Synthesis and Application of Lactosylated, 99m Tc Chelating Albumin for Measurement of Liver Function 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-Bice ˆtre, F-94275, AP-HP, CHU de Bice ˆtre, Le Kremlin-Bice ˆtre, 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, Ho ˆpital 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 99m Tc-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 pro- teins. 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 local- ized 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). 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 99m Tc-DTPA- galactosylalbumin ( 99m Tc-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 Sigma- Aldrich company, and solvents from SDS (France). 4-Nitro- phenylcarbonate 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 regula- tions was purchased from LFB (France) as a 40 mg/mL solution in normal saline (Vialebex). Vasculocis (i.e., 99m Tc human albumin) was obtained from Cisbio IBA (France). Disposable aseptic 0.22 µm filters were purchased from Millipore Company. The 99 Mo/ 99m Tc-generators were purchased from Mallinckrodt Company. The ITLC SG plates were purchased from PALL Corporation (France). Tin salts were purchased from Sigma- Aldrich Company (99.995% purity). Bicinchonic acid assay for protein quantitation was purchased from Pierce by Perbio Scientific. Quantichrom Calcium Assay Kit for DTPA deter- mination was from Gentaur (Belgium). Grafting on HSA, preparation of the ready to use mixture with SnCl 2 , and lyophilization were performed under sterile conditions. Analyti- * Corresponding author. Fax: +33(0)145212112; e-mail: philippe. chaumet-riffaud@bct.aphp.fr. † Universite ´ Paris-Sud 11, EA4046. ‡ AP-HP, CHU de Bice ˆtre. § Unite ´ de Pharmacologie Chimique et Ge ´ne ´tique, U640 INSERM, UMR8151 CNRS. | Universite ´ Paris 7, U733 INSERM. ⊥ AP-HP, Ho ˆpital Bichat. # Morelos State Autonomous University. Bioconjugate Chem. 2010, 21, 589–596 589 10.1021/bc900275f  2010 American Chemical Society Published on Web 03/04/2010