0883-2897/9163.00+ 0.00 Copyright c 1991Pergamon Press plc
Nucl. Med. Biol. Vol. 18, No. 2, pp. 241-246, 1991 ht. J. Radiar. Appl. Instrum. Part B
Printed in Great Britain. All rights reserved
Distribution and Stability in the Rat of a 76Br/1251-labelled Polypeptide, Epidermal Growth Factor STEPHEN
SCOTT-ROBSON’, JACEK CAPALA’, JirRGEN CARLSSON’, PETTER MALMBORG’ and HANS LUNDQVIST-‘.*
‘Department of Radiation Sciences, Uppsala University, Box 535, S-751 21 Uppsala and *The Svedberg Laboratory, Uppsala University, Box 533, S-751 21 Uppsala, Sweden (Received
4 May 1990)
A positron-emitting isotope of bromine, ‘“Br, with a half-life of 16.2 h, was produced using the reaction na’Br(p, xn)76Kr. Labelling of mouse epidermal growth factor (EGF) with r6Br was optimized, using the chloramine-T method, obtaining a maximal radiochemical yield of 53%. In tests with receptor-rich, cultured glioma cells, [76Br]EGF and [‘*‘I]EGF bound equally well. A study of the distribution and stability of [76Br]EGF and [“‘I]EGF in normal rat was carried out. The distribution of both radioisotopes was similar, however, the percentage of 76Brbound to the high molecular weight fraction in the plasma, liver and kidney was greater than that of ‘251.
Introduction
In our initial studies, we selected epidermal growth factor (EGF) as a suitable macromolecule. EGF is a Radiohalogenated compounds are widely used in small polypeptide of 53 amino acid residues, and a biochemical and medical research, in diagnostic promolecular weight of 6 x IO3 daltons (Da) (Cohen, cedures and to some extent in therapy. The most popular radioisotopes are those of iodine such as lz31, 1986). EGF is of special interest because the EGF receptor is overexpressed in some tumours such as “‘1 and 13’1 because of their availability, suitable gliomas and squamous carcinomas (Mendelsohn, half-lives and the comparative ease with which many 1988). The EGF receptor is a potential candidate if organic compounds can be labelled. toxic agents are to be targeted against such tumours There are, however, advantages in using bromine as (Epenetos et al., 1985). Thus, it is of interest to analyse a halogen label (Maziere and Loch, 1986). Firstly, the whether the distribution of EGF, as seen through the C-Br bond in a bromine-containing organic comPET technique, can give any information about pound is generally somewhat stronger than the C-I in the clinical situation. bond for the iodine-analogue. Secondly, bromine is overexpression The aims of the present investigation were: freely distributed in the extracellular space and unlike iodine, has no large specific uptake in the thyroid or (1) to optimize the bromination of EGF as a the gastrointestinal tract. In order to follow a given function of pH and time, biological process, a convenient physical half-life is (2) to establish that [76Br]EGF retained its bindrequired. Bromine-76 has a physical half-life of 16.2 h ing specificity, and emits positrons in 57% of its decays. Thus, it may (3) to compare the distribution and stability of lend itself to PET studies of the metabolism of large [76Br]EGF and [rZ51]EGF in the normal rat. biomolecules, such as antibodies, whose rate of circulation in the human body is of the order of days. Materials and Methods It has been shown, using the chloramine-T method, Preparation of aqueous 76Brthat maximum radiochemical yields with bromine and the amino acids, tyrosine, uracil and cytosine, were Bromine-76 was produced at the Gustaf Werner obtained at low pH (Hadi et al., 1979; Petzold and Cyclotron at The Svedberg Laboratory, Uppsala Coenen, 1981). It was suggested that this simple University using the DatBr(p,xn)76Kr reaction. Targets method may be suitable for the radiobromination of of NaBr were irradiated for 6 h with I DA of 60 MeV proteins. protons. Both “Kr and 76Kr were produced, which decayed to “Br and 76Br respectively. To avoid *Author for correspondence. contamination by “Br, the radiochemical separation 241
242
STEPHEN SCOTT-ROBSON er
of the target was carried out 7 h after the end of bombardment. Preparations of up to 100 MBq ‘%~ were produced in sterile water at a concentration of 200 MBq/ml. Further details are given by Lundqvist et al. (1979). Radiolabelling
of EGF with 76Br and lz51
EGF was labelled with radioactive iodine using the chloramine-T (CAT) method (Hunter and Greenwood, 1962). Briefly, 5 pg of EGF in 50 PL of 0.5 M phosphate buffer, pH 7.5, were mixed with 20 MBq lz51, The reaction was initiated by the addition of 10 PL CAT (2 mg/mL). After 1 min, the reaction was terminated by adding 25 PL of sodium metabisulphite (2 mg/mL). Iodinated EGF and free radioactivity were separated on a G-25 Sephadex column. Prior to use, the column was pre-coated with phosphate buffered saline (PBS) containing 1 mg/mL bovine serum albumin (BSA). For cell experiments, Ham’s F-10 medium was used to elute the column and fractions of 0.5 mL were collected manually. For animal studies, physiological NaCl solution (9 mg/mL) was used to elute the column. The bromination of EGF was optimized as a function of pH. To 1 pg of EGF in 2OpL 0.5 M buffer was added 5OpL of aqueous ‘B-. The reaction was initiated by adding 1OpL of CAT (2 mg/mL). After 3 min, the reaction was terminated by adding 25 PL of sodium metabisulphite (2 mg/mL). The pH of the buffer of the EGF and CAT solutions was the same and its range varied between 3.5 and 7.5. The buffers used were citric acid-Na,HPO, in the pH range of 3.5-5.5 and a phosphate buffer in the range of 6.5-7.5. The pH of the sodium metabisulphite solution was 7.5. Separation of protein bound and free radioactivity was carried out as above. The same method was used to optimize the radiobromination as a function of reaction time. The pH of the buffer of the EGF and CAT solutions was 3.5. lZ51was purchased from Amersham International, U.K. G-25 Sephadex columns were purchased from Pharmacia, Stockholm, Sweden and Ham’s F-10 medium was purchased from Flow Laboratories, Scotland. Mouse epidermal growth factor (EGF), BSA and other chemicals were purchased from Sigma Chemical Co., St Louis, U.S.A.
al
activities, 0.4 and 1.0 MBq/pg, for ‘6Br and “? respectively. “Control experiments” were performed in which a further 12 samples of glioma cells were incubated in the presence of 1 fig of non-radioactive EGF. After careful washing (6 times for 1 min) in serum free medium the samples were trypsinized and measured in a liquid scintillation counter to obtain the radioactivity associated with the cells. Animal studies
The animal studies were conducted according to the rules set by the Swedish animal ethical committee. Sixteen male Sprague-Dawley rats (ALAB, Sollentuna, Sweden) weighing 3 15 + 15 g (mean f SD) were used. Food and water were available ad libitum. The epigastric vein was injected with both [76Br]EGF and [“‘IJEGF. The injection volume into each animal was 0.5 mL over a 5 s period and was taken from a stock solution of pooled [‘(‘Br]EGF and [‘251]EGF. The injected volume contained 0.5 MBq of [12’I]EGF and between 0.11 and 0.74 MBq of [76Br]EGF in about 70 pmol EGF. The 76Br radioactivity varied since the experiments were carried out over 3 days. Four rats were killed at intervals of 0.5, 1, 2 and 4 h. Prior to killing, samples of cardiac blood were taken and directly after killing, parts of the liver, kidney, small intestine, colon, muscle and testes were removed, weighed and measured for retained radioactivity. For analysis of the nature of the radioactivity in liver and kidney, 1 g of these organs was homogenized with 5 mL of NaCl solution (9 mg/mL). The blood and organs were centrifuged (lOOOg, 10 min) and 1 mL of each supernatant was eluted on a precoated Sephadex column to obtain the percentage of radioactivity in the high and low molecular fractions (HMF and LMF respectively). Samples were counted using a well-type y counter. Correction was made for the half-life of 76Brand its contribution to the count in the “‘1 window. In order to normalize measurements to differences in administered radioactivity and animal weight, the results are presented as “relative concentration” where: Relative concentration Measured radioactivity (cps)/ organ weight (g) = Administered
Administrution
qf the labelled EGF in cell experiments
To study [76Br]EGF binding specificity, a comparison was made with [“‘I]EGF. Using the methods described, EGF was labelled with each radioisotope with reaction times 0.5, 1, 2, 3, 5 and 10 min. The pH of the buffer used in the radiobromination was 5.5. Twelve samples of malignant glioma U 343 MG aC1 2:6 cells (Nister et a/., 1987) with about 250,000 EGF receptors per cell (Werner et al., 1988) were incubated for 20 min in media with the labelled EGF. Each dish contained about 0.2pg EGF with specific radio-
radioactivity (cps)/ animal weight (g)
When relative concentrations of HMF or LMF are given the organ weight in the definition above is multiplied with the HMF or LMF respectively.
Results The radiochemical yield obtained following the radiobromination of EGF at different pH and reaction times had a maximum of 53% at a reaction
Labelling of EGF with 76Br time of 3 min and at pH 3.5. However, the radiochemical yield decreased only slightly for pH changes up to 6.5. We chose a buffer with pH = 5.5 (radiochemical yield of 48%) for the following studies, since macromolecules are more likely to retain their biological activity if the labelling procedure is carried out at a more neutral pH. In the glioma cell culture study, the mean value of the ratio of the radioactivity associated with the cells in the sample divided by the radioactivity associated with the cells in the control (presaturated with nonradioactive EGF) for each reaction time was 9.3 k 2.4 and 7.9 &-1.9 for 76Br and “‘1 respectively. The binding specificity of [76Br]EGF and [“‘I]EGF to the EGF receptor, obtained for reaction times up to 10 min, was independent of the reaction time. Since there were no significant differences in the results when using [76Br]EGF or [“‘I]EGF, it was concluded that the binding specificities were the same. The results of the animal studies are shown in Figs 1-4. The relative concentrations in the plasma, liver, kidney and the percentage of the injected radioactivity in urine at intervals 0.5, 1, 2 and 4 h after i.v. injection are shown in Fig. 1. In all cases, the relative concentration of 76Br is greater than that of ‘251. The relative concentrations in the small intestine, colon, muscle and testes at intervals 0.5, 1, 2 and 4 h after i.v. injection are shown in Fig. 2. In these
a
243
organs, the relative concentration of 76Br is smaller than that of ‘**I at 0.5 h, but larger at 4 h. The relative concentrations of HMF and LMF in the plasma at intervals 0.5, 1, 2 and 4 h after i.v. injection are shown in Fig. 3. It was seen that the relative concentration of HMF of both radioisotopes in the plasma could be described by exponential functions with about the same slope (2.31 h for 76Br and 2.07 h for ‘2sI), but with different amplitudes. The relative concentration of LMF of 76Br in the plasma increased as a function of time, while the relative concentration of LMF of “‘1 in the plasma increased to a maximum between I and 2 h after iv. and decreased thereafter. The relative concentration of HMF in the liver and kidney at intervals 0.5, I, 2 and 4 h after i.v. injection is shown in Fig. 4. It was seen that the clearance of relative conentration of HMF from both organs could be described by exponential functions with different slopes but with the same amplitude at zero time. The biological half-lives obtained for 76Br were 1.19 h in the liver and 1.99 h in the kidney. Corresponding values for 12’1were 0.61 and 0.75h.
Discussion Radiolabelled polypeptides and proteins such as antibodies, for in viva studies have been labelled mainly with iodine (‘231, “‘1 and 13’1)or “‘In for
b
TIME (hr) Fig. I. The relative concentration of radioactivity in (a) plasma, (b) liver and (c) kidney is given at 0.5, I, 2 and 4 h respectively. The percentage of injected radioactivity contained in urine at respective times is given in (d). n , lz51; q 76Br.
244
STEPHEN SCOTT-ROBKIN
et a(.
TIME (hr)
TIME (hr)
TIME (hr)
TIME (hr)
Fig. 2. The relative concentrations of radioactivity in (a) small intestine, (b) colon, (c) muscle and (d) testes is given at 0.5, 1, 2 and 4 h respectively. n , i2sI; q, 76Br. imaging with the y camera (Mather, 1986). Radioiodination of proteins is well established, but none of the iodine isotopes have physical properties which are ideal for y camera studies. Recently, it has become popular to label antibodies with “‘In via conjugated chelating groups since, in many cases, a stable in vivo label is obtained. “‘In has a suitable half-life of 2.81 days and convenient photon energies for imaging.
TIME (hr) Fig. 3. The relative concentration of HMF-bound and LMF-bound radioactivity in plasma as a function of time; (a) corresponds to 76Br in LMF, (b) 12’1in LMF, (c) HMF-bound 76Brand (d) HMF-bound ‘2sI. The clearance of both HMF 76Brand 12JImay be described by exponential functions as shown.
To our knowledge, PET has been used only occasionally for the study of protein and polypeptide distribution, but should offer some specific advantages compared with y camera or single photon emission computed tomography (SPECT) studies. These advantages are: (i) better spatial resolution since PET offers a spatial resolution of about 68 mm while Spect has a spatial resolution in the order of 15mm; (ii) better signal-to-background ratio. This is especially true compared with y camera studies since PET provides tomographic sections. In addition, better quantitative corrections for attenuation and scatter can be made in PET investigations, compared with SPECT; (iii) better quantified radioactivity distribution obtained with PET allows a correct subtraction of e.g. blood-borne radioactivity which generally is the main cause of background in antibody studies. Suitable positron emitting isotopes of iodine and indium were considered since their stability and effects during the labelling procedure are well known. Due to its half-life of 4.15 days ‘24Iis of interest, but, its physical properties are less suitable, since its decay contains only 25% positrons (Sharma ef al., 1988). The only possible positron emitter of indium is “0In,
Labelling of EGF with 76Br
a
I TIME (hr)
1:
0
1
2
3
4
5
TIME (hr) Fig. 4. The relative concentration of HMF-bound radioactivity in (a) liver and (b) kidney respectively. The clearance of both 76Br and lZ51may be described by exponential functions as shown.
whose half-life of 69 min is however too short for most macromolecular studies. Other positron emitting radionuchdes have been proposed (Larsson, 1984). We considered also other isotopes of halogens since there should be an analogy with iodine-labelling when it concerns the simplicity, stability and effects of labelling. The use of “F for PET studies with its half-life of 1lOmin gives a practical investigation time window of 6-7 h. This is probably an adequate time window to image antibodies acting in the vascular space but, for the extravascular/intracellular space where membrane transport is involved, the half-life is much too short. The radionuclide studied in this paper, 76Br,is easy to produce with high radionuclidic purity using the ““Br(p, xn)76Kr reaction and a yield of about 18MBq/pAh (De Jong et al., 1979). The decay of 76Brcontains 57% positrons and its half-life of 16.2 h provides a reasonable time window of 2 days. EGF was chosen as a model protein since there is an on-going project at our institute using EGF as a carrier for cytotoxic agents, labelling procedures with iodine and in vitro biological tests had therefore already been established. The normal in vivo distri-
245
bution in rat, studied with [‘*‘I]EGF has previously been carried out showing rapid and dominant uptakes in the liver and kidney (Kim et al., 1988, 1989; Mateo de Acosta ef al., 1989). There is a need for in in vivo detection technique if EGF is to be used for tumour diagnosis and/or therapy. The use of 76Brfor PET studies is thought to give valuable information concerning the in vivo distribution of EGF, as well as providing experience and information for its possible future use as a label of antibodies in PET. The chloramine-T method of labelling proteins with iodine was chosen for its simplicity. Higher radiochemical yields may have been obtainable using an enzymatic method such as chloroperoxydase (Maziere and Loc’h, 1986). The chloramine-T method was found to work well with 76Brand optimal labelling conditions differed only slightly from those of iodine. The cell binding studies showed that the 76Br-labelled EGF bound to the EGF receptor. Our results are in accord with those previously reported. It is known that the clearance of EGF from blood is rapid and that 2.5 min after injection, 66% of the label is found in the liver and kidney (Jorgensen et al., 1988). The degradation of EGF was already significant 0.5 h after injection. The relative concentration of 76Brat 0.5 h was higher than that of “‘1 in all organs except for the small intestine and colon, the latter which are known to concentrate free iodine. Since bromine is evenly distributed in the extracellular space, the higher relative concentration of 76Brwas due to larger fraction of 76Brbeing bound in high molecular fractions in the other organs. The clearance of the labelled HMF from liver and kidney was exponential for 76Br and izsI, but it was more rapid for ‘25I. If these curves were extrapolated to the injection time, the same relative concentration values were obtained for the two labels indicating that the initial rapid blood clearance phase was the same for [76Br]EGF and [iZSI]EGF. Up to 4 h after injection, the label was still bound in the HMF. This HMF in plasma could not be EGF since the rapid clearance from the blood and the relatively long clearance time from the organs should completely empty the labelled EGF pool. It is also clear that a greater fraction of the 76Br radioactivity is bound to the HMF than IzsI. That is reasonable, since the covalent binding of C-Br is stronger than that of C-I. Iodine is also an important trace element and some specific enzyme systems exist for its release from proteins. Since brom.ne, as far as we know is not an essential trace element, the enzymatic dehalogenation of bromine compounds may be slower than that of iodine or non existent. Free iodine is distributed in the body with high specific uptakes in the thyroid and in the gastrointestinal tract, while free bromine is evenly distributed in the extracellular space. A more stable bromine label should give a slower release of free bromine. The clearance of bromine from plasma is also slower than that of iodine. The two factors together might explain the
246
time course
STEPHEN
SCOTT-ROBSON et al.
of 76Br and ‘*‘I in the LMF of plasma,
where “‘1 is high at 0.5 h and attains a maximum at 1 h, while 76Br starts at a fairly low value at 0.5 h and increases steadily during the time of the experiment. The relative concentration of 76Br in the low molecular form is twice that of ‘25Iat 4 h. A higher signal but also a higher background is thus to be expected at later times when using bromine as label instead of iodine. Due to the even distribution of bromide this
background may, if needed, be subtracted in PET investigations by taking plasma samples. We are presently analysing data concerning the distribution of [76Br]EGF in the rhesus monkey using the PET technique. Acknowledgement-This
work was supported by the Swedish Cancer Society, Grant Nos 1l76-B90-03XA, 90: 307 and 980-B90-02XB. 90: 237.
References Cohen, S. Epidermal growth factor. Biosci. Rep. 6: 1017; 1986.
De Jong, D.; Kooiman, H.; Beenboer, J. Th. 76Brand “Br from decay of cyclotron produced 76Kr and “Kr. Inr. J. Appl. Radial. Isor. 30: 786; 1979.
Epenetos, A. A.; Courtney-Luck, N.; Pickering, D.; Hookert, G.; Durbin, H.; Lavender, J. P.; McKenzie, C. C. Antibody guided irradiation of brain glioma by arterial infusion of radioactive monoclonal antibody against epidermal growth factor receptor and blood group A antigen. Br. Med. J. 290: 1463; 1985. Hadi, U. A. M.; Malcolme-Lawes, D. J.; Oldham, G. Rapid radiohalogenations of small molecules. 1n1. J. Appl. Radial. Isot. 30: 709; 1979.
Hunter, W. M.; Greenwood, F. C. Preparation of iodine13I labelled human growth hormone of high specific activity. Nature 194: 495; 1962. Jorgensen, P. E.; Poulsen, S. S.; Nex0, E. Distribution of i.v. administered epidermal growth factor in the rat. Regularory Peptides 23: 161; 1988.
Kim. D. C.; Sugiyama, Y.: Satoh, H.; Fuwa, T.; Iga. T.; Hanano, M. Kinetic analysis of in z?ro receptor dependent binding of human epidermal growth factor by rat tissues. J. fharm. Sci. 77: 200: 1988. Kim, D. C.; Sugiyama, Y.; Fuwa, T.; Sakamoto. S.; Iga. T.; Hanano. M. Kinetic analysis of the elimination process of human epidermal growth factor (hEGF) in rats. Biochem. Pharmacol. 38: 241; 1989.
Larsson. B. Labelled macromolecules for studies of cellular receptors in rioo. Medical Applications qf C_vclotrons III, Ann. Unit,. Turkuensis D: 17: 87; 1984. Lundqvist, H.; Malmborg. P.; Langstrom, B.; Chiengmai. S. N. Simple production of “Br- and ‘221- and their use in the ladelling of [“Br]BrUdR and [‘?‘I]IUdR. Inr. J. Appl. Radial. Isot. 30: 39; 1979.
Mateo de Acosta, C.; Justiz, E.; Skoog. L.; Lage. A. Biodistribution of radioactive epidermal growth factor in normal and tumor bearing mice. Anticancer Res. 9: 87; 1989.
Mather, S. J. Radioiodinated monoclonal antibodies: a critical review. Appl. Radial. Isor. 37: 725; 1986. Maziere, B.; Loc’h. C. Radiopharmaceuticals labelled with bromine isotopes. Appl. Radial. Isor. 37: 703: 1986. Mendelsohn, J. Growth factor receptors as targets for antitumour therapy with monoclonal antibodies. Monoclonal Antibody Ther. 45: 147; 1988.
Nister, M.; Wedell. B.; Betsholtz, C.; Bywater. M.: Pettersson, M.; Westermark, B.; Mark, J. Evidence for progressional changes in the human malignant glioma line U-343MGa; analysis of karyotype and expression of genes encoding the subunit chains of platelet-derived growth factor. Cancer Res. 47: 4953; 1987. Petzold, G.; Coenen, H. H. Chloramine-T for “no carrieradded” labelling of aromatic biomolecules with bromine75, 77. J. Labelled Compd. Radiopharm. 18: 1319; 1981. Sharma. H. L.: Zweit. J.: Downev. S.: Smith. A. M.: Smith. A. G. Prodiction of li41 for p&itrbn emission tomogra: phy. Seventh Int. S.vmp. Radiopharmaceutical Chem isrry-Abstract 165. Werner, M. H.; Humphrey, P. A.; Bigner, D. D.; Bigner, S. H. Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines. Acfa Neuropathol. 77: 196; 1988.
Nucl. Med. Biol. Vol. 18, No. 2, pp. 241-246, 1991 ht. J. Radiar. Appl. Instrum. Part B
Printed in Great Britain. All rights reserved
Distribution and Stability in the Rat of a 76Br/1251-labelled Polypeptide, Epidermal Growth Factor STEPHEN
SCOTT-ROBSON’, JACEK CAPALA’, JirRGEN CARLSSON’, PETTER MALMBORG’ and HANS LUNDQVIST-‘.*
‘Department of Radiation Sciences, Uppsala University, Box 535, S-751 21 Uppsala and *The Svedberg Laboratory, Uppsala University, Box 533, S-751 21 Uppsala, Sweden (Received
4 May 1990)
A positron-emitting isotope of bromine, ‘“Br, with a half-life of 16.2 h, was produced using the reaction na’Br(p, xn)76Kr. Labelling of mouse epidermal growth factor (EGF) with r6Br was optimized, using the chloramine-T method, obtaining a maximal radiochemical yield of 53%. In tests with receptor-rich, cultured glioma cells, [76Br]EGF and [‘*‘I]EGF bound equally well. A study of the distribution and stability of [76Br]EGF and [“‘I]EGF in normal rat was carried out. The distribution of both radioisotopes was similar, however, the percentage of 76Brbound to the high molecular weight fraction in the plasma, liver and kidney was greater than that of ‘251.
Introduction
In our initial studies, we selected epidermal growth factor (EGF) as a suitable macromolecule. EGF is a Radiohalogenated compounds are widely used in small polypeptide of 53 amino acid residues, and a biochemical and medical research, in diagnostic promolecular weight of 6 x IO3 daltons (Da) (Cohen, cedures and to some extent in therapy. The most popular radioisotopes are those of iodine such as lz31, 1986). EGF is of special interest because the EGF receptor is overexpressed in some tumours such as “‘1 and 13’1 because of their availability, suitable gliomas and squamous carcinomas (Mendelsohn, half-lives and the comparative ease with which many 1988). The EGF receptor is a potential candidate if organic compounds can be labelled. toxic agents are to be targeted against such tumours There are, however, advantages in using bromine as (Epenetos et al., 1985). Thus, it is of interest to analyse a halogen label (Maziere and Loch, 1986). Firstly, the whether the distribution of EGF, as seen through the C-Br bond in a bromine-containing organic comPET technique, can give any information about pound is generally somewhat stronger than the C-I in the clinical situation. bond for the iodine-analogue. Secondly, bromine is overexpression The aims of the present investigation were: freely distributed in the extracellular space and unlike iodine, has no large specific uptake in the thyroid or (1) to optimize the bromination of EGF as a the gastrointestinal tract. In order to follow a given function of pH and time, biological process, a convenient physical half-life is (2) to establish that [76Br]EGF retained its bindrequired. Bromine-76 has a physical half-life of 16.2 h ing specificity, and emits positrons in 57% of its decays. Thus, it may (3) to compare the distribution and stability of lend itself to PET studies of the metabolism of large [76Br]EGF and [rZ51]EGF in the normal rat. biomolecules, such as antibodies, whose rate of circulation in the human body is of the order of days. Materials and Methods It has been shown, using the chloramine-T method, Preparation of aqueous 76Brthat maximum radiochemical yields with bromine and the amino acids, tyrosine, uracil and cytosine, were Bromine-76 was produced at the Gustaf Werner obtained at low pH (Hadi et al., 1979; Petzold and Cyclotron at The Svedberg Laboratory, Uppsala Coenen, 1981). It was suggested that this simple University using the DatBr(p,xn)76Kr reaction. Targets method may be suitable for the radiobromination of of NaBr were irradiated for 6 h with I DA of 60 MeV proteins. protons. Both “Kr and 76Kr were produced, which decayed to “Br and 76Br respectively. To avoid *Author for correspondence. contamination by “Br, the radiochemical separation 241
242
STEPHEN SCOTT-ROBSON er
of the target was carried out 7 h after the end of bombardment. Preparations of up to 100 MBq ‘%~ were produced in sterile water at a concentration of 200 MBq/ml. Further details are given by Lundqvist et al. (1979). Radiolabelling
of EGF with 76Br and lz51
EGF was labelled with radioactive iodine using the chloramine-T (CAT) method (Hunter and Greenwood, 1962). Briefly, 5 pg of EGF in 50 PL of 0.5 M phosphate buffer, pH 7.5, were mixed with 20 MBq lz51, The reaction was initiated by the addition of 10 PL CAT (2 mg/mL). After 1 min, the reaction was terminated by adding 25 PL of sodium metabisulphite (2 mg/mL). Iodinated EGF and free radioactivity were separated on a G-25 Sephadex column. Prior to use, the column was pre-coated with phosphate buffered saline (PBS) containing 1 mg/mL bovine serum albumin (BSA). For cell experiments, Ham’s F-10 medium was used to elute the column and fractions of 0.5 mL were collected manually. For animal studies, physiological NaCl solution (9 mg/mL) was used to elute the column. The bromination of EGF was optimized as a function of pH. To 1 pg of EGF in 2OpL 0.5 M buffer was added 5OpL of aqueous ‘B-. The reaction was initiated by adding 1OpL of CAT (2 mg/mL). After 3 min, the reaction was terminated by adding 25 PL of sodium metabisulphite (2 mg/mL). The pH of the buffer of the EGF and CAT solutions was the same and its range varied between 3.5 and 7.5. The buffers used were citric acid-Na,HPO, in the pH range of 3.5-5.5 and a phosphate buffer in the range of 6.5-7.5. The pH of the sodium metabisulphite solution was 7.5. Separation of protein bound and free radioactivity was carried out as above. The same method was used to optimize the radiobromination as a function of reaction time. The pH of the buffer of the EGF and CAT solutions was 3.5. lZ51was purchased from Amersham International, U.K. G-25 Sephadex columns were purchased from Pharmacia, Stockholm, Sweden and Ham’s F-10 medium was purchased from Flow Laboratories, Scotland. Mouse epidermal growth factor (EGF), BSA and other chemicals were purchased from Sigma Chemical Co., St Louis, U.S.A.
al
activities, 0.4 and 1.0 MBq/pg, for ‘6Br and “? respectively. “Control experiments” were performed in which a further 12 samples of glioma cells were incubated in the presence of 1 fig of non-radioactive EGF. After careful washing (6 times for 1 min) in serum free medium the samples were trypsinized and measured in a liquid scintillation counter to obtain the radioactivity associated with the cells. Animal studies
The animal studies were conducted according to the rules set by the Swedish animal ethical committee. Sixteen male Sprague-Dawley rats (ALAB, Sollentuna, Sweden) weighing 3 15 + 15 g (mean f SD) were used. Food and water were available ad libitum. The epigastric vein was injected with both [76Br]EGF and [“‘IJEGF. The injection volume into each animal was 0.5 mL over a 5 s period and was taken from a stock solution of pooled [‘(‘Br]EGF and [‘251]EGF. The injected volume contained 0.5 MBq of [12’I]EGF and between 0.11 and 0.74 MBq of [76Br]EGF in about 70 pmol EGF. The 76Br radioactivity varied since the experiments were carried out over 3 days. Four rats were killed at intervals of 0.5, 1, 2 and 4 h. Prior to killing, samples of cardiac blood were taken and directly after killing, parts of the liver, kidney, small intestine, colon, muscle and testes were removed, weighed and measured for retained radioactivity. For analysis of the nature of the radioactivity in liver and kidney, 1 g of these organs was homogenized with 5 mL of NaCl solution (9 mg/mL). The blood and organs were centrifuged (lOOOg, 10 min) and 1 mL of each supernatant was eluted on a precoated Sephadex column to obtain the percentage of radioactivity in the high and low molecular fractions (HMF and LMF respectively). Samples were counted using a well-type y counter. Correction was made for the half-life of 76Brand its contribution to the count in the “‘1 window. In order to normalize measurements to differences in administered radioactivity and animal weight, the results are presented as “relative concentration” where: Relative concentration Measured radioactivity (cps)/ organ weight (g) = Administered
Administrution
qf the labelled EGF in cell experiments
To study [76Br]EGF binding specificity, a comparison was made with [“‘I]EGF. Using the methods described, EGF was labelled with each radioisotope with reaction times 0.5, 1, 2, 3, 5 and 10 min. The pH of the buffer used in the radiobromination was 5.5. Twelve samples of malignant glioma U 343 MG aC1 2:6 cells (Nister et a/., 1987) with about 250,000 EGF receptors per cell (Werner et al., 1988) were incubated for 20 min in media with the labelled EGF. Each dish contained about 0.2pg EGF with specific radio-
radioactivity (cps)/ animal weight (g)
When relative concentrations of HMF or LMF are given the organ weight in the definition above is multiplied with the HMF or LMF respectively.
Results The radiochemical yield obtained following the radiobromination of EGF at different pH and reaction times had a maximum of 53% at a reaction
Labelling of EGF with 76Br time of 3 min and at pH 3.5. However, the radiochemical yield decreased only slightly for pH changes up to 6.5. We chose a buffer with pH = 5.5 (radiochemical yield of 48%) for the following studies, since macromolecules are more likely to retain their biological activity if the labelling procedure is carried out at a more neutral pH. In the glioma cell culture study, the mean value of the ratio of the radioactivity associated with the cells in the sample divided by the radioactivity associated with the cells in the control (presaturated with nonradioactive EGF) for each reaction time was 9.3 k 2.4 and 7.9 &-1.9 for 76Br and “‘1 respectively. The binding specificity of [76Br]EGF and [“‘I]EGF to the EGF receptor, obtained for reaction times up to 10 min, was independent of the reaction time. Since there were no significant differences in the results when using [76Br]EGF or [“‘I]EGF, it was concluded that the binding specificities were the same. The results of the animal studies are shown in Figs 1-4. The relative concentrations in the plasma, liver, kidney and the percentage of the injected radioactivity in urine at intervals 0.5, 1, 2 and 4 h after i.v. injection are shown in Fig. 1. In all cases, the relative concentration of 76Br is greater than that of ‘251. The relative concentrations in the small intestine, colon, muscle and testes at intervals 0.5, 1, 2 and 4 h after i.v. injection are shown in Fig. 2. In these
a
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organs, the relative concentration of 76Br is smaller than that of ‘**I at 0.5 h, but larger at 4 h. The relative concentrations of HMF and LMF in the plasma at intervals 0.5, 1, 2 and 4 h after i.v. injection are shown in Fig. 3. It was seen that the relative concentration of HMF of both radioisotopes in the plasma could be described by exponential functions with about the same slope (2.31 h for 76Br and 2.07 h for ‘2sI), but with different amplitudes. The relative concentration of LMF of 76Br in the plasma increased as a function of time, while the relative concentration of LMF of “‘1 in the plasma increased to a maximum between I and 2 h after iv. and decreased thereafter. The relative concentration of HMF in the liver and kidney at intervals 0.5, I, 2 and 4 h after i.v. injection is shown in Fig. 4. It was seen that the clearance of relative conentration of HMF from both organs could be described by exponential functions with different slopes but with the same amplitude at zero time. The biological half-lives obtained for 76Br were 1.19 h in the liver and 1.99 h in the kidney. Corresponding values for 12’1were 0.61 and 0.75h.
Discussion Radiolabelled polypeptides and proteins such as antibodies, for in viva studies have been labelled mainly with iodine (‘231, “‘1 and 13’1)or “‘In for
b
TIME (hr) Fig. I. The relative concentration of radioactivity in (a) plasma, (b) liver and (c) kidney is given at 0.5, I, 2 and 4 h respectively. The percentage of injected radioactivity contained in urine at respective times is given in (d). n , lz51; q 76Br.
244
STEPHEN SCOTT-ROBKIN
et a(.
TIME (hr)
TIME (hr)
TIME (hr)
TIME (hr)
Fig. 2. The relative concentrations of radioactivity in (a) small intestine, (b) colon, (c) muscle and (d) testes is given at 0.5, 1, 2 and 4 h respectively. n , i2sI; q, 76Br. imaging with the y camera (Mather, 1986). Radioiodination of proteins is well established, but none of the iodine isotopes have physical properties which are ideal for y camera studies. Recently, it has become popular to label antibodies with “‘In via conjugated chelating groups since, in many cases, a stable in vivo label is obtained. “‘In has a suitable half-life of 2.81 days and convenient photon energies for imaging.
TIME (hr) Fig. 3. The relative concentration of HMF-bound and LMF-bound radioactivity in plasma as a function of time; (a) corresponds to 76Br in LMF, (b) 12’1in LMF, (c) HMF-bound 76Brand (d) HMF-bound ‘2sI. The clearance of both HMF 76Brand 12JImay be described by exponential functions as shown.
To our knowledge, PET has been used only occasionally for the study of protein and polypeptide distribution, but should offer some specific advantages compared with y camera or single photon emission computed tomography (SPECT) studies. These advantages are: (i) better spatial resolution since PET offers a spatial resolution of about 68 mm while Spect has a spatial resolution in the order of 15mm; (ii) better signal-to-background ratio. This is especially true compared with y camera studies since PET provides tomographic sections. In addition, better quantitative corrections for attenuation and scatter can be made in PET investigations, compared with SPECT; (iii) better quantified radioactivity distribution obtained with PET allows a correct subtraction of e.g. blood-borne radioactivity which generally is the main cause of background in antibody studies. Suitable positron emitting isotopes of iodine and indium were considered since their stability and effects during the labelling procedure are well known. Due to its half-life of 4.15 days ‘24Iis of interest, but, its physical properties are less suitable, since its decay contains only 25% positrons (Sharma ef al., 1988). The only possible positron emitter of indium is “0In,
Labelling of EGF with 76Br
a
I TIME (hr)
1:
0
1
2
3
4
5
TIME (hr) Fig. 4. The relative concentration of HMF-bound radioactivity in (a) liver and (b) kidney respectively. The clearance of both 76Br and lZ51may be described by exponential functions as shown.
whose half-life of 69 min is however too short for most macromolecular studies. Other positron emitting radionuchdes have been proposed (Larsson, 1984). We considered also other isotopes of halogens since there should be an analogy with iodine-labelling when it concerns the simplicity, stability and effects of labelling. The use of “F for PET studies with its half-life of 1lOmin gives a practical investigation time window of 6-7 h. This is probably an adequate time window to image antibodies acting in the vascular space but, for the extravascular/intracellular space where membrane transport is involved, the half-life is much too short. The radionuclide studied in this paper, 76Br,is easy to produce with high radionuclidic purity using the ““Br(p, xn)76Kr reaction and a yield of about 18MBq/pAh (De Jong et al., 1979). The decay of 76Brcontains 57% positrons and its half-life of 16.2 h provides a reasonable time window of 2 days. EGF was chosen as a model protein since there is an on-going project at our institute using EGF as a carrier for cytotoxic agents, labelling procedures with iodine and in vitro biological tests had therefore already been established. The normal in vivo distri-
245
bution in rat, studied with [‘*‘I]EGF has previously been carried out showing rapid and dominant uptakes in the liver and kidney (Kim et al., 1988, 1989; Mateo de Acosta ef al., 1989). There is a need for in in vivo detection technique if EGF is to be used for tumour diagnosis and/or therapy. The use of 76Brfor PET studies is thought to give valuable information concerning the in vivo distribution of EGF, as well as providing experience and information for its possible future use as a label of antibodies in PET. The chloramine-T method of labelling proteins with iodine was chosen for its simplicity. Higher radiochemical yields may have been obtainable using an enzymatic method such as chloroperoxydase (Maziere and Loc’h, 1986). The chloramine-T method was found to work well with 76Brand optimal labelling conditions differed only slightly from those of iodine. The cell binding studies showed that the 76Br-labelled EGF bound to the EGF receptor. Our results are in accord with those previously reported. It is known that the clearance of EGF from blood is rapid and that 2.5 min after injection, 66% of the label is found in the liver and kidney (Jorgensen et al., 1988). The degradation of EGF was already significant 0.5 h after injection. The relative concentration of 76Brat 0.5 h was higher than that of “‘1 in all organs except for the small intestine and colon, the latter which are known to concentrate free iodine. Since bromine is evenly distributed in the extracellular space, the higher relative concentration of 76Brwas due to larger fraction of 76Brbeing bound in high molecular fractions in the other organs. The clearance of the labelled HMF from liver and kidney was exponential for 76Br and izsI, but it was more rapid for ‘25I. If these curves were extrapolated to the injection time, the same relative concentration values were obtained for the two labels indicating that the initial rapid blood clearance phase was the same for [76Br]EGF and [iZSI]EGF. Up to 4 h after injection, the label was still bound in the HMF. This HMF in plasma could not be EGF since the rapid clearance from the blood and the relatively long clearance time from the organs should completely empty the labelled EGF pool. It is also clear that a greater fraction of the 76Br radioactivity is bound to the HMF than IzsI. That is reasonable, since the covalent binding of C-Br is stronger than that of C-I. Iodine is also an important trace element and some specific enzyme systems exist for its release from proteins. Since brom.ne, as far as we know is not an essential trace element, the enzymatic dehalogenation of bromine compounds may be slower than that of iodine or non existent. Free iodine is distributed in the body with high specific uptakes in the thyroid and in the gastrointestinal tract, while free bromine is evenly distributed in the extracellular space. A more stable bromine label should give a slower release of free bromine. The clearance of bromine from plasma is also slower than that of iodine. The two factors together might explain the
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SCOTT-ROBSON et al.
of 76Br and ‘*‘I in the LMF of plasma,
where “‘1 is high at 0.5 h and attains a maximum at 1 h, while 76Br starts at a fairly low value at 0.5 h and increases steadily during the time of the experiment. The relative concentration of 76Br in the low molecular form is twice that of ‘25Iat 4 h. A higher signal but also a higher background is thus to be expected at later times when using bromine as label instead of iodine. Due to the even distribution of bromide this
background may, if needed, be subtracted in PET investigations by taking plasma samples. We are presently analysing data concerning the distribution of [76Br]EGF in the rhesus monkey using the PET technique. Acknowledgement-This
work was supported by the Swedish Cancer Society, Grant Nos 1l76-B90-03XA, 90: 307 and 980-B90-02XB. 90: 237.
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Nister, M.; Wedell. B.; Betsholtz, C.; Bywater. M.: Pettersson, M.; Westermark, B.; Mark, J. Evidence for progressional changes in the human malignant glioma line U-343MGa; analysis of karyotype and expression of genes encoding the subunit chains of platelet-derived growth factor. Cancer Res. 47: 4953; 1987. Petzold, G.; Coenen, H. H. Chloramine-T for “no carrieradded” labelling of aromatic biomolecules with bromine75, 77. J. Labelled Compd. Radiopharm. 18: 1319; 1981. Sharma. H. L.: Zweit. J.: Downev. S.: Smith. A. M.: Smith. A. G. Prodiction of li41 for p&itrbn emission tomogra: phy. Seventh Int. S.vmp. Radiopharmaceutical Chem isrry-Abstract 165. Werner, M. H.; Humphrey, P. A.; Bigner, D. D.; Bigner, S. H. Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines. Acfa Neuropathol. 77: 196; 1988.
