title: Optimizing the bioavailability of small molecular optical imaging probes by conjugation to an albumin affinity tag.
abstract: Small molecular imaging probes are often found to be rapidly cleared from the circulation. In order to improve signal to noise ratio (SNR) by high probe accumulation in the target tissue we intended to prolong the presence of the probes in the circulation by exploiting inherent transport mechanisms. Human serum albumin (HSA) is playing an increasingly important role as a drug carrier in clinical settings and drugs directly bound to albumin or attached to albumin binding moieties have been successfully developed for treatment approaches. To optimize the bioavailability of existing fluorescent probes, a hydrophobic affinity tag is installed, which enhances albumin binding. In a first experiment an endothelin-A receptor (ETAR) probe is modified by inserting a trivalent linker, attaching an albumin affinity tag and labeling the conjugate with the fluorescent dye Cy 5.5. The spectroscopic properties of the conjugate are examined by photometer- and fluorometer measurements in comparison to a probe without albumin binding tag. Albumin binding was proven by agarose gel electrophoresis. The affinity towards ETAR was confirmed in vitro by cell binding assays on human fibrosarcoma cells (HT-1080) and in vivo by murine xenograft imaging studies. In vitro, the modified probe retains high target binding in the absence and presence of albumin. Binding could be blocked by predosing with ETAR antagonist atrasentan, proving specificity. The in vivo examinations in comparison to the established probe showed a reduced renal elimination and a prolonged circulation of the tracer resulting in significantly higher signal intensity (SI) at the target and a higher signal-to-noise ratio (SNR) between 3h and 96 h after injection. In summary, we designed a small molecular, non-peptidic fluorescent probe which targets ETAR and reversibly binds to serum albumins. The reversible binding to albumin enhances the biological half-life of the probe substantially and enables near infrared optical imaging of subcutaneous tumors for several days. This approach of reversibly attaching probes to serum albumin may serve as a tool to optimize tracer distribution for more precise target characterization in molecular imaging experiments.
authors.affiliation: Dept. of Clinical Radiology, University Hospital Muenster, Muenster, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Hahnenkamp
authors.affiliation: Dept. of Clinical Radiology, University Hospital Muenster, Muenster, Germany.
authors.forename: Wael
authors.initials: W
authors.lastname: Alsibai
authors.affiliation: Clinic for Radiology, St. Franziskus Hospital Muenster, Muenster, Germany; Interdisciplinary Center for Clinical Research (IZKF), University of Muenster, Muenster, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Bremer
authors.affiliation: Dept. of Clinical Radiology, University Hospital Muenster, Muenster, Germany. Electronic address: carsten.hoeltke@uni-muenster.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Höltke
mesh_terms: D000345:Affinity Labels / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D000818:Animals; D001682:Biological Availability; D002232:Carbocyanines / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D045744:Cell Line, Tumor; D005260:Female; D005456:Fluorescent Dyes / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D006801:Humans; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods; D015335:Molecular Probes / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D009369:Neoplasms / Q000378:metabolism*; D044022:Receptor, Endothelin A / Q000378:metabolism*; D012709:Serum Albumin / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D015335:Molecular Probes; D044022:Receptor, Endothelin A; D012709:Serum Albumin
keywords: Albumin; Bioavailability; Drug carrier; Endothelin axis; Molecular imaging; Optical imaging