Labeling proteins with Tc-99m via hydrazinonicotinamide (HYNIC): optimization of the conjugation reaction.

At present there is considerable interest in labeling peptides with Tc-99m for the development of target specific radiopharmaceuticals for imaging purposes. In the present study the conjugation of the bifunctional coupling agent succinimidyl-hydrazinonicotinamide (S-HYNIC) was studied and optimized in a series of peptides [molecular weight (MW) 6.5-14.3 kDa]. Aprotinin (MW 6.5 kDa), cytochrome C (MW 12.4 kDa), alpha-lactalbumin (MW 14.2 kDa), and lysozyme (MW 14.3 kDa) were conjugated with S-HYNIC via the epsilon amino groups of their lysine residues. The effects of molar conjugation ratio, reaction temperature, pH, and protein concentration were studied. Reaction products were analyzed both with respect to the HYNIC-substitution ratio (spectrophotometrically) as well as to the labeling efficiency silica gel-instant thin layer chromatography (SG-ITLC) and molecular size fast performance liquid chromatography (FPLC). The effects of conjugation on biological activity were studied in three proteins binding to receptors on leukocytes: interleukin-8 (MW 8.5 kDa), interleukin-1alpha (MW 17 kDa), and interleukin-1 receptor antagonist (MW 17 kDa). The labeling efficiency of aprotinin, cytochrome c, alpha-lactalbumin, and lysozyme conjugated under optimal conjugation conditions exceeded 90%. Specific activities obtained were up to 7.5 MBq/microg. Conjugation was most efficient at 0 degrees C (as compared to 20 and 40 degrees C), at pH 8.2 (as compared to 6.0, 7.2, and 9.5), and at protein concentrations > or = 2. 5 mg/mL. In general, efficiency increased with increasing molar conjugation ratio (protein-HYNIC-ratio 1:3 < 1:6 < 1:15<1:30). For the receptor binding proteins, biological activity was preserved only under the mildest conjugation conditions. For each of these proteins an inverse relation between labeling efficiency and receptor binding capacity was found. Labeling proteins with (99m)Tc using S-HYNIC is easy, rapid, and efficient, and preparations with high specific activity can be obtained. However, biological activity of proteins may be lost at high HYNIC-substitution ratios. With the proteins tested here a careful balancing of reaction conditions resulted in acceptable, although suboptimal, labeling efficiencies and preservation of biological activity.

A novel method to label liposomes with 99mTc by the hydrazino nicotinyl derivative.

UNLABELLED: In this study a new 99mTc labeling method for polyethyleneglycol (PEG)-coated liposomes is described. The in vitro and in vivo characteristics were compared with the conventional 99mTc-HMPAO-labeled PEG-coated liposomes. METHODS: PEG-coated liposomes were labeled with 99mTc by the hydrazino nicotinyl (HYNIC) derivative of distearoylphosphatidyl-ethanolamine (DSPE) and compared with PEG-coated liposomes labeled with 99mTc-HMPAO. In vitro stability tests were performed. Biodistribution and imaging characteristics of both liposomal preparations were determined in rats with Staphylococcus aureus infection in the left calf muscle. RESULTS: Per liposome, 230 hydrazine groups were incorporated. The labeling efficiency of the 99mTc-HYNIC liposomes was greater than 95%, so no postlabeling purification was required, in contrast to the 99mTc-HMPAO liposomes. The 99mTc-HYNIC liposomes showed greater in vitro stability than the conventional 99mTc-HMPAO liposomes. Abscess uptake of the 99mTc-HYNIC liposomes was significantly greater (1.74+/-0.38%ID/g versus 1.26+/-0.29%ID/g, 24 h postinjection, P < 0.03). Furthermore, kidney uptake of the 99mTc-HYNIC liposomes was one third of the uptake of the 99mTc-HMPAO liposomes (0.79+/-0.07%ID/g versus 2.47+/-0.35%ID/g, 24 h postinjection, P < 0.0001). CONCLUSION: This new 99mTc-HYNIC-based labeling method for liposomes is rapid, efficient and easy to perform. Most importantly, the 99mTc-labeled liposomes have an improved stability and in vivo characteristics. The new labeling method is a major step forward toward a radiopharmaceutical for infection imaging that can be prepared in a one-step procedure within 15 min at room temperature and thus can be applied in every routine clinical practice.

Technetium-99m-labeled liposomes to image experimental colitis in rabbits: comparison with technetium-99m-HMPAO-granulocytes and technetium-99m-HYNIC-IgG.

UNLABELLED: Scintigraphic techniques are routinely used for the evaluation of the extent and severity of inflammatory bowel disease. Currently, the radiopharmaceutical of choice is 99mTc-hexamethyl propyleneamine oxime (HMPAO)-leukocytes. We studied the imaging potential of two recently developed 99mTc-labeled agents, polyethylene glycol (PEG)-coated liposomes and hydrazinonicotinate (HYNIC) IgG, in a rabbit model of acute colitis, and compared them with that of 99mTc-labeled, granulocyte-enriched (>90%), white blood cells. METHODS: Acute colitis was induced in rabbits by retrograde instillation of trinitrobenzene sulfonic acid. After 48 hr, 37 MBq of each radiopharmaceutical was administered intravenously. Gamma camera images were taken at 0, 1, 2, 4, 10 and 24 hr. At 4 and 24 hr postinjection, groups of rabbits were killed, and the uptake of the radiolabel in the dissected tissues was determined. For each affected 5-cm segment, the colitis index (CI, affected-to-normal-colon-uptake ratio) was calculated and correlated to the macroscopically scored severity of inflammation. RESULTS: All three agents visualized the colitis lesions within 1 hr postinjection. The CI correlated with the severity of the abnormalities. With increasing severity, the CI at 4 hr postinjection for liposomes was 3.89+/-0.73, 4.41+/-0.47 and 5.76+/-0.65; for IgG 1.67+/-0.08, 3.92+/-0.44 and 6.14+/-0.65; and for granulocytes 2.90+/-0.09, 6.15+/-0.96 and 9.36+/-3.35. For liposomes, the CI further increased during 24-hr postinjection to 6.56+/-0.84, 8.50+/-0.53 and 10.61+/-1.34, respectively. The CI for the other two agents did not change significantly with time. CONCLUSION: In this rabbit model, 99mTc-labeled granulocytes, IgG and liposomes all rapidly visualized colonic inflammation. Granulocytes and liposomes showed the highest CI. Technetium-99m-labeled PEG-liposomes may be an attractive alternative for labeled leukocytes to image inflammatory bowel disease, because they can be prepared off the shelf and no handling of blood is required.

Dosimetric analysis of chimeric monoclonal antibody cMOv18 IgG in ovarian carcinoma patients after intraperitoneal and intravenous administration.

In this study the potential of intraperitoneal (i.p.) and intravenous (i.v.) administration of chimeric iodine-131-labelled MOv18 IgG for radioimmunotherapy was determined. The dosimetry associated with both routes of administration of cMOv18 IgG was studied in patients. Eight patients suspected of having ovarian carcinoma received 150 MBq 131I-cMOv18 IgG i.p. Blood and urine were collected and serial gamma camera images were acquired. Another group of four patients received 7.5 MBq 131I-cMOv18 IgG i.v. For all patients, tissue biopsies were obtained at surgery. Activity in the blood after i.p. administration was described by a bi-exponential curve with a mean uptake and elimination half-life of 6.9+/-3.2 h and 160+/-45 h, respectively. For i.v. infusion the mean half-life for the elimination phase was 103+/-12 h. Cumulative excretion in the urine was 17%+/-3% ID and 21%+/-7% ID in 96 h for i.p. and i.v. administration, respectively. Scintigraphic images after i.p. administration showed accumulation in ovarian cancer lesions, while all other tissues showed decreasing activity with time. Tumour uptake determined in the ovarian cancer tissue specimens ranged from 3.4% to 12.3% ID/kg for i.p. administration and from 3.6% to 5.4% ID/kg for i.v. administration. Dosimetric analysis of the data indicated that 1.7-4.3 mGy/MBq and 1.7-2.2 mGy/MBq can be guided to solid or ascites cells after i.p. and i.v. administration, respectively. Assuming that an absorbed dose to the bone marrow of 2 Gy will be dose limiting, a total activity of 4.1 GBq 131I-cMOv18 IgG can be administered safely via the i.p. route and 3.5 GBq via the i.v. route. At this maximal tolerated dose, a maximum absorbed dose to 1-g tumours in the peritoneal cavity of 18 and 8 Gy can be reached after i.p. and i.v. administration, respectively. For the i. p. route of administration, dose estimates for the tumour are even higher when the electron dose of the peritoneal activity is also taken into account: total doses to the tumour of 30 Gy and 22 Gy will be absorbed at the tumour surface and at 0.2 mm depth, respectively. In conclusion, therapeutic tumour doses can be achieved with 131I-cMOv18 IgG in patients with intraperitoneal ovarian cancer lesions with no normal organ toxicity. The i.p. route of administration seems to be preferable to i.v. administration.

Scintigraphic evaluation of experimental colitis in rabbits.

UNLABELLED: Scintigraphic techniques are frequently used for evaluation of inflammatory bowel disease. The radiopharmaceutical of choice is labeled leukocytes. In this study, two new agents, 111In-labeled polyethylene glycol-coated liposomes and 111In-labeled human nonspecific gamma globulin (immunoglobulin G; IgG), were compared with 111In-leukocytes in a rabbit model of colitis. METHODS: In rabbits, acute colitis was induced by colonic instillation of trinitrobenzene sulfonic acid at 25 cm from the anal sphincter. After 24 hr, 15 MBq of the radiopharmaceuticals was injected intravenously in groups of four rabbits. Twenty-four hours after injection, the animals were killed and macroscopic abnormalities were scored in seven consecutive affected colonic segments of 5 cm each (0 = normal, 1 = inflammation, 2 = ulcers). The ex vivo uptake was measured in the normal ascending colon and the affected colonic segments. The colitis index (CI, affected-to-normal colon-uptake ratio) was calculated. RESULTS: Histologically, an acute, patchy, transmural colitis was observed at the site of instillation and the distal colon. The CI of all agents in colitis lesions correlated with the severity of the abnormalities. With increasing severity, the CI for liposomes was 1.86 +/- 0.24, 4.88 +/- 0.42 and 7.42 +/- 0.54 (r2 = 0.68, p < 0.001); for leukocytes 1.77 +/- 0.32, 3.10 +/- 0.58 and 5.54 +/- 0.83 (r2 = 0.31, p < 0.01); for IgG 1.60 +/- 0.29, 2.81 +/- 0.21 and 2.65 +/- 0.21 (r2 = 0.29, p < 0.02). CONCLUSION: Indium-111-labeled-leukocytes, -IgG and -liposomes all show increased uptake in inflamed colonic tissue. Indium-111-liposomes showed the highest CI, which correlates best with the morphological abnormalities. Indium-111-leukocytes and 111In-liposomes are superior to 111In-IgG for this indication.

Targeting of renal cell carcinoma with iodine-131-labeled chimeric monoclonal antibody G250.

PURPOSE: Pharmacokinetics, biodistribution, immunogenicity, and imaging characteristics of iodine 131 (131I)-labeled chimeric monoclonal antibody (mAb) G250 (cG250) were studied in patients with renal cell carcinoma (RCC) to determine the therapeutic potential of this antibody. PATIENTS AND METHODS: Sixteen patients with RCC received a single intravenous (IV) infusion of 6 mCi 131I-labeled cG250. Five protein dose levels were investigated (2 to 50 mg). Planar scintigraphic images were acquired, and normal tissue biopsies and tumor samples were obtained of surgery (7 days postinjection). The immunogenicity of cG250 was investigated using a sandwich enzyme-linked immunosorbent assay (ELISA) and dosimetric analysis was performed. RESULTS: In all patients with antigen-positive tumors (n = 13), the primary tumors and all known metastases were clearly visualized. Overall uptake, expressed as the percentage of the injected dose (%ID), in the primary tumors ranged from 2.4 to 9.0. Focally, 131I-cG250 uptake as high as 0.52% ID/g was observed. However, intratumoral uptake was highly heterogeneous. 131I-cG250 uptake in nontumorous tissues remained low. Dosimetric analysis showed that up to .48 Gy/mCi was guided to the primary tumors. Selected "hot areas" within these tumors received up to .72 Gy/mCi. A bone metastasis received .23 Gy/mCi and regional lymph node metastases received .20 Gy/mCi. Minimal human antichimeric antibody (HACA) levels were detected in two of 16 patients. CONCLUSION: 131I-cG250 tumor uptake is among the highest reported in clinical studies with antitumor antibodies in solid tumors. Since tumor-sterilizing levels may be guided to the tumor when high doses 131I-cG250 are administered (> 100 mCi) and cG250 appears to be immunosilent, cG250 is a promising vehicle for radioimmunotherapy in RCC.

Optimization of technetium-99m-labeled PEG liposomes to image focal infection: effects of particle size and circulation time.

UNLABELLED: In previous studies we have shown that liposomes sterically stabilized with polyethylene glycol (PEG), preferentially localize in infectious and inflammatory foci. In this study, we further optimized the formulation of PEG liposomes for infection imaging in a rat model. METHODS: The biodistribution and imaging characteristics of different liposomal formulations labeled with 99mTc were determined in rats with S. aureus infection of the left calf muscle. The influence of liposomal size (mean diameter varying from 90 nm to 220 nm) as well as circulation time (modulated by inclusion of 0-10 mole% phosphatidylserine) were studied. RESULTS: The smallest liposomes displayed improved characteristics for infection imaging: 90-nm liposomes revealed the highest abscess uptake (1.6% +/- 0.4% ID/g, 24 hr postinjection) in combination with the lowest splenic accumulation (6.9% +/- 0.7% ID/g, 24 hr postinjection) as compared to the larger sized preparations. Enhanced abscess-to-blood ratios (4.0 versus 1.3 at 24 hr postinjection) were obtained by including 1.0 mole% phosphatidylserine in the lipid bilayer of the PEG liposomes. However, enhanced blood clearance of these liposomes reduced their absolute abscess uptake. CONCLUSION: These results indicate that the in vivo behavior of PEG liposomes can be modulated to optimize their characteristics for infection imaging.

Retention of technetium-99m in infectious foci in rats after release from technetium-99m labelled human non-specific polyclonal immunoglobulin G: a dual-label study with hydrazinonicotinamido and iminothiolano immunoglobulin.

In an effort to contribute to the understanding of the mechanism of uptake of technetium-99m labelled non-specific polyclonal human immunoglobulin G (hIgG) in inflammatory lesions we compared the tissue distribution of double-labelled 99mTc-hydrazinonicotinamido (HYNIC) hIgG-14C and 99mTc-iminothiolano hIgG-14C in groups of five Wistar rats with a Staphylococcus aureus infection of the left calf muscle between 2 h p.i. and 24 h p.i. The stability of the two double-labelled hIgG preparations was evaluated in vitro and in plasma in vivo by high-performance liquid chromatography (HPLC) analysis. At 24 h after injection of 99mTc-HYNIC-hIgG-14C the abscess uptake of 99mTc (1.5% ID/g+/-0.2% ID/g) was significantly higher (P<0.01) than the 14C uptake (1.0% ID/g+/-0.1% ID/g). After injection of 99mTc-iminothiolano hIgG-14C no significant difference (P=0.08) was found between the abscess uptake of the two radionuclides at 24 h p.i. (99mTc: 0.8% ID/g+/-0.1% ID/g; 14C: 0.90% ID/g+/-0.09% ID/g). HPLC analysis of plasma samples revealed release of 99mTc from both double-labelled immunoglobulin preparations. This phenomenon was more pronounced for iminothiolano hIgG than for HYNIC hIgG (43% vs 18%). In most tissues other than abscesses significant differences were also found between the 99mTc and the corresponding 14C uptake. Our results demonstrate that the chemical form in which 99mTc is bound to hIgG severely influences its release from hIgG and its retention in infections.

Labelled Stealth liposomes in experimental infection: an alternative to leukocyte scintigraphy?

Indium-111 (111In) and technetium-99m (99Tcm) Stealth liposomes were compared with 111In- and 99Tcm-labelled white blood cells (WBC) in experimental infection in a rabbit model. Preformed polyethylene glycol-coated liposomes and separated WBC were radiolabelled with either 111In-oxine or 99Tcm-hexamethylpropyleneamine oxime (99TcM-HMPAO). After the intravenous administration of one of the four radiopharmaceuticals to rabbits with focal Staphylococcus aureus infection, scintigraphic images were recorded at various time points post-injection and the biodistribution of the radiopharmaceuticals was determined. At 4 h post-injection, uptake of 111In-WBC in the abscess was significantly higher than that of the three other products. AT later time points, 111In-WBC, 111In-liposome and 99Tcm-liposome uptake in the abscess were similar. In contrast, a 20 h post-injection, uptake of 99Tcm-WBC was significantly lower. The abscess-to-background ratios showed a similar pattern to the absolute abscess uptake: initial high values for 111In-WBC, a more gradual increase over time of the liposome preparations to the level of 111In-WBC and persistently low values for 99Tcm-WBC. Clearance from the blood of both labelled WBC preparations was significantly faster and splenic uptake significantly higher compared with those of the labelled liposomes. In conclusion, given the similar in vivo characteristics of labelled liposomes and labelled WBC, labelled liposomes may be an attractive replacement for labelled WBC, providing a continuously available, high-quality, 99Tcm-labelled radiopharmaceutical that can be prepared easily without any need to handle blood.

Detecting infection and inflammation with technetium-99m-labeled Stealth liposomes.

UNLABELLED: The performance of 99mTc Stealth liposomes was investigated in various rat models. METHODS: Preformed polyethyleneglycol-containing liposomes with encapsulated reduced glutathione, were radiolabeled using the lipophilic 99mTc-HMPAO. The labeled liposomes were intravenously administered to rats with focal S. aureus or E. coli infection, or turpentine-induced inflammation. For comparison, Tc-99m-nanocolloid- and 99mTc-labeled nonspecific IgG were tested. In rats with Pneumocystis carinii pneumonia (PCP), Tc-99m-liposomes were directly compared to In-111 labeled nonspecific IgG. RESULTS: Technetium-99m-liposomes accumulated in the infectious and inflammatory muscle foci over 24 hr (0.59% injected dose per gram tissue (%ID/g) for S. aureus; 1.18 %ID/g for turpentine). Abscess-to-muscle ratios increased to values as high as 24.0, 41.7 and 44.5 for the respective models at 24 hr postinjection. Technetium-99m-liposomes visualized the foci as early as 1 hr postinjection. Technetium-99m-IgG visualized S. aureus infection, but abscess-to-muscle ratios and abscess uptake at the later time points were significantly lower. Technetium-99m-nanocolloid failed to visualize any of the muscle foci. In PCP however, 99mTc-liposomes did not show preferential localization in the infection. The control agent 111In-IgG showed a significant, two-fold increase in lung uptake. CONCLUSION: Technetium-99m-Stealth liposomes preferentially accumulated in abscesses, leading to very high target-to-nontarget ratios. This property appears to be related to a process based on uptake of long-circulating particles. In a specific type of infection, i.c. PCP, 99mTc-liposomes did not accumulate in diseased lung tissue, thus mimicking the in vivo behavior of labeled leukocytes.

Biodistribution of 111In-labelled IgG and IgM in experimental infection.

Both the protein used and the conjugation method are factors which may be relevant for targeting infection with 111In-labelled proteins. In this study, human immunoglobulin G (IgG), conjugated to either DTPA or LiLo, and LiLo conjugated human immunoglobulin M (IgM) were evaluated. In rats with Staphylococcus aureus calf muscle infection, biodistribution was determined 6, 24 and 48 h after the injection of 111In-DTPA-IgG, 111InLiLo-IgG or 111In-LiLo-IgM. Absolute abscess uptake of 111In-LiLo-IgG was significantly higher than that of 111In-DTPA-IgG (P < 0.05). Since blood clearance of 111In-LiLo-IgG was initially significantly slower (P < 0.01), the higher abscess uptake did not result in higher abscess-to-background ratios. 111In-LiLo-IgG accumulated to a greater extent in the liver (P < 0.001). 111In-DTPA-IgG showed higher uptake in the kidneys and bone marrow (P < 0.001 and P < 0.01, respectively). Although decreasing over time, 111In-LiLo-IgM showed reasonable abscess uptake and rapid blood clearance, resulting in higher abscess-to-background ratios compared with 111In-LiLo-IgG (P < 0.01). However, liver and spleen uptake were three- to four-fold higher than that of 111In-LiLo-IgG (P < 0.001). Compared with DTPA-conjugation, chelation with LiLo has a minor influence on abscess targeting of 111In-labelled IgG. However, differences in blood clearance and organ uptake do occur. 111In-LiLo-IgM shows high relative accumulation in abscesses as well as high liver and spleen uptake. 111In-LiLo-IgM appears promising for imaging infection outside the trunk region.

Technetium-99m labelled hydrazinonicotinamido human non-specific polyclonal immunoglobulin G for detection of infectious foci: a comparison with two other technetium-labelled immunoglobulin preparations.

Recently a new linker - hydrazinonicotinate (HYNIC) - was introduced for labelling of proteins and peptides with technetium-99m. HYNIC and other linkers have been used for labelling of human non-specific polyclonal immunoglobulin G (hIgG) with 99mTc for the detection of infections. In this study we compared the tissue distribution of three different 99mTc-hIgG preparations in groups of five Wistar rats with a focal intramuscular infection with Staphylococcus aureus. We compared 99mTc-HYNIC-hIgG with 99mTc-hIgG labelled via the so-called Schwarz method (reduction of disulphide bonds) and with the 99mTc-labelled commercially available Technescan-HIG. Unlike the HYNIC linker, in the two other labelling methods free sulph-hydryl groups are involved in the binding of 99mTc. High-performance liquid chromatography analysis of the labelled preparations and of plasma samples revealed aggregate or polymer formation in all three agents; this was least pronounced in the product labelled by means of the Schwarz method. The tested preparations did not show signs of degradation in vitro. The difference in linker chemistry was reflected in the tissue distribution. Thus the biodistribution of 99mTc-HYNIC-hIgG was significantly different from the distribution of the two other preparations: abscess (1.4%+/-0.2%ID/g), muscle, liver, spleen, plasma, lung, bone marrow, and small intestine concentrations were higher at 24 h p.i.; kidney uptake (1.19%+/-0.08%ID/g) was significantly lower. The abscess-to-plasma and the abscess-to-muscle ratios (0.5 and 11, respectively), however, were in the same range for the three preparations tested. Quantitative analysis of the scintigraphs revealed that the total body clearance of 99mTc-HYNIC-hIgG was significantly slower than for the other agents. The abscess uptake of 99mTc-HYNIC-hIgG as a percentage of the remaining body activity was significantly higher. Based on its high abscess uptake, its low uptake in the kidneys and the high percentage of its abscess uptake in relation to the remaining body activity, we conclude that 99mTc-HYNIC-hIgG seems superior to the two other preparations tested for the detection of infections.

Dissociation of indium from indium-111-labelled diethylene triamine penta-acetic acid conjugated non-specific polyclonal human immunoglobulin G in inflammatory foci.

Several investigators have reported retention of indium-111 in infectious foci after intravenous injection of 111In-labelled immunoglobulin G (IgG). With this study we intended to test the hypothesis that, upon administration of 111In-diethylene triamine penta-acetic acid (DTPA-IgG), 111In is retained in the infectious foci after dissociation from IgG. Therefore we measured the tissue distribution of double-labelled 111In-DTPA-IgG-(carbon-14) in rats with a focal infection and compared the results with corresponding data for DTPA-IgG-(14C). DTPA-conjugated IgG was labelled with 111In via citrate transchelation. 111In-DTPA-IgG and DTPA-IgG were labelled with 14C through methylation. High-performance liquid chromatography (HPLC) and instant thin-layer chromatography analysis were performed to test the in vitro stability of the labelled proteins. Young Wistar rats with a Staphylococcus aureus infection of the left calf muscle were injected intravenously with 0.2 ml of a solution containing either 0.4 MBq 111In and 30 kBq 14C or 30 kBq 14C labelled to 80 micrograms IgG. Groups of five rats were sacrificed at 2, 6, 24, and 48 h. p.i. Activity uptake was determined for plasma, urine, abscess, muscle and various other tissues. Averages and standard deviations were calculated for groups of five rats. HPLC analysis was performed on plasma and urine samples taken up to 48 h p.i. The radiochemical purity of the IgG preparations was > 95%. The labelled preparations appeared stable in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased kidney uptake of technetium-99m-labelled Fab' fragments in ovarian carcinoma bearing nude mice using a cleavable chelator.

A new 99mTc labelling method using a cleavable chelator, RP-1, was developed. In this study Balb/c mice with ovarian carcinoma xenografts received various Fab' fragments labelled with 99mTc either directly or via RP-1. Kidney uptake was significantly lower for the RP-1 linked conjugates. Tumour uptake showed no significant differences between RP-1 conjugates and directly labelled preparations. In conclusion, with the use of the cleavable linker RP-1, kidney uptake can be reduced significantly resulting in a lower radiation dose to the kidneys.

Evaluation of indium-111-polyclonal immunoglobulin G to quantitate atherosclerosis in Watanabe heritable hyperlipidemic rabbits with scintigraphy: effect of age and treatment with antioxidants or ethinylestradiol.

Scintigraphic detection of atherosclerotic lesions using 111In-polyclonal IgG was studied. In Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for hypercholesterolemia with spontaneous atherosclerosis, aged WHHL rabbits incorporated more 111In-IgG into atherosclerotic lesions than young WHHL or control NZW rabbits. This result is in agreement with histological analysis. However, due to the low ratio of lesion-incorporated radioactivity to circulating radioactivity, in vivo gamma imaging of atherosclerosis with 111In-IgG scintigraphy was unsuccessful. Interventional agents, Probucol or vitamin E, used for 28 days to reduce the amount of autoantibodies produced against biological modified low-density lipoproteins did not produce differences in 111In-IgG incorporation into the aorta ex vivo. Ethinylestradiol, used for 28 days, exhibited similar incorporation with decreased serum cholesterol by 45%. Although atherosclerosis histology and lesion surfaces of WHHL rabbits are similar to those in adult humans, it is obvious that noninvasive gamma imaging with polyclonal 111In scintigraphy is not reliable for serial evaluation of the extent of atherosclerosis. Our results emphasize the need to develop pharmaceuticals to image atherosclerosis.

Intraoperative bone and bone marrow sampling: a simple method for accurate measurement of uptake of radiopharmaceuticals in bone and bone marrow.

Accurate estimation of bone marrow uptake of radiopharmaceuticals is of crucial importance for accurate whole body dosimetry. In this study, a method for obtaining normal bone marrow and bone during routine surgery without inconvenience to volunteers is suggested and compared to an indirect method. In five volunteers (group 1), 4 MBq 111In-labelled human polyclonal IgG (111In-IgG) was administered 48 h before placement of a total hip prosthesis. After resection of the femoral head and neck, bone marrow was aspirated from the medullary space with a biopsy needle. In five patients, suspected of having infectious disease (group 2), bone marrow uptake was calculated according to a well-accepted method using regions of interest over the lumbar spine, 48 h after injection of 75 MBq 111In-IgG. Bone marrow uptake in group 1 (4.5 +/- 1.3% D kg-1) was significantly lower than that in group 2 (8.5 +/- 2.1% D kg-1) (P < 0.01). Blood and plasma activity did not differ significantly for both groups. This method provides a system for directly and accurately measuring uptake and retention in normal bone marrow and bone of all radiopharmaceuticals at various time points. It is a safe and simple procedure without any discomfort to the patient. Since small amounts of activity are sufficient, the radiation dose to the patient is low.