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.

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.

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.

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.

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.

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.

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.

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.

Preservation of ischemic myocardial function and integrity with targeted cytoskeleton-specific immunoliposomes.

OBJECTIVES: We sought to demonstrate preservation of myocardial function and integrity after targeted cytoskeleton-specific immunoliposome (CSIL) treatment of globally ischemic Langendorff instrumented hearts and a time response to treatment. BACKGROUND: Cell membrane lesion sealing of hypoxic cardiocytes in culture with CSIL has been reported. METHODS: Langendorff-perfused isolated rat hearts were subjected to global ischemia (25 min). Either CSIL or placebo administration (1-min ischemia) was followed by 30 min of reperfusion. Immunoglobulin G liposomes (IgG-L) or CSIL was also infused at 5, 10, and 20 min of ischemia, reperfused, and then prepared for histochemical staining and electron microscopy. RESULTS: Recovery of left ventricular developed pressure (LVDP) of ischemic hearts treated with CSIL at 1 min of ischemia, assessed at 5 min of reperfusion (98 +/- 14%), was similar to that of sham-operated hearts (100%) but was significantly greater than that of placebo-treated hearts (12 +/- 7%, p = 0.01). The LVDP of hearts treated with CSIL at 5, 10, and 20 min was significantly greater than that with IgG-L at corresponding times (p < 0.03). Histochemical integrity and ultra-structural myocardial integrity were consistent with the functional data. CONCLUSIONS: Preservation of myocardial viability ex vivo was achieved with CSIL therapy. The extent of preservation is proportional to the time of initiation of therapy. Beneficial effects were observed even when CSIL therapy was initiated at 20 min of global ischemia. Therefore, delayed CSIL intervention after the onset of ischemia may augment preservation of myocardial viability during reperfusion therapy.

Smooth muscle cell proliferation index correlates with 111In-labeled antibody Z2D3 uptake in a transplant vasculopathy swine model.

UNLABELLED: Transplant vasculopathy is a major cause of morbidity and mortality in heart transplantation. The proliferation of coronary vascular smooth muscle cells is a hallmark of transplant vasculopathy. The goal of this study was to detect coronary vascular smooth muscle cell proliferation in a swine model by imaging regions of uptake of a monoclonal antibody (Z2D3) labeled with 111In. METHODS: Coronary-to-right carotid artery transplantation was performed in 10 Yucatan minipigs with coronary arteries from farm pigs as donors. In 5 of these experiments, the right carotid artery was also grafted to the left carotid artery as a homograft. In 1 farm pig, the left and right carotid arteries were switched. After 44 +/- 22 days (mean +/- SE), animals were injected with 5-bromo-2-deoxyuridine (BrDU) and 111In-Z2D3 F(ab')2. Approximately 24 h later, the pigs underwent planar and SPECT imaging. After the imaging session, the pigs were sacrificed and the vessels were removed. Ex vivo autoradiography of all grafts was performed. Next, the tissues were immersion fixed, paraffin embedded, sectioned, and stained for histologic or immunohistologic examination. Quantitative morphometry was performed. A smooth muscle cell proliferation index, calculated as (BrDU- and actin-stained cells/actin-stained cells) x 100, was correlated with in vivo and ex vivo radiotracer uptake. RESULTS: Patency or neovascularization was demonstrated in 10 of 10 allografts and 5 of 6 homografts. Ten of the scans were positive for focal tracer uptake in the neck in the area corresponding to the graft site, and 6 were negative. Actin- and BrDU-stained cells were seen in the media of allografts and in the recanalized lumen of occluded homografts. A smooth muscle cell proliferation index of 30 was used as a cutoff for scan positivity, on the basis of previous work. Analysis by the chi2 test indicated significant concordance (P < 0.01). Ex vivo vessel count ratios were significantly correlated with the smooth muscle cell proliferation index (r2 = 0.528, P < 0.01). CONCLUSION: The use of monoclonal antibody Z2D3 tagged with 111In allows the detection of proliferating smooth muscle cells and correlates with the intensity of cell proliferation. This diagnostic method could allow early noninvasive detection of transplant vasculopathy.

Bispecific enzyme-linked signal-enhanced immunoassay with subattomole sensitivity.

A bispecific enzyme-linked signal-enhanced immunoassay (BiELSIA) was developed with markedly increased sensitivity. Antimyosin, the detection antibody, was linked to the signal probespecific antibody. Probes consisted of diethylenetriamine pentaacetic acids attached to polylysine modified with up to seven or eight horseradish peroxidase (HRP) units. Each bispecific antibody bound two polymer probes, providing twice the signal. Using BiELSIA in a competitive inhibition immunoassay format with an average of 1.5, 3, 4.5, 6, and 7.5 HRP units per polymer-probe, the sensitivity of standard enzyme-linked immunosorbent assay (10(13) mole) was increased to 10(15), 10(18), 10(19), 10(20), and 10(-21) mol (< or = 1,000 molecules), respectively. BiELSIA detected cardiac myosin heavy chain fragments in sera of patients obtained at the time of emergency department admission for acute myocardial infarction, but not in normal sera. This technology should be applicable for detection of cancer, human immunodeficiency virus, prion, and other antigens that are present in concentrations too low for detection by current immunoassays.

Uptake of 111In-Z2D3 on SPECT imaging in a swine model of coronary stent restenosis correlated with cell proliferation.

UNLABELLED: Small targets such as cell proliferation in the coronary arteries may potentially be detected with single-photon imaging using high-radiotracer-specific activity. We hypothesized that an antibody linked to polymers to increase specific radioactivity can be visualized on SPECT images and that counts in the target will correlate with the strength of the biologic signal. METHODS: Twenty-four stents were placed using the balloon overexpansion technique in the coronary arteries of 14 juvenile domestic swine. One week later, the animals received 74 MBq of (111)In-diethylenetriaminepentaacetic acid-polylysine Z2D3-F(ab')(2), and SPECT imaging was performed at 24 h. The coronary vessels were removed, and the stented vessels were processed with plastic embedding and sectioning. Medial and neointimal areas, percentage of vessel stenosis, and cell proliferation indices were quantified using a 5-bromo-2-deoxyuridine (BrdU) labeling index. Reconstructed SPECT images were interpreted for tracer uptake in coronary vessels. RESULTS: Sixteen of the vessels were positive on SPECT imaging and 10 were negative. The percentage injected dose was 0.85 +/- 0.28 x 10(-3) in scan-positive vessels and 0.34 +/- 0.11 x 10(-3) in scan-negative vessels (P < 0.001). The medial-plus-neointimal proliferative index was 42 +/- 11 in scan-positive vessels and 11 +/- 11 in scan-negative vessels (P < 0.0001). The percentage stenoses were 21% +/- 22% versus 19% +/- 15% (not statistically significant). When individual values for the stented-to-control vessel counts were plotted against BrdU labeling index, a significant relationship was found (r(2) = 0.441; P = 0.0014). CONCLUSION: These data indicate that small targets relevant to human coronary vascular disease may be detected using polymer-modified radiolabeled antibodies.

Early detection of infarct in reperfused canine myocardium using 99mTc-glucarate.

UNLABELLED: 99mTc-Glucarate is an infarct-avid imaging agent with the potential for very early detection of myocardial infarction. The purposes of this study using a canine model were to determine (a) the time course of (99m)Tc-glucarate uptake and clearance from necrotic and normal myocardium; (b) the (99m)Tc-glucarate necrotic-to-normal activity ratio over time; (c) the time course of detectable scan positivity after intravenous administration of the tracer; and (d) the relationship of infarct size determined by triphenyltetrazolium chloride (TTC) staining versus (99m)Tc-glucarate imaging ex vivo. METHODS: A 90-min left circumflex coronary artery (LCx) occlusion was followed by 270 min of reperfusion at 100% baseline flow in 6 open-chest, anesthetized dogs. (99m)Tc-Glucarate (555 MBq [15 mCi]) was injected 30 min after reperfusion and was followed by 240 min of gamma-camera serial imaging. Microspheres were injected during baseline, occlusion, tracer injection, and before the dogs were euthanized. Creatine kinase assays were performed to assess developing injury. Ex vivo gamma-camera imaging was performed. Blood flow and tracer activity were determined by well counting. TTC stain was used to mark infarct areas, which were sized using computerized digital planimetry. RESULTS: Hemodynamics demonstrated no significant change from baseline at any time for any parameter except LCx flow, which was significantly depressed during occlusion. The mean infarct size +/- SEM was 10.7% +/- 2% of total left ventricle. Blood (99m)Tc-glucarate clearance was triexponential and rapid. Qualitative image analysis revealed a well defined hot spot after 30 min, which remained well defined through 240 min after injection (150 and 360 min after occlusion, respectively). Images were quantitatively abnormal with hot spot-to-normal zone activity ratios of >/=2:1 within 10 min of tracer administration (130 min after occlusion), reaching 8:1 at 240 min after tracer administration (360 min after occlusion). There was a linear correlation between infarct size determined by (99m)Tc-glucarate and TTC staining (r = 0.96; slope = 0.87). CONCLUSION: (99m)Tc-Glucarate marks acute myocardial infarct very early after occlusion and appears to accurately assess infarct size when compared with TTC staining.

ATP-containing immunoliposomes specific for cardiac myosin.

The application of ATP-loaded liposomes has been shown effective against ischemic damage in several tissues. In this study, we have prepared ATP-containing liposomes capable of specific recognition of component (myosin) specific for ischemic myocardium. ATP-containing immunoliposomes specific towards cardiac myosin were obtained by the attachment of the monoclonal anti-cardiac myosin 2G4 antibody to the surface of ATP-containing PEGylated liposomes prepared by the freezing-thawing method. Since intracellular myosin is exposed only in the areas containing ischemically compromised cells with damaged plasmic membranes, such liposomes are expected to target these areas both in vitro and in vivo. The attachment of the antibody did not provoke their ATP release from the liposomes and only minimally influenced liposome size and size distribution. Liposome-attached anti-myosin 2G4 antibody preserved its specific activity; and anti-myosin antibody-bearing, ATP-loaded liposomes bound efficiently to the monolayer of myosin in ELISA. The preparation of myosin-specific ATP-loaded immunoliposomes represented an important step in the development of targeted delivery systems capable of providing energy support to ischemic myocardium in vivo.

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.

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.