Naseem Nossiff

Targeted accumulation of polyethylene glycol-coated immunoliposomes in infarcted rabbit myocardium.

The less than optimal accumulation of immunoliposome‐associated reagents at target sites has often been attributed to the rapid in vivo clearance of immunoliposomes from the blood. In an attempt to overcome the drawback of rapid clearance and use the targeting potential of immunoliposomes, we have prepared long‐circulating, 111In‐labeled immunoliposomes. Targeting properties and enhanced circulation times were demonstrated in a rabbit model of acute experimental myocardial infarct. The specificity of liposomes for newly exposed intracellular cardiac myosin at the necrotic sites was achieved by incorporating monoclonal antimyosin antibody. Extended circulation times were achieved by cocoating the antimyosin‐liposomes with polyethylene glycol (PEG). The half‐life of the immunoliposomes was 40 min, which increased to 200 min with 4% mol PEG and to approximately 1000 min with 10% mol PEG. The degree of binding of modified immunoliposomes at the target sites was also dependent on the concentration of PEG incorporated at the liposome surface. This study demonstrates the accumulation of long‐circulating targeted liposomes at the area of acute rabbit experimental myocardial infarction.— Torchilin, V. P.; Klibanov, A. L.; Huang, L.; ODonnell, S.; Nossiff, N. D.; Khaw, B. A. Targeted accumulation of polyethylene glycolcoated immunoliposomes in infarcted rabbit myocardium. FASEB J. 6: 2716‐2719; 1992.

Colloidal magnetic resonance contrast agents: effect of particle surface on biodistribution

Abstract Surface structure of dextran-coated superparamagnetic particles is studied as a biodistribution-defining factor. Colloidal particle and polymer molecule of the same size and similar outer structure are shown to possess similar biokinetics and biodistribution. A dense dextran layer on the particle surface is identified as a structure responsible for particle accumulation in the lymph nodes.

Mion-ASF: Biokinetics of an MR receptor agent

Receptor-directed MR contrast agents are currently being designed to improve sensitivity and specificity of MR imaging and to provide for functional MR imaging. In the current study we have synthesized a conjugate of asialofetuin (ASF), a bovine plasma protein with a known, high affinity for the hepatic asialoglycoprotein receptor, and a well defined, single crystal superparamagnetic label (monocrystalline iron oxide nanoparticle, MION). MION-ASF is cleared from the circulation more than 300 times faster than MION, has a 3.7 times higher hepatic accumulation, increases liver R2 relaxivity 2.8-fold compared to MION, and accumulates in hepatocytes unlike MION, which accumulates only in macrophages. Competition assays indicate that receptor-mediated hepatocyte uptake can be competitively blocked and that this effect can be demonstrated by imaging. These studies indicate that sensitive iron oxide based probes can be developed for functional MR imaging.

MR Imaging of Slow Axonal Transport in Vivo

Three magnetopharmaceuticals based on a monocrystalline iron oxide nanocompound (MION) are evaluated as potential contrast agents for demonstrating axonal transport in vivo by magnetic resonance (MR) imaging. One agent has a strong positive charge, one has a strong negative charge, and the third is covalently linked to wheat germ agglutinin, a plant lectin with a high affinity for axon terminals. All three agents were tagged with rhodamine, and fluorescence microscopy was used to determine their fate after administration and to validate the imaging results. Following injection into or near various neural structures in the motor and visual systems of rats, MR images were obtained at multiple times up to 11 days later, and the imaged tissues were processed for subsequent histological examination. Similar results were obtained with all three agents. Axonal transport was not seen by MR imaging or fluorescence microscopy when the agents were injected into the calf muscles, the vitreous of the eye, or the superior colliculus. However, bidirectional axonal transport was shown unequivocally by both methods after injection directly into the site of a focal crush injury to the sciatic nerve. The nerve, which otherwise is isointense with surrounding tissues on MR images, appeared as a uniformly hypointense structure having a length approximately in proportion to the time from injection to imaging. By 11 days, the course of the nerve was traceable from its component roots in the cauda equina to its bifurcation into the tibial and common peroneal nerves in the leg. A transport rate of about 5 mm/day was calculated, which is consistent with the mechanism of slow transport. MION-based magnetopharmaceuticals thus can be used to demonstrate slow axonal transport, and thereby visualize peripheral nerves, in vivo by MR imaging.

Modification of monoclonal antimyosin antibody: Enhanced specificity of localization and scintigraphic visualization in acute experimental myocardial infarction

Positively charged polymers have been shown to interact nonselectively with cells in vitro by means of an electrostatic binding to a negatively charged cell surface. We reasoned that, if a net negative charge could be introduced onto an antibody molecule, some of the nonspecific antibody interactions with cells could be avoided without affecting the function of the antibody combining site. An important result would be improved target-to-background ratios should such antibodies be used as in vivo imaging agents. To test this hypothesis, we first reacted bifunctional chelators such as diethylenetriamine pentaacetic acid with cationic polylysine polymers to permit radiolabeling with 111In, then rendered the polymer completely anionic by reacting the residual epsilon amino groups with succinic anhydride. These modified polymers were then covalently linked either to monoclonal antimyosin antibody or to its Fab fragment by means of a water soluble carbodiimide. The immunoreactivity of the antibody-polymer conjugates was not significantly diminished. 111In-labeled antimyosin Fab modified with succinylated polylysine permitted visualization of experimental myocardial infarcts as early as 30 min after intravenous injection. An inverse exponential relationship was observed between the distribution of 201Tl and that of polymer-modified antimyosin Fab. 111In-labeled succinylated polymer administered by itself did not localize in the infarct. These observations suggest that anionically modified antibodies may enhance the specificity of antibody imaging.

In vivo targeting of acute myocardial infarction with negative-charge, polymer-modified antimyosin antibody: Use of different cross-linkers

BackgroundCell surfaces and intercellular matrixes contain acidic residues, making them negatively charged. Antibodies are basic, positively charged glycoproteins. Therefore the potential for nonspecific ionic interaction exists, which could increase the background activity. Modification of antibodies with negatively charge-modified polymers have been shown to reduce this nonspecific background activity. This study was performed to investigate the appropriateness of different cross-linkers used covalently to link the chelating negatively charge-modified polylysine to antimyosin Fab (AM-Fab). The cross-linking was performed through peptide (AM-I) or thioether (AM-II) bonds. The in vitro evaluation of the immunointegrity and the in vivo assessment were performed to investigate the potential for reduction of nontarget background activity. Furthermore, the role of the charge of the polymers (whether completely negatively charge modified by succinylation [AM-IIs] or only partially negatively charge modified [AM-IIns]) was also assessed.Methods and ResultsAll polymer-modified preparations (AM-I, AM-IIs, and AM-IIns) retained the immunoreactivities relative to the unmodified or conventional diethylenetriaminepentaacetic acid-coupled AM-Fab as assessed by radioimmunoassay or enzyme-linked immunosorbent assay. These polymer-modified preparations labeled with 111In were assessed in 13 rabbits with acute experimental myocardial infarction. Acute infarcts were produced by 40 minutes of left anterior descending coronary artery occlusion followed by reperfusion. At between 10 and 30 minutes of reperfusion, 10.4±1.8 mBq 111In-AM-I (10 to 20 μg; n=7) or 11.4±2.3 mBq 111In-AM-II (n or ns) (20 to 25 μg; n=6) was administered intravenously. Gamma imaging was performed in the left lateral position and arterial blood samples were withdrawn serially for the next 3 hours. At the end of the final imaging session, AM-I uptake was determined to be 1.09%±0.11% (mean percent injected dose per gram myocardium ± SEM) in 20 infarcted myocardial segments from seven rabbits, compared with 0.031%±0.003% in 20 normal myocardial segments (infarct/normal myocardial ratio 53.9±18.41). The mean percent injected dose of 111In-labeled thioether-linked AM-Fab preparations in nine infarcted myocardial segments from each group was 0.067%±0.008% (infarct/normal myocardial ratio 9.0±1.5) and 0.144%±0.011% (infarct/normal myocardial ratio 10.2±1.9) with AM-IIs (n=3) and AM-IIns (n=3), respectively (p<0.0001). The non-target organ distribution of the AM-I and AM-IIs was similar. AM-IIns preparation resulted in high non-target organ activities.ConclusionThis study shows that the charge of the antibody can be manipulated favorably by cross-linking with negatively charged polymers, which results in the reduced in vivo non-target organ activities. Charge modification does not adversely affect the apparent affinity of the antibody. However, the type of cross-linkers used may significantly influence the in vivo stability of the modified antibody preparations for target organ visualization. These data may find potential application in future clinical imaging protocols.

A DRUG SYSTEM (PDH) FOR INTERVENTIONAL RADIOLOGY : SYNTHESIS, PROPERTIES, AND EFFICACY

RATIONALE AND OBJECTIVESnThe authors synthesized and tested a novel hydrogel system proposed for use in extra- and intravascular radiologic interventions, such as chemoembolizations and embolizations, and as a vehicle for sustained drug release.nnnMATERIALSnThe material was specifically designed to meet the prerequisites of biodegradation, biocompatibility, low immunogenicity, low toxicity, and easy use. The material consists of a protein backbone cross-linked with activated bifunctional polyethyleneglycol (PEG) derivatives (PEG-derivatized hydrogel, [PDH]) to which are attached therapeutic (e.g., doxorubicin, a chemotherapeutic agent = PDH-dx) or diagnostic labels (e.g. Gd-DTPA).nnnRESULTSnPDH-dx effectively reduced the risk of local tumor recurrence in a rat model when implanted locally after surgical tumor removal. After administration, PDH is degraded by proteases release from macrophages; implantations of 1 mL samples into paraspinal muscles of rats were completely absorbed within 4 weeks and its constituents were metabolized. Antibody titers (total Ig response) against the PDH were not detectable 1 week after implantation, whereas protein control substances elicited a strong response.nnnCONCLUSIONSnPDH and its derivatives are relatively nontoxic, biodegradable materials for use in radiologic interventions and as a vehicle for sustained drug release.

Correlation of immunoreactivity and polymer formation to DTPA modification of a monoclonal antibody

The protocol used for coupling of monoclonal antibodies with mixed anhydride of DTPA for subsequent radiolabeling with indium-111 affects the integrity of the immunoreactivity of the antibody preparations. To analyze the effect of minor methodological variations on coupling characteristics, a two-step addition of DTPA to antimyosin antibody with gentle mixing was compared to a single addition with vigorous stirring. The molar ratios of DTPA to antibody were also varied. The polymer formation was assessed by SDS-PAGE and immunoreactivity was assessed by solid phase radioimmunoassay using human heart myosin as the antigen. The immunoreactivity was significantly decreased in the two-step, gentle-mixing method where polymer formation was evident. The one-step, vigorous-stirring method of DTPA incorporation produced no polymerization and no loss of immunoreactivity.