Radiolabeled constructs for evaluation of the asialoglycoprotein receptor status and hepatic functional reserves.
ABSTRACT Transplantation of isolated hepatocytes may eventually replace a whole liver transplantation for the treatment of selected liver metabolic disorders and acute hepatic failure. To understand the behavior of transplanted hepatocytes, methods for longitudinal assessment of functional activity and survival of hepatocyte transplants must be developed. Targeting of asialoglycoprotein receptor (ASGPr) with various radiolabeled or Gd-labeled constructs of asialofetuin (AF) is expected to allow noninvasive and quantitative assessments of the ASGPr status in functioning hepatocytes before and after the transplant. Six new constructs of (125)I-, (99m)Tc-, (153)Gd-, and (111)In-radiolabeled AF with distinct stabilities and clearance rates were prepared and evaluated in vitro in mice, rat, porcine, and human hepatocytes, and in vivo in mice and rats. The blood and organ clearance rates, as well as liver and spleen uptake, were measured. Even extensive chemical modifications of AF with poly-l-lysine and various chelating agents do not appear to diminish AF's binding to ASGPr. Binding to isolated hepatocytes and the in vivo liver uptake studies indicate unimpaired functional activity of AF as evidenced by the rapid (<10 min) and nearly complete hepatic extraction of AF constructs from the systemic circulation. The catabolic processing and elimination of AF constructs from liver depend on the chemical modification used in the preparation of a given reagent. Radioiodinated AF has by far the shortest postabsorption (5.1 min +/- 0.05 min) and elimination half-lives (2.8 +/- 0.06 h) in liver. In comparison, the AF construct prepared by conjugation of DTPA- and 2-iminothiolane-substituted p-Lys with N-sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (SMPB)-modified AF (AF-SMPB-Traut-p-Lys-((111)In-DTPA)(20)(-)(30)) has a hepatic postabsorption time of 9.1 +/- 0.1 min and an elimination half-life of 44.3 +/- 3.08 h, whereas [(99m)Tc]technetium-labeled AF appears to be permanently retained in liver. These differences in rates of liver uptake and clearance of catabolized radiolabeled AF can be used to determine functional activity of liver and transplanted hepatocytes.
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ABSTRACT: Existing methods for the prediction of human clearance of therapeutic proteins involve the use of allometry approaches. In general, these approaches have concentrated on the role of body weight, with only occasional attention given to more specific physiological parameters. The objective of this study was to develop a mechanism-based model of hepatic clearance (CL(H)), which combines a single-species scaling approach with liver physiology, for predicting CL(H) of selected glycoprotein derivate therapeutics, and to compare the outcome of this novel method with those of two empirical methods obtained from the literature - namely, the single-exponent theory and multiple-species allometry. Thus, this study was designed as an explanatory study to verify if the addition of physiological information is of benefit for extrapolating clearance of selected therapeutic proteins from one species to another. Five glycoprotein derivate therapeutics that are known to be principally eliminated by asialoglycoprotein receptors (ASGPRs) under in vivo conditions were selected. It was assumed that the interspecies differences in CL(H) reported for these compounds are reflected by the interspecies differences in the abundance of these receptors. Therefore, key scaling factors related to these differences were integrated into one model. Fourteen extrapolation (prediction) scenarios across species were used in this study while comparing the single-species model, based on physiology, with the single-exponent theory. In addition, the physiological model was compared with multiple-species allometry for three proteins. In general, the novel physiological model is superior to the derived allometric methods. Overall, the physiological model produced a predicted CL(H) value with levels of accuracy of 100% within 3-fold, 100% within 2-fold and about 82% within 1.5-fold, compared with the observed values, whereas the levels of accuracy decreased to 93%, 77% and 53%, respectively, for allometry. The proposed physiological model is also superior to allometry on the basis of the root mean square error and absolute average fold error values. It has been demonstrated that interspecies differences in the abundance of ASGPRs principally govern interspecies variations in CL(H) of compounds that are principally eliminated by ASGPRs. Overall, the proposed physiological model is an additional tool, which should facilitate investigation and prediction of human CL(H) of specific glycoproteins solely on the basis of clearance data determined in a single preclinical species.Clinical Pharmacokinetics 10/2011; 50(10):665-74. · 5.49 Impact Factor
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ABSTRACT: Identification by molecular imaging of key processes in handling of transition state metals, such as copper (Cu), will be of considerable clinical value. For instance, the ability to diagnose Wilson’s disease with molecular imaging by identifying copper excretion in an ATP7B-dependent manner will be very significant. To develop highly effective diagnostic approaches, we hypothesized that targeting of radiocopper via the asialoglycoprotein receptor will be appropriate for positron emission tomography, and examined this approach in a rat model of Wilson’s disease. After complexing 64Cu to asialofetuin we studied handling of this complex compared with 64Cu in healthy LEA rats and diseased homozygous LEC rats lacking ATP7B and exhibiting hepatic copper toxicosis. We analyzed radiotracer clearance from blood, organ uptake, and biliary excretion, including sixty minute dynamic positron emission tomography recordings. In LEA rats, 64Cu-asialofetuin was better cleared from blood followed by liver uptake and greater biliary excretion than 64Cu. In LEC rats, 64Cu-asialofetuin activity cleared even more rapidly from blood followed by greater uptake in liver, but neither 64Cu-asialofetuin nor 64Cu appeared in bile. Image analysis demonstrated rapid visualization of liver after 64Cu-asialofetuin administration followed by decreased liver activity in LEA rats while liver activity progressively increased in LEC rats. Image analysis resolved this difference in hepatic activity within one hour. We concluded that 64Cu-asialofetuin complex was successfully targeted to the liver and radiocopper was then excreted into bile in an ATP7B-dependent manner. Therefore, hepatic targeting of radiocopper will be appropriate for improving molecular diagnosis and for developing drug/cell/gene therapies in Wilson’s disease.American Journal of Nuclear Medicine and Molecular Imaging 09/2014; 4(6):537-547. · 3.25 Impact Factor
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ABSTRACT: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of ven occlusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury.International journal of radiation oncology, biology, physics 12/2013; · 4.59 Impact Factor