Bioconjugate Chem. 2010, 21, 589–596
Synthesis and Application of Lactosylated, Measurement of Liver Function
99m
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Tc Chelating Albumin for
Philippe Chaumet-Riffaud,*,†,‡ Ivan Martinez-Duncker,# Anne-Laure Marty,§ Cyrille Richard,§ Alain Prigent,†,‡ Frederic Moati,†,‡ Laure Sarda-Mantel,|,⊥ Daniel Scherman,§ Michel Bessodes,§ and Nathalie Mignet§ Universite´ Paris-Sud 11, EA4046, Kremlin-Biceˆtre, F-94275, AP-HP, CHU de Biceˆtre, Le Kremlin-Biceˆtre, F-94275, Unite´ de Pharmacologie Chimique et Ge´ne´tique, U640 INSERM, UMR8151 CNRS, Universite´ Paris Descartes, Paris, France, F-75006, Universite´ Paris 7, U733 INSERM, CRB3, Faculte´ Xavier Bichat, Paris, France, AP-HP, Hoˆpital Bichat, Paris, F-75018, and Faculty of Science, Morelos State Autonomous University, Cuernavaca, Mexico. Received June 24, 2009; Revised Manuscript Received December 15, 2009
Neogalactosylated and neolactosylated albumins are currently used as radiopharmaceutical agents for imaging the liver asialoglycoprotein receptors, which allows the quantification of hepatic liver function in various diseases and also in healthy liver transplant donors. We developed an original process for synthesizing a chelating neolactosylated human albumin using maleimidopropyl-lactose and maleimidopropyl-diethylene triamine pentaacetic acid (DTPA) derivatives. The lactosylated protein (LACTAL) conjugate showed excellent liver uptake compared to nonlactosylated protein and a very high signal-to-noise ratio, based on functional assessment of biodistribution in mice using 99mTc-scintigraphy.
INTRODUCTION The evaluation of hepatic function is required to determine the physiological status of the liver, particularly for patients with liver disease whose treatment of choice is hepatectomy or liver transplantation (1). Therefore, determination of the hepatic function is crucial to predict patient outcome before and after hepatectomy or liver transplantation (2), as well as for healthy liver donor assessment. Asialoglycoproteins are internalized into hepatocytes via the asialoglycoprotein receptors which recognize galactose and N-acetyl-galactosamine moieties bound to proteins. The number of asialoglycoprotein receptors on the hepatocytes of patients with liver disease is reduced and is thus considered a good indicator for the evaluation of liver function (3, 4). In addition, noninvasive quantification of the liver uptake of radiolabeled asialoglycoproteins also provides useful information, especially in terms of spatial distribution of hepatic function. The asialoglycoprotein receptors are localized mainly on liver parenchymal cells (5) and play a major role in the hepatic clearance of serum proteins (6). Most of the receptors (90%) are located on sinusoidal faces of hepatocytes with only 10% on lateral faces (7). Non-hepatic cells such as endothelial and Kupffer cells have no or few asialoglycoprotein receptors (8), and hepatocytes are the most represented cells in the liver. Therefore, targeting the asialoglycoprotein receptors results in targeting mainly hepatocytes. The measurement of the hepatic functional reserve by kinetic studies of liver binding and blood clearance of galactosylated ligands to the asialoglycoprotein receptors is validated and directly reflects the number of functional hepatocytes (9, 10). * Corresponding author. Fax: +33(0)145212112; e-mail: philippe.
[email protected]. † Universite´ Paris-Sud 11, EA4046. ‡ AP-HP, CHU de Biceˆtre. § Unite´ de Pharmacologie Chimique et Ge´ne´tique, U640 INSERM, UMR8151 CNRS. | Universite´ Paris 7, U733 INSERM. ⊥ AP-HP, Hoˆpital Bichat. # Morelos State Autonomous University.
Quite a few reports of procedures for the preparation of a modified human serum albumin (HSA) have been described for use as a liver radiopharmaceutical agent (11-14). For instance, de Graaf et al. (11) recently showed that 99mTc-DTPAgalactosylalbumin (99mTc-GSA) scintigraphy combined with SPECT (single photon emission computed tomography) is a feasible noninvasive method to assess hepatic functional volume in normal rat liver, as well as during liver regeneration in a rat model with partial hepatectomy. However, these procedures have a number of limitations: long reaction times, reduction of structural disulfide bonds, alterations of the neat charge of the protein, or reactions involving toxic reagents such as sodium cyanoborohydride for reductive amination. Therefore, the aim of our study was to develop an alternate process to prepare a DTPA-lactosyl albumin (LACTAL), using a quick single-step reaction involving maleimido-derivatized reagents, and perform preclinical studies to validate its use as a radiopharmaceutical for liver function imaging (15).
MATERIAL AND METHODS Material. Chemical reagents were obtained from SigmaAldrich company, and solvents from SDS (France). 4-Nitrophenylcarbonate Wang resin was obtained from Novabiochem. 3-(Maleimido) propionic acid N-hydroxysuccinimide ester and lactosylamine were obtained using described procedures (16, 17). Human serum albumin compliant with European safety regulations was purchased from LFB (France) as a 40 mg/mL solution in normal saline (Vialebex). Vasculocis (i.e., 99mTc human albumin) was obtained from Cisbio IBA (France). Disposable aseptic 0.22 µm filters were purchased from Millipore Company. The 99Mo/99mTc-generators were purchased from Mallinckrodt Company. The ITLC SG plates were purchased from PALL Corporation (France). Tin salts were purchased from SigmaAldrich Company (99.995% purity). Bicinchonic acid assay for protein quantitation was purchased from Pierce by Perbio Scientific. Quantichrom Calcium Assay Kit for DTPA determination was from Gentaur (Belgium). Grafting on HSA, preparation of the ready to use mixture with SnCl2, and lyophilization were performed under sterile conditions. Analyti-
10.1021/bc900275f 2010 American Chemical Society Published on Web 03/04/2010
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cal HPLC was performed on a Merck-Hitachi gradient pump equipped with a L-6200A intelligent pump and a UV-visible detector L-4000 Merck. Electrophoresis of proteins was performed on NuPage 10% Bis-Tris Gel from InVitrogen. 1-(β-D-Lactosyl)-3-maleimidopropionamide or Maleimidopropyl-Lactose (1). β-D-lactosylamine (100 mg; 0.25 mmol, ref 16) was dissolved in DMF (2 mL), 3-(maleimido)propionic acid N-hydroxysuccinimide ester (73 mg; 0.28 mmol) was added and the mixture was stirred overnight at room temperature. The reaction medium was evaporated and the residue precipitated in acetone. The white solid obtained by centrifugation was washed several times with acetone and dried to give 1 (73 mg; 60%). This product is hygroscopic and should be stored accordingly. MS: m/z 515 for [M + Na]. 1H NMR (400 MHz, DMSO, δ ppm): 8.55 (d, J ) 12 Hz, 1H), 7.01 (s, 2H), 5.12 (d, J ) 4 Hz, 1H), 5.02 (d, J ) 8 Hz, 1H), 4.80 (d, J ) 4 Hz, 1H), 4.76-4.71 (m, 2H), 4.66 (dd, J ) 4 Hz, 1H), 4.55-4.52 (m, 2H), 4.2 (d, J ) 8 Hz, 1H), 3.69 (dd, J ) 4 Hz, J ) 12 Hz, 1H), 3.63-3.44 (m, 6H), 3.35-3.29 (m, 8H), 3.10 (m, 1H). 13 C NMR (100 MHz, DMSO, δ ppm): 170.61; 169.71; 134.42; 103.67; 80.36; 79.02; 76.29; 75.53; 75.41; 73.09; 71.92; 70.47; 68.00; 60.26. Anal. Calcd for C19H28N2O13; H2O: C 44.70, H 5.88, N 5.49. Found: C 44.24, H 6.08, N 4.88. {Carboxymethyl-[2-(carboxymethyl-{2-[carboxymethyl({2-[3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionylamino]ethylcarbamoyl}-methyl)-amino]-ethyl}-amino)-ethyl]-amino}acetic acid or Maleimidopropyl-DTPA (3). 4-Nitrophenyl carbonate Wang resin (1 g; 0.54 mmol/g) was swollen in N-methylpyrrolidone (NMP) and ethylenediamine was added in excess (0.36 mL; 10 equiv). It was left under gentle shaking overnight. It was then thoroughly rinsed with NMP and a solution of diethylene triamine pentacetic dianhydride (1.9 g; 5.4 mmol; 10 equiv) in NMP (20 mL) was added, followed by triethylamine (0.8 mL; 10 equiv). The suspension was shaken overnight. The resin was washed with NMP (3 × 10 mL), H2O (1 × 10 mL; contact 30 min), absolute ethanol (3 × 10 mL), and ether (2 × 10 mL), and dried in air. A solution of trifluoroacetic acid (TFA) (9 mL), H2O (1 mL), and triisopropyl silane (200 µL) was added to the dry resin and allowed to react 3 h at 30 °C. The eluate was concentrated and the residue washed with ether to give (2) as a white powder (0.268 g; 84% crude). The powder was dissolved in DMSO (1 mL), then 3-(maleimido)propionic acid N-hydroxysuccinimide ester (0.14 g; 1.1 equiv) and triethylamine (0.14 mL; 2 equiv) were added. The mixture was left at room temperature overnight then diluted with ethyl acetate to induce precipitation. The precipitate was centrifuged, rinsed with ethyl acetate and ether, and dried to give 3 (0.16 g; 51% overall yield; hygroscopic). MS: m/z 587. 1 H NMR (400 MHz, DMSO, δ ppm): 7.28 (s, 2H), 4.55 (m, 2H), 4.40-4.10 (m, 7H), 3.86-3.57 (m, 9H), 3.15 (m, 2H), 2.92 (m, 2H). 13C NMR (100 MHz, D2O, δ ppm MeOH): 176.72; 174.00; 172.77; 170.6; 134.65; 57.59; 57.36; 57.29; 57.11; 52.10; 51.35; 38.88; 38.67; 34.87; 34.60. Anal. Calcd for C23H34N6O12; H2O: C 45.69, H 5.96, N 13.90. Found C 45.59, H 5.74, N 13.30. LACTAL Synthesis. Instruments and solutions were sterilized using a Getinge Vertical Sterilizer AVOR 130. Buffer was degassed by sonication in a US cleaning bath (Branson 1510). Iminothiolane (6 mg, 64 µmol in 200 µL PBS/EDTA 5 mM pH 7.8), maleimido-propyl-lactose (3.3 mg, 6.8 µmol in 100 µL PBS/EDTA 5 mM), and buffered maleimido-propyl-DTPA (2 mg, 3.4 µmol in 100 µL PBS/EDTA 5 mM) were added to the solution of HSA (100 µL, 4 mg, 59 nmol, 3.2 µmol NH2). All components were gently mixed during 1 h at room temperature. Then, the mixture was diluted to 4 mL with NaCl 0.9% (degassed with N2 and filtered under 0.22 µm) and
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transferred to a centrifuge ultrafiltration system with a molecular weight cutoff of 30 kDa. The mixture was centrifuged at 4000 rpm (3000 g) during 10 min, until the filtrate did not contain any DTPA. The absence of residual DTPA reagent was controlled by taking a sample of the filtrate (180 µL) and adding an excess of EuCl3, 6H2O (20 µL, 1 g/L). Measurement of the europium signal by time-resolved fluorescence at 616 nm with a spectrofluorimeter (Victor2, Perkin-Elmer) indicated the diminution of the DTPA content as washes proceeded. The total protein amount recovered from the experiment was quantified by the BCA reagent. The modified HSA (10 µL of a solution estimated to be at 1 mg/mL) was mixed with 200 µL of the BCA reagent and incubated for 30 min at 37 °C. The concentration was obtained from a standard concentration range of unmodified HSA taken at 562 nm. 98 ( 1% of protein was recovered from the process. The amount of lactose coupled to the protein was determined by the method of Dubois (18). The modified HSA (10 µL, 1 mg/mL) was introduced in a glass vial, then phenol (5% in water, 190 µL) and concentrated sulfuric acid (200 µL) were added. The vial was heated at 90 °C during 5 min then left to cool down at room temperature. Measurements were done at 490 nm; the concentration was calculated from a standard concentration range of lactose and indicated 30 ( 3 lactose/ HSA (mol/mol). The amount of DTPA coupled to the protein was determined by the Quantichrom Calcium Assay Kit (Gentaur). The modified HSA (100 µL, 1 mg/mL) was introduced in a 1 mL cuvette, then CaCl2 (20 µL, 1 M) was added, and the volume was completed with 500 µL of the calcium working solution provided by the kit. After 3 min, the optical density was measured at 612 nm giving the free calcium concentration from which the amount of DTPA could be deduced. 5 ( 1 DTPA/ HSA (mol/mol) was found. The modified and unmodified albumin were analyzed by HPLC using a Vydac column C4-214TP5415 (4.6 mm × 150 mm) (Interchim, Montluc¸on, France), eluted with a gradient from 90/10 A/B to 10/90 A/B during 20 min with a flow rate of 1 mL/min (solvent A: H2O + 0.1% TFA, solvent B: acetonitrile) and detection at 220 nm. HSA elution time: 12.1 ( 0.1 min. LACTAL elution time: 11.8 ( 0.1 min. (Despite the small difference in elution time, a shoulder was clearly observed when a mixture of the two compounds were injected.) Electrophoresis (MOPS buffer, 150 V) was performed with LACTAL and HSA and compared with the SeeBlue Plus2 PreStained Standard (Invitrogen, 4-250 kDa). The modified protein appeared as a single band. The estimated mass for LACTAL was 93 kDa, which corresponded to 29 lactose units and 5 DTPA (Figure 1). Preparation of LACTAL Lyophilisate and Labeling. The pH of a modified protein solution (500 µL, 15 mg/mL) was carefully adjusted to 1.5 with HCl 1 N, then a solution of stannous chloride in HCl 1 N (10 mg/mL, 20 µL, 28 µg/mg protein) was added and the mixture was left 20 min protected from light at room temperature. The volume was adjusted to 1 mL and aliquoted in 5 × 2 mL serum vials (200 µL, 1.44 mg/ vial), frozen in liquid nitrogen, and lyophilized. The system was then equilibrated to atmospheric pressure by nitrogen. The vials were sealed under nitrogen using a glovebox. The lyophilized product was redissolved in NaCl solution (0.9%; 1 mL). A saline solution of 99mTc (pertechnetate) freshly eluted from a 99Mb generator was added (5-7 × 106 Bq/200 µg prot), and the mixture was incubated for 30 min at 50 °C. Radio-TLC chromatograms were obtained using a BIOSCAN Miniscan system. The radiochemical purity was checked using ITLC methods to determine the amounts of unbound and hydrolyzed 99mTc. ITLC/Umezawa (ethanol/10% ammonium
Lactosylated
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Tc Chelating Albumin
Figure 1. Gel electrophoresis of HSA and LACTAL with the Invitrogen NuPAGE Novex kit (MOPS buffer, 150 V).
acetate ) 1:1) was used to check the labeling efficiencies. Unbound 99mTc moved to the solvent front, and LACTALbound 99mTc stayed at the origin on ITLC/Umezawa. The solution of 99mTc-LACTAL was stable at room temperature for 20 h. Biodistribution Studies. A gamma scintillation counter Wizard 1480 automatic model from Perkin-Elmer Company was used for counting radioactivity of mouse organs in biodistribution studies. Male mice (26-28 g) were injected via the tail vein with 0.55 MBq of 99mTc-labeled ligand. Animals were anesthetized and sacrificed at planned time-points (three per time point; 10, 30, and 90 min). Blood and organ samples (heart, blood, kidney, spleen, lung, and liver) were collected, weighed with an electronic balance, and counted in a gamma scintillation counter. In Vivo Imaging Studies. A small-animal dedicated gamma camera from BIOSPACE Lab (France) equipped with a lowenergy high-resolution parallel collimator was used for imaging rats (field of view: 8 cm). Following anesthesia with pentobarbital, male Wistar rats (n ) 9, 220-240 g) were injected intravenously through the penis vein with 200 µL of tracer (5-10 MBq). The rats were then placed on their back on the gamma camera where posterior planar acquisitions were recorded.
RESULTS Synthesis of the Maleimidopropyl-Lactose. D-Lactose was first reacted with ammonium carbamate in methanol/water to give the carbamate salt of the amino-lactoside which was treated just before use with triethylamine to give the 1-aminosugar as described in the literature (16). This product was reacted with
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3-(maleimido)-propionic acid N-hydroxysuccinimide ester (17) to give the final maleimidopropyl-lactose 1 (Scheme 1). This product could be stored for months at 4 °C in dry atmosphere. Synthesis of the Maleimidopropyl-DTPA. For a better regioselective reaction of the multifunctional DTPA, we developed a solid-phase synthesis (Scheme 2). 4-Nitrophenylcarbonate Wang resin was reacted with excess ethylene diamine in NMP. The reaction progress could be monitored in UV by the release of 4-nitrophenol (410 nm). After thorough washings, DTPA anhydride was added in excess and the mixture swirled for 1 h. End of reaction was monitored by negative Kaiser and TNBS tests. Successive washings with NMP, water, methanol, and diethyl ether were performed to eliminate the excess of DTPA, hydrolyze the remaining anhydride function, and dry the resin. The resin was dried in air, and a mixture of trifluoroacetic acid/water/triisopropylsilane was added as a degrafting reagent. The solution was thoroughly evaporated to give pure ethylenediamine-DTPA (2) which was condensed without further purification with 3-(maleimido)-propionic acid N-hydroxysuccinimide ester to give maleimidopropyl-DTPA (3). Synthesis of the Modified Albumin (LACTAL). In preliminary studies, the modified protein was prepared in two separated steps: first, introduction of the thiol functions with iminothiolane (19), then addition of the maleimido-lactose (1) and maleimido-DTPA (3). This allowed quantitation of the intermediate thiol groups with the Ellman reagent. However, this first step was unreliable, leading to unreproducible amounts of thiol groups which was probably due to rearrangement of the iminothiolane moiety with time (20) or oxidation. The protocol was then modified to a single-step process (Scheme 3). The successive addition of iminothiolane, lactose, and DTPA derived maleimide compounds allowed achieving the reproducible HSA modification. The lactose and DTPA derivatives (1 and 3) were introduced on the ε-amino groups of the protein lysines without significant changes in the ionic balance of the protein (21); the presence of EDTA avoided thiol oxidation. There was no evidence in favor of the formation of aggregates which could induce changes in surface properties of the protein molecule. Stability of the Solution and of the Lyophilisate. Stability of the solution of LACTAL was checked after a one-year storage at 4 °C and 32 months for the lyophilized kit. There were no differences from freshly prepared material, no aggregates were observed, and the imaging properties were identical. In Vivo Imaging in Rats. The biodistribution of the tracer was studied through the completion of an acquisition in list mode over an extended period up to 2 h in some rats. Figure 2 shows a sequence of 15 dynamic images of 2 min each, corresponding to the first 30 min post iv administration. Just after injection, the distribution of LACTAL resembled an intravascular tracer and the anatomical regions showing high activity at this stage corresponded to highly vascularized organs. Then, the images highlighted an early and predominantly hepatic uptake with a plateau that was reached between 2-6 min and continued for
Scheme 1a
a Synthesis of maleimidopropyl-lactose (1): i, NH4OH, NH2-COO-; NH4+; ii, Et3N, MeOH (0.5), EtOH/water (1:0.4), see ref 16; ii, 3-(maleimido)propionic acid N-hydroxysuccinimide ester, DMF.
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Scheme 2a
a Synthesis of maleimidopropyl-DTPA (3): i, ethylene diamine, NMP; ii, DTPA anhydride, triethylamine; iii, TFA, triisopropylsilane; iv, 3-(maleimido)propionic acid N-hydroxysuccinimide ester, Et3N, DMSO.
Scheme 3. One-Step Preparation of LACTAL: Human Serum Albumin Modified by Simultaneous Introduction of Maleimidopropyl-Lactose and -DTPA Derivatives
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Figure 2. Posterior planar scintigraphic acquisition was started immediately after intravenous injection of 200 µL of 99mTc labeled LACTAL (5-10 MBq). List-mode data were acquired for at least 30 min for all animals. Using Biospace software, the list-mode data were reformated into fifteen 2 min frames. The last image on the bottom right is a summed image (0-30 min).
at least 20 min in this animal species. After 30 min, the liver uptake began to decrease, and analysis of dynamic images such as those in Figure 3 (12 images of 5 min each between 30 and 90 min after iv injection) referred to a disposal of the product by the bile ducts. An important passage in the gastrointestinal tract most likely in the form of degradation products was also visible. The last picture showed predominant radioactivity in the digestive tube and some traces in the bladder indicating the modes of degradation and elimination of the tracer. Biodistribution in Mice. The biodistribution of LACTAL in mice is illustrated in Table 1. The activities were measured in different organs and expressed as the percentage of the injected dose per gram of organ. Non-lactosylated 99mTc human serum albumin (Vascolucis) was used as a control. The biodistribution data confirmed the results of the visual analysis of scintigraphic acquisitions. The liver uptake was very early and intense. Splenic fixation was much lower, confirming the specificity of the radiolabeled molecule for ASGP hepatic receptors. Blood levels were very low after 10 min reflecting the very high plasma clearance; a slight rise to late time is likely to reflect intrahepatic degradation of the tracer leading to release of 99mTc labeled catabolites in the bloodstream. Contrary to what was observed with LACTAL, the activity in hepatic tissue of the control (Vascolucis) one hour after the injection was much lower than the blood activity. These results are consistent with the fact that this tracer remains mainly in the intravascular compartment (Table 1).
DISCUSSION Although the indocyanine green clearance test is widely used to assess the hepatic functional reserve before hepatectomy, discrepancies between indocyanine green clearance values and histological observations in the liver are noted in given instances. Volumetric measurements may not be relevant in patients with advanced chronic liver disease requiring major liver resection (22). In a similar way, iminodiacetic acid 99mTc labeled derivatives are good tracers of the hepatic biliary clearance and are mostly dedicated to the diagnosis of gall bladder dysfunctions by cholescintigraphy (23). These tracers are not specifically targeted to the asialoglycoprotein receptors. Previously reported work indicated that the measurement of the hepatic functional reserve could be very useful before hepatectomy as well as for the evaluation of the posttransplantation residual hepatic functional reserve in donors. It could also be useful for the follow-up and functional prognosis of the hepatic graft, staging, and preoperative evaluation. Furthermore, a precise assessment of functional hepatic reserve would also be useful for the follow-up and prognosis of hepatic function in cirrhotic patients, the evaluation of surgical resection extension in patients with hepatocellular carcinoma, and prognosis in acute hepatitis. The preoperative estimation of the maximal removal rate of 99mTc GSA radioligand in the predicted liver remnant has proved useful for determining the hepatectomy procedure (24). There are at least 5 different glycosylated tracers identified, based on a literature search. 99mTc-Galactosyl-neoglycoalbumin
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Figure 3. Posterior planar scintigraphic acquisition was performed between 30 and 90 min after intravenous injection of 200 µL of LACTAL (5-10 MBq). Using Biospace software, the list-mode data were reformated into twelve 5 min dynamic frames. Table 1. Biodistribution Results after 10 min (n ) 3), 30 min (n ) 3), and 90 min (n ) 3) following Intravenous Injection of LACTAL or Vasculosis (60 min, n ) 3) into Healthy Micea Lactal [t ) 10 min] Lactal [t ) 30 min] Lactal [t ) 90 min] Vasculosis 60 min a
99m
Tc labeled
99m
Tc-Labeled
heart
liver
spleen
kidneys
blood
lung
0.88 ( 0.24 0.26 ( 0.10 0.52 ( 0.12 4.13 ( 1.05
51.3 ( 4.27 36.7 ( 6.68 24.3 ( 3.19 5.10 ( 1.55
3.77 ( 0.35 0.25 ( 0.15 1.74 ( 1.16 3.31 ( 0.84
3.89 ( 0.59 1.21 ( 0.54 2.82 ( 0.48 6.77 ( 1.92
1.98 ( 0.60 0.59 ( 0.24 0.82 ( 0.03 35.3 ( 4.04
1.76 ( 0.55 0.34 ( 0.16 0.72 ( 0.09 6.17 ( 2.03
Results were expressed as the percentage of injected dose per gram of organ (% of ID/g of organ) at the different times.
(99mTc-NGA) was prepared by the covalent coupling of 2-imino2-ethyloxymethyl-1-thiogalactose to human serum albumin (13) (about 24 galactose units per albumin molecule). Labeling of this compound with 99mTc was achieved by an electrolytic method. Biodistribution studies in rabbits demonstrated that the liver is the only site with 99mTc-NGA uptake (25). As for 99mTcGSA (11), preclinical studies demonstrated its diagnostic efficiency, the safety and the stability of 99mTc-NGA, but the labeling method is difficult to implement in most clinical departments. This is why a diethylenetriaminepentaacetic acid conjugated neogalactosylated albumin was developed later on (26). The reaction is very quick and yields HSA derivatives containing 4.5 to 7 DTPA per mole of protein. The combination of galactosylated albumin to DTPA enabled the production of an instant kit (27). Numerous publications are available concerning the use of this radiopharmaceutical, which is distributed only in Japan and not commercially available in European countries. Besides, the chemical process for synthesis is a derivatization with cyanomethyl-thiogalactose which may lead to toxic byproduct. 67 Ga-desferoxamine-galactosyl-neoglycoalbumin (67Ga-DFNGA) is a galactosylated human serum albumin molecule. It is similar to NGA, as described above except for a covalent coupling of a desferoxamin molecule. Thus, DF-NGA indirectly binds to 67Ga. This radiomarker has demonstrated high liver uptake in rabbits, but it has never been administered to humans (14). 99mTc-Neolactosylated-albumin (99mTc-LSA) is the only glycosylated human albumin synthesized by conjugating lactose
to HSA using a reductive amination process (12). However, the use of sodium cyanoborohydride in this reaction is questionable for human use. Direct labeling with 99mTc was possible due to the adjunction of medronate and stannous chloride to the reduced LSA. We have developed a new synthetic process for a lactosylated albumin which involves a simultaneous set of reactions with the protein. Thiol functions were generated by reaction of iminothiolane with the ε-amines of lysines, in the presence of the maleimides derivatives (lactose and DTPA). This avoided the formation of byproduct induced by the rearrangement of the iminothiol moiety (20) or by oxidation. The same conjugation chemistry was used for the introduction of both the ligand (lactose) and the cryptant (DTPA), leading to reliable and reproducible results and the control of the two entity ratio. This new process involved five simple steps: (1) synthesis of a maleimide derivative of the targeting ligand (in the example, lactose) and of a technetium chelate (diethylene triamine pentaacetic acid, DTPA); (2) introduction of thiol functions by reaction of iminothiolane on the ε-amino groups of the protein lysines and simultaneous covalent grafting of the ligand and chelate moieties on the protein carrier (this smoothly provided functionalization without significant changes in the ionic balance of the protein (21)); (3) purification of the modified protein by ultrafiltration; (4) lyophilization after addition of the correct amount of stannous chloride (for technetium reduction); (5) prior to injection, association with technetium or another radioligand or contrast agent by chelation. This chemistry can easily be
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Tc Chelating Albumin
adapted to the conjugation of many other ligands, including sugars, peptides or vitamins, and other markers can be introduced into the DTPA cryptants for other modes of detection (i.e., magnetic resonance imaging or fluorescence). Furthermore, the reaction times are considerably shorter than the abovementioned protocols thus avoiding bacteria growth. Asialoglycoproteins are internalized into hepatocytes via a receptor that recognizes the terminal galactose and N-acetylgalactosamine residues of carbohydrate chains. The uptake and the intracellular processing of 125I and 99m Tc NGA were studied in an isolated perfused rat liver model (28). After a 1 min pulse administration of iodine NGA, 40% of the radioactivity was taken up at first pass by the liver, and after 15-20 min, 82% of the 125I was released at the sinusoidal pole of the hepatocyte, predominantly as small catabolites. In contrast, only 4% of 99mTc galactosylated albumin taken up by the liver reappeared in the perfusate, and 40% of the radioactivity was found in the bile one hour after injection. Our results confirmed that liver captation was very high after first pass (80% of injected dose) and that the majority of the injected activity was eliminated by the biliary track. The residual activity in the liver was 34% ID after 90 min. Unlike colloidal tracers, the liver uptake was not accompanied by a splenic uptake suggesting a specific hepatocyte captation as also shown by Kim et al. (29). No or very little activity was visible in the urinary tract.
CONCLUSION A receptor-specific ligand for the asialoglycoprotein hepatic receptor was developed using a straightforward, cyanide-free conjugation with human serum albumin. The pharmaceutical validation of the process will need complementary studies. Imaging and biodistribution studies demonstrated hepatic targeting and rapid blood clearance for this tracer. 99mTc-LACTAL accumulated mainly in the liver, and there was negligible uptake in the spleen. Kinetic parameters provided by these experiments will be useful for the design and the implementation of supplementary non clinical studies and phase I trials in healthy volunteers. In addition, this process could enable efficient and fast modification of protein templates with other ligands and chelated nuclei for use in nuclear medicine or magnetic resonance imaging of different targets.
ACKNOWLEDGMENT We thank the CNRS for a one-year support for a valorisation engineer (M. A-L.). We would like to thank Mr. Abdelhakand Jallane for his technical assistance in biodistribution studies and Dr. Anne Gruaz-Guyon for her scientific advice.
LITERATURE CITED (1) Clavien, P. A., Petrowsky, H., De Oliveira, M. L., and Graf, R. (2007) Strategies for safer liver surgery and partial liver transplantation. New. Engl. J. Med. 356, 1545–1559. (2) Ishikawa, M., Yogita, S., Miyake, H., Fukuda, Y., Harada, M., Wada, D., and Tashiro, S. (2007) Differential diagnosis of small hepatocellular carcinoma and borderline lesions and therapeutic strategy. Hepato-gastroenterol. 49, 1591–1596. (3) Sawamura, T., Nakada, H., Hazama, H., Shiozaki, Y., Sameshima, Y., and Tashiro, Y. (1984) Hyperasialoglycoproteinemia in patients with chronic liver diseases and/or liver cell carcinoma. Asialoglycoprotein receptor in cirrhosis and liver cell carcinoma. Gastroenterology 87, 1217–1221. (4) Stockert, R. J. (1995) The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol. ReV. 75, 591–609. (5) Aschwell, G., and Morell, A. G. (1981) The role of surface carbohydrates in the hepatic recognition and transport of circulating proteins. AdV. Enzymol. 41, 99–128.
Bioconjugate Chem., Vol. 21, No. 4, 2010 595 (6) Aschwell, G., and Streer, C. J. (1981) Hepatic recognition and catabolism of serum glycoprotein. JAMA 246, 2358–2364. (7) Stockert, R. J., and Morell, A. G. (1983) Hepatic binding protein: the galactose-specific receptor of mammalian hepatocytes. Hepatology 3, 750–757. (8) Matsuura, S., Nakada, H., Sawamura, T., and Tashiro, Y. (1982) Distribution of an asialoglycoprotein receptor on rat hepatocyte cell surface. J. Cell Biol. 95, 864–875. (9) Lee, J. (1998) Quantitative evaluation of liver function with hepatic receptor scintigraphy using Tc-99m galactosylated serum albumin (GSA). Korean J. Nucl. Med. 32, 305–313. (10) Miki, K., Kubota, K., Inoue, Y., Vera, D. R., and Makuuchi, M. (2001) Receptor measurements via Tc-GSA kinetic modeling are proportional to functional hepatocellular mass. J. Nucl. Med. 42, 733–737. (11) de Graaf, W., Vetela¨inen, R. L., de Bruin, K., van Vliet, A. K., van Gulik, T. M., and Bennink, R. J. (2008) 99mTc-GSA scintigraphy with SPECT for assessment of hepatic function and functional volume during liver regeneration in a rat model of partial hepatectomy. J. Nucl. Med. 49, 122–128. (12) Jeong, J. M., Hong, M. K., Lee, J., Son, M., So, Y., Lee, D. S., Chung, J. K., and Lee, M. C. (2004) 99mTc-Neolactosylated human serum albumin for imaging the hepatic asialoglycoprotein receptor. Bioconjugate Chem. 15, 850–855. (13) Vera, D. R., Stadalnik, R. C., and Krohn, K. A. (1985) Technetium-99m galactosyl-neoglycoalbumin: preparation and preclinical studies. J. Nucl. Med. 26, 1157–1167. (14) Vera, D. R. (1992) Gallium-labeled deferoxamine-galactosylneoglycoalbumin: a radiopharmaceutical for regional measurement of hepatic receptor. J. Nucl. Med. 33, 1160–1166. (15) Scherman, D., Bessodes, M., Chaumet-Riffaud, P., and Mignet, N. (2008)New conjugates for therapeutic purposes, and/ or as a diagnostic and/or imaging agent, and methods for preparing the same. PCT/FR2007/052517; WO/2008/074960 A3 (16) Likhosherstov, L. M., Novikova, O. S., and Shibaev, V. N. (2002) New efficient synthesis of β-glucosylamines of mono and disaccharides with the use of ammonium carbamate. Dokl. Chem. 383, 89–92. (17) Nielsen, O., and Buchardt, O. (1991) Facile synthesis of reagents containing a terminal maleimido ligand linked to an active ester. Synthesis 819–821. (18) Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. (19) Weber, C., Reiss, S., and Langer, K. (2000) Preparation of surface modified protein nanoparticles by introduction of sulfhydryl groups. Int. J. Pharm. 211, 67–78. (20) Singh, R., Kats, L., Bla¨ttler, W. A., and Lambert, J. M. (1996) Formation of N-substituted 2-iminothiolanes when amino groups in proteins and peptides are modified by 2-iminothiolane. Anal. Biochem. 236, 114–125. (21) Jue, R., Lambert, J. M., Pierce, L. R., and Traut, R. R. (1978) Addition of sulfhydryl groups to Escherichia Coli ribosomes by protein modification with 2-iminothiolane (methyl 4-mercaptobutyrimidate). Biochemistry 17, 5399–5405. (22) Vauthey, J. N., Chaoui, A., Do, K. A., Bilimoria, M. M., Fenstermacher, M. J., Charnsangavej, C., Hicks, M., Alsfasser, G., Lauwers, G., Hawkins, I. F., and Caridi, J. (2000) Standardized measurement of the future liver remnant prior to extended liver resection: Methodology and clinical associations. Surgery 127, 512–519. (23) Krishnamurthy, G. T., and Krishnamurthy, S. (2002) Hepatic bile entry into and transit pattern within the gallbladder lumen: a new quantitative cholescintigraphic technique for measurement of its concentration function. J. Nucl. Med. 43, 901–908. (24) Kwon, A.-H., Matsui, Y., Kaibori, M., and Ha-Kawa, S. K. (2006) Preoperative regional maximal removal rate of technetium-99m-galactosyl human serum albumin (GSA-Rmax) is useful for judging the safety of hepatic resection. Surgery 140, 379–386.
596 Bioconjugate Chem., Vol. 21, No. 4, 2010 (25) Kudo, M., Vera, D. R., Stadalnik, R. C., Trudeau, W. L., Ikekubo, K., and Todo, A. (1990) In vivo estimates of hepatic binding protein concentration: correlation with classical indicators of hepatic functional reserve. Am. J. Gastroenterol. 85, 1142– 1148. (26) Kudo, M., Todo, A., Ikekubo, K., Hino, M., Yonekura, Y., Yamamoto, K., and Torizuka, K. (1991) Functional hepatic imaging with receptor-binding radiopharmaceutical: clinical potential as a measure of functioning hepatocyte mass. Gastroenterol. Jpn. 26, 734–741. (27) Kubota, Y., Kojima, M., Hazama, H., Kawa, S., Nakazawa, M., Nishiyama, Y., Nakagawa, S., Murase, T., Okuno, H., et al. (1986) A new liver function test using the asialoglycoproteinreceptor system on the liver cell membrane. I. Evaluation of liver
Chaumet-Riffaud et al. imaging using the technetium-99m-neoglycoprotein. Kaku Igaku 23, 899–905. (28) Gore, S., Morris, A. I., Gilmore, I. T., Maltby, P. J., Thornback, J. R., and Billington, D. (1991) Differences in the intracellular processing of the radiolabel following the uptake of iodine-125- and technetium-99m-neogalactosyl albumin by the isolated perfused rat liver. J. Nucl. Med. 32, 506–511. (29) Kim, S., Jeong, J. M., Hong, M. K., Jang, J.-J., Lee, J., Lee, D. S., Chung, J.-K., and Lee, M. C. (2008) Differential receptor targeting of liver cells using 99mTc- neoglycosylated human serum albumins. Arch. Pharm. Res. 31, 60–66. BC900275F
