evaluation of copper(ii)-pyruvaldehyde bis (n-4-methylthiosemicarbazone) for tissue blood flow...

European Journal of

Nuclear Medicine Original article

Evaluation of copper(ll)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) for tissue blood flow measurement using a trapped tracer model Helen Young, Paul Carnochan, Jamal Zweit, John Babich*, Simon Cherry**, Robert Ott

Joint Department of Physics, Royal Marsden Hospital and Institute of Cancer Research, Downs Road, Sutton, Surrey SM2 5PT, UK

Received 2 July and in revised form 8 December 1993

Abstract. Copper(II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) (Cu-PTSM) labelled with 62'64Cu is a promising radiotracer for the study of blood flow using positron emission tomography (PET). We have investigated the application of a simple trapped tracer model to measurements of tissue 64Cu-PTSM uptake combined with continuous arterial sampling. A dual-tracer method was used to compare blood flow estimated by 64Cu- PTSM with values derived from measurements using cobalt-57 microspheres in the rat. Prolonged retention of 64Cu-PTSM following intravenous administration was initially confirmed in both normal tissues and tumours. After intraventricular 64Cu-PTSM infusion, cumulative arterial 64Cu activity increased progressively, and after extraction in n-octanol was found to plateau to levels corresponding with those reached following administration of 57C0 microspheres. Rapid and species-dependent rates of 64Cn-PTSM decomposition to non-extractable 64Cu complexes were found in rat and human blood in vitro (70%_+6% and 43%+5% respectively at 16 rain), demonstrating the need for immediate processing of arterial samples. Close agreement was found between blood flow estimated by 64Cu-PTSM and 57Co microsphere methods in tissues of low to moderate flow: muscle (0.01, 0.08, 0.07 ml/min per gram; mean difference, mean 64Cu, mean eTCo), brain (0.09, 0.52, 0.43 ml/min per gram) and kidney (-0.16, 2.29, 2.45 ml/min per gram). Estimates of cardiac output also compared fa- vourably between the two methods (5.7, 59.8, 54.1 ml/min). We conclude that a simple tissue trapping model may be suitable for the derivation of blood flow estimates using 62'64Cu-PTSM, PET imaging and continuous arterial blood sampling.

*Present address: Division of Nuclear Medicine, Massachusetts General Hospital, Boston, MA 02114, USA **Present address: Division of Nuclear Medicine and Biophysics, UCLA School of Medicine, Los Angeles, CA 90024, USA

Correspondence to: R Carnochan

Key words: Cu-PTSM - Blood flow - Microspheres - Arterial sampling

Eur J Nucl Med (1994) 21:336-341

Introduction

Copper(II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) (Cu-PTSM) is being evaluated as a tracer for tissue blood flow measurement using positron emission tomography (PET). Cu-PTSM may be labelled with 64Cu, having a half-life of 12.7 h, or with the relatively short-lived (9.8 min) 62Cu produced from a 62Zn/62Cu generator system [1]. The relatively long half-life of 64Cu is useful for developmental work whereas 62Cu provides for repeat PET imaging at short intervals, facilitat- ing the design, for example, of multiple tracer studies or pharmacological challenge tests. These radionuclides permit imaging at PET centres remote from a cyclotron, which is normally required to produce the short half-life radiotracers commonly used for PET imaging.

Cu-PTSM is a highly lipophilic copper complex [2, 3] with a molecular weight of 308 Da, well below the reported limit for efficient penetration of the blood-brain barrier [4]. Following i.v. administration tissue uptake of Cu-PTSM is rapid, and prolonged retention in several tissues has been reported [2, 5-7]. These findings have also been demonstrated in tumours [8]. Tissue retention of Cu-PTSM is thought to result from reductive decomposition of the copper (II) complex by intracellular sul- phydryl groups, with subsequent liberation of copper (I) and entrapment by binding to intracellular macromole- cules [9].

The high extraction efficiency and prolonged tissue retention of Cu-PTSM may be compared with the prop- erties of other "tissue-trapped" tracers such as radiolab- elled microspheres. Good correlation has been found between Cu-PTSM and microsphere trapping in the heart

European Journal of Nuclear Medicine Vol. 21, No. 4, April 1994 - © Springer-Verlag 1994

and k idney [5, 10]. It m a y therefore be poss ib le to app ly a s imple t issue t rapp ing m o d e l [11] to der ive b lood f low values f rom measu remen t s o f in tegra ted arter ial Cu- P T S M concent ra t ion and t issue uptake. However , Cu- P T S M is rap id ly m e t a b o l i s e d to non- l ipoph i l i c complex - es in the b lood [6, 12], po ten t i a l ly l ead ing to overes t ima- t ion o f ar ter ia l concentra t ions . So lven t ex t rac t ion us ing n-oc tano l has recen t ly been shown to be a useful me thod for the separa t ion o f C u - P T S M f rom such complexes , and m a y be used to measure the f ree ly exchangeab le f ract ion o f C u - P T S M in a b lood sample [12].

The a ims o f this s tudy were (1) to conf i rm high ini t ia l uptake and t issue re tent ion o f 64Cu-PTSM over per iods cons is ten t wi th the i m a g e acquis i t ion t ime o f a p ro to type large area mul t iwi re p ropor t iona l chamber pos i t ron camera M U P - P E T [13], (2) to invest igate the s tabi l i ty o f 64Cu-PTSM in who le b l o o d and (3) to evaluate the use o f a s imple t issue t rapp ing m o d e l to der ive b l o o d f low values us ing measu remen t s o f 64Cu-PTSM uptake c o m b i n e d with b lood sampl ing .

Materials and methods

Preparation of 64Cu-PTSM. No carrier-added 64Cu was produced by the 64Ni(d,2n)6'*Cu reaction [14]. 64Cu-PTSM was prepared by buffering an aqueous solution of 64CUC12 in 0.3 M HCI with two equivalents of 3 M sodium acetate (pH 4.6). To this was added an ethanolic solution of H2-PTSM ligand (50-100 lal, 0.1 ~g/tal). The radiochemical purity of the labelled complex was determined by thin-layer chromatography (TLC) using ethyl acetate, giving an Rf value of 0.6-0.7 with an average labelling yield of 94%. The octanol/water partition coefficient (logp) was on average 1.61. For all studies 64Cu-PTSM solutions were diluted with 0.9% saline to a final radioactive concentration of 5-20 MBq/ml and ethanol concentration of approximately 5 %.

Preparation of 57Co microspheres. Cobalt-57 labelled polymeric microspheres (diameter:15 ~m, NEN Dupont) were washed twice in 0.9% NaC1 and resuspended to a final concentration of 2.5 x 106 spheres/ml. The microspheres were suspended in an 83% w/v glucose solution to prevent settling [15] and 0.05% Tween-80 was included to minimise particle aggregation. The average radioactiv- ity per microsphere was determined according to the method of Warren and Ledingham [16] for each batch of microspheres used.

64Cu-PTSM tissue retention. The biodistribution of 64Cu-PTSM as a function of time was determined in adult hooded rats bearing HSN tumours. Rats weighing 190-230 g were implanted with a suspension of 1.0 x 106 HSN tumour cells by deep intramuscular injection into the lateral thigh. Experiments were carried out after 14 days of tnmour growth, at a size of approximately 5-10 mm in diameter. Animals were anaesthetised with a 1% mixture of halo- thane and oxygen delivered using a commercial vaporiser system (Halovet, IMS Ltd., UK). Following surgical exposure of the right jugular vein, 64Cu-PTSM (0.4 MBq, 0.2 ml per rat) was administered by rapid i.v. infusion. At various times post-infusion, animals were killed by cervical dislocation whilst under anaesthesia. Tissue of interest were dissected out, weighed and counted using an autogamma counter (MR80, Kontron, UK). Tissue uptake of 6gcu-PTSM was expressed as %injected dose/g normalised to a rat weight of 200 g.

337

Stability of 64Cu-PTSM. The stability of 64Cu-PTSM was studied in vitro using human and rat whole blood and plasma. 100 lal of 64Cu-PTSM (11.2 MBq/ml) was added to 1 ml of plasma or whole blood at room temperature (21°C). Samples of 100-~tl were taken at 0.25, 0.5, 1, 2, 4, 8, 12, 16 and 20 min, added to 1 ml n-octanol, vortexed and centrifuged (11 000 g for 5 min). The octanol and aqueous layers were then separated and ?'-counted. The non-com- plexed 64Cu-PTSM fraction was determined for each sample as the ratio of the counts in the octanol layer to the total.

Bloodflow measurement. A dual-tracer method was used to compare cardiac output and tissue blood flow estimated by 64Cu- PTSM and 57Co microsphere distribution methods. Adult hooded rats weighing 225-260 g were anaesthetised using alphaxalone/al- phadolone (Saffan, Pittman-Moore, UK). Following surgical exposure of the superficial vessels of the neck and left medial thigh, cannulae primed with heparinised saline were placed into the right carotid artery (polythene 0.4 x 0.8 mm, ID x OD, Portex, UK) and femoral artery (PTFE 0.4 x 0.6 ram, Adtech Ltd., UK). The carotid cannula was advanced until the tip was within the left ventricle of the heart, and the tip of the femoral cannula was positioned at the level of the aortic bifurcation. Immediately prior to radiotracer infusion, free-flow arterial sampling was commenced via the femoral cannula, and 10-s fractions collected into precooled, weighed vials containing 1 ml n-octanol. Sequential infusions of 0.2 ml 57Co microsphere suspension and 0.2 ml 64Cu-PTSM solution (4 MBq) were then administered via the carotid cannula over a pe- riod of 1 min, and arterial sampling continued for a further 90 s. Animals were killed by rapid injection of potassium chloride solution after a total time of 2.5 rain had elapsed. The arterial fractions were vortexed and centrifuged (11 000 g for 5 min), and the whole sample ?'-counted by a dual-channel method to independently assess 64Cu and 57Co activity. The octanol layer was drawn off and counted in the same way. Organs of interest were then removed from each rat, weighed and )'-counted. Satisfactory intravascular mixing of microspheres was assessed by comparison of left and right "kidney counts; where differences >10% were found, data were excluded from further analysis. Tissue uptake of 64Cu and 57Co was expressed as %injected dose/g normalised to a rat weight of 200 g, and cumulative time-activity curves were derived from the blood sample measurements. Individual arterial sample flow rates were estimated using mean values of sample weight, assuming a blood density of 1.05 g/ml. The average arterial sampling rate during tracer infusion was approximately 0.4 ml/min.

Perfusion model. For a radiotracer which exhibits minimal back- flux after tissue extraction (i.e. is chemically "trapped"), the tissue uptake at time T may be expressed as:

T

Qtiss = Ftiss X Etiss X fC,(t)dt, (1) 0

where Qass is tissue uptake in cpm/g, Ftis~ is tissue blood flow in ml/min per gram, Et~s~ is flow-dependent tissue extraction of tracer and C a is arterial blood concentration of tracer in cpndml.

An analogous expression may be derived for the "whole-body" uptake of radiotracer [ 17]:

T

Qwu = CO x Ewb x f Ca(t)dt, (2) 0

where Q,~b is whole-body tracer uptake in cpm, CO is cardiac output in ml/min and Ewu is whole-body "average" fractional extraction of tracer.

European Journal of Nuclear Medicine Vol. 21, No. 4, April 1994

Tissue 64Cu-PTSM uptake (%dose/g) a

2 min 5 min 15 min 30 rain 60 rain

Turnout 1.01 (0.40) 0.64 (0.31) 1.37 (0.46) 1.56 (0.31) 1.52 (0.58) Heart 4.37 (0.56) 4.86 (0.55) 3.74 (0.84) 3.86 (0.61) 2.81 (0.27) Brain 3.13 (0.31) 3.38 (0.55) 2.97 (0.54) 3.09 (0.75) 2.61 (0.36) Lung 4.88 (1.06) 6,09 (1.73) 4.34 (1.63) 2.49 (0.41) 2.32 (0.28) Liver 1.82 (0.19) 1.98 (0.29) 2.19 (0.05) 2.11 (0.17) 1.91 (0.20) Spleen 2.66 (0.65) 1,62 (0.58) 1.40 (0.17) 1.02 (0.12) 0.86 (0.05) Kidney 6.73 (0.79) 6,56 (0.80) 7.58 (0.32) 8.36 (1.44) 9.40 (0.55) Muscle 0.20 (0.02) 0.17 (0.03) 0.18 (0.04) 0.15 (0.04) 0.16 (0.05)

an=5, values normalised to 200-g rat, mean (1 SD)

1.0 Following complete tissue extraction of exchangeable radiotracer, Qwb may then be expressed in terms of the amount of tracer injected,Q~.j, by:

Qwb = Qi,j x (1 - B), (3)

where B is the fraction of tracer retained in the circulation in the form of non-lipophilic complexes. It should be noted that an extra term (1 - L) may be added to Eq. 3 for the case of intravenous administration of tracer to account for the fraction L extracted in the lungs via the pulmonary circulation.

Equations 1-3 may be combined to derive expressions for tissue blood flow and cardiac output using arterial blood sampling as an artificial "reference organ". For the case of the reference organ, E~s s is unity; therefore cardiac output is given by:

Fref X Qinj x (1 - B) CO - Ewb X Qref ' (4)

where Fre f is arterial sample flow rate in ml/min and Qref is inte- grated arterial sample activity in cpm.

Tissue blood flow is then given by:

Fref X Qtiss Ftiss - Qref X Etiss" (5)

For the calculation of CO and Ftiss it was assumed that B=0 and Ewb=Etiss = 1.

Statistical analysis. The method of mean differences [18] was used to compare estimates of tissue perfusion and cardiac output by 64Cu-PTSM and 57C0 microspheres, which assumes neither measurement is unequivocally correct but provides an assessment of agreement between the two. The difference (64Cu - 57C0) was plotted against the mean (64Cu + 57C0)/2 for each organ. It the values derived by the two methods agree, one would expect the differences to be normally distributed within two standard deviations of a mean of zero. A systematic error in one of the measurements results in a mean difference greater or less than zero. Student's t- test was used to assess the significance of the mean difference values, using the null hypothesis (64Cu - 57Co)=0.

R e s u l t s

The uptake o f 64Cu-PTSM within t issues o f in teres t is sum mar i zed in Table 1. P ro longed t issue re ten t ion was found for both turnout and norma l t issues over the per i-

1 ,... 0 0 .8 0 ',,= U U

0

~ 0.6

i--- -~ I:L ~ 0.4 I

k _

x 0.2

338

Table 1. Biodistribution of 64Cu-PTSM at various times following i.v. administration in the rat

0 5 10 15 20

t i m e (rain]

Fig. 1. Room temperature stability of 64Cu-PTSM in rat whole blood (solid circles), rat plasma (open circles), human whole blood (solid squares) and human plasma (open squares) (mean _+1 SD, n=6)

od o f observat ion. The progress ive r ise in k idney uptake after 15 min is cons is ten t wi th renal c learance of tracer. No increase in l iver uptake was evident . A n apparent fal l in t racer up take was obse rved in the spleen, and m a y re- f lect the k inet ics o f b lood cel ls conta in ing t rapped 64Cu complexes .

R a p i d convers ion of 64Cu-PTSM to complexes not ex- t rac ted by n-oc tano l was found in rat and human whole b lood (Fig. 1), but not in p l a s m a f rom ei ther species. The rate o f complex fo rmat ion was found to be greater in rat b lood.

Fo l l owing in t ravent r icu lar infus ion of 57Co microspheres, the cumula t ive arter ial sample act ivi ty rap id ly reaches a plateau, as shown in Fig. 2. However , a cont in- ued r ise in act ivi ty was found for 64Cu-PTSM, which was at t r ibuted to the rec i rcu la t ion of non- l ipophi l i c 64Cu complexes . Us ing n-oc tanol ex t rac t ion to remove 64Cu- P T S M resul ted in c lose ag reemen t be tween p la teau ac- t ivi ty values der ived us ing the two tracers. F r o m the cor- rec ted data it can be inferred that t issue ext rac t ion of


1.5

O

~. 1.0

'~ O

. ~ 0 .5 ,,i-,

E O

0 ~ - - I ~ ~ ] I I

0 15 30 4 5 6 0 75 90

time from start of infusion (sec) Fig. 2. Typical arterial sample cumulative activity curve for 57C0 microspheres (open squares), total 64Cu (solid circles) and 64Cu extracted in n-octanol (open circles)

339

64Cu-PTSM is virtually complete within 30 s post infusion, equivalent to approximately two mean circulation times in the rat. Levels of non-exchangeable circulating 64Cu complexes were estimated using the final blood fractions, and were found to be 0.351%___0. 029% injected dose/ml (mean ___ 1 SD) (0.419%_0.022%dose/ml total 64Cu).

Tissue uptake and blood flow values estimated by 64Cu-PTSM and 57Co microspheres are summarized in Table 2, and the influence of the solvent extraction procedure on blood flow estimation is illustrated in Fig. 3. Significant disagreement between the two methods was found only in heart and liver. Cardiac output values estimated by 64Cu-PTSM (59.8 ml/min _13.6, mean _+ 1 SD) and 57Co microspheres (54.1+16.9) were also in agreement (mean difference 5.7 ml/min +15.3; t=0.84, NS).

Apparent differences were noted in the distribution of 64Cu-PTSM uptake between the tissue retention and

Table 2. Tissue uptake at 2 . i n and blood flow estimates compared for 64Cu-PTSM and 57C0 microsphere methods

Tissue Uptake (%dose/g) ~ Blood flow (ml/min per gram)

64Cu 57C0 64Cu 57C0 64Cu_57C0

Heart 8.43 (1.15) 15.30 (5.21) 4.09 (1.33) 6.55 (1.90) -2.46 (0.69)** Brain 1.12 (0.20) 1.03 (0.26) 0.52 (0.14) 0.43 (0.17) 0.09 (0.18)* Lung 1.75 (0.44) 2.28 (2.04) 0.73 (0.11) 1.01 (1.14) -0.28 (1.13)* Liver 2.05 (0.83) 0.30 (0.14) 1.01 (0.57) 0.14 (0.03) 0.87 (0.55)** Spleen 1.53 (0.74) 2.73 (1.06) 0.77 (0.54) 1.15 (0.53) -0.36 (0.84)* Kidney 4.79 (0.78) 5.99 (1.29) 2.29 (0.88) 2.45 (0.65) -0.16 (1.33)* Muscle 0.17 (0.05) 0.18 (0.07) 0.08 (0.01) 0.07 (0.03) 0.01 (0.03)*

"n=5, values normalised to 200-g rat, mean (1 SD) * P >0.05, not significant; ** P <0.05

60 =

~. 40

" ~ 2o

0 O ,,t

- 2 0

- 4 0 ®

- 8 o

CARDIAC OUTPUT

. . . . . . . . . . . . _ ~ . . . . . . . . . . . . . . Et

[ ] A

I I I I . . . . . . . . . . . . . . . . . .

. . . . . . - f f - ~ . . . . . . . . . .

20 3o 40 so 60 70 Bo go

m e a n [(e lCLI" I -STCo)/2] (ml/min)

MUSCLE 0.10

._c 0.08

,EE 0.06 E 0.04

%" o.o2 o o.oo

(.) -0,02

-0.04

-0.06

-0,08

-0.10 1=, 0.02

0.5 7 BRAIN

c 0.4

o.3 • E 0.2 ~ . . . . . . . . . . . . . . . . 11" . . . . . . . . . . .

~ 0.1J m • A

I - o . o .

c~ -o.1 ~ . . . . ~ . . . . . [ ] . . . . - • " . . . . . . . • - - - /

/ -o., 1 []

/

011 013 0"5 0 ' 7

m e a n [ [ a 4 O u + 5 " C o ] / 2 ] ( m l / m i n / g )

._=

B -~

A ~ • ~,

. . . . u _ _ ~ . . . . . . . . . . _~_ . . . . . . . . . 8 c

ca

0 " 0 5 0 . 0 9 0 . 1 2

m e a n [ ( = 4 C u + S r C o ) / 2 ] I m l / m i n / g )

0 -

- 2 -

-6

- 8

HEART

I A . . . . . ~ . . . . . . . . . . . . . . . . . . . m - -

c

n

I k 4 6

m e a n [ [ e 4 C u + 5 7 C o ) / 2 ] ( m l / m i n / g )

Fig. 3. Difference against mean for cardiac output and tissue blood flow estimated by 57C0 microspheres and 64Cu-PTSM. Solid squares: solvent-extracted 64Cu (A shows mean difference, B represents -+1 SD); open squares: total 64Cu (C shows mean difference)


340

blood flow experiments (Tables 1, 2). Apart from the ex- pected decreased lung uptake where intraventricular tracer infusion was used, these were attributed to the dif- ferent anaesthetic regimens used for the two studies. Saffan was chosen as anaesthetic for the blood flow studies in order to minimise blood pressure depression during the procedure (own unpublished work), thus en- suring the stability of flee-flow arterial sampling.

Discussion

Our tissue retention findings support those of earlier re- ports [2, 3, 8], confirming that 64Cu-PTSM uptake re- mains constant in tumour and several normal tissues for periods compatible with MUP-PET imaging. If similar findings were demonstrated in humans, blood flow estimates could be derived using a single PET image combined with arterial blood sampling.

The estimation of arterial 64Cu-PTSM concentration has been investigated using a paired tracer technique with 57Co microspheres, and found to be influenced sig- nificantly by the recirculation of non-exchangeable 64Cu complexes. A straightforward correction method based on solvent extraction of 64Cu-PTSM using n-octanol has been demonstrated. Chromatographic analysis of n-octanol extracts has been reported [12] to show no evidence of lipophilic Cu-PTSM metabolites.

The rate of 64Cu-PTSM decomposition to non-lipophilic complexes in blood has been assessed and found to be species dependent, thus confirming the findings of Mathias et al. [12], who also showed complex formation to be temperature dependent. This is likely to be of little significance in practice, since tissue extraction of 64Cu- PTSM is relatively fast (two mean circulation times being approximately 2 rain in man). Rapid solvent extraction of blood samples is strongly recommended, however, which may be facilitated by withdrawal into a syringe preloaded with n-octanol.

Application of a trapped tracer model to estimate tissue blood flow appears satisfactory for tissues of low to moderate flow. For high-flow tissues such as myocardi- urn, flow will be underestimated as a result of reduced tracer extraction (Et~ <1 in Eq. 5). Flow-dependent re- duction of myocardial Cu-PTSM uptake has previously been demonstrated by Shelton et al. [5]; however, this effect is not apparent in the recently reported findings of Herrero et al. [19]. Further investigation of computation- al techniques for determining true myocardial blood flow values is therefore warranted. The estimation of cerebral blood flow in areas of high flow should also be made with caution, as demonstrated by Mathias et al. [6] in a comparative study using oxygen-15 labelled water.

Hepatic blood flow estimates were higher using 64Cu- PTSM measurements than by the microsphere method, reflecting a contribution to liver 64Cu-PTSM uptake via the portal circulation. Although no evidence of liver me- tabolism of 64Cu-PTSM was found in our study, the indi-

rect influence of splanchnic blood flow upon the estimation of liver flow appears to preclude reliable interpreta- tion.

In summary, our data suggest that the trapped tracer model provides a suitable basis for blood flow estimation with Cu-PTSM in tissues of low to moderate flow, when combined with a single measurement of tissue uptake and continuous arterial sampling. This approach provides an alternative to rapid dynamic PET imaging, and is therefore particularly well suited to large-area positron cameras such as MUP-PET.

Acknowledgement. The authors gratefully acknowledge the finan- cial support of the Cancer Research Campaign UK and the Medi- cal Research Council UK. We would also like to thank Dr. G. Pot- ter for providing samples of H2-PTSM ligand, and the team of the Nuffield Cyclotron for performing the irradiations of 64Ni.

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