hematocrit values (fig. 8) were similar

in all groups and followed the same dynamic curve. Baseline hematocrit values were 37±2.65% in group M, 41±1.32% in group SH, 41.27±U4% in group P, 41.1±1.58% 111 group PSH. After hemorrhagic shock hematocrit decreased to 29.33±2.03% in group M, 30±2% in group SH, 31.45±l.S5% in group P and 30.67±1.83% in group PSH. Therapy decreased hematocrit values further by dilutional effect: 23±3. 1 6% in group M, 22.6±l.S% in group SH, 25±l.09% in group P and 22.56±1.97% in group PSH.

Hemoglobin values (fig. 9) also

decreased together with hematocrit,

following the same curve.

Showed results indicate that after a loss of blood between 31.28±1.36 and 41.7±6.31 % of blood volume, MAP decreases to critical values (below 45 mmHg), hematocrit drops with 7.67 to 11 % and hemoglobin concentration decreases with 3.1 to 3.9 g/dl., This decreases tissue perfusion and induces the change in cell metabolism to anaerobic. Because respiratory system is working well and animals were ventilated with 100% oxygen, but tissue perfusion is highly decreased and there is not enough hemoglobin to bind oxygen, partial pressure of oxygen in arterial blood remains unchanged and slightly increases. Oxygen accumulates in the blood but it cannot get entirely to the tissue. This decreases the oxygen

Base defflclt

20.0<1,-----.--------------

15.0<1 !---------

10.011

0.00

.",'

.'.0IIl..___.-~------------ ..

Fig. 7: Actual base deficit (rnmol/L)

"'

''''''~_''

Fig. 8: Hematocrit (%)

1----···- - - - - -----

Fig. 9. Hemoglobin concentration (g/dl.)

35

consume which leads to a decrease in carbon dioxide production. To compensate acidosis, lactate accumulates in the blood. PH decreases, being more acid and the base deficit highly increases.

Fluid administration of Perftoran and PSH increases MAP, decreases hematocrit and hemoglobin levels, inducing dilution anemia. Although dilution anemia OCCUlTed, oxygenation was efficient as showed by the increase of peo2, MAP and P02 and decrease of ischemia indicators such lactic acid and base deficit.

CONCLUSIONS

y Perft0Fan iFlG'feaseSt'bxygei1ad0~:l:a:(id:rest'ores<bl06d pressurealmost to normal values, disregarding the i!lZ}E~e!,0f!hemat9c,rit andhemoglobinx

., Perftoran associated with NaCI 2.9% solution has the same effect, but in this dose is not so efficient as Perftoran. A increase in Perftoran dose together with a increase of NaCl concentration to 7% will probably be more efficient and follows to be tested;

, LV. administration should be considered for a greater increase of MAP.

BIBLIOGRAPHY

I. Dunham eM., J.H. Siegel, L. Weireter, et al, 1991. Oxygen debt and metabolic acidemia as quantitative predictors of mortality and the severity of ischemia insult in hemorrhagic shock, Crit Care Med 19, p.231- 243,

2. Lee H.B., M.D. Blaufox, 1985, Blood Volume in the Rat. Journal of Nuclear Medicine, vol. 25, p. 72-76.

3. Maevsky G., L. Ivanitsky, O. Bogdanova, N. Axenova, E. Karrnen, R. Zhibuti, S. Scnina, S.Y. Pushkin, LA. Maslennikov, A. Orlov, l. Marinicheva, 2005, Artif Cells Blood Substitutes Biotechuol., 33, p.37- 46, cited by. Lowe K. e, 2006, Blood substitutes: from chemistry to clinic, J. Mater. Chern. 16, p.4189-4196.

4. Maevsky G.,L. lvanitsky, B. L Isla1110v. Y. Y. Moroz, L. A. Bogdanova, N. B. Karmen, S. Y. Pushkin, L A.

Maslennikov, 2005, in Blood Substitutes, ed. R. W. Winslow, Academic Press, London, p. 288, cited by Lowe K.e, 2006, Blood substitutes: from chemistry to clinic, J_ Mater. Chern., 16, p.4189-4 [96.

5. Schuszler Larisa, S. Bolte, C. lgna, V. Vanatu, 2005, Contracararca Hipotensiunii intraanestezice prin pcrfuzia cu solutie de NaCI 7,5% la caine, Lucr. St. Med, Vet. Bucharest, 48. p. 520-523.

6. Valentin L. Ordodi, Felix A. Mic, Ani A. Mic, Dorel Sandesc, Virgil Paunescu, 2005, A simple deviee for intubation of rats, Lab Animal, vol. 34, no. S, p.1-3.

7. Valentin L. Ordcdi, V. Pauncscu, Ani A. Mic.Lazar Gabor. Mihai Ionac, Octavian Toma, Dorel Sand esc, Felix A. Mic, 2006, Pressure-controlled Rat Ventilator With Electronically Preset Respirations, Artif Organs, vol. 30, no. 12, p.966-969.

8. Velasco LT., Y. Ponticri, M. Rocha e Silva, O.U. Lopes, 1980, Hyperosmotic NaC1 and severe hacmorrhagic shock, Am J Physio1 Heart Circ Physiol, 239, p.H664-H673.

9, *** - Arterial Blood Gas, Operational Medicine, Ed. Brookeside Associates, 2001.

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