Bioequivalence decision for nanoparticular iron complex drugs for parenteral administration based on their disposition.

Although parenteral iron products have been established to medicinal use decades before, their structure and pharmacokinetic properties are not fully characterized yet. With its' second reflection paper on intravenous iron-based nano-colloidal products (EMA/CHMP/SWP/620008/2012) the European Medicine Agency provided an extensive catalogue of methods for quality, non-clinical and pharmacokinetic studies for the comparison of nano-sized iron products to an originator (EMA, 2015). For iron distribution studies, the reflection paper assumed the use of rodents. In our tests, we used a turkey fetus model to investigate time dependent tissue concentrations in pharmacological and toxicological relevant tissues liver, heart and kidney. We found turkey embryos to be a suitable alternative to rodents with high discriminatory sensitivity. Clear differences were found between equimolar doses of iron products with hydroxyethyl amylopectin, sucrose, dextrane and carboxymaltose shell. A linear dose dependency for the tissue accumulation was also demonstrated.

Imaging of isoproterenol-induced myocardial injury with 18F labeled fluoroglucaric acid in a rat model.

Positron emission tomography (PET) of myocardial infarction (MI) by infarct avid imaging has the potential to reduce the time to diagnosis and improve diagnostic accuracy. The objective of this work was to synthesize 18F-labeled glucaric acid (FGA) for PET imaging of isoproterenol-induced cardiomyopathy in a rat model. METHODS: We synthesized 18F-FGA by controlled oxidation of 18F-fluorodeoxy glucose (FDG), mediated by 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) in presence of NaBr and NaOCl in highly-buffered reaction conditions. After ascertaining preferential uptake of 18F-FGA in necrotic as compared to normal H9c2 myoblasts, the biodistribution and circulation kinetics of 18F-FGA was assessed in mice. Moreover, the potential of 18F-FGA to image myocardial damage was investigated in a rat model of isoproterenol-induced cardiomyopathy. Isoproterenol-induced myocardial injury was verified at necropsy by tissue staining and plasma cardiac troponin levels. RESULTS: Synthesis of radiochemically pure 18F-FGA was accomplished by a 5 min, one step oxidation of 18F-FDG. Reaction yield was quantitative and no side-products were detected. Biodistribution studies showed rapid elimination from the body (ke = 0.83 h-1); the major organ of 18F-FGA accumulation was kidney. In the rat model, isoproterenol-treatment resulted in significant increase in cardiac troponin. PET images showed that the hearts of isoproterenol-treated rats accumulated significant amounts of 18F-FGA, whereas healthy hearts showed negligible uptake of 18F-FGA. Target-to-nontarget contrast for 18F-FGA accumulation became significantly more pronounced in 4 h images as compared to images acquired 1 h post-injection. CONCLUSION: 18F-FGA can be easily and quantitatively synthesized from ubiquitously available 18F-FDG as a precursor. The resultant 18F-FGA has a potential to serve as an infarct-avid agent for PET imaging of MI. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: 18F-FGA/PET will complement existing perfusion imaging protocols in therapeutic decision making, determination of revascularization candidacy and success, differentiation of ischemia from necrosis in MI, discrimination of myocarditis from infarction, and surveillance of heart transplant rejection.

Iron sucrose: assessing the similarity between the originator drug and its intended copies.

Iron sucrose (IS) is a complex nanocolloidal intravenous suspension used in the treatment of iron-deficiency anemia. Follow-on IS products (iron sucrose similars (ISSs)) have obtained marketing authorization by the generic pathway, implying that identical copies of IS may be manufactured. However, recent prospective and retrospective clinical studies showed discrepancies in clinical outcomes, which might be related to differences in physicochemical properties. The aim of this work is to measure and compare the physicochemical properties of IS and three ISSs available in the market using innovative analytical procedures. The comprehensive elucidation of size, size distribution, morphology, and stability of these complex drugs revealed very significant differences between the products. This study serves to provide the basis to define critical quality attributes that may be linked to differences in clinical outcome and thus may contribute to an adequate regulatory approach for IS and its follow-on products.

Complexity of intravenous iron nanoparticle formulations: implications for bioequivalence evaluation.

Intravenous iron formulations are a class of complex drugs that are commonly used to treat a wide variety of disease states associated with iron deficiency and anemia. Venofer® (iron-sucrose) is one of the most frequently used formulations, with more than 90% of dialysis patients in the United States receiving this formulation. Emerging data from global markets outside the United States, where many iron-sucrose similars or copies are available, have shown that these formulations may have safety and efficacy profiles that differ from the reference listed drug. This may be attributable to uncharacterized differences in physicochemical characteristics and/or differences in labile iron release. As bioequivalence evaluation guidance evolves, clinicians should be educated on these potential clinical issues before a switch to the generic formulation is made in the clinical setting.

Investigations of (99m)Tc-labeled glucarate as a SPECT radiotracer for non-small cell lung cancer (NSCLC) and potential tumor uptake mechanism.

This study attempted to evaluate the feasibility of (99m)Tc-labeled glucarate ((99m)Tc-GLA) imaging in non-small cell lung cancer (NSCLC) and the potential tumor uptake mechanism. Cell lysates from two NSCLC cell lines, H292 and H1975, were immunoblotted with anti-glucose transporter 5 (GLUT5) antibody for Western blotting. Thereafter, the two cell lines were used to examine cellular uptake of (99m)Tc-GLA with or without fructose. SPECT/CT imaging studies were performed on small animals bearing H292 and H1975 tumors. Biodistribution studies were also conducted to achieve accurate tissue uptake of this tracer in two tumor models. Hematoxylin & eosin (H&E) staining and GLUT5, Ki67 and cytokeratin-7 (CK-7) immunohistochemistry (IHC) analysis were further investigated on tumor tissues. In Western blotting, H292 cells showed higher levels of GLUT5 compared to the H1975 cells. Meanwhile, the in vitro cell assays indicated GLUT5-dependent uptake of (99m)Tc-GLA in H292 and H1975 cells. The fructose competition assays showed a significant decrease in (99m)Tc-GLA uptake by H292 and H1975 cells when fructose was added. The (99m)Tc-GLA accumulation was as much as two-fold higher in H292 implanted tumors than in H1975 implanted tumors. (99m)Tc-GLA exhibited rapid clearance pharmacokinetics and reasonable uptake in human NSCLC H292 (1.69±0.37 ID%/g) and H1975 (0.89±0.06 ID%/g) implanted tumors at 30min post injection. Finally, the expression of GLUT5, Ki67 and CK-7 on tumor tissues also exhibited positive correlation with the in vitro cell test results and in vivo SPECT/CT imaging results in xenograft tumors. Both in vitro and ex vivo studies demonstrated that the uptake of (99m)Tc-GLA in NSCLC is highly related to GLUT5 expression. Imaging and further IHC results support that (99m)Tc-GLA could be a promising SPECT imaging agent for NSCLC diagnosis and prognosis evaluation.

Comparative study of the oral absorption of microencapsulated ferric saccharate and ferrous sulfate in humans.

PURPOSE: Our objective was to compare the absorption of microencapsulated ferric saccharate (MFS) and ferrous sulfate (FS) in a fortified milk product, using a crossover design. METHODS: Seventeen non-iron-deficient healthy adults from both sexes participated in the study. On each intervention day (days 1 and 8), after an overnight fast, the volunteers consumed one type of product (test or control) and blood sampling was carried out at different times. The interventions days were separated by 7-day washout periods. This study was double blinded, crossover and randomized for nature of the test meals. The primary outcomes of the study were total serum iron and transferrin saturation. RESULTS: No significant differences could be observed in serum iron concentration during the 6-h postprandial study due to the type of milk product consumed, and there was neither an effect of time nor an interaction between the type of milk product and time. Transferrin saturation significantly increased after the intake of both products (P < 0.005), reaching a peak value between hours 2 and 4. No significant differences were detected between MFS and FS, indicating that iron absorption from MFS is equivalent to absorption from FS. CONCLUSIONS: MFS is a new ingredient that allows the fortification of a wide range of food products, including heat-processed and non-acidic products with similar absorption to FS, designed to produce neither organoleptic changes nor off-color development during storage of fortified food.

Hydroxyurea could be a good clinically relevant iron chelator.

Our previous study showed a reduction in serum ferritin of ß-thalassemia patients on hydroxyurea therapy. Here we aimed to evaluate the efficacy of hydroxyurea alone and in combination with most widely used iron chelators like deferiprone and deferasirox for reducing iron from experimentally iron overloaded mice. 70 BALB/c mice received intraperitonial injections of iron-sucrose. The mice were then divided into 8 groups and were orally given hydroxyurea, deferiprone or deferasirox alone and their combinations for 4 months. CBC, serum-ferritin, TBARS, sTfr and hepcidin were evaluated before and after iron overload and subsequently after 4 months of drug therapy. All animals were then killed. Iron staining of the heart and liver tissue was done using Perl's Prussian Blue stain. Dry weight of iron in the heart and liver was determined by atomic absorption spectrometry. Increased serum-ferritin, TBARS, hepcidin and dry weight of iron in the liver and heart showed a significant reduction in groups treated with iron chelators with maximum reduction in the group treated with a combination of deferiprone, deferasirox and hydroxyurea. Thus hydroxyurea proves its role in reducing iron from iron overloaded mice. The iron chelating effect of these drugs can also be increased if given in combination.

Biodistribution and predictive hepatic gene expression of intravenous iron sucrose.

INTRODUCTION: We have examined iron biodistribution and hepatic gene expression in rats following administration of the generic Iron Sucrose Azad (ISA) or the reference iron sucrose drug Venofer®. METHODS: ISA and Venofer® were administered intravenously to normal, non-anemic, male rats at 15 mg/kg (a supra-therapeutic dose-level). To evaluate biodistribution, tissue iron levels were determined over 28 days for plasma, liver, spleen, bone marrow, heart, kidney, lung and stomach using a validated ICP-MS method. Hepatic gene expression was evaluated by microarray analysis of mRNA from samples taken 24 h after drug administration. RESULTS: Iron concentration/time profiles for plasma and tissues were quantitatively similar for ISA and Venofer. Following administration, circulating iron levels briefly exceeded transferrin binding capacity and there was a transient increase in hepatic iron. Bone marrow iron levels remained elevated throughout the study. No increases in tissue iron levels were observed in the heart, stomach or lungs. Spleen iron levels increased over the course of the study in treated and control rats. Small, transient increases were recorded in the kidneys of treated rats. The effects of ISA and Venofer® on hepatic gene transcription were similar. Principal components analysis showed that there was no systematic effect of either treatment on transcriptional profiles. Only a small number of genes showed significant modulation of expression. No transcriptional pattern matches with toxicity pathways were found in the ToxFX database for either treatment. No modulation of key genes in apoptosis, inflammation or oxidative stress pathways was detected. DISCUSSION: These findings demonstrated that the biodistribution of administered iron is essentially similar for Iron Sucrose Azad and Venofer®, that iron sucrose partitions predominantly into the liver, spleen and bone marrow, and that hepatic gene expression studies did not provide any evidence of toxicity in animals treated at a supra-therapeutic dose-level.

Nanoparticle iron medicinal products - Requirements for approval of intended copies of non-biological complex drugs (NBCD) and the importance of clinical comparative studies.

Currently, most countries apply the standard generic approach for the approval of intended copies of originator nanoparticle iron medicinal products, requiring only demonstration of bioequivalence to a reference medicinal product by bioavailability studies. However, growing evidence suggests that this regulatory approach is not appropriate. Clinical and non-clinical studies have shown that intended copy preparations of nanoparticle iron medicinal products can differ substantially from the originator product in their efficacy and potentially in their safety profile. An adapted regulatory pathway (separate from the standard generic approach) with defined data requirements is needed for approval of intended copies of iron medicinal products. Here, we discuss the difficulties involved in assessing therapeutic equivalence of nanoparticle iron medicinal products and suggest key concepts of a regulatory approach. Standardized non-clinical comparative studies are necessary but, as demonstrated in the reported clinical data, they may not be sufficient to demonstrate a comparable efficacy and safety profile. Validated, prospective, comparative clinical studies might be needed, in addition to non-clinical studies, in order to enable appropriate assessment of therapeutic equivalence. Furthermore, including brand names in addition to the International Non-proprietary Names (INNs) in safety reports could enable effective safety monitoring of intended copies and originator products.

Evaluation of 99mTc-glucarate as a breast cancer imaging agent in a xenograft animal model.

INTRODUCTION: The use of [(99m)Tc]glucarate has been reported as an infarct-avid agent with the potential for very early detection of myocardial infarction. [(99m)Tc]Glucarate has also been postulated as an agent for non-invasive detection of tumors. The aim of our study was to develop a Glucarate kit and evaluate [(99m)Tc]glucarate as a potential cancer imaging agent in female SCID mice bearing human MDA-MB-435 breast tumors. METHODS: Glucarate in a kit formulation was labeled with (99m)Tc and evaluated for radiolabelling efficiency and radiochemical purity. The Glucarate kit stability was assessed by monthly quality controls. The pharmacokinetics of [(99m)Tc]glucarate were determined in female SCID mice bearing MDA-MB-435 human breast carcinoma tumors at 0.5, 1, 2, 4 and 24 h. Nuclear imaging studies were performed with a micro-single photon emission tomography (SPECT)/computed tomography (CT) system at 2 h post injection, while magnetic resonance imaging (MRI) was employed for tumor morphology analysis and metastatic deposit localization. RESULTS: The Glucarate kits exhibited a stable shelf life of 6 months. [(99m)Tc]Glucarate was obtained with radiochemical purity greater than 95%. Biodistribution studies demonstrated moderate tumor uptake coupled with high renal clearance. Tumor-to-muscle ratios were 4.85 and 5.14 at 1 and 4 h post injection. MRI analysis showed tumors with dense cellular growth and moderate central necrosis. [(99m)Tc]Glucarate uptake in the primary MDA-MB-435 shoulder tumors and metastatic lesions were clearly visualized with micro-SPECT/CT imaging. CONCLUSIONS: Selective tumor uptake and rapid clearance from nontarget organs makes [(99m)Tc]glucarate a potential agent for breast cancer imaging that awaits validation in a clinical trial.

(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.

MRP-1 protein expression and glutathione content of in vitro tumor cell lines derived from human glioma carcinoma U-87-MG do not interact with 99mTc-glucarate uptake.

The main cause of the multidrug resistance (MDR) of glioma cells is the overexpression of MRP-1, often associated with high levels of glutathione (GSH). We investigated whether MRP-1-related GSH content can influence (99m)Tc-glucarate entry by comparing its uptake with that of (99m)Tc-sestamibi (MIBI), an MRP- 1 probe, in an in vitro model of a sensitive cell line (U-87-MG) and a resistant derived cell line expressing MRP-1 (U-87-MG-R). Drug resistance was assessed by immunoblotting, GSH measurement, and Alamar Blue assay. To correlate MDR phenotype with tracer accumulation, uptakes were performed with and without modulators and after GSH depletion. Similar accumulation of (99m)Tc-glucarate was observed in the two cell lines, and the use of MDR reversals did not enhance its uptake. Our results clearly demonstrate that (99m)Tc-glucarate uptake is not related to MRP-1 expression or GSH levels. In contrast, (99m)Tc- MIBI accumulation is inversely proportional to the cell MDR phenotype. The combination of (99m)Tc-glucarate and (99m)Tc-MIBI may be a useful tool for the noninvasive detection of malignant sites and their chemoresistance status.

99mTc glucarate high-resolution imaging of drug sensitive and drug resistant human breast cancer xenografts in SCID mice.

BACKGROUND AND AIM: Previous studies have showed that 99mTc labelled glucarate (GLA) might be an agent for non-invasive detection of breast tumours. In xenografted BT20 breast tumours, GLA was found to have higher uptake than 99mTc sestamibi (MIBI). It is unclear whether GLA can localize in all cell line breast cancer xenografts, as well as breast tumours with multidrug resistance (MDR). The present study aimed to investigate the properties of GLA in detecting drug sensitive and drug resistant MCF7 breast cancer xenografts in mice by using dynamic single photon emission computed tomography (SPECT) imaging. METHODS: MCF7/S cells are drug sensitive breast carcinoma cells. MCF7/D40 cells are 40-fold more resistant to doxorubicin compared to MCF7/S. Subcutaneous tumours were grown in SCID mice for 10-14 days after injection of 1 x 10(6) cells into the right thigh. Anaesthetized mice with MCF7/S (MIBI, n=9; GLA, n=8) and MCF7/D40 (MIBI, n=6; GLA, n=5) tumours were imaged using a high-resolution SPECT system called FASTSPECT. Dynamic images were acquired for 2 h after intravenous injection of GLA or MIBI. Expression of MDR P-glycoprotein (Pgp) in the tumours was demonstrated in the MCF7/D40 tumours by western blotting, not in the MCF7/S tumours. RESULTS: The xenografted tumours were visualized unequivocally within 10-30 min in GLA images and remained detectable for at least 2 h after injection. Drug resistant tumours, from which MIBI was rapidly expelled, retained GLA as readily as did drug sensitive tumours. The biodistribution data of GLA demonstrated significantly higher accumulation (%ID/g) compared to MIBI. CONCLUSION: MCF7 tumour xenografts can be detected by 99mTc glucarate imaging. More importantly, 99mTc glucarate can potentially localize drug resistant breast tumours.

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.

Myocardial kinetics of Tc-99m glucarate in low flow, hypoxia, and aglycemia.

BACKGROUND: Technetium-99m glucarate is a myocardial infarct-avid imaging agent. Recent conflicting and inconclusive reports have suggested that the agent may be taken up by ischemic but viable myocardium. The purposes of this study were (1) to determine conclusively whether there is Tc-99m glucarate uptake in ischemic viable myocardium and (2) to investigate the potential mechanisms for such uptake by studying components of ischemia, namely, low flow, hypoxia, and aglycemia. METHODS AND RESULTS: Rat hearts were isolated and perfused in a modified Langendorff preparation with a crytalloid perfusate. Tc-99m glucarate was studied in control (n = 6), low-flow (n = 5), hypoxic (n = 5), and aglycemic (n = 5) conditions. The experimental protocol consisted of 20-minute baseline (12 mL/min flow) and 20-minute treatment (low flow at 1 mL/min, hypoxia, or aglycemia), followed by tracer uptake (20 minute) and washout (20 minutes). Activity was monitored with a sodium iodide detector. The tracer was delivered continuously over a 20-minute uptake period. The injected dose was 150 micro Ci (5.6 MBq). Hemodynamics were monitored throughout. Triphenyltetrazolium chloride staining was used to assess myocardial viability. There was no evidence of myocardial necrosis. Low flow tended to delay tracer uptake compared with control for the first 10 minutes, but this did not reach statistical significance. Low flow increased end fractional retention significantly compared with control (mean +/- SEM, 59.0% +/- 0.9% peak vs 41.2% +/- 1.4%, respectively; P <.05). Hypoxia resulted in a trend toward increased uptake; however, this was significant only at one early time point during the uptake phase. Retention in the hypoxia group was similar to control. Tc-99m glucarate uptake was significantly increased in aglycemia from 16 minutes to peak compared with control (1.36% +/- 0.71% injected dose per gram vs 0.91% +/- 0.37% injected dose per gram, respectively; P <.05). Aglycemia produced significantly higher end fractional retention compared with control (51.6% +/- 1.8% peak vs 41.2% +/- 1.4%, respectively; P <.05). CONCLUSIONS: Tc-99m glucarate myocardial retention is increased in the setting of ischemia, even in the absence of necrosis. This increased retention is not due to hypoxia. Furthermore, the retention is only partially explained by tissue hypoglycemia. Thus low flow per se appears to have a role in this increased retention, probably as a result of delayed flow-dependent washout.

Accumulation of technetium-99m glucarate: in vitro cell cultures and in vivo tumour models.

99mTc-glucarate is an investigational radiopharmaceutical which has been shown to accumulate in acute cerebral and myocardial injuries and in some tumours. In the present work, a survey of possible factors affecting the cellular accumulation of 99mTc-glucarate was carried out in cell lines and strains in vitro and in murine tumours in vivo. Accumulation was enhanced under hypoxic conditions in 12 of the 16 human and murine cell lines and strains studied, and inhibited in the presence of nitroimidazoles. At temperatures lower than 37 degrees C, accumulation was reduced, but a hypoxic/aerobic differential was maintained. Aerobic accumulation of 99mTc-glucarate was enhanced by cyanide. In transplanted tumours in mice, 99mTc-glucarate showed high tumour/muscle and tumour/blood ratios at early times after injection. Pharmacological enhancement of the extent of hypoxia by the administration of hydralazine or nitro-L-arginine resulted in significantly increased accumulation of 99mTc-glucarate in the tumour. The in vitro and in vivo properties of 99mTc-glucarate suggest that it may be useful for tumour imaging in the clinic, although the exact mechanism(s) by which it localizes in tumours remains unknown.

99mTc-glucarate for detection of isoproterenol-induced myocardial infarction in rats.

Infarct-avid radiopharmaceuticals are necessary for rapid and timely diagnosis of acute myocardial infarction. The animal model used to produce infarction implies artery ligation but chemical induction can be easily obtained with isoproterenol. A new infarct-avid radiopharmaceutical based on glucaric acid was prepared in the hospital radiopharmacy of the INCMNSZ. 99mTc-glucarate was easy to prepare, stable for 96 h and was used to study its biodistribution in rats with isoproterenol-induced acute myocardial infarction. Histological studies demonstrated that the rats developed an infarct 18 h after isoproterenol administration. The rat biodistribution studies showed a rapid blood clearance via the kidneys. Thirty minutes after 99mTc-glucarate administration the standardised heart uptake value S(h)UV was 4.7 in infarcted rat heart which is six times more than in normal rats. ROIs drawn over the gamma camera images showed a ratio of 4.4. The high image quality suggests that high contrast images can be obtained in humans and the 96 h stability makes it an ideal agent to detect, in patients, early cardiac infarction.

Inhibition of liver metastases and tumor cell invasion in spontaneous liver metastasis model (LMFS) by sodium D-glucaro-delta-lactam (ND2001).

We examined the inhibitory effect of sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam: ND2001) upon liver metastases of the LMFS tumor. A permanent cell line, LMFS, was established from a spontaneously occurring murine retroperitoneal tumor of BALB/c mouse origin, and after a subcutaneous injection, the LMFS cells proliferated at the inoculation site (100% take) with liver metastases. ND2001 had little effect on the cell growth, cell cycle and phagokinesis of the LMFS cells in vitro. However, when the invasive activity was measured by the Boydem chamber method, the number of LMFS cells was reduced, with inhibition rates of 98.0%. After the LMFS cells treated with ND2001 in vitro, the numbers of hepatic metastases of subcutaneous inoculation of treated cells were reduced dose-dependently, and those of intravenous inoculation were not found by microscopical study. When the LMFS tumor-bearing mice were treated with ND2001 (0, 30, 100 mg/kg/d) from day 1, ND2001 (30 mg/kg) inhibited the liver metastases with a rate of 56.4%, and when given from day 15, ND2001 (100 mg/kg) inhibited with a rate of 47.5%. But ND2001 showed neither cytocidal nor anti-tumor activity. Combination therapy of primary tumor resection and ND2001 administration revealed that preoperative use of ND2001 was more effective in preventing liver metastases. These results suggested that ND2001 might have a potential use as an anti-metastatic agent for operative patients without metastasis.

Differentiation of myocardial ischemia and necrosis by technetium 99m glucaric acid kinetics.

BACKGROUND: Current clinical approaches may not always be helpful in the early differentiation of necrotic tissue from ischemic viable myocardium in patients with acute myocardial infarction. Tc-99m-glucaric acid is a carbohydrate ligand that may permit differentiation of necrosis from ischemia. However, the myocardial kinetics of Tc-99m-glucaric acid have not been well defined early after myocardial injury. The aim of this study was to evaluate the effect of necrosis in comparison to postischemic injury alone on the kinetics of Tc-99m-glucaric acid with the use of an isolated perfused rat heart model. METHODS AND RESULTS: Three groups of hearts were studied: group I: control (n = 6); group II: postischemia (15 minutes of no flow with complete reperfusion, n = 6); and group III: necrosis (90 minutes of no flow to induce necrosis with complete reperfusion, n = 6). Tc-99m-glucaric acid (1.3 +/- 0.6 mCi/L of buffer) was perfused for 30 minutes to evaluate tracer accumulation. Then tracer-free buffer was perfused for 45 minutes to evaluate clearance. The peak accumulation relative to the control group mean was significantly increased (p < 0.01) in group III (necrosis) (254% +/- 75%) compared with control (100% +/- 10%) and compared with postischemia (108% +/- 26%). On kinetic data analysis, the monoexponential clearance rate constant: (kc) was significantly reduced with necrosis (control: kc = 20.2 +/- 14.0 x 10(-4) sec-1; postischemia: kc = 22.3 +/- 15.2 x 10(-4) sec-1; and necrosis: kc = 1.2 +/- 0.3 x 10(-4) sec-1; p < 0.05). A retention fraction was calculated from the activity after 45 minutes of clearance corrected for the peak activity for each group. The necrotic group had significant myocardial retention in comparison to control and postischemia (control: 12% +/- 8%; postischemia: 14% +/- 16%; necrosis: 64% +/- 10%; p < 0.01). CONCLUSIONS: The accumulation and retention of Tc-99m-glucaric acid is markedly increased in the presence of myocardial necrosis in comparison to control and postischemic myocardial injury. In this model, Tc-99m-glucaric acid is capable of defining the presence of necrotic myocardial injury in comparison to postischemic injury alone. This agent may have potential application for the early differentiation of ischemic from necrotic myocardium in acute myocardial infarction.

Avidity of technetium 99m glucarate for the necrotic myocardium: in vivo and in vitro assessment.

BACKGROUND: Similar to other 99mTc-based infarct-avid agents, 99mTc-glucarate localizes in myocardial infarcts. Whether severely ischemic viable myocytes sequester 99mTc-glucarate is uncertain. To assess the infarct specificity, in vitro and in vivo studies were performed. METHODS AND RESULTS: H9C2 embryonic rat cardiocytes cultured under normoxia (N) or hypoxia (H) for 24 hours in 7.5 muCi 99mTc-glucarate were compared with necrotic cardiocytes. Mean H/N ratio (3.0 +/- 0.004, mean +/- SD) was significantly less than that of the necrotic/N ratio (39.9 +/- 6.5, p < 0.01). Reperfused myocardial infarction (MI) in 4 dogs confirmed by 201Tl (0.5 to 1.0 mCi) scintigraphy were imaged serially with simultaneously injected mixture of 99mTc-glucarate and 111In-antimyosin Fab. Infarcts were detected scintigraphically within 4 to 10 minutes with 99mTc-glucarate. 111In-antimyosin required more than 1 hour. Myocardial distribution at 5 hours showed a direct correlation between 99mTc-glucarate and 111In-antimyosin uptake (r = 0.99, p < 0.0001). Both 99mTc-glucarate (r = -0.777, p < 0.0001) and 111In-antimyosin (r = -0.775, p < 0.0001) were inversely related to 201Tl distribution. CONCLUSIONS: The near perfect correlation between 99mTc-glucarate and 111In-antimyosin uptake (r = 0.99) in reperfused canine MI and the insignificant glucarate uptake by viable cardiocytes in vitro attest to the avidity of 99mTc-glucarate for the necrotic myocardium and favor its use as a specific early marker of myocyte necrosis in acute MI.