865

Brain and Tissue Distribution ofPolyethylene Glycol-ConjugatedSuperoxide Dismutase in Rats

Kenshi Yoshida, MD; Gregory F. Burton, PhD; Jerry S. McKinney, BS;Harold Young, MD; and Earl F. Ellis, MD

Background and Purpose: The purpose of this study was to determine the distribution of polyethyleneglycol-conjugated superoxide dismutase in the brain, cerebrospinal fluid, and various organs.

Methods: Distribution of iodine-125-labeled polyethylene glycol-conjugated superoxide dismutase wasdetermined in three groups of male Sprague-Dawley rats: a normotensive sham control group (n=9) andgroups given l23I-labeled polyethylene glycol-conjugated superoxide dismutase either 30 minutes before(n=10) or 30 minutes after (n=7) norepinephrine-induced hypertensive injury.

Results: In the first 30 minutes after intravenous administration, polyethylene glycol-conjugatedsuperoxide dismutase plasma activity declined to 70% of the initial value and then decreased negligiblybetween 30 and 90 minutes. Levels of '"l-labeled polyethylene glycol-conjugated superoxide dismutase innormotensive animals were low in the brain and cerebrospinal fluid and highest in kidney. Brain levelsof polyethylene glycol-conjugated superoxide dismutase were elevated only in those rats that received itbefore hypertensive injury, however, cerebrospinal fluid levels were elevated in animals receiving the drugeither before or after hypertensive injury.

Conclusion: Our results suggest that the blood-brain barrier becomes more permeable to polyethyleneglycol-conjugated superoxide dismutase only during the hypertensive period but that the blood-cerebrospinal fluid barrier sustains more permanent injury. We suggest that the therapeutic effectivenessof polyethylene glycol-conjugated superoxide dismutase in hypertensive brain injury is due to its actionin the vascular wall or to its extracellular activity in the cerebrospinal fluid. (Stroke 1992^23:865-869)

KEY WORDS • blood-brain barrier • free radicals • neuronal damage • rats

We and other investigators have attempted totherapeutically use an antioxidant agent or afree radical scavenging enzyme such as su-

peroxide dismutase (SOD) because there is strongevidence supporting the involvement of oxygen freeradicals in the pathogenesis of various kinds of braininjury.'-8 When SOD is injected into the circulation, itspresumed ideal locus of action should be the intracel-lular space, because this is thought to be the site ofgeneration of free radicals.579-11 However, SOD is alarge, water-soluble molecule that is therefore unlikelyto cross many barriers, including the blood-brain bar-rier.12-13 Additionally, native SOD has a circulatingplasma half-life of only 6-10 minutes.1314 To increasethe circulating half-life, SOD has been conjugated withpolyethylene glycol (PEG), and studies indicate thatPEG-SOD has a half-life of approximately 40 hours in

From the Departments of Pharmacology and Toxicology(J.S.M., E.F.E.), Department of Surgery, Division of Neurosur-gery (K.Y., H.Y.), and Department of Microbiology and Immu-nology (G.F.B.), Medical College of Virginia, Virginia Common-wealth University, Richmond, Va.

Supported by National Institutes of Health grants NS-27214 andNS-12587. E.F.E. is the recipient of a Javits Neuroscience Inves-tigator Award.

Address for correspondence: Dr. Earl F. Ellis, Box 613, MCVStation, Richmond, VA 23298-0613.

Received October 15, 1991; accepted February 3, 1992.

the circulation.1516 While conjugation increases half-life, it also increases the molecular weight and maypresent increased problems in terms of extravasculardistribution. This large molecular weight may not be asmuch of a hindrance to tissue access after injury be-cause the integrity of various barriers may be broached,resulting in an increased tissue distribution of PEG-SOD. We therefore surmise that PEG-SOD may beable to enter the brain more readily after various kindsof insults, such as traumatic brain injury,1718 cerebralischemia,1920 or acute hypertension.21-22

A few studies have examined the pharmacokinetics ofSOD in the central nervous system after its peripheraladministration.131416 Chan et al7 have reported thatSOD does not enter the brain when given peripherally.Very recently Haun et al23 reported that intravenousPEG-SOD did not increase SOD activity in the braindespite global ischemia and reperfusion. Although thereport of Haun et al clearly shows that there are notlarge increases in PEG-SOD after global ischemia andreperfusion, the lower limit of sensitivity for their SODassay may have prevented them from detecting verysmall increases in activity.24-26 The aim of the presentstudy was to address the tissue distribution of iodine-125-labeled PEG-SOD. It was hoped that by radioac-tively labeling the PEG-SOD we would increase thelower limit of sensitivity for detection of its tissuedistribution. We also investigated whether distributionof 125I-PEG-SOD was altered by acute norepinephrine-induced hypertension.

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866 Stroke Vol 23, No 6 June 1992

Group

Control (PEG-SOO, no HT)

Pre-lnjury (PEG-SOO -> HT)

Post-injun/ (HT -<• PEG-SOO)HTi

r-30

HT

(60 min post HT)

(120 min post HT)

0t

PEG-SOD

PEG-SOD - (2000 U/kg + 5 (iCi/kg, l.v.)

I I I+ 3 0 + 6 0 + 9 0

Time of PEG-SOD circulation (min)

HT - 5 mln hypertension

FIGURE 1. Diagram of exper-imental protocol for each group.Control group: Animals weregiven polyethylene glycol-super-oxide dismutase (PEG-SOD)but did not receive hypertensiveinjury. Preinjury group: Ani-mals were &m pEG-SOD 30minutes before norepinephrine-induced hypertensive injury.Postinjury group: Animals weregiven PEG-SOD 30 minutes af-ter norepinephrine-induced hy-pertensive injury. HT, 5-minuteduration of norepinephrine-induced hypertension.

Materials and MethodsIodination of PEG-SOD (Sigma Chemical Co., St.

Louis, Mo.) was performed using a modification of theprocedure of Beckman et al.15 Briefly, 15 /xg PEG-SOD(in 100 fi\ Dulbecco's phosphate-buffered saline) wasmixed with 2 mCi 125I (Amersham, Arlington Heights,111.) and added to a glass culture tube previously coatedwith l,3,4,6-tetrachloro-3a,6«-diphenylglycouril (Iodo-Gen, Pierce Chemical Co., Rockford, 111.). The reactionmixture was incubated at room temperature for 20minutes, and the reaction was stopped by removing theliquid phase. Unbound iodine was removed by adding theiodine-protein mixture to an Eppendorf tube containingSephadex G-25 (Pharmacia, Piscataway, N.J.), centrifug-ing for 30 seconds at 900g, and collecting the superna-tant. The PEG-SOD was labeled to a specific activity of21-28 nOJug protein. The iodinated PEG-SOD retained78% of its superoxide scavenging capacity, as determinedby the cytochrome C reduction assay for superoxide.24

The material was stored at 4°C until used.A total of 26 male Sprague-Dawley rats weighing

300-400 g were used. After anesthesia was induced with50 mg/kg i.p. pentobarbital, the animals were tracheos-tomized and ventilated with room air. The ventilatoryrate and volume were adjusted so that the end-expira-tory CO2 was maintained at approximately 30 mm Hg. Afemoral artery and vein were cannulated for bloodsampling and intravenous infusion. Blood gases and pHwere analyzed to ensure adequate and consistent venti-lation. The temperature of all animals was maintainedat 37°C with a heating pad. The animals were placed ina prone position, and a rostrocaudal midline incisionwas made in the scalp and a needle inserted to collectcerebrospinal fluid (CSF) from the cisterna magna.Samples showing any evidence of blood were discarded.

Hypertension was produced by constant intravenousadministration of norepinephrine using an infusionpump. The norepinephrine stock solution was in 0.9%saline, and the infusion speed was varied from 0.068 to0.34 ml/min to elevate mean arterial blood pressure to180 mm Hg for 5 minutes. It took approximately 30Aig/kg per minute to maintain this pressure for thisperiod. The total infusion volume for the 5 minutes ofhypertension did not exceed 1.2 ml.

l25I-PEG-SOD (5 /iCi/kg) plus unlabeled PEG-SOD(2,000 IU/kg) was given in 0.2 ml saline through the

femoral vein. The total weight of PEG-SOD to achievethis dose was less than 1 mg per animal. Therefore,there is no significant drug effect on osmolality. Plasmasamples (0.1 ml) were collected through the arterial lineat 1, 5, 30, 60, and 90 minutes after PEG-SOD infusion.All animals were killed at 90 minutes after administra-tion of PEG-SOD.

The animals were divided into three experimentalgroups (Figure 1). One group received only 125I-PEG-SOD. Another group received 125I-PEG-SOD 30 minutesafter hypertensive injury, and a third group received125I-PEG-SOD 30 minutes before hypertensive injury. At90 minutes after injection of PEG-SOD, a CSF samplewas collected by cisternal puncture. The CSF sample wasexamined visually, and any sample with evidence of bloodwas discarded because the presence of blood would causeabnormally high 125I-PEG-SOD levels. Animals werethen perfused with saline through the left ventricle at apressure of 100 mm Hg to eliminate intravascular 12iI-PEG-SOD. The brain and various organs were thenremoved and the 125I counted with a gamma counter.Exogenous PEG-SOD concentration in each sample wasestimated from the radioactive counts in the tissue by useof the specific activity of total PEG-SOD infused. Resultswere examined by analysis of variance followed by aFisher least significant difference test. Data are ex-pressed as mean±SEM. A value of p<0.05 was consid-ered to be statistically significant.

ResultsThe control physiological parameters shown in Table

1 indicate that the three groups of rats were notsignificantly different. Changes in plasma PEG-SODconcentration in the control, preinjury, and postinjurygroups are shown in Figure 2. Although the averageplasma levels of PEG-SOD in the control group arelower than those of the other groups, there was nostatistically significant difference in the plasma levelsbetween groups over the 90-minute period. The plasmaconcentration of PEG-SOD fell more dramatically inthe first 30 minutes and was relatively stable between 30and 90 minutes after injection. At 90 minutes afterinjection, the plasma PEG-SOD level was approxi-mately 60-70% of that at 1 minute after injection.

Figure 3 shows the tissue concentration of PEG-SODin various organs at 90 minutes after intravenous injec-

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Yoshida et al Superoxide Dismutase Distribution 867

TABLE 1. Control Physiological Parameters in Rats by Group

Group

Variable

Mean arterial pressure (mm Hg)PaOj (mm Hg)PacOj (mm Hg)Blood pH

Control(PEG-SOD, no HT)

(«=9)

100.6±8.794.4±4.132.4±0.6

7.399±0.02

Preinjury(PEG-SOD-»HT)

(n = 10)

102.4±5.496.1±4.131.8±0.4

7.406±0.01

Postinjury(HT—PEG-SOD)

(n=7)

102.7±8.59O5±2.730.6±0.8

7.392±0.01

Physiological variables measured at baseline in control, preinjury, and postinjury groups. Values are mean±SEM.PEG-SOD, polyethylene glycol-superoxide dismutase; HT, 5-minute duration of norepinephrine-induced hypertension.

tion of PEG-SOD into the control group. The kidneyclearly had the highest concentration of PEG-SOD(16.4±1.4 IU/g wet wt), followed by the liver (3.5±0.7IU/g wet wt) and intestine (3.3 ±1.9 IU/g wet wt). Insharp contrast, the concentration of PEG-SOD in brainand CSF was quite low, 0.029±0.004 IU/g wet wt and0.052±0.010 IU/ml, respectively.

Figure 4 shows that the brain PEG-SOD concentra-tion was increased only in the group that receivedPEG-SOD before the hypertensive injury. It also showsthat, unlike the brain response, CSF PEG-SOD waselevated in both groups that received hypertensiveinjury. Table 2 shows the brain-to-plasma and CSF-to-plasma ratios for PEG-SOD in the control, preinjury,and postinjury groups. Again, it can be clearly seen thatthe brain-to-plasma ratio is similar in the control andposttreatment groups and elevated approximatelythreefold in the group given radiolabeled PEG-SODbefore hypertensive injury. The ratios also show that thepermeability of the blood-cerebrospinal fluid barrier toPEG-SOD increased approximately eightfold in thegroup treated before injury and 11-fold in the grouptreated after injury.

50-

15

Plasma PEG-SOD ConcentrationFollowing IV Administration

control (PEG-SOO, no HT), n-9pre-tnjury (PEG-SOD-»HT), n-10post-injury (HT -»PEG-SOD), n-7

20 40 60 80 100

Time post PEG-SOD (min)

FIGURE 2. Graph showing plasma polyethylene gtycol-super-axide dismutase (PEG-SOD) concentration after intravenousadministration. Values are mean±SEM. All animals were givenPEG-SOD (2,000 IUI kg, 5 uCi/kg) intravenously. HT, 5-minuteduration of norepinephrine-induced hypertension.

Discussion

Odlind et al14 reported the distribution pattern of125I-labeled bovine SOD in rats after intravenous infu-sion using whole-body autoradiography. They showedthat SOD, with a half-life of approximately 6 minutes,rapidly disappears from the circulation, with subsequentdominant localization of 125I-SOD in the kidney andurinary tract. Our current results are in agreement withthose of Beckman et al15 and show that conjugation ofSOD with PEG results in a dramatic increase in circu-lating half-life of SOD. Our study showed a markeddecline in the concentration of PEG-SOD within 30minutes after intravenous injection, which likely repre-sents its distribution and equilibrium with the varioussystemic organs. After 30 minutes, this first phase ofdistribution was complete, and plasma levels remainedrelatively stable between 30 and 90 minutes afterinfusion.

Our results for distribution of PEG-SOD are similarto those of Odlind et al14 in that we also show dominantlocalization of PEG-SOD to the kidney. Although thekidney is less than 2% of the rat body weight, it appearsto be the major organ for distribution of PEG-SODafter intravenous injection. However, the role of theliver cannot be discounted because its mass and percentbody weight are relatively high compared with those ofthe kidney.

Tissue PEG-SOD Concentration 90 minutesAfter IV Administration (2000 U/kg, n=7-9)

f

Kidney Uver Intestine Stdn Lung Muscle Heart Braki

FIGURE 3. Bar graph showing tissue polyethylene glycol-superoxide dismutase (PEG-SOD) concentration 90 minutesafter intravenous administration. Values are mean±SEM foreach organ in control group (n=7-9).

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868 Stroke Vol 23, No 6 June 1992

PEG-SOD Concentration in Brain and CSF

0.10-,

0.00

-1.0

n=8 n=5 n=7 n=6 n=6 n=6control pre-injury post-injury

(PEG-SOD, no HT) (PEG-SOD -> HT) (HT -> PEG-SOD)

FIGURE 4. Bar graph showing brain andcerebrospinal fluid (CSF) polyethylene glycol-superoxide dismutase (PEG-SOD) concen-tration 90 minutes after intravenous admin-istration. Values are mean±SEM. BrainPEG-SOD concentration in postinjury groupwas not significantly elevated. Brain PEG-SOD concentration in preinjury group showssignificant elevation compared with those inother groups. PEG-SOD concentration inCSF in both groups subjected to hypertensiveinjury shows significant elevation comparedwith that in control group, a, p<0.05 versuscontrol or postinjury groups; b, p<0.05 versuscontrol. HT, 5-minute duration of norepi-nephrine-induced hypertension.

In agreement with previous studies, our current re-sults show that in control animals there is no majoruptake of PEG-SOD into the brain or CSF.1314 Inanimals in which iodinated PEG-SOD was presentduring the hypertensive challenge, analysis of the brainshowed an approximate 2!/2-fold increase in the brainlevel of PEG-SOD. This increase in PEG-SOD level inthe brain in the pretreatment but not the posttreatmentgroup is similar to the results that we have previouslyreported with 125I-labeled human serum albumin andhypertensive challenge; that is, permeability increasesonly during the hypertensive phase. Therefore, whenPEG-SOD, like human serum albumin, is administeredafter the hypertensive challenge, there is no increase inbrain content of these two substances.2127

The results with CSF PEG-SOD levels offer a differ-ent interpretation with respect to the effect of hyper-tension on the blood-CSF barrier. Figure 4 shows thatPEG-SOD activity at 90 minutes after administrationwas increased in the CSF sample irrespective of whetherthe PEG-SOD was given before or after hypertensiveinjury. This implies that unlike the blood-brain barrier,the blood-CSF barrier is more lastingly injured by theacute hypertension. This results in a dramatic increasein PEG-SOD sampled at the cisterna magna in both thepretreatment and posttreatment groups. From theseresults we infer that the tissue that forms the CSF we

sampled is injured and compromised beyond the time ofthe hypertensive insult.2829

These findings shed light on the interpretation of oneof our previous studies, wherein we examined the effectof PEG-SOD on brain edema induced by acute hyper-tension.27 We reported that treatment after acute hy-pertension with PEG-SOD, at the same dose used in thecurrent study, was able to reduce hypertension-inducedbrain edema. It should be emphasized that edemacaused by acute hypertension is minor, causing less than1% increase in water content. Whether PEG-SODwould substantially reduce edema in more severe mod-els of edema, such as that caused by cold injury, is lesscertain. The results of the current study suggest that ifthe site of therapeutic action of PEG-SOD is extravasrcular, the PEG-SOD may access this compartmentthrough the CSF-forming organ, the choroid plexus.28'29

Alternatively, of course, it may be suggested that thetherapeutic site of action is entirely intravascular andthat any effect of PEG-SOD to reduce brain edema isdue to the action of PEG-SOD on the vessel wall.23

The main source of oxygen radical formation afterinjury is thought to be intracellular; however, we havepreviously provided evidence that after experimentalfluid percussion brain injury or acute hypertension,oxygen radicals enter the extracellular space through ananion channel.30 In reality, it is not known whether

TABLE 2. Brain-to-Plasma and CSF-to-Plasma Ratios of PEG-SOD in Rats by Group

Group

Brain or CSFPlasma

Control(PEG-SOD only)

1.51 ±0.26(«=8)

2.30±0.20("=5)

Preinjury(PEG-SOD^HT)

3.35±0.65*(n=7)

18.2±3.93t(n=7)

Postinjury(HT-»PEG-SOD)

1.07±0.24(«=6)

25.9±7.19t(«=5)

IU per gram brainIU per milliliter plasma

IU per milliliter CSF .IU per milliliter plasma

Values are mean±SEM. CSF, cerebrospinal fluid; PEG-SOD, polyethylene glycol-superoxide dismutase; HT,5-minute duration of norepinephrine-induced hypertension.

Brain-to-plasma ratio in control and postinjury groups were not significantly different. Brain-to-plasma ratio inpreinjury group shows significant elevation compared with those in other group; *p<0.05 versus control or postinjurygroup. CSF-to-plasma ratio in preinjury and postinjury groups shows significant elevation compared with control group;tp<0.05 versus control.

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Yoshida et at Superoxide Dismutase Distribution 869

radical damage to cells is greater intracellularly orextracellularly. The possibility exists that when radicalsleave the cell, they attack the outer cell membrane,where defense mechanisms may be more limited. SincePEG-SOD is not likely to readily enter cells its main siteof action may be on radicals which transiently existoutside the cell.

Another factor to consider is the specific cellular siteat which brain 125I-PEG-SOD is located. Our studiesshow that very small amounts of PEG-SOD are found inthe brain when considered on a whole-brain basis.However, we do not know exactly where the 125I-PEG-SOD is localized. If the 125I-PEG-SOD were concen-trated exclusively in one particular tissue, its localconcentration might reach a more effective scavenginglevel. If, for example, the PEG-SOD were only in thevascular wall or endothelium, its local concentrationmight effectively be 100-fold greater. This is an impor-tant question to be resolved in future studies.

Our results using iodinated PEG-SOD confirm thework of Haun et al23 indicating that at least for globalischemia/reperfusion and acute hypertensive injury,PEG-SOD does not appear to enter the brain in largequantities. However, with the current radiolabel tech-nique we were also able to sample CSF and show thatthe level of SOD activity in CSF was significantlyelevated. This points out the importance of samplingboth brain and CSF for SOD activity. In addition, ourstudy suggests that finding little or no increase inwhole-brain SOD activity does not necessarily meanthat SOD is not increased in the CSF bathing the brain.Although the current studies indicate little passage ofPEG-SOD across the blood-brain barrier after hyper-tensive injury, this does not rule out the possibility thatmore dramatic types of brain injury, such as traumaticinjury, may be associated with a greater opening of theblood-brain barrier and increased passage of PEG-SOD into the brain parenchyma.

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K Yoshida, G F Burton, J S McKinney, H Young and E F Ellisin rats.

Brain and tissue distribution of polyethylene glycol-conjugated superoxide dismutase

Print ISSN: 0039-2499. Online ISSN: 1524-4628 Copyright © 1992 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Stroke doi: 10.1161/01.STR.23.6.865

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