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Gedda, L., Olsson, P., Ponten, J., and Carlsson, J. (1996) Development and in vitro studies of epidermal growth factor-dextran conjugates for boron ne...
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Bioconjugate Chem. 1999, 10, 938−946

Effects of Dextranation on the Pharmacokinetics of Short Peptides. A PET Study on mEGF Qinghai Zhao,† Vladimir Tolmachev,*,† Jo¨rgen Carlsson,† Hans Lundqvist,† Johanna Sundin,†,‡ Jan-Christer Janson,§,| and Anders Sundin‡ Biomedical Radiation Sciences, Box 535, Uppsala University, S-751 21 Uppsala, Sweden, Department of Diagnostic Radiology, Uppsala University and Uppsala University PET-Centre, Uppsala University Hospital, S-751 85 Uppsala, Sweden, Pharmacia Biotech AB, S-751 82 Uppsala, Sweden, and Centre of Surface Biotechnology, Uppsala Biomedical Centre, Box 577,S- 751 23 Uppsala, Sweden. Received January 27, 1999; Revised Manuscript Received June 1, 1999

The effects of dextranation on the biodistribution of mouse epidermal growth factor (mEGF, 6 kDa) were assessed. By reductive amination, mEGF was coupled to 13 and 46 kDa dextran. The two dextranated conjugates and free mEGF were labeled with the positron-emitting nuclide 76Br (T1/2 ) 16 h). After intravenous administration to Sprague Dawley rats, the radioactivity biodistribution was evaluated by positron emission tomography (PET) and by measurements of dissected tissues. The dextranation prolonged the retention time in blood, especially when the dextran chain was long. [76Br]mEGF-dextran conjugates were shown to have significantly, more than 5 times, lower kidney accumulation than the nonconjugated [76Br]mEGF. In conclusion, dextranation affects the biodistribution of mEGF in vivo giving a prolonged circulation time, a decreased uptake in kidney, and an increased spleen accumulation.

INTRODUCTION

Epidermal growth factor (EGF) is a short (53 residues, 6 kDa) regulatory protein, which is involved in growth and maturation of epithelial tissues in homeostasis as well as wound healing. The binding of EGF to the extracellular part of the EGF-receptor, EGFR, induces the signal pathway, ultimately leading to DNA replication and cell proliferation (13). The EGFR is overexpressed in some tumors and may therefore serve as a potential diagnostic and therapeutic target in some malignancies such as gliomas, bladder cancers, squamous cell carcinomas, and some forms of adenocarcinomas (1418). EGF injected intravenously (iv) has a fast blood clearance. Only 7% of the radioactivity remained in blood 2.5 min after iv administration of [125I]EGF (20). Intravenously injected EGF is taken up by the liver. It is also to a high extent accumulated in the kidney, which is typical for short peptides such as octreotide, vasoactive intestinal peptide (VIP), or single chain Fv (19-23). Even in animals with tumors expressing high numbers of EGF receptors, the dominating EGF uptake is in the liver and kidney (21). Studies of human lung cancer have, however, shown a higher uptake in tumors expressing EGF receptors than in receptor negative tumors (34). Glycosylation has been shown to improve the stability and prolong the retention time of proteins in vivo in mice after iv injection (24, 25). In this study, we used dextran for protein glycosylation. Dextran is a glucose polymer * To whom correspondence should be addressed. Phone: (46) 18-4713840. Fax: (46) 18-4713432. E-mail: [email protected]. † Biomedical Radiation Sciences. ‡ Department of Diagnostic Radiology. § Pharmacia Biotech AB. | Centre of Surface Biotechnology.

in which glucose units are bound to each other by R1-6 linkages. The branching of the dextran structure is approximately 5% and comprises mostly R1-3 linkages. By sequential degradation, which quantitatively removes nonreducing end groups, it has been found that about 40% of the side chains are only one glucose (1, 2). For clinical application, dextran is usually produced by fermentation using the microbes Leuconostoc mesenteroides NRRL B-512 (F) or the B-512 strain (1, 3, 4). In the clinic, dextran is used worldwide as the plasma volume expander of choice, applied to treat shock caused by hemorrhage, burns, surgery, or trauma (3-5). An interesting application of dextran is its conjugation to biologically active substances with the aim to prolong their biological half-life and to increase their stability in vivo (6, 7). Dextran may also act as a carrier for various cytotoxic drugs and radioisotopes in targeted therapy and may, in addition, reduce the antigenicity of biomolecules (8-12). The primary aim of this paper was to study general changes in the kinetics of dextranated molecules. However, a secondary interest was to see if dextranated EGF could be modified to increase the circulation time and to lower the normal tissue uptake, thus improving the targeting properties. EGF was chosen as a suitable biomolecule since it is known to have a dominating and fast receptor-mediated liver uptake and that small changes in the uptake kinetic should be possible to perceive fairly easily. The mEGF-dextran conjugates were prepared by reductive amination, in which the end aldehyde group of dextran reacted with the only amino group on the N terminal of mEGF (26). A positronemitting nuclide, 76Br (T1/2 )16 h), was used to label mEGF and the mEGF-dextran conjugates in order to apply positron emission tomography (PET) for the assessment. PET was used since it can follow fast kinetic changes in vivo. It also has a spatial resolution, which

10.1021/bc990011l CCC: $18.00 © 1999 American Chemical Society Published on Web 09/24/1999

Pharmacokinetics of Dextranated mEGF

makes it possible to distinguish the liver uptake from any other organ in the rat. Excised tissues were also measured for radioactivity content in the body. MATERIAL AND METHODS

Conjugation. Freeze-dried dextran weighting of 13 kDa (D13) and 46 kDa (D46) was obtained by fractionation of Dextran T10 or T40 (Pharmacia Biotech AB, Sweden) by gel filtration on Sephacryl 100 HR (Pharmacia Biotech AB, Sweden). Sodium cyanoboronhydride (NaCNBH3, Merck-Schuchardt, Germany), mEGF (tissue culture grade, Janssen Biochimica, Belgium), and 125I (Amersham Laboratories, England) were used as received from the manufacturer. The mEGF-dextran conjugates were produced by reductive amination as previously reported (26). Dextran D13 or D46 (20 mg, 1.54 and 0.43 µmol, respectively) was dissolved in 100 µL mEGF (25 µg, 4.17 nmol) in 0.05 M phosphate buffer solution, pH 8.0. To this, NaCNBH3 (8 mg, 127 µmol) was added and the reaction was allowed to continue for 24 h at room temperature during continuous stirring. The reductive amination reaction mixture was applied to a Sephadex G-50 Fine (Pharmacia HR 10/ 30) column (Pharmacia Biotech AB, Sweden), and the conjugate was eluted with distilled water at a flow rate of 0.25 mL/min and a fraction volume of 0.5 mL. The fractions containing conjugate were pooled and freeze dried. Bromination. Br-76 was produced at the Uppsala PET Centre, Uppsala, Sweden, by the 76Se(p,n)76Br reaction using 76Se-enriched Cu2Se as target. Radiobromine was separated from target material by dry distillation (27). About 150-200 MBq 76Br was obtained in 100 µL of ethanol after separation. The ethanol was evaporated, and freeze-dried mEGF-dextran conjugate (1520 µg) or mEGF (20 µg) dissolved in 30 µL of citratephosphate buffer (pH 4, 0.05 M) was added. Adding 10 µL of Chloramine-T (4 mg/mL in 0.05 M citrate-phosphate buffer, pH 4.0) started labeling. After the solution was mixed for 3 min, the reaction was quenched with 20 µL of sodium bisulfite (4 mg/mL in 0.05 M citratephosphate buffer, pH 4.0). Purification was performed on a NAP-5 column (Sephadex G-25, Pharmacia Biotech AB, Sweden) equilibrated and eluted with PBS. Fractions of 0.2 mL were collected and measured for radioactivity. The high-molecular-weight fractions were pooled and used in the cell test and animal experiment. Cell Test. A subclone (U-343MGaC12:6) of a glioma cell line U-343 was grown in HAM’S F-10 culture media (Kebo, Stockholm, Sweden), supplemented with FCS, L-glutamine, penicillin, and streptomycin, in culture dishes. Brominated mEGF-dextran conjugates and mEGF, respectively, were mixed in culture media or in the culture media containing 1 µg/mL mEGF. The incubation experiments were carried out in triplicates. After 90 min of incubation at 37 °C, the cultures were washed 6 × 1 min with HAM’S F-10 media and trypsinized. The number of cells was determined in a cell counter (Coulter ZM, Coulter Electronics Ltd, U.K.) and analyzed for cell-associated radioactivity in a γ-counter (1480 Wizard γ-counter, Wallac, Finland). Animals. Fourteen Sprague Dawley male rats weighing 350 ( 120 g (mean ( SD) from BK Universal AB (Stockholm, Sweden) were used. After arrival at the animal department, the rats were allowed to condition for 10 days before the experiment. For anesthesia, the rats were injected intraperitoneally with 0.24-0.3 mL/ 100 g body weight of thiobutabarbital sodium salt 5 mg/ mL (Inactin, RBI, MA).

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PET Imaging. Through a groin incision, the right femoral vein was isolated and cannulated with a plastic catheter to ensure iv injection of the tracers. The rats, placed in a plastic multicompartment holder, were positioned in a Scanditronix GE 2048 PET scanner (Scanditronix, Uppsala, Sweden), which simultaneously produces 15 contiguous 6.5 mm tomographic slices and has an in-plane resolution of 5-6 mm. Before injection, a 10 min transmission scan was performed to enable attenuation corrections of the emission scan. After a rapid iv bolus of 3-10 MBq of [76Br]mEGF and [76Br]mEGFdextran conjugates, a dynamic sequence was started and continued for 1 or 2 h, respectively. Tomograms were reconstructed using a 128 × 128 image matrix and a 6 mm Hanning filter. Data were corrected for attenuation and scattered radiation. The regional radioactivity concentration measured by PET was divided by injected radioactivity per gram of body weight to provide images of standardized uptake values (SUV). Regions of interest (ROI), representing liver, heart, blood, kidney, muscle, and urinary bladder, were drawn manually in the SUV images and time-activity curves were calculated and plotted. Pharmacokinetic Analysis. Clearance of radioactivity from blood was analyzed by fitting a dual exponential function to data, SUV ) Ae-at + Be-bt, using the leastsquares method where A and B are constants, a and b are clearance rates, and t is time after injection. The impact of dextranation to the blood kinetics was tested statistically. To accentuate the early, rapidly changing blood kinetics, a constant steady-state radioactivity level appearing in all the blood curves was subtracted. The time integral of the resulting radioactivity curve, divided by the maximum value, was calculated for each animal. The obtained values, the transit times, for each experimental group were tested by a twosamples Student’s t-test. The early uptake phase in the liver, i.e., the first minutes after injection, was analyzed by a simple two compartment model assuming that all radioactivity passing the liver was trapped in the liver. This can mathematically be described as

CL(t) ) k1*

∫0tCb(S) dS + Cb(t)

where CL is the radioactivity concentration in liver, Cb is the radioactivity concentration in blood, k1 is the rate constant (min-1), and  is the blood volume. The late phase of the liver uptake, i.e., the part where the liver starts to lose radioactivity was analyzed by fitting data by the least-squares method to a simple exponential function, SUV ) Ae-at, where A is a constant, a is the clearance rate of radioactivity, and t is the time after injection. Kinetic analysis was also performed according to Patlak et al. (28) where the transport rate of ligand into the tissues, generated on a pixel by pixel basis was presented as images. The radioactivity concentration in blood obtained from a ROI over the heart blood pool was used as the input function. In the Patlak graphs, Y ) Ci/Cb was plotted against X ) ∫Cb(t) dt/Cb(t), where Ci ) tissue radioactivity concentration and Cb ) radioactivity concentration in the blood at time t. This graphical representation is, after onset of a dynamic equilibrium, equivalent to a situation, where the blood radioactivity concentration is constant throughout the examination. For a radiolabeled compound that is trapped in the tissue, the graph shows a linear increase over time, whereas a

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Table 1: Receptor Binging Test for [76Br]mEGF and [76Br]mEGF-Dextran Conjugatea cell associated radioactivity (cpm/105 cells)

76Br-mEGF 76Br-mEGF-dextran

presaturated receptors

not presaturated receptors

3130 (410) 5570 (310)

22 630 (1400) 12 240 (920)

a Test was performed on a glioma cell line U-343. For presaturation of the EGFR, 1 µg/mL mEGF was added. Cell were incubated with labeled compounds during 90 min, than washed three times. Data are presented as mean values from three dishes and maximum errors.

compound equilibrating in the tissue gives a constant value after a while. In a situation where the compound is heavily degraded and the radioactivity concentration in blood, at later times, may be dominated by labeled metabolites, the graphical presentation can show a negative slope after the initial equilibrium. Tissue Measurements. After PET scanning, the animals were killed at different times after tracer injection. Samples of blood and urine were collected and various organs were excised, weighed, and measured for radioactivity in the γ-counter. The accumulation index (AI) of each tissue was calculated, after decay correction, according to the formula

AI )

(tissue radioactivity/tissue weight) (injected radioactivity/body weight)

Assuming a tissue density of 1 g/cm3, AI corresponds to the SUV. The use of AI for studies of biodistribution of small molecules enables comparison of data obtained from animals with different body weight and, to a certain extent, even between different species, e.g., mice and rats. Chromatography. Serum and urine were analyzed by gel filtration with a Sephadex G-50 Fine (1 × 50 cm) column coupled with a FPLC system (Pharmacia Biotech AB, Uppsala, Sweden). The column was eluted with PBS with a flow rate of 0.5 mL/min. Fractions of 1 mL were collected and measured for radioactivity content. By calibrating the column with a conjugate (>10 kDa), mEGF (6 kDa) and salt, the high-molecular-weight (HMW) position, the mEGF position, and the lowmolecular-weight (LMW) position, respectively, were defined in the chromatogram. Since the conjugates were desalted before the animal experiment and the fractionation range for globular protein with Sephadex G-50 Fine is within (1.5 × 103)-(3 × 104) kDa, the radioactivity eluted at the salt position was considered to represent degraded fragments of mEGF. RESULTS

Radiolabeled Conjugates. The labeling yield of bromination varied from 15 to 30%. To ensure that the EGF-receptor binding ability of the different preparations was retained before starting the biodistribution experiments, each batch was tested by incubating the [76Br]mEGF and [76Br]mEGF-dextran conjugates with U343 cells as described in the Material and Methods. The data concerning cell-binding tests are given in Table 1. The results of the tests demonstrated specific binding of conjugates to EGF-receptor-expressing cells, since it can be displaced by nonradioactive mEGF. PET Pharmacokinetics. The blood pharmacokinetics of the different preparations measured by PET are presented in Figure 1 for the first 10 min of the experi-

Figure 1. The kinetic standard uptake values (SUVs) of [76Br]mEGF (solid line), [76Br]mEGF-dextran D13 (dotted line), and [76Br]r-mEGF-dextran D46 (dashed line) in blood evaluated by PET. The experiment animals were Sprague Dawley male rats. The curve fittings were done with a formula SUV ) Aeat + Bebt to evaluate the uptake within 10 min PI. [76Br]mEGF, [76Br]mEGF D13, and [76Br]mEGF D46 have designated b values of 0, -0.06, and -0.01, respectively. Each curve represents the result of one rat.

ments. All curves showed a marked two-phase behavior with an early rapid phase (mean rate constant a ) 2.28, 1.85, and 0.58 min-1 for the [76Br]mEGF, [76Br]mEGFD13, and [76Br]mEGF-D46, respectively) and a slow phase (mean clearance rate b ) 0, 0.06, and 0.01 min-1 for the [76Br]mEGF, [76Br]mEGF-D13, and [76Br]mEGFD46, respectively). The transit times in blood for [76Br]mEGF, [76Br]mEGF-D13, and [76Br]mEGF-D46 were found to be 0.68 ( 0.18, 1.97 ( 0.37, and 5.14 ( 1.15 min (mean ( standard deviation), respectively. The differences between the different values were proved to be statistically significant in a paired t-test, giving the results [76Br]mEGF minus [76Br]mEGF-D13 (p < 0.007), [76Br]mEGF minus [76Br]mEGF-D46 (p < 0.01), and [76Br]mEGF-D13 minus [76Br]mEGF-D46 (p < 0.02). The liver pharmacokinetics of the three brominated compounds measured by PET are shown in Figure 2. There was a fast radioactivity uptake in the liver, which leveled out after a few minutes. After 20 min, there was a pronounced decrease in the SUV values indicating a loss of radioactivity from the liver for all three ligands although the loss was most rapid for the mEGF. The mean clearance rate of this radioactivity between 20 and 60 min was calculated to be 0.01, 0.007, and 0.006 min-1 for [76Br]mEGF, [76Br]mEGF-D13, and [76Br]mEGF-D46, respectively. The liver pharmacokinetics plotted according to the Patlak method is seen in Figure 3. All three plots show a pronounced decrease after a few minutes, which indicated a considerable influence of labeled metabolites in the analyzes. The first half-minute was analyzed using a simple linear model to compare the initial liver increase of the three ligands. The mean increase was found to be 2.14, 1.84, and 2.23 min-1 for [76Br]mEGF, [76Br]mEGFD13, and [76Br]mEGF-D46, respectively. The uptake in the kidneys was also rapid and rather high for all three ligands. However, the variation in the pharmacokinetic pattern varied considerably between animals. The uptake of [76Br]mEGF reached rapidly a plateau but showed no significant decrease with time. Radioactivity appeared in the urinary bladder 20-40 min PI for all three [76Br]compounds. The kinetic pattern was similar for all three ligands although uptake values varied greatly (Figure 4). Table 2 summarizes the PET results of the kinetic studies in which different mathematical models have been applied.

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Figure 2. The kinetic standard uptake values (SUVs) of [76Br]mEGF (a), [76Br]mEGF-dextran D13 (b), and [76Br]mEGF-dextran D46 (c) in liver evaluated by PET. The experiment animals are Sprague Dawley rats. The curve fittings were done with a formula SUV ) Aeat between 20 and 60 min PI to evaluate the clearance after 20 min PI. Each curve represents the result of one rat.

Figure 3. Patlak plot of [76Br]mEGF (a), [76Br]mEGF-dextran D13 (b), and [76Br]mEGF-dextran D46 (c) in liver of PET study according to a two-compartment model. The experiment animals are Sprague Dawley rats. The curve fittings were done with the formula Ci/Cb ) A + BX between 0 and 0.5 Patlak time where X ) ∫Cb(t) dt/Cb(t). Each curve represents the result of one rat.

found in the serum chromatograms (Figure 5, panels a-c), not even when mEGF without dextran was administered. A radioactivity peak corresponding to mEGF was found in urine after the injection of [76Br]mEGF. This was not the case for the dextranated compounds (Figure 5, panels d-f). DISCUSSION

Figure 4. The kinetic standard uptake values (SUVs) of [76Br]mEGF (filled circles), [76Br]mEGF-dextran D13 (open circles), and [76Br]mEGF-dextran D46 (filled triangles)in urinary bladder evaluated by PET. The experiment animals are Sprague Dawley male rats. Each curve represents the result of one rat.

Measurements on Excised Tissues. The 76Br accumulation at different times postinjection (PI) was measured in excised organs and tissues after intravenous injection of [76Br]mEGF, [76Br]mEGF-dextran D13, and [76Br]mEGF-dextran D46 (Table 3). High radioactivity concentrations were measured in the urine. At 1 h PI, the dextranated conjugates were compared with mEGF. The AI for all three compounds was found to be high in the liver. The spleen showed high AI values for the dextranated compounds only, whereas the opposite was true for the kidney. In blood, AI ranged 1.4-2.4 with the lowest value for [76Br]mEGF. The small intestine and colon showed higher AI for [76Br]mEGF than for the dextranated compounds. In all other tissues the AI was low for all three preparations. Chromatographic Analysis. The chromatograms of serum and urine sample are shown in Figure 5. Two hours PI, the ratio of LMW to HMW in serum was significantly lower for [76Br]mEGF-dextran D46 than for [76Br]mEGF-dextran D13 and [76Br]mEGF. No radioactive peaks corresponding to the mEGF position were

The aim of this study was to investigate if dextranation of a peptide changed its in vivo pharmacokinetics and biodistribution. EGF administered iv into rats was expected to be a sensitive test system since EGF is known to be effectively removed from the circulation by receptormediated trapping in the liver and through kidney excretion. Even a small change in the pharmacokinetic pattern due to dextranation should thus be detected. Due to the fast pharmacokinetics of mEGF, PET was used since this technique allows measurements with high temporal and spatial resolution. During the first minutes PI when free intact ligand still circulated in plasma, the measuring time was 10 s/time frame. The spatial resolution of about 6-7 mm made it possible to study the pharmacokinetics separately in liver and kidney in the rat. However, due to the limited spatial resolution, PET generally tends to underestimate the radioactivity uptake in small organs (30). Also, the 6.5 mm tomographic slice may only include a part of an organ, which also will change the absolute value of the measurements due to partial volume effect. In the measurements of kidney radioactivity in vivo, the spatial resolution of PET is insufficient to discriminate the radioactivity related to urine from that related to renal tissue. Thus, PET allows accurate pharmacokinetic measurements although absolute quantification of tracer uptake in the small organs of the rat may be less dependable. Human EGF and mEGF are both 53 amino acid long peptides, with 37 amino acids in common, and with three

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Table 2: Pharmacokinetics Evaluation by PET of Intravenous Administered [76Br]mEGF, [76Br]mEGF-dextran (D13), and [76Br]mEGF-Dextran (D46) Conjugates in Sprague Dawley Ratsa tissue/conjugate

time

blood [76Br]mEGF [76Br]mEGF-D13 [76Br]mEGF-D46 liver [76Br]mEGF [76Br]mEGF-D13 [76Br]mEGF-D46 liver [76Br]mEGF [76Br]mEGF-D13 [76Br]mEGF-D46 a

formula

A

a

B

b

0-10 min PI

SUV ) Ae-at + Be-bt

7.9 6.8 4.1

2.28 1.85 0.58

1.6 3.2 4.0

0 0.06 0.01

20-60 min PI

SUV ) Ae-at

12.2 6.5 11.3

0.01 0.007 0.006

0-0.5 P time*

Ci/Cb ) A + BX

0.006 0.009 0.074

2.14 1.84 2.23

P time is Patlak time; X is Patlak time, X ) ∫Cb(t) dt/Cb(t). Data represent the mean values of three curve fittings for each compound.

Table 3: Accumulation Index (AI) for [76Br]mEGF and [76Br]mEGF-Dextran Conjugates 1 h after Intravenous Administration in Sprague Dawley Ratsa

blood urinary bladder kidney spleen pancreas liver stomach small intestine colon heart lung muscle bone skin testicle brain thyroid submandibular gland

[76Br]mEGF

[76Br]mEGFD13

[76Br]mEGFD46

2.4 (0.2) 3.0 (0.4) 8.7 (1.5) 1.5 (0.1) 1.8 (0.2) 5.6 (0.2) 1.8 (0.2) 2.9 (0.6) 1.5 (0.1) 1.0 (0.1) 1.9 (0.1) 0.5 (0.0) 1.2 (0.1) 1.2 (0.1) 0.8 (0.0) 0.4 (0.1) 1.4 (0.2) 1.8 (0.1)

1.4 (0.1) 2.1 (0.4) 0.1 (0.1) 7.3 (1.6) 0.5 (0.1) 6.2 (0.1) 0.6 (0.0) 0.7 (0.1) 0.4 (0.0) 0.5 (0.0) 1.5 (0.7) 0.2 (0.0) 0.6 (0.2) 0.5 (0.0) 0.3 (0.0) 0.1 (0.0) 0.4 (0.1) 0.5 (0.0)

2.0 (0.0) 1.0 (0.4) 1.6 (0.1) 7.6 (0.0) 0.5 (0.0) 6.0 (0.4) 0.5 (0.0) 1.1 (0.2) 0.4 (0.0) 0.6 (0.1) 0.9 (0.0) 0.2 (0.0) 0.5 (0.0) 0.5 (0.1) 0.3 (0.0) 0.1 (0.0) 0.4 (0.1)

a Data are presented as mean values and maximum errors. Values lower than 0.05 are shown as zero. The number of rats killed and measured was two, four and three for [76Br]mEGF, [76Br]mEGF-dextran D13 ([76Br]mEGF-D13), and [76Br]mEGFdextran D46 ([76Br]mEGF-D46), respectively.

disulfide bonds in the same relative positions. Also, they both have approximately the same binding affinity to the human EGF-receptor (29). In the present study, mEGF was used since it has one single free amino group, the amino terminus (no lysine residues), which can be coupled by reductive amination to a unique position on dextran, the aldehyde group on the reducing end of the dextran chain. This yielded a well-defined complex with a 1:1 ratio of mEGF to dextran. After bromination, mEGF and conjugates retained their binding ability to the human glioma cancer cell line U343. PET Pharmacokinetics of [76Br]mEGF. The analysis of the blood clearance indicated a rapid mEGF turnover (Figure 1), in good agreement with the fast accumulation of radioactivity in liver (Figure 2a). The liver pharmacokinetics during this early phase was just an integral of the blood curve. The liver uptake reached a plateau 2-3 min PI. After this time point, the radioactivity in the blood probably represented merely labeled metabolites and not mEGF. This is also supported by the liver Patlak-plot, which after leveling out showed a pronounced decrease (Figure 3a). The liver uptake plateau, which probably lasted for about 20 min, reflects the receptor binding and the internalization process. After about 20 min, an exponential radioactivity decrease

probably reflected the excretion of labeled metabolites from the liver. The kidney also showed a rapidly increasing radioactivity uptake, which after a few minutes leveled out to a plateau and decreased only slightly during the next hour. Since the mEGF most likely was trapped quantitatively in the liver and in kidney, the high uptakes in these organs reflect their high blood perfusion. The findings of a rapid clearance from the circulation and a high accumulation in liver and kidney of [76Br]mEGF are in agreement with previous results. It has been reported that, 2.5 min after iv injection of [125I]EGF into rats, 52% was found in liver and 14% in kidney (31). This high accumulation of EGF in liver and kidney was also shown in a study using iv injected [76Br]/[125I]EGF into rats (23). In this latter study it was claimed that the liver and kidney were the major organs responsible for the elimination and metabolism of hEGF from the plasma. The contribution of the liver and kidney to the total body clearance was estimated to approximately 70 and 20%, respectively (32). Moreover, in tumor-bearing mice, 125I-labeled EGF accumulated in receptor-positive tumors, but to a much lesser extent than in the liver. One hour after iv injection, the tumor-to-liver ratio was less than 0.5 (21). Pharmacokinetic Analyses of Dextranated mEGF. Compared with free mEGF, dextranation somewhat prolonged the retention time of the conjugates in the circulation (Figure 1). The conjugate with the longer dextran chain also showed the longest retention time, indicating a relation between the dextran chain length and blood residence time. The liver uptake of dextranated ligands was equally fast for all three compounds, and the maximal uptake was reached within 2-3 min (Figure 2). The rate constants obtained in the detailed analysis of the first minute were equally high and probably just reflected the blood perfusion of the liver. We concluded that the extraction of the ligand from blood to liver is close to 100% for all three ligands. The dextranated ligands thus retain their receptor binding ability although small changes in receptor affinity cannot be excluded. More refined studies with preparations of different specific radioactivity might show if such changes occur. After the maximal liver uptake, the accumulation pattern varied. The dextranated ligands showed not an extended plateau as for native EGF but a two-phase exponential decrease in radioactivity. The first phase might represent dextranated ligand returning into circulation, which is supported by the prolonged retention times in blood. Thus, it is possible that the dextranation may prevent some of the ligand being internalized. The

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Figure 5. The chromatograms obtained by eluting serum and urine samples through Sephadex G-50 Fine column (1 × 50 cm) were shown. I, II, and III represent high molecular weight (conjugate), mEGF, and low molecular weight (salt) elution positions. The serum (a, b, and c) and urine (d, e, and f) samples from the rats administered with [76Br]mEGF (a, d), [76Br]mEGF-dextran D13 (b, e), and [76Br]mEGF-dextran D46 (c, f) were analyzed by gel filtration. Each dotted, dashed, or solid line in the chromatogram represents the sample from one rat.

second phase may represent ligand, which was internalized and undergoing degradation. However, the rate of radioactivity loss here was less than that of free mEGF, which indicates that dextran can partly protect an internalized molecule from degradation. Earlier, we studied the influence of dextranation of EGF on in vitro binding to EGFR-expressing tumor cells (41). It was shown that dextranation somewhat decreases the binding affinity to EGFR (dissociation constants were 6.6 × 10-10 and 7.1 × 10-9 for [125I]mEGF and [125I]mEGF-D14, respectively). Still, the affinity after dextranation remained high. It was also noticed that dextranation slows down the internalization, but the mechanism for this process is not fully understood. We propose the following model. It is known that the activation of EGFRassociated protein kinase required dimerization of receptors (42). According to the model presented by Heldin and O ¨ stman (41), one EGF molecule binds to one receptor with one epitope and to the second receptor with another epitope. The other EGF molecule would then bind in a reciprocal manner. The presence of bulky dextran moiety might preclude the binding to the second receptor or to complicate the binding of the second EGF molecule. Thus, the internalization might be hampered. On the other hand, it has been shown for antibodies that a bivalent attachment to antigens is essential to make a binding

nearly irreversible while a monovalent lead to dissociation (43). This might be also the truth for binding of EGF to its receptors. Such a model can explain both the delay of internalization and the release of a part of the conjugate back into blood stream. At least a part of the ligand returning into circulation may have lost its receptor-binding ability since the plasma chromatography showed labeled high molecular complexes (Figure 5, panels b and c). The nature of this inactivation will be discussed in the chapter dealing with the results of chromatographic analysis. The observation that the release of radioactivity associated with dextranated EGF is slower that in the case of free ligand is worth attention. Peptides and proteins are after internalization rapidly degraded in the lysosomes (29). The radioactivity, mainly in the form of halogenated tyrosine, leaks out from the cells. This will reduce the tumor-to-background ratio in diagnostic procedures and the tumor dose during targeted therapy. It has been demonstrated earlier in vitro that conjugation to dextran improves intracellular retention of mEGFassociated radioactivity (12). The results in this PET study indicate that this protective effect of dextran was true also in vivo. However, an even better retention effect can be obtained if the dextran part of the molecule is

944 Bioconjugate Chem., Vol. 10, No. 6, 1999

radiohalogenated since dextran will remain intracellularly for a longer time (35). Due to the fast liver uptake, the fast blood clearance and the rapid appearance of labeled metabolites in blood, the Patlak graphical analysis was not applied to calculate the transport rate constants, but used in order to make a normalized qualitative comparison between the different labeled compounds. For the liver, the Patlak plot during the first minute showed the same slope for all three ligands, indicating a similar receptor-binding rate. After reaching equilibrium, free EGF showed a more pronounced negative slope (Figure 3) and was probably more affected by the influence of labeled metabolites than that of the dextranated ligands. However, the [76Br]mEGF curve still reached a higher level than the curves for the conjugates. This was probably due to the faster release of receptor-bound dextranated ligands, which was also seen in the direct comparison of SUV in the PET measurements. Well Counter Measurements. Consistent with the PET results, the well counter measurements showed high accumulation of radioactivity in liver, spleen, kidney, and urine, whereas the accumulation in the other organs was low (Table 3). An interesting finding of this study was that the dextranation of EGF decreased the uptake in the kidney. This result may have implications for future studies on EGF and targeted radiotherapy since the kidney is one of the dose-limiting critical organs. It is assumed nowadays that renal uptake of short proteins is facilitated by positively charged amino acids reacting with negative “receptors” on the surface of proximal tubuli (36). The most successful attempts to decrease renal uptake of short peptides are associated with the use of lysine and its derivatives to block these negative patches (37-39). However, it has been reported that similar reduction of labeled protein uptake in kidney may be achieved by acetylation of free amino groups (40), which dramatically decreases an isoelectric point of antibody fragment. The coupling of a bulky dextran to the mEGF might have reduced the binding of this molecule, due to the sterical hindrance, to the negative regions of renal tubuli. Another possible explanation to the reduced reabsorption by the kidney could be an increased hydrophilicity of the conjugate. By contrast, in the spleen, the dextranation of mEGF resulted in increased AI values. This was most likely explained by phagocytosis of the [76Br]mEGF-dextran by the reticular endothelial system cells in the spleen, a phenomenon that has previously been shown for nonconjugated dextran (31). The finding is consistent with the PET measurements where the spleen could only be visualized when using the D46 conjugate. Size-Exclusion Chromatography. The serum from the rats administered with [76Br]mEGF did not show any peak corresponding to the mEGF itself. Surprisingly, there was a peak found at the high-molecular-weight position in the chromatogram (Figure 5a). It has earlier been not shown that EGF binds to some component in plasma but the chromatogram suggests that such a complex was formed. The possibility of mEGF aggregation during bromination may be excluded since [76Br]mEGF showed a single peak corresponding to the mEGF position by gel filtration on Sephadex G-50 Fine (data now shown). A high-molecular-weight peak in the same position was found as well when [125I]mEGF was incubated with blood plasma, taken from Sprague Dawley rats. When separated, the substance in this peak did not bind to EGFR expressing cells in vitro. It is possible that binding of EGF to some of blood component should limit

Zhao et al.

the action of the growth factor to paracrine mode and prevent the activation of cell division far away from the site where it is required. It is also possible that the highmolecular-weight peaks obtained after chromatography of blood samples after injection of [76Br]mEGF-dextran conjugates rather belong to such complex than free conjugate. The size-exclusion system, which was used for the analysis, is not able to resolve such high-molecularweight compounds. A formation of a nonactive complex can explain why nonbound conjugate remains in the circulation. We believe that the formation of highmolecular weight complex of EGF with blood components deserves a thorough study in a future. In urine, a main peak corresponding to mEGF was seen, but the high-molecular-weight peak seen in plasma was usually not present, i.e., it was only seen to a small extent in some animals. Since the urine content was integrated over the whole experiment, this can be interpreted as that mEGF was excreted in urine early, but the compound corresponding to the high-molecularweight peak was not. The small high-molecular-weight peaks seen in some urine samples were probably caused by leakage of blood into the urinary bladder during dissection. Other possible explanations may include extreme extension of the urinary bladder during prolonged anesthesia during PET scanning or bleeding in connection with intraperitoneal anesthesia. In urine from rats administered with [76Br]mEGFdextran conjugates, the main peaks were still of highmolecular-weight indicating that the kidneys excreted the intact ligand. Probably, longer dextran chains (>70 kDa) have to be used in order to prevent or considerably decrease the urinary excretion and thus prolong the circulation time of the conjugates even more. To conclude, dextranation affects the biodistribution of mEGF in vivo giving a prolonged circulation time, decreased uptake in kidney, and increased spleen accumulation. ACKNOWLEDGMENT

The authors thank laboratory engineers Veronika Asplund and Ulla Johansson and the staff at the Uppsala University PET center for valuable technical assistance. Financial support was given by the Swedish Cancer Society, the Swedish Medical Research Council and the Swedish Institute. LITERATURE CITED (1) Cote, G. L., and Robyt, J. F. (1982) Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B-1355 that synthesizes an alternating (1 goes to 6), (1 goes to 3)-alpha-D-glucan. Carbohydr. Res. 101, 57-74. (2) Larm, O., Lindberg, B., and Svensson, S. (1971) Studies on the length of the side chains of the dextran elaborated by Leuconostoc mesenteroides NRRL B-512. Carbohydr. Res. 20, 39-48. (3) Steinmann, E., Duckert, F., and Gruber, U. F. (1975) The value of dextran 70 in the prevention of thromboembolism in general surgery, orthopedics, urology and gynecology. A review of the literature. Schweiz. Med. Wochenschr. 105, 1637-1649. (4) Atik, M. (1967) Dextran 40 and dextran 70. A review. Arch. Surg. 94, 664-672. (5) Dubick, M. A., and Wade, C. E. (1994) A review of the efficacy and safety of 7.5% NaCl/6% dextran 70 in experimental animals and in humans. J. Trauma. 36, 323-330.

Pharmacokinetics of Dextranated mEGF (6) Baudys, M., Letourneur, D., Liu, F., Mix, D., Jozefonvicz, J., and Kim S. W. (1998) Extending insulin action in vivo by conjugation to carboxymethyl dextran. Bioconjugate Chem. 9, 176-183. (7) Takakura, Y., Fujita, T., Hashida, M., Maeda, H., and Sezaki, H. (1989) Control of pharmaceutical properties of soybean trypsin inhibitor by conjugation with dextran. II: Biopharmaceutical and pharmacological properties. J. Pharm. Sci. 78, 219. (8) Sjostrom, A., Bue, P., Malmstrom, P. U., Nilsson, S., and Carlsson, J. (1997) Binding, internalization and degradation of EGF-dextran conjugates in two human bladder-cancer cell lines. Int. J. Cancer. 70, 383-389. (9) Lindstrom, A., and Carlsson, J. (1993) Penetration and binding of epidermal growth factor-dextran conjugates in spheroids of human glioma origin. Cancer Biother. 8, 145158. (10) Gedda, L., Olsson, P., Ponten, J., and Carlsson, J. (1996) Development and in vitro studies of epidermal growth factordextran conjugates for boron neutron capture therapy. Bioconjugate Chem. 7, 584-591. (11) Carlsson, J., Gedda, L., Gronvik, C., Hartman, T., Lindstrom, A., Lindstrom, P., Lundqvist, H., Lovqvist, A., Malmqvist, J., and Olsson, P., et al. (1994) Strategy for boron neutron capture therapy against tumor cells with overexpression of the epidermal growth factor-receptor. Int. J. Radiat. Oncol. Biol. Phys. 30, 105-115. (12) Olsson, P., Lindstrom, A., and Carlsson, J. (1994) Internalization and excretion of epidermal growth factor-dextranassociated radioactivity in cultured human squamouscarcinoma cells. Int. J. Cancer. 56, 529-537. (13) Carpenter, G., and Cohen, S. (1990) Epidermal growth factor. J. Biol. Chem. 265, 7709-7712. (14) Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, I., Soreq, H., Whittle, N., Waterfield, M. D., Ullrich, A., and Schlessinger, J. (1985) Amplification and overexpression of the EGF receptor gene in primary human glioblastomas. J. Cell. Sci. Suppl. 3, 161-172. (15) Messing, E. M., and Reznikoff, C. A. (1992) Epidermal growth factor and its receptor: markers of-and targets forchemoprevention of bladder cancer. J. Cell. Biochem. Suppl. 16I, 56-62. (16) Sauter, G., Haley, J., Chew, K., Kerschmann, R., Moore, D., Carroll, P., Moch, H., Gudat, F., Mihatsch, M. J., and Waldman, F. (1994) Epidermal-growth-factor-receptor expression is associated with rapid tumor proliferation in bladder cancer. Int. J. Cancer 57, 508-514. (17) Filmus, J., Trent, J. M., Pollak, M. N., and Buick, R. N. (1987) Epidermal growth factor receptor gene-amplified MDA468 breast cancer cell line and its nonamplified variants. Mol. Cell. Biol. 7, 251-257. (18) Hernandez Sotomayor, S. M., Arteaga, C. L., Soler, C., and Carpenter, G. (1993) Epidermal growth factor stimulates substrate-selective protein-tyrosine-phosphatase activity. Proc. Natl. Acad. Sci. U.S.A. 90, 7691-7695. (19) Burwen, S. J., Barker, M. E., Goldman, I. S., Hradek, G. T., Raper, S. E., and Jones, A. L. (1984) Transport of epidermal growth factor by rat liver: evidence for a nonlysosomal pathway. J. Cell Biol. 99, 1259-1265. (20) Jorgensen, P. E., Poulsen, S. S., and Nexo, E. (1988) Distribution of iv administered epidermal growth factor in the rat. Regul. Pept. 23, 161-169. (21) Mateo de Acosta, C., Justiz, E., Skoog, L., and Lage, A. (1989) Biodistribution of radioactive epidermal growth factor in normal and tumor bearing mice. Anticancer Res. 9, 8792. (22) Yanai, S., Sugiyama, Y., Iga, T., Fuwa, T., and Hanano, M. (1990) Kinetic analysis of the downregulation of epidermal growth factor receptors in rats in vivo. Am. J. Physiol. 258, C593-598. (23) Scott Robson, S., Capala, J., Carlsson, J., Malmborg, P., and Lundqvist, H. (1991) Distribution and stability in the rat of a 76Br/125I-labeled polypeptide, epidermal growth factor. Int. J. Rad. Appl. Instrum. B 18, 241-246. (24) Fujita, T., Nishikawa, M., Tamaki, C., Takakura, Y., Hashida, M., and Sezaki, H. (1992) Targeted delivery of

Bioconjugate Chem., Vol. 10, No. 6, 1999 945 human recombinant superoxide dismutase by chemical modification with mono- and polysaccharide derivatives. J. Pharmacol. Exp. Ther. 263, 971-978. (25) Mikolajczyk, S. D., Meyer, D. L., Fagnani, R., Hagan, M. S., Law, K. L., and Starling, J. J. (1996) Dextran modification of a Fab′- β-lactamase conjugate modulated by variable pretreatment of Fab′ with amine-blocking reagents. Bioconjugate Chem. 7, 150-158. (26) Zhao, Q., Gottschalk, I., Carlsson, J., Arvidsson, L.-E., Oscarsson, S., Medin, A., Ersson, B., and Janson, J.-C. (1997) Preparation and Purification of an End to End coupled mEGF-dextran conjugate. Bioconjugate Chem. 8, 927-934. (27) Tolmachev, V., Lo¨vqvist, A., Einarsson, L., Schultz, J., and Lundqvist, H. Production of 76Br by a low-energy cyclotron. Appl. Radiat. Isot. 49, 1537-1540. (28) Patlak, C., Blasberg, R., and Fenstermacher, J. (1983) Graphic evaluation of blood-to-brain transfer constants from multiple-time uptake data. J. Cerebr. Blood. Flow. Metab. 3, 1-7. (29) Carpenter, G., and Cohen, S. (1976) 125-I labeled human growth factor. J. Cell Biol. 71, 159-171. (30) Bergstrom, M., Litton, J., Eriksson, L., Bohm, C., and Blomqvist, G. (1982) Determination of object contour from projections for attenuation correction in cranial positron emission tomography. J. Comput. Assist. Tomogr. 6, 365372. (31) Jorgensen, P. E., Rasmussen, T. N., Skov Olsen, P., Raaberg, L., Seier Poulsen, S., and Nexo, E. (1990) Renal uptake and excretion of epidermal growth factor from plasma in the rat. Regul. Pept. 28, 273-281. (32) Kim, D., Sugiyama, Y., Fuwa, T., Sakamatomo, S., Iga, T., and Hanano, M. (1989) Kinetic analysis of the elimination process of human epidermal growth factor (hEGF) in rats. Biochem. Pharm. 38, 241-249. (33) Renfrew, C. A., and Hubbard, A. L. (1991) Sequental Processing of epidermal growth factor in early and late endosomas of rat liver. J. Biol. Chem. 266, 4348-4356. (34) Cuartero-Plaza, A., Martinez-Miralles, E., Rossel, R., Vadell-Nadal, C., Farre., Magi., and Real., F. R. (1996) Radiolocalization of suamous lund carcinoma with 131Ilabeled epidermal growth factor. Clin. Cancer Res. 2, 13-20. (35) Zhao, Q., Blomquist, E., Bolander, H., Gedda, L., Hartvig, P., Janson, J.-C., Lundqvist, H., Mellstedt, H., Nilsson, S., Nister, M., Sundin, A., Tolmachev, V., Westlin, J.-E., and Carlsson, J. (1998) Conjugate chemistry, iodination and cellular binding of mEGF-dextran-tyrosine: preclinical tests in preparation for clincal trials. Int. J. Mol. Med. 1, 693702. (36) Behr, T. M., Goldenberg, D. M., and Becker, W. (1998) Reducing the renal uptake of radiolabeled antibody fragments and peptides for diagnosis and therapy: present status, future prospects and limitations. Eur. J. Nucl. Med. 25, 201-212. (37) Bernard, B. F., Krenning, E. P., Breeman, W. A., Rolleman, E. J., Bakker, W. H., Visser, T. J., Macke, H., and de Jong, M. J. (1997) D-lysine reduction of indium-111 octreotide and yttrium-90 octreotide renal uptake. J. Nucl. Med. 38, 19291933. (38) De Jong, M., Bakker, W. H., Breeman, W. A., Bernard, B. F., Hofland, L. J., Visser, T. J., Srinivasan, A., Schmidt, M., Behe, M., Macke, H. R., and Krenning, E. P. (1998) Preclinical comparison of [DTPA0] octreotide, [DTPA0, Tyr3] octreotide and [DOTA0, Tyr3] octreotide as carriers for somatostatin receptor-targeted scintigraphy and radionuclide therapy. Int. J. Cancer 7, 406-411. (39) Behr, T. M., Becker, W. S., Sharkey, R. M., Juweid, M. E., Dunn, R. M., Bair, H. J., Wolf, F. G., and Goldenberg, D. M. (1996) Reduction of renal uptake of monoclonal antibody fragments by amino acid infusion. J. Nucl. Med. 37, 829833. (40) Tarburton, J. P., Halpern, S. E., Hagan, P. L., Sudora, E., Chen, A., Fridman, D. M., and Pfaff, A. E. (1990) Effect of acetylation on monoclonal antibody ZCE-025 Fab′: distribution in normal and tumor-bearing mice. J. Biol. Response Modif. 9, 221-230.

946 Bioconjugate Chem., Vol. 10, No. 6, 1999 (41) Zhao, Q., and Carlsson, J. (1997) In vitro characterization of an end-end coupled mEGF-dextran conjugate using a cultured human glioma cell line. Int. J. Oncol. 11, 1263-1269. (42) Heldin, C.-H., and O ¨ stman, A. (1996) Ligand-induced dimerization of growth factor receptors: variation on the theme. Cytokine Growth Factor Rev. 7, 3-10.

Zhao et al. (43) Kyriakos, R. J., Shih, L. B., Ong, G. L., Patel, K., Goldenberg, D. M., and Mattes, M. J. (1992) The fate of antibodies bound to the surface of tumor cells in vitro. Cancer Res 52, 835-842.

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