Imaging doxorubicin and polymer-drug conjugates of doxorubicin-induced cardiotoxicity with bispecific anti-myosin-anti-DTPA antibody and Tc-99m-labeled polymers.

BACKGROUND: Radiolabeled anti-myosin imaging is well-established for imaging doxorubicin-induced cardiotoxicity. However, to enable imaging of drug-induced cardiotoxicity in small experimental animals, pretargeting with bispecific anti-myosin-anti-DTPA-Fab-Fab' and targeting with high-specific radioactivity Tc-99m-DTPA-succinylated-polylysine (DSPL) was developed. METHODS: Mice were injected biweekly with 10 mg/kg Dox or its equivalent as D-Dox-PGA. Tc-99m-DSPL myocardial activity after pretargeting with bsAb-Fab-Fab' was determined after gamma imaging performed at day 7 for Dox-treated mice and day 39 for all others. RESULTS: Mice treated with 10 mg/kg Dox lost 10% total body weight in 1 week and 20% after a second dose. Pretargeted mice treated with 30 mg/kg cumulative D-Dox-PGA dose showed no loss of body weight for the duration of the study. Cardiotoxicity was confirmed by gamma imaging and scintillation counting (1.9 ± 0.25 [mean% ID/g ± SD]) after 1 dose of Dox. Mice injected with 3 × 10 mg/kg Dox equivalent as D-Dox-PGA (0.4 ± 0.04, P < .01) and untreated 2 control groups (0.20 ± 0.05 and 0.19 ± 0.04, P < .01) showed significantly lower myocardial anti-myosin radioactivity relative to the 10 mg/kg Dox group. CONCLUSION: Pretargeting with bsAb-Fab-Fab' and targeting with Tc-99m labeled high-specific activity polymers enabled early visualization of doxorubicin induce cardiotoxicity in mice. Tolerated dose of D-Dox-PGA was greater than to 30 mg/kg Dox-equivalent dose with minimal cardiotoxicity.

Iron overload exacerbates age-associated cardiac hypertrophy in a mouse model of hemochromatosis.

Cardiac damage associated with iron overload is the most common cause of morbidity and mortality in patients with hereditary hemochromatosis, but the precise mechanisms leading to disease progression are largely unexplored. Here we investigated the effects of iron overload and age on cardiac hypertrophy using 1-, 5- and 12-month old Hfe-deficient mice, an animal model of hemochromatosis in humans. Cardiac iron levels increased progressively with age, which was exacerbated in Hfe-deficient mice. The heart/body weight ratios were greater in Hfe-deficient mice at 5- and 12-month old, compared with their age-matched wild-type controls. Cardiac hypertrophy in 12-month old Hfe-deficient mice was consistent with decreased alpha myosin and increased beta myosin heavy chains, suggesting an alpha-to-beta conversion with age. This was accompanied by cardiac fibrosis and up-regulation of NFAT-c2, reflecting increased calcineurin/NFAT signaling in myocyte hypertrophy. Moreover, there was an age-dependent increase in the cardiac isoprostane levels in Hfe-deficient mice, indicating elevated oxidative stress. Also, rats fed high-iron diet demonstrated increased heart-to-body weight ratios, alpha myosin heavy chain and cardiac isoprostane levels, suggesting that iron overload promotes oxidative stress and cardiac hypertrophy. Our findings provide a molecular basis for the progression of age-dependent cardiac stress exacerbated by iron overload hemochromatosis.

An in vitro demonstration of overcoming drug resistance in SKOV3 TR and MCF7 ADR with targeted delivery of polymer pro-drug conjugates.

Drug resistance is a common phenomenon that occurs in cancer chemotherapy. Delivery of chemotherapeutic agents as polymer pro-drug conjugates (PPDCs) pretargeted with bispecific antibodies could circumvent drug resistance in cancer cells. To demonstrate this approach to overcome drug resistance, Paclitaxel (Ptxl)-resistant SKOV3 TR human ovarian- and doxorubicin (Dox)-resistant MCF7 ADR human mammary-carcinoma cell lines were used. Pre-targeting over-expressed biotin or HER2/neu receptors on cancer cells was conducted by biotinylated anti-DTPA or anti-HER2/neu affibody - anti-DTPA Fab bispecific antibody complexes. The targeting PPDCs are either D-Dox-PGA or D-Ptxl-PGA. Cytotoxicity studies demonstrate that the pretargeted approach increases cytotoxicity of Ptxl or Dox in SKOV3 TR or MCF7 ADR resistant cell lines by 5.4 and 27 times, respectively. Epifluorescent microscopy - used to track internalization of D-Dox-PGA and Dox in MCF7 ADR cells - shows that the pretargeted delivery of D-Dox-PGA resulted in a 2- to 4-fold increase in intracellular Dox concentration relative to treatment with free Dox. The mechanism of internalization of PPDCs is consistent with endocytosis. Enhanced drug delivery and intracellular retention following pretargeted delivery of PPDCs resulted in greater tumor cell toxicity in the current in vitro studies.

Design and In Vitro Evaluation of Bispecific Complexes and Drug Conjugates of Anticancer Peptide, LyP-1 in Human Breast Cancer.

PURPOSE: LyP-1, a nine-amino-acid tumor homing peptide, selectively binds to its cognate receptor, p32. Overexpression of p32 in certain tumors should allow use of LyP-1 as a targeting agent for the delivery of therapeutic or diagnostic agents. Peptide conjugates are developed for enhanced pre-targeting of MDA-MB-231 breast cancer cells with peptide-antibody bispecific complexes and targeting with multiple-drug/-fluorophore-conjugated nano-polymers. METHODS: LyP-1-anti-DTPA bispecific antibody complexes (LyP-1-bsAbCx) were generated by conjugation of anti-DTPA antibody and LyP-1. LyP-1-doxorubicin (Dox), Dox-DTPA-succinyl-polylysine (Dox-DSPL), Dox-DSPL-LyP-1, DTPA-Dox-poly glutamic acid (D-Dox-PGA) or DTPA-rhodamine conjugated polylysine (DSPL-RITC) were prepared. In vitro therapeutic efficacy and targeting by immunofluorescence in MDA-MB-231 breast cancer cells were assessed with Dox-LyP-1. Immunofluorescence visualization of cancer cells was evaluated after pretargeting with LyP-1-bsAbCx and targeting with DSPL-RITC. RESULTS: Cytotoxicity of Dox-LyP-1 conjugates was significantly greater than free doxorubicin (p < 0.0001). For fluorescent-labeled LyP-1, internalization occurred in 30 min in tumor cells. Fluorescence intensity of two-step targeted cells showed that pretargeting with LyP-1-bsAbC, followed by targeting with DSPL-RITC was greater than non-pretargeted DSPL-RITC (p < 0.05). CONCLUSIONS: Peptide-conjugates are effective targeting agents for MDA-MB-231 breast cancer cells in culture. LyP-1-bsAbCx and Dox-LyP-1 conjugates may allow development of novel targeted cancer therapy and diagnosis.

Bispecific antibody complex pre-targeting and targeted delivery of polymer drug conjugates for imaging and therapy in dual human mammary cancer xenografts: targeted polymer drug conjugates for cancer diagnosis and therapy.

INTRODUCTION: Doxorubicin, a frontline chemotherapeutic agent, limited by its cardiotoxicity and other tissue toxicities, was conjugated to N-terminal DTPA-modified polyglutamic acid (D-Dox-PGA) to produce polymer pro-drug conjugates. D-Dox-PGA or Tc-99 m labeled DTPA-succinyl-polylysine polymers (DSPL) were targeted to HER2-positive human mammary carcinoma (BT-474) in a double xenografted SCID mouse model also hosting HER2-negative human mammary carcinoma (BT-20). METHODS: After pretargeting with bispecific anti-HER2-affibody-anti-DTPA-Fab complexes (BAAC), anti-DTPA-Fab or only phosphate buffered saline, D-Dox-PGA or Tc-99 m DSPL were administered. Positive therapeutic control mice were injected with Dox alone at maximum tolerated dose (MTD). RESULTS: Only BT-474 lesions were visualized by gamma imaging with Tc-99 m-DSPL; BT-20 lesions were not. Therapeutic efficacy was equivalent in mice pretargeted with BAAC/targeted with D-Dox-PGA to mice treated only with doxorubicin. There was no total body weight (TBW) loss at three times the doxorubicin equivalent MTD with D-Dox-PGA, whereas mice treated with doxorubicin lost 10% of TBW at 2 weeks and 16% after the second MTD injection leading to death of all mice. CONCLUSIONS: Our cancer imaging and pretargeted therapeutic approaches are highly target specific, delivering very high specific activity reagents that may result in the development of a novel theranostic application. HER/2 neu specific affibody-anti-DTPA-Fab bispecific antibody pretargeting of HER2 positive human mammary xenografts enabled exquisite targeting of polymers loaded with radioisotopes for molecular imaging and doxorubicin for effective therapy without the associating non-tumor normal tissue toxicities.

In vitro demonstration of enhanced prostate cancer toxicity: pretargeting with Bombesin bispecific complexes and targeting with polymer-drug-conjugates.

BACKGROUND: Bombesin has been used to target Bombesin receptor, a growth receptor, which is over-expressed in many cancers, including prostate cancer. Polymer-anti-neoplastic-drug-conjugates (PDC) were also developed to reduce non-specific toxicity and increase tumor toxicity utilizing the enhanced permeability and retention effect, benefitting treatment of large tumors with well-established vasculature. PURPOSE: If PDCs were delivered by targeted delivery to cancer cells, tumor toxicity would be enhanced and non-specific toxicity decreased. METHODS: Cardiocyte toxicity was assessed in H9c2 cardiocytes with doxorubicin (Dox) or N-terminal DTPA-modified-Doxorubicin-loaded-polyglutamic acid polymers (D-Dox-PGA). Therapeutic efficacy of targeted D-Dox-PGA after pretargeting with Bombesin-conjugated anti-DTPA-antibody Bispecific Complexes (Bom-BiSpCx) was compared to that of Dox in PC3 cells. Bom-BiSpCx was generated by thioether bond between Bombesin to Anti-DTPA antibody. RESULTS: D-Dox-PGA was demonstrated to have less cardiocyte toxicity (IC50 = 20 µg/ml) than free Dox (1.55 µg/ml, p < 0.001). However, after pre-targeting of human prostate cancer PC3 cells with Bom-BiSpCx and targeting with D-Dox-PGA, IC50 (13.2 µg/ml) was about two times less than that of Dox (28.5 µg/ml, p < 0.0001). DISCUSSION: Targeted delivery of PDCs having lower cardiocyte toxicity enabled higher efficiency cancer cell therapy. CONCLUSION: This study may allow development of very efficient targeted prostate cancer pro-drug therapy.

Imaging small human prostate cancer xenografts after pretargeting with bispecific bombesin-antibody complexes and targeting with high specific radioactivity labeled polymer-drug conjugates.

PURPOSE: Pretargeting with bispecific monoclonal antibodies (bsMAb) for tumor imaging was developed to enhance target to background activity ratios. Visualization of tumors was achieved by the delivery of mono- and divalent radiolabeled haptens. To improve the ability to image tumors with bsMAb, we have combined the pretargeting approach with targeting of high specific activity radiotracer labeled negatively charged polymers. The tumor antigen-specific antibody was replaced with bombesin (Bom), a ligand that binds specifically to the growth receptors that are overexpressed by many tumors including prostate cancer. Bomanti- diethylenetriaminepentaacetic acid (DTPA) bispecific antibody complexes were used to demonstrate pretargeting and imaging of very small human prostate cancer xenografts targeted with high specific activity ¹¹¹In- or 99mTc-labeled negatively charged polymers. METHODS: Bispecific antibody complexes consisting of intact anti-DTPA antibody or Fab' linked to Bom via thioether bonds (Bom-bsCx or Bom-bsFCx, respectively) were used to pretarget PC-3 human prostate cancer xenografts in SCID mice. Negative control mice were pretargeted with Bom or anti-DTPA Ab. 111In-Labeled DTPA-succinyl polylysine (DSPL) was injected intravenously at 24 h (7.03 ± 1.74 or 6.88 ± 1.89 MBq ¹¹¹In-DSPL) after Bom-bsCx or 50 ± 5.34 MBq of 99mTc-DSPL after Bom-bsFCx pretargeting, respectively. Planar or single photon emission computed tomography (SPECT)/CT gamma images were obtained for up to 3 h and only planar images at 24 h. After imaging, all mice were killed and biodistribution of 111In or 99mTc activities were determined by scintillation counting. RESULTS: Both planar and SPECT/CT imaging enabled detection of PC-3 prostate cancer lesions less than 1-2 mm in diameter in 1-3 h post 111In-DSPL injection. No lesions were visualized in Bom or anti-DTPA Ab pretargeted controls. 111In-DSPL activity in Bom-bsCx pretargeted tumors (1.21 ± 0.36 %ID/g) was 5.4 times that in tumors pretargeted with Bom or anti-DTPA alone (0.22 ± 0.08, p = 0.001). PC-3 xenografts pretargeted with Bom-bsFCx and targeted with 99mTc-DSPL were visualizable by 1-3 h. Exquisite tumor uptake at 24 h (6.54 ± 1.58 %ID/g) was about 15 times greater than that of Bom pretargeted controls (0.44 ± 0.17, p = 0.002). CONCLUSION: Pretargeting prostate cancer with Bom-bsCx or Bom-bsFCx enabled fast delivery of high specific radioactivity ¹¹¹In- or 99mTc-labeled polymer-drug conjugates resulting in visualization of lesions smaller than 1- 2 mm in diameter within 3 h.

Palmitoyl ascorbate liposomes and free ascorbic acid: comparison of anticancer therapeutic effects upon parenteral administration.

PURPOSE: To evaluate and compare anticancer therapeutic effect of palmitoyl ascorbate liposomes (PAL) and free ascorbic acid (AA). METHODS: Liposomes incorporating palmitoyl ascorbate (PA) were prepared and evaluated for PA content by HPLC. To elucidate mechanism of action of cell death in vitro, effect of various H(2)O(2) scavengers and metal chelators on PA-mediated cytotoxicity was studied. Effect of various combinations of PAL and free AA on in vitro cytotoxicity was evaluated on 4T1 cells. In vivo, PAL formulation was modified with polyethylene glycol; effect of PEGylation on in vitro cytotoxicity was evaluated. Biodistribution of PEG-PAL formulation was investigated in female Balb/c mice bearing murine mammary carcinoma (4T1 cells). In vivo anticancer activity of PEG-PAL (PEG-PAL equivalent to 20 mg/kg of PA injected intravenously on alternate days) was compared with free AA therapy in same model. RESULTS: PEG-PAL treatment was significantly more effective than free AA treatment in slowing tumor growth. CONCLUSIONS: Nanoparticle formulations incorporating PA can kill cancer cells in vitro. The mechanism of PA cytotoxicity is based on production of extracellular reactive oxygen species and involves intracellular transition metals.

Bispecific antibody complex pre-targeted delivery of polymer-drug conjugates for cancer therapy.

The pretargeting approach using bispecific affibody-antibody complex (BAAC) and targeting of chemotherapeutic drug loaded polymers have been used in breast cancer cell cultures to demonstrate targeted chemotherapy and reduce toxicity to non-pretargeted cancer and cardiac cells. HER2/neu-positive BT-474 and -negative BT-20 human mammary carcinoma cell lines were pretargeted with BAAC and targeted with multi-doxorubicin (Dox) loaded polyglutamic acid (PGA) site specifically modified with diethylene triamine pentaacetic acid (DTPA) (D-Dox-PGA) at the N-terminal of PGA. Toxicity to embryonic cardiocytes and human mammary carcinoma cells were assessed. BAAC was prepared by covalent conjugation of anti-HER2/neu affibody and anti-DTPA Fab via the thioether linkage. N-terminal DTPA modified polyglutamic acid was conjugated with doxorubicin via the amide bonds. Reduction in cardiotoxicity and IC50 of D-Dox-PGA and free Dox were determined in embryonic cardiocyte H9C2 cultures. Enhanced targeted tumor toxicity was demonstrated in BT-474 human mammary carcinoma cell line pretargeting with BAAC followed by targeting with D-Dox-PGA and compared to D-Dox-PGA alone with no pretargeting or free Dox. No enhanced targeted tumor toxicity was observed in HER2/neu negative BT-20 cells. IC50 of D-Dox-PGA and free Dox on embryonic cardiocytes was 15.75 and 1.20 µg/ml, respectively. When BT-474 and BT-20 cells were pretargeted with BAAC followed by targeting with D-Dox-PGA, higher tumor cell-killing was seen only in BT-474 cells. Pretargeting with BAAC resulted in greater tumor cell death in BT-474 human breast cancer cells due to specific targeted delivery of D-Dox-PGA than cancer cells treated with D-Dox-PGA without pretargeting or treatment with free doxorubicin. In vitro targeted delivery of polymer drug conjugate resulted in highly specific, targeted HER2/neu positive BT-474 cancer cell death. Such a pretargeting and targeting approach using prodrug polymers may allow development of very efficient, lower non-target toxicity, and image-guided targeted therapy since these polymers can also be labeled with radioisotopes.

Pretargeted gamma imaging of murine metastatic melanoma lung lesions with bispecific antibody and radiolabeled polymer drug conjugates.

INTRODUCTION: Bispecific monoclonal antibodies (bsMAbs) have been developed as a pretargeting tool to reduce background activity, thereby increasing target to background (T : B) ratios. To enhance visualization of small lesions in vivo, we have used the pretargeting approach of bsMAb and negatively charged polymers radiolabeled with high-specific radioactivity. METHODS: Imaging of metastatic melanoma lesions localized in lung tissues pretargeted with bsMAb and targeted with high-specific radioactivity polymers was carried out. The bsMAb was prepared by covalent conjugation of an antinucleosomal antibody (2C5) recognizing a nucleosomal pan cancer antigen and an anti-diethylene triaminepentaacetic acid antibody (6C31H3) by means of thioether linkage. BsMAb was injected intravenously 10 days after the initiation of the induction of murine melanoma metastasized to the lungs. The next day, 37 MBq 99mTc-diethylene triaminepentaacetic acid-succinylated polylysine were injected intravenously and in-vivo imaging was carried out after the injection. In-vivo and ex-vivo target (T) to background (B) activity ratios were assessed by computer planimetry and biodistribution studies. RESULTS: Lesions were visualized unequivocally in 3 h by gamma scintigraphy. Ex-vivo gamma-scintillation counting corrected for the lesion mass showed that the mean lesion activity was 24.85 ± 13.53 percent injected dose per gram when pretargeted with bsMAb, whereas it was 0.977 ± 0.465 percent injected dose per gram (P<0.01) in the control group injected only with radioactive polymers also corrected similarly. CONCLUSION: The use of bsMAb complexes and 99mTc-diethylene triaminepentaacetic acid-succinylated polylysine enabled early in-vivo visualization of small metastatic melanoma lesions in the lungs.

99mTc-glucarate kinetics differentiate normal, stunned, hibernating, and nonviable myocardium in a perfused rat heart model.

PURPOSE: (99m)Tc-glucarate is an infarct-avid imaging agent. However, patients may have mixtures of normal, irreversibly injured, stunned, and hibernating myocardium. The purposes were to determine (99m)Tc-glucarate uptake and clearance kinetics in these four conditions, and its ability to determine the extent of injury. METHODS: Twenty-two perfused rat hearts were studied: controls (n = 5), stunned (n = 5; 20-min no-flow followed by 5-min reflow), hibernating (n = 6; 120-min low flow at 4 ml/min), and ischemic-reperfused (n = 6; 120-min no-flow followed by reflow). (99m)Tc-glucarate was then infused. Tracer activity was monitored using a NaI scintillation detector and a multichannel analyzer. Creatine kinase, electron microscopy, and triphenyltetrazolium chloride determined viability. RESULTS: (99m)Tc-glucarate 10-min myocardial uptake was significantly greater in ischemic-reperfused (2.50 +/- 0.09) (cpm, SEM) than in control (1.74 +/- 0.07), stunned (1.68 +/- 0.11), and hibernating (1.59 +/- 0.11) (p < 0.05). Tracer retention curves for ischemic-reperfused were elevated at all time points as compared with the other groups. (99m)Tc-glucarate 60-min myocardial uptake was significantly greater in ischemic-reperfused (7.60 +/- 0.63) than in control (1.98 +/- 0.15), stunned (1.79 +/- 0.08), and hibernating (2.33 +/- 0.15) (p < 0.05). The 60-min well-counted tracer activity ratio of ischemic-reperfused to control was 9:1 and corroborated the NaI detector results. Creatine kinase, triphenyltetrazolium chloride, and electron microscopy all demonstrated significantly greater injury in ischemic-reperfused compared to the other groups. An excellent correlation was observed between viability markers and tracer activity (r = 0.99 triphenyltetrazolium chloride; r = 0.90 creatine kinase). CONCLUSION: (99m)Tc-glucarate activity continually and progressively increased in irreversibly injured myocardium. (99m)Tc-glucarate uptake was strongly correlated with myocardial necrosis as determined by three independent assessments of viability. There were minimal and similar (99m)Tc-glucarate uptakes in control, stunned, and hibernating myocardium.

Design of targeted cardiovascular molecular imaging probes.

Molecular imaging relies on the development of sensitive and specific probes coupled with imaging hardware and software to provide information about the molecular status of a disease and its response to therapy, which are important aspects of disease management. As genomic and proteomic information from a variety of cardiovascular diseases becomes available, new cellular and molecular targets will provide an imaging readout of fundamental disease processes. A review of the development and application of several cardiovascular probes is presented here. Strategies for labeling cells with superparamagnetic iron oxide nanoparticles enable monitoring of the delivery of stem cell therapies. Small molecules and biologics (e.g., proteins and antibodies) with high affinity and specificity for cell surface receptors or cellular proteins as well as enzyme substrates or inhibitors may be labeled with single-photon-emitting or positron-emitting isotopes for nuclear molecular imaging applications. Labeling of bispecific antibodies with single-photon-emitting isotopes coupled with a pretargeting strategy may be used to enhance signal accumulation in small lesions. Emerging nanomaterials will provide platforms that have various sizes and structures and that may be used to develop multimeric, multimodal molecular imaging agents to probe one or more targets simultaneously. These platforms may be chemically manipulated to afford molecules with specific targeting and clearance properties. These examples of molecular imaging probes are characteristic of the multidisciplinary nature of the extraction of advanced biochemical information that will enhance diagnostic evaluation and drug development and predict clinical outcomes, fulfilling the promise of personalized medicine and improved patient care.

Targeting very small model lesions pretargeted with bispecific antibody with 99mTc-labeled high-specific radioactivity polymers.

BACKGROUND: Two-step targeting with bispecific antibody and Tc-labeled high-specific radioactivity polymers was used for molecular imaging of two very small model lesions in rats. METHODS AND RESULTS: Sprague-Dawley rats (group I) were injected with surrogate antigen-coated beads (SA beads) in the right hind leg or unmodified beads in the contralateral hind leg. In group II, femoral artery de-endothelialization was induced in the left hind leg and sham operation was performed in the contralateral hind leg. Bispecific antibody Z2D3 F(ab')2-anti-DTPA F(ab')2 was injected intravenously 24 h after SAB injection or 1 week after endothelial denudation. Tc-labeled polymers were injected intravenously 24 h later and gamma-images obtained at 2 and 24 h in group I or approximately 2.5 h in group II. Lesions were visualized by 2 h. In group I, SA beads-specific uptake in muscles was significantly greater than with unmodified beads (P<0.015). In group II, lesions were visualized by 2.5 h after radiopolymer injection with uptake activity 2.1+/-0.6 times greater than in the contralateral side from in-vivo images (P<0.004) and 1.8+/-0.7 times by gamma-scintillation counting (P<0.04). CONCLUSION: Pretargeting with Z2D3 bispecific antibody for the localization of radiolabeled polymers enabled successful in-vivo gamma-imaging of very small lesions in two rat models of extravascular and intravascular targets. Biodistribution data confirmed that pretargeting with bispecific antibody enabled targeted visualization of two different very small model lesions by in-vivo gamma-imaging.

Cytoskeletal-antigen specific immunoliposomes: preservation of myocardial viability.

Pathological conditions such as hypoxia and inflammation can lead to the development of cell membrane-lesions. The presence of these membrane-lesions leads to egress of intracellular macromolecules as well as exposure of intracellular microenvironment to the extracellular milieu resulting in necrotic cell death. An intracellular structure that becomes exposed to the extracellular environment is myosin, a cytoskeletal antigen. We had hypothesized that cell viability can be preserved in nascent necrotic cells if the cell membrane lesions were sealed and the injurious conditions removed. Cell membrane lesion sealing and preservation of cell viability were achieved by the application of Cytoskeletal-antigen Specific ImmunoLiposomes (CSIL) as molecular "Band-Aid" that initially plugs the holes with subsequent sealing of the lesions. Anti-myosin antibody was chosen as the cytoskeleton-antigen specific antibody to develop CSILs, because antimyosin antibody is highly specific for targeting myosin exposed through myocardial cell membrane lesions in various cardiomyopathies. Liposomes are biocompatible lipid bilayer vesicles that have been used in many biological applications for several decades. This chapter will be limited to the description of CSIL therapy to ex vivo studies in adult mammalian hearts. Due to page limitations, cell culture, gene delivery and in vivo studies will not be included. Therapeutic efficacy of CSIL in preservation of myocardial viability as well as function (by left ventricular developed pressure measurements) as assessed in globally ischemic Langendorff instrumented hearts is both dose and time dependent. This approach of cell membrane lesion repair and sealing may have broader applications in other cell systems.

External magnet improves antitumor effect of vinblastine and the suppression of metastasis.

The use of magnetic drug targeting (MDT) to selectively deliver chemotherapeutic drugs to tumor cells is a widely investigated approach; however, the notion of targeting tumor endothelial cells by this method is a fairly new concept. Positively-charged (cationic) liposomes have an extraordinarily high affinity for tumor vessels, but heterogeneous targeting is frequently observed. In order to improve on the overall efficiency of targeting tumor vessels, we investigated the use of an externally applied magnetic field together with magnetic cationic liposomes (MCLs) for cancer treatment. We examined the antitumor effect of the chemotherapeutic agent vinblastine loaded in MCLs, using a murine model of melanoma. Two hours following i.v. administration of MCLs, we observed significant tumor vascular uptake with use of an external magnet (15.9 +/- 6.3%) compared to no magnet (5 +/- 1.3%). The administration of vinblastine-loaded MCLs with the magnet produced a significant antitumor effect, reducing the presence of tumor nodules in preferential sites of metastasis compared to untreated and free drug control groups. CD31 immunostaining revealed a decrease in the general length of tumor blood vessels, altered vascular morphology and interruptions in the tumor vascular lining for the vinblastine-loaded MCL groups. Drug-loaded MCLs with magnetic fields may represent a promising combination approach for cancer treatment.

Myocardial uptake of 99mTc-annexin-V and 111In-antimyosin-antibodies after ischemia-reperfusion in rats.

OBJECTIVES: Phosphatidylserin exposure on cell surfaces occurs early during apoptosis and is detected in vivo by using (99m)Tc-annexin-V (ANX). Cardiomyocyte membrane disruption is detected in vivo by using (111)In-antimyosin-antibodies (AM). We aimed to determine if ANX and AM allow evaluation of the time-course of these two distinct cell death events after myocardial ischemia-reperfusion. METHODS: Coronary tying (20 min) followed by reperfusion (IR) was performed in 31 rats. Twelve of the rats were injected with ANX, 11 with AM, and eight with both tracers. Myocardial uptake of tracers was studied 1-2 h, 4 h, or 24 h after IR by scintigraphy (ANX, n = 14) and autoradiography (all cases), and compared to histology and Apostain staining. RESULTS: Scintigraphy was positive in all rats 2 h after IR and in three of five rats at 24 h. On autoradiography, ANX activity was intense in myocardial lesions as early as 1 h post-IR, whereas AM activity was mild at 2 h then increased at 4 h post-IR. ANX and AM uptakes evolved from mid-myocardium to endocardial and epicardial regions from 2 h to 24 h post-IR. Apostain staining was significant in myocardial lesions (p < 10(6) compared to six sham-operated rats). On histology, myocardial lesion was characterized by interstitial oedema, myocytes necrosis, and dramatic thinning at 24 h. CONCLUSION: These data suggest that ANX and AM allow temporal and regional evaluations of PS exposure and membrane disruption, respectively, during myocytes death after 20-min myocardial ischemia followed by reperfusion. Also, (i) apoptosis starts very early in injured myocardium, (ii) myocyte necrosis occurs later (3-4 h post-reperfusion), and (iii) most dead cells are removed from mid-myocardium between 6 h and 24 h after reperfusion.

Dose-response to cytoskeletal-antigen specific immunoliposome therapy for preservation of myocardial viability and function in langendorff instrumented rat hearts.

BACKGROUND: Treatment with Cytoskeketal-antigen Specific ImmunoLiposomes (CSIL) has resulted in the preservation of cell and organ viability and function. The current study investigates whether CSIL-intervention is dose-dependent in Langendorff instrumented adult rat hearts undergoing global ischemia. METHOD AND RESULTS: Rat hearts undergoing experimental global ischemic insult for 25 minutes were treated with CSIL, IgG-liposomes (IgG-L), plain-liposomes (PL) or placebo. Infarct sizes were assessed by histochemical staining method and quantitated by computer planimetry. Mean infarct size of CSIL treated globally ischemic rat hearts was about 5 times smaller than that of control hearts (P

Cytoskeletal-antigen specific immunoliposome-targeted in vivo preservation of myocardial viability.

Preservation of cell viability using Cytoskeletal-antigen Specific ImmunoLiposomes (antimyosin-CSIL) has been demonstrated in cell cultures. The current study utilized the same CSIL intervention for myocardial protection in an in vivo rabbit model of acute myocardial infarction. Rabbit hearts with experimental left ventricular myocardial infarction were treated with CSIL; control liposomes, [(CL), IgG-liposomes (IgG-L) or plain liposomes (PL)], or vehicle (placebo). Mean myocardial infarct size in rabbit hearts treated in vivo with CSIL was 5 times smaller than in those treated with non-specific CL or vehicle. Treatment of ischemic adult myocardium with CSIL results in significant preservation of myocardial viability by dramatically decreasing the infarct size relative to CL or placebo treatment. Immunohistochemical myocardial preservation of CSIL-treated hearts was confirmed by the lack of contraction band necrosis using histological H&E stains relative to controls. Electrocardiographic confirmation of reduction in myocardial injury after CSIL therapy relative to controls was also observed. Application of CSIL technology to non-cardiac tissues would confirm a broader applicability of this cell membrane lesion sealing technology.

Imaging experimental atherosclerotic lesions in ApoE knockout mice: enhanced targeting with Z2D3-anti-DTPA bispecific antibody and 99mTc-labeled negatively charged polymers.

UNLABELLED: In vivo molecular imaging may be improved if specific radioactivity at the target site could be increased while maintaining low background activity. Bispecific antibody complexes and (99m)Tc-labeled negatively charged chelating polymers that react specifically with the capture arm of the bispecific antibody complex were used to demonstrate the feasibility of imaging very small atherosclerotic lesions in ApoE knockout mice. METHODS: Left femoral artery denudation in ApoE(-/-) mice on a hyperlipidemic diet was used to induce accelerated atherosclerotic lesions. Approximately 40 microg of bispecific antibodies were injected intravenously after 2 wk of endothelial denudation. The next day, approximately 15.0 MBq (99m)Tc-DTPA-succinyl-polylysine (2 microg; DTPA is diethylenetriaminepentaacetic acid) were injected intravenously. RESULTS: In vivo gamma-images showed that lesions were observed unequivocally by 2-3 h. Sham-operated right femoral regions showed no radiotracer accumulation. Ex vivo gamma-scintillation counting corrected for sham-operated nonspecific activity and lesion mass showed that the mean lesion activity was 10.10 +/- 6.76 %ID/g (percentage injected dose per gram), whereas nonspecific human IgG bispecific control (NSB control) also corrected similarly was 0.939 +/- 0.877 %ID/g (P < 0.03). Atherosclerotic lesions were confirmed by immunohistochemical staining. Computer planimetry of immunohistograms showed the mean lesion size to be 2.64 +/- 2.46 mg. CONCLUSION: Use of bispecific antibody complexes and (99m)Tc-DTPA-succinyl-polylysine enabled in vivo visualization of very small atherosclerotic lesions in ApoE knockout mice.

(99m)Tc-glucarate imaging for the early detection of infarct in partially reperfused canine myocardium.

PURPOSE: (99m)Tc-glucarate is an imaging agent developed for the detection of acutely infarcted myocardium. The purposes of the current study were to (1) determine whether (99m)Tc-glucarate can detect acute infarct in the setting of only partial minimal reperfusion, (2) study the persistence and time course of scan positivity following coronary occlusion and intravenous tracer injection, (3) assess the ability of (99m)Tc-glucarate to determine infarct size, and (4) compare these data with previous results obtained using a 100% reperfusion model. METHODS: Six dogs underwent left circumflex (LCx) coronary occlusion for 90 min, followed by 10% epicardial blood flow reperfusion. Fifteen mCi (555 MBq) (99m)Tc-glucarate was injected intravenously 30 min later. Serial gamma camera images were acquired over 240 min. Microsphere blood flow determinations were performed at baseline, during occlusion, during tracer administration, and just before euthanasia. Ex vivo gamma camera images were obtained. Triphenyltetrazolium chloride (TTC) staining was performed to assess infarct size. RESULTS: Qualitatively, (99m)Tc-glucarate images showed a well-defined "hot spot" in all six dogs by 30 min after tracer injection (150 min following coronary occlusion), which persisted for 240 min following tracer administration. Quantitatively, there was a significant increase in the LCx/LAD (left anterior descending) counts ratio beginning 10 min after tracer administration (130 min after occlusion), and continuing to 240 min after tracer administration. Tracer retention was 12.0+/-0.9% for the LAD and 39.0+/-4.1% for the LCx hot spot zone (p<0.05) at 240 min after (99m)Tc-glucarate injection. The correlation coefficient was 0.90 for infarct size by TTC versus (99m)Tc-glucarate. CONCLUSION: In the setting of only partial minimal coronary reperfusion following infarction, (99m)Tc-glucarate myocardial uptake is delayed and less intense compared with the setting of complete reperfusion. Nevertheless, infarcts can still be reliably detected in dogs using qualitative in vivo imaging, and significant abnormalities in quantitative parameters are observed. Thus, (99m)Tc-glucarate imaging may be useful for the clinical detection and relative sizing of acute myocardial infarction, even in the setting of only minimal coronary reperfusion.