Effects of a 99mTc-labeled murine immunoglobulin M antibody to CD15 antigens on human granulocyte membranes in healthy volunteers.

UNLABELLED: An injectible, 99mTc-labeled, murine immunoglobulin M antibody to stage-specific embryonic antigen-1 has been developed that can localize infections by binding to CD15 glycoproteins expressed on the cell membranes of human granulocytes in vivo after systemic administration. The purpose of this study was to measure its clinical effects on healthy people. METHODS: Multiple blood samples were aspirated before and after the intravenous administration of about 125 microg antibody labeled with approximately 370 MBq (10.0 mCi) 99mTc in 10 healthy human volunteers. Complete blood cell counts were performed at each time point. Whole-body scans were acquired contemporaneously with a dual-head gamma camera. The fraction of the administered dose at each time point was quantified in 18 regions of interest. Statistical analyses included paired t tests. RESULTS: Administration was associated with a transient decrease in the concentration of red and white blood cells in the whole blood. The effect always began within 3 min of administration. Its nadir was always reached 15-20 min after administration. There was full recovery with mild overcompensation in about an hour. The hematocrit dropped by a mean of 3.8% (P<0.002), whereas the total white blood cell count fell 44.0%+/-3.1% (P<0.001). The effect was most pronounced on the number of circulating granulocytes, which fell from 5.7+/-2.1 to 3.2+/-1.3x10(3)/microL blood. The drop paralleled a decrease in the percentage of whole blood radioactivity bound to the white blood cell membranes, which peaked at 50.4%+/-7.6% at 3 min after injection and then fell to 26.1%+/-9.3% over the next 30+/-13.4 min before recovering to 40.7%+/-8.2% at 2 h. Image analysis showed that the effect was temporally associated with an increase in the amount of radioactivity within the liver and the spleen. Recovery was associated with a decrease in hepatosplenic radioactivity. No evidence of cell destruction or agglutination could be detected. CONCLUSION: This study confirmed that administration of this radiolabeled antibody is associated with a transient decrease in the number of circulating granulocytes. However, there also seems to be a secondary hemodilutionlike effect on all blood components that has not been reported previously. The effect appears to be clinically silent and very short-lived.

Clinical aspects of local and regional tumor therapy with 188Re-RC-160.

Somatostatin-receptors have been found to be overexpressed in a variety of neuro-endocrine and epithelial cancers. While the introduction of a long-acting somatostatin-analogue, octreotide, exerted mainly anti-cancer activity in neuro-endocrine tumors, no convincing results have been demonstrated in other cancers. RC-160, another somatostatin-analogue has been selected because of its high receptor affinity and its anti-cancer activity. 188Re is a generator produced radionuclide with favourable gamma and beta-emission, allowing diagnostic and therapeutic application. The results of in vivo biodistribution and therapeutic outcome following systemic, intralesional and intracavitary application in animal studies employing 188Re-RC-160 are summarized. Safety considerations, dosimetry estimates and applicable indications are outlined. The clinical impacts of this radiopharmaceutical in cancer management are discussed.

Pre-clinical experience with Re-188-RC-160, a radiolabeled somatostatin analog for use in peptide-targeted radiotherapy.

The clinical potential of radiolabeled peptides such as octreotide and VIP has been widely established for tumor localization. Radiotherapy based on the tumor binding potential of the peptides and the radiotoxic effects of beta- or a-emitting radionuclides is an extension of such applications. Rhenium-188 (T1/2 16.9 hr, beta-max 2.1 MeV) coupled to the analogue RC-160 has been used to establish the feasibility of treating tumors with radiolabeled peptides, and our experience with this approach is summarized. In three different experimental tumor models (human prostate, mammary gland, and small cell lung carcinomas) in nude mice, treatment resulted in significant reduction or elimination of tumor burden. Two routes of administration were used: intra-lesional injection (prostate carcinoma) and intra-cavity injection (mammary and SCLC). Re-188-labeled negative control peptides bound to tumor cells to a low extent and did not exhibit therapeutic benefit. RC-160 by itself did not result in therapeutic benefit. Tumors which did not bind Re-188-RC-160 did not evidence a therapeutic benefit. Uncoupled Re-188 (control) was rapidly excreted via the urinary bladder and did not accumulate in either tumors or normal tissues even following direct injection. Instant radiolabeling kits containing 200 micrograms of RC-160 were labeled with < 3000 MBq of Re-188 in 30 minutes with no need for subsequent purification. These studies establish the conceptual feasibility of targeted radiotherapy based on the local or regional administration of radiolabeled peptides.

Radiolabeling of epidermal growth factor with 99mTc and in vivo localization following intracerebral injection into normal and glioma-bearing rats.

High grade gliomas may have amplified expression of the epidermal growth factor receptor (EGFR) gene c-erb-B, which often is associated with increased expression of transmembrane EGFR. The purpose of the present study was to develop a method for labeling EGF with 99mTc and to determine whether the resulting radioligand would localize, following intracerebral injection, in rats bearing EGFR-positive gliomas. EGF has a relatively low molecular mass (approximately 6 kDa) compared to monoclonal antibodies, and this has allowed smaller bioconjugates, which should diffuse more rapidly within the brain and more effectively target disseminated glioma cells, to be constructed. In the present study, EGF has been labeled with either 131I or 99mTc, and in vitro uptake of the resulting radioligand has been investigated using C6EGFR rat glioma cells, which had been transfected with the EGFR gene. Cellular uptake of 131I radioactivity peaked after approximately 30 min of incubation with [131I]EGF, following which time it declined, while 99mTc radioactivity continued to increase over a 6 h incubation with [99mTc]-EGF. To determine if radiolabeled EGF had in vivo tumor-localizing properties, C6EGFR glioma cells were implanted stereotactically into the brains of Fischer rats. Four weeks later, either 99mTc- or 131I-labeled EGF was injected intracerebrally into normal or glioma-bearing animals using the same stereotactic coordinates. External gamma scintigraphy revealed that 131I radioactivity disappeared rapidly from the brain regions of tumor-bearing animals compared to 99mTc, approximately 50% of which remained in the tumor for up to 12 h. In contrast, only approximately 20% remained in the brains of non-tumor-bearing animals after 6 h. These studies are the first to describe a method for radiolabeling EGF with 99mTc and to detect it by external scintigraphy in the brains of tumor-bearing animals.

Targeting peptides for pleural cavity tumor radiotherapy: specificity and dosimetry of Re-188-RC-160.

Re-188-RC-160, a radiolabeled somatostatin analogue, is being explored for its potential as a local/regionally administered radiotherapeutic agent targeting somatostatin receptor-positive tumors. In studies in vitro and in vivo, Re-188-RC-160 was found to bind to somatostatin receptor-positive cells (NCI-H69, human small cell lung carcinoma), but not to binding-negative cells (Raji, Burkitt's lymphoma). The comparative binding of Re-188-labeled RC-160 [cyclic NH2-(D)-Phe-Cys-Try-(D)-Trp-Lys-Val-Cys-Trp-NH2] or CTOP [cyclic NH2-(D)-Phe-Cys-Try-(D)-Trp-Orn-Thr-Pen-Thr-ol], a mu-opioid receptor antagonist used a negative control compound, was also determined in vitro and in vivo using NCI-H69 cells as targets. Re-188-RC-160 demonstrated higher net binding in vitro and in vivo compared to Re-188-RC-CTOP, and in vitro its binding could be reduced by excess unlabeled RC-160 whereas binding of Re-188-CTOP could not be reduced. Using another somatostatin receptor-positive human tumor line, ZR-75-1, a substantial amount of the cell-bound Re-188-RC-160 was found to be internalized. Does estimates for Re-188-RC-160 in animals containing NCI-H69 tumors indicated that with three serial injections therapeutic doses greater than 60 Gy can be achieved.

Biodistribution and clearance of 99mTc-labeled Arg-Gly-Asp (RGD) peptide in rats with ischemic acute renal failure.

Based on the previous demonstration of a renoprotective effect of arginine-glycine-aspartic acid (RGD) peptides in acute renal failure, experiments were designed to test the distribution and renal accumulation of the peptide. To accomplish this goal, in this study, RGD peptide was radiolabeled and its biodistribution and renal accumulation was determined in rats with ischemic acute renal failure (ARF). 99mTc-RGD with or without 111In-DTPA were injected intravenously in control and ARF rats. Various organs were dissected at different times after injection and subjected to gamma-scintillation counting and autoradiography (ARG). Blood clearance of 99mTc-RGD was rapid, with t1/2 < 10 min, and unchanged in ARF compared with control rats. Kidneys retained the largest portion of the injected dose in both control and ARF rats, as detected using scintillation counting and whole-body ARG (10.56 +/- 1.05% and 10.12 +/- 3.16% injected dose/g wet weight, respectively). Renal ARG revealed a significant increase in binding to the cortex in ARF kidneys, compared with that of control kidneys. Given the differences in renal blood flow and GFR in control and postischemic kidneys, the next series of experiments was performed with two radiopharmaceuticals, 99mTc-RGD and 111In-DTPA. The ratio of 99mTc-RGD:111In-DTPA was increased more than three-fold in ARF kidneys compared with control kidneys (2.7 +/- 0.15 versus 0.8 +/- 0.19, respectively). The results indicate that (1) RGD peptide undergoes a rapid clearance predominantly via the renal route; (2) despite a significant reduction in the renal perfusion, 99mTc-RGD peptide accumulates in the postischemic kidney; (3) this is consistent with the hypothesis on the involvement of RGD-recognizing integrins in the development of tubular obstruction in renal ischemia.

Clinical radiopharmacy: principles and practices.

On the average, radiopharmacists spend about 17.2% of their time in clinical activities if their practice setting is in an institution, and about 8.5% of their time if their practice setting is in a centralized nuclear pharmacy. A recent survey of radiopharmacists was conducted to determine: (1) the percentage of time they spend engaged in selected activities, and (2) the specific clinical activities in which they are involved. A few radiopharmacists spend as much as 50% of their time in clinical activities, but most spend only 5% to 20% of their time. Some of the clinical activities involve direct interactions with patients, such as explaining the reasons for administering the radioactive material or actually administering the dose. Other clinical activities are indirect, such as reviewing charts before or after studies and making recommendations to other health care professionals. About half of the pharmacists surveyed see a need for increasing their clinical activities. The need to maximize the time involved in providing pharmaceutical care is discussed and several patient-care activities/responsibilities are proposed.

Experimental radiotherapy of receptor-positive human prostate adenocarcinoma with 188Re-RC-160, a directly-radiolabeled somatostatin analogue.

The therapeutic potential of the somatostatin analogue RC-160 radiolabeled with 188Re was evaluated in nude mice bearing xenografts of human prostate adenocarcinoma. 188Re-RC-160 was selectively retained in both DU-145 and PC-3 tumors following direct intra-tumor injection at all time points examined (2, 6 and 24 hr post-injection). Unbound 188Re-RC-160 was rapidly excreted via the hepatobiliary system and, with the exception of the gastrointestinal tract, very little normal organ uptake was found at any time point examined. Negative control compounds, 188Re-perrhenate and 188Re-mercaptoacetyl-triglycine (188Re-MAG3), were essentially washed out of the tumor by 6 hr post-injection and were rapidly excreted through the kidneys. 131I-RC-160, used as reference compound, had a biodistribution in tumor-bearing animals similar to that of 188Re-RC-160. In PC-3 xenografts, 188Re-RC-160 gave a dose-dependent therapeutic response (stasis or regression) even in animals with relatively large tumor masses (greater than 600 mm3), whereas the macro-aggregated form of 188Re-RC-160 did not. Long-term studies with 188Re-RC-160 demonstrated a protracted reduction of tumor volume and a positive effect on animal survival. Neither RC-160 by itself nor a 188Re-labeled peptide, unrelated to somatostatin (PA-22-2, a laminin peptide), demonstrated the reduction in tumor mass observed with 188Re-RC-160. 188Re-RC-160 shows potential as a new clinical agent for treatment of somatostatin-receptor-positive cancers.

Re-188 labelled antibodies.

Monoclonal antibodies can be directly labelled with 188Re using a simple one-step radiolabelling kit. Using B72.3 as a model antibody, the formulation was optimized and kits were made and tested and compared to data previously reported for the same antibody labelled with other radioisotopes. Labelling with Re-188 was carried out with the eluate of a W-188/Re-188 generator from Oak Ridge National Laboratory. Fresh generator eluate was added to the pre-reduced lyophilized antibody and the mixture allowed to incubate overnight at room temperature. The radiochemical purity, immunoreactive fraction, and biodistribution in normal and LS174T tumor bearing nude mice was determined. The radiochemical purity was 88 +/- 7%, the immunoreactive fraction was 68.46 +/- 3.8%. The immunoreactive fraction was higher than any previously reported for this antibody when labelled with other radioisotopes. At 48 h, 7.9 +/- 2.4% of the injected dose per gram was found in the tumor. The biodistribution and tumor uptake of Re-188 labelled B72.3 was similar to that previously reported for Re-186 and In-111 labelled B72.3.

Quantitation of radiolabeled antibody binding to cells by thin-layer chromatography.

Thin layer chromatography (TLC) was used to monitor binding of radiolabeled antibodies to cells. Labeled antibodies were reacted with cells and aliquots chromatographed on serum-blocked, ITLC strips. The cell-antibody complexes remain at the origin and unbound antibody migrates with the solvent front. The antibody binding was estimated from the ratio of radioactivity at the origin compared to the total applied. Separations are completed in about 10 min. This method does not use centrifugation or wash steps, and provides an inexpensive and self-contained system to evaluate radioligand binding. Cell binding assay results using this method are approximately the same as those obtained using bead- or cell-type assays.

Affinity thin-layer chromatography test of the immunoreactive fraction of radiolabeled antibodies.

A simple, rapid and self-contained system for assaying the immunoreactive fraction of radiolabeled antibodies was developed using affinity thin-layer chromatography (ATLC). ATLC combines use of solid-phase-bound antigen and conventional TLC. The technique is an improvement over existing means of measuring immunoreactive fraction (bead-type or cell-type assays) in that it has neither wash steps nor centrifugation steps, yet provides results essentially identical to those obtained with the more time-consuming assays. ATLC is accomplished using chromatography strips that are coated with antigen material in a discrete region near the origin. The antigen-coated strips are then blocked in serum, air-dried and stored. For use, radiolabeled antibody is spotted at the origin, and the strip is developed using a buffered solvent. Immunoreactive antibody binds to the antigen at or near the origin, while radioactivity not associated with immunoreactive antibody migrates with the solvent front. Antigen-negative strips (serum-blocked only) are used to measure "nonspecific" binding. The ATLC development time is about 16 min, and the results can be obtained in about 30 min. The assay described in this report uses antigens from colon tumor and is suitable for use with B72.3 and other colon cancer-reactive antibodies.

Biological distribution of 99mTc-labeled YIGSR and IKVAV laminin peptides in rodents: 99mTc-IKVAV peptide localizes to the lung.

Two laminin-derived peptides containing either YIGSR or IKVAV (single amino acid code) sequences were radiolabeled with 99mTc and their biological distribution evaluated in rodents. Both 99mTc-peptides cleared rapidly from the circulation though the kidney, and to a lesser extent, through the liver. 99mTc-YIGSR peptide did not accumulate in any organ examined in normal, tumored, and emphysemic mice. The 99mTc-IKVAV peptide localized within 10 min to the lung of normal animals, resulting in lung-to-blood ratios of approximately 23:1. The 99mTc-IKVAV peptide localized to lung after submicron filtration and after intraperitoneal injection, suggesting that particulates do not major role in localization. Pre-incubation of 99mTc-IKVAV peptide in whole blood decreased lung localization, suggesting that margination of radiolabeled cells does not play a major role in the lung localization. When 99mTc-IKVAV was injected into mice with tumored lungs (melanoma), the lung uptake was markedly increased (up to 20% injected dose higher than control lungs) at all time points examined (10, 30, and 120 min). When 99mTc-IKVAV was injected into mice with genetic emphysema, the lung uptake was markedly decreased at all time points. The localization of the 99mTc-IKVAV-containing peptide to the lung is consistent with a receptor-based mechanism.

Comparison of duplex ultrasonography and ascending contrast venography in the diagnosis of venous thrombosis.

The application of duplex ultrasonography to the diagnosis of venous thrombosis requires validation by comparison of the duplex findings with the results of ascending contrast venography. In this study, 2534 veins were examined by both methods with contrast venography results serving as the standard for comparison. In this setting, duplex ultrasonography proved to be 100% sensitive and 99% specific for venous thrombosis. Duplex ultrasonography is as reliable as venography in the diagnosis of venous thrombosis and has no associated risks or known complication. In addition, duplex ultrasonography provides information regarding pathologic anatomy that is comparable to the detail provided by high-quality venography. The authors conclude that duplex ultrasonography should be the diagnostic method of choice for evaluating patients with suspected venous thrombosis.

Abscess scintigraphy with 99mTc-human immunoglobulin (IgG) using a one-step labeling method.

It was shown earlier that non-specific human gamma globulin (IgG) labeled with 111In can be used as an agent for abscess localization. We describe experimental results with 99mTc-IgG in animals bearing abscesses and tumors using a one-step labeling method with 99mTc. We studied this compound in several animal models: mice bearing turpentine abscesses and subcutaneously transplanted sarcomas, in rats with turpentine or E. coli abscesses and intracerebrally implanted gliomas and in rabbits with E. coli or turpentine abscesses. Blood clearance was studied in dogs. It was found that the absolute concentration of 111In-IgG in abscess and tumor was higher than that of 99mTc-IgG. However, the abscess-to-tumor ratio was higher for 99mTc-IgG. The 99mTc-IgG images were of high quality and abscesses could be detected as early as 30 min post-injection (p.i.). It appears that 99mTc-IgG has many potential advantages over 111In-IgG because of better physical properties of 99mTc, simpler preparation, lower cost and greater availability and the possibility of using higher 99mTc doses.

Imidazoles as well as thiolates in proteins bind technetium-99m.

99mTc is widely thought to directly bind proteins through thiolate groups of cysteine residues, resulting in Tc-cysteinyl-protein bonds. Chemical reduction of disulfide bonds in proteins is widely used to generate thiolates with the goal of increasing 99mTc binding. This strategy is used because most proteins contain no thiolates, but many do contain disulfide bonds. In this study, we have evaluated the hypothesis that imidazole groups of histidine are also involved in direct 99mTc binding to proteins. Human gamma-globulin was used as the model protein in these studies. The immunoglobulin was used (a) without reduction or was (b) treated with stannous ions to reduce disulfide bonds thereby increasing thiolate concentration. These proteins were used to evaluate the hypothesis that imidazole as well as thiolate groups bind Tc. The proteins were evaluated by (a) using free amino acids to compete with proteins for 99mTc and (b) by chemical modification of amino acid side chains. In addition, peptides known to contain either cysteine or histidine, but not both, were also successfully directly labeled with 99mTc. These results indicate that in proteins (and peptides) imidazole-containing groups as well as thiolate-containing groups bind Tc.

Similarity of copper and technetium binding sites in human IgG.

Kits for direct labeling of IgG with 99mTc were used without modification for the preparation of [67Cu]IgG. The IgG was pre-treated to generate thiolate groups which would bind 67Cu. The direct labeling of reduced IgG with 67Cu was highly efficient, resulting in approx. 95% 67Cu binding. Non-reduced IgG (negative control) had labeling efficiencies of less than 10%. IgG pre-exposed to Cu(II) had reduced amounts of 99mTc bound to it. The results demonstrate a direct relationship between copper- and 99mTc-binding sites in IgG.

Duplex ultrasonographic insonation and visualization of intracerebral arteries.

Until now, clinical, noninvasive interrogation of intracranial vessels has consisted only of insonation via transcranial Doppler. Such devices have utilized a 2.0 MHz, continuous wave probe with Doppler spectral waveform display. Clincial aapplication of these techniques has required precise location of cranial "windows" and has been hampered by the extreme anatomic variability of both cranial bony structures and intracerebral arteries. The lack of simultaneous intracranial arterial visualization has limited the clinical pplicability of transcranial Doppler technology. Recently, the authors have utilized a 2.25 MHz curved phased array probe with a pulsed Doppler to image and insonate simultaneously the intracerebral arteries. Colorflow imaging of both near- and far-field regions is the necessary first step for vessel localization and identification. Once this is accomplished, the image of each artery in turn is amplified and gray scale tuning is employed to permit direct visualization of the arterial walls and lumen. Pulsed Doppler waveform analysis is perfomred simultaneously and along the entire visible artery length. In this manner the arteries of both right and left hemispheres are examined in detail. We have found the duplex technique to be superior to the use of Doppler alone in the examination on intracerebral vessels. The ability to visualize and insonate simultaneously eliminates the uncertainty caused by anatomic variation. These advantages, long applied to the evaluation of peripheral vessels, are now available for use in the diagnosis of intracranial arterial disease.

Direct labeling of proteins with 99mTc.

The direct labeling of antibodies and antibody fragments to form a highly stable bond between technetium and the sulfide groups of proteins is now well established. To optimize this reaction, the antibody protein must have sufficient reactive sulfides available to accept that technetium metal ions that are formed by the reduction of pertechnetate in the presence of a weak complexing agent. The reactive sulfide groups are provided by first reducing a small fraction of the disulfide bridges in the antibody protein or by starting with Fab' fragments, which already have reactive sulfide groups. When the antibody protein has been appropriately reduced, and the reactive sulfide groups protected by a metal ion with a lower binding affinity than technetium, such as tin or zinc, very high labeling yields of high-affinity-bonded 99mTc can be achieved. This can be accomplished without loss of immunoreactivity, measured as either affinity or immunoreactive fraction. Side reactions can produce radiochemical impurities such as low-affinity, bound 99mTc; 99mTc colloids; 99mTc peptides or antibody aggregates; or 99mTc-complexes. Also, pertechnetate ions may be an impurity if the sodium pertechnetate solution added to the reduced antibodies is not completely reduced. The specifics of minimizing these side reactions have not been extensively discussed in the prior literature; however, it is clear that appropriate reduction of the protein prior to labeling and complete removal of the reducing agent, particularly if it contains reactive sulfide groups or is toxic, are critical. One- or two-step 99mTc-labeling kits for preparing 99mTc-labeled antibody or antibody fragments are rapidly being introduced for use in clinical nuclear medicine studies. These direct labeling methods employ a common sequence of chemical reactions, although the reducing agents for both the antibody and the [99mTc]pertechnetate may vary. Different 99mTc transfer agents may be used, but all transfer agents have the common feature of quickly forming weak to moderately strong complexes with reduced technetium. Most use Sn(II) to reduce the pertechnetate, although other reducing agents can be used.

Quality control test for immunoreactivity of radiolabeled antibody.

A rapid method for the measurement of the immunoreactive fraction of a radiolabeled monoclonal antibody or antibody fragment has been developed. This may be used as a quality control test prior to patient administration of the radiolabeled antibody preparation. The test employs solid phase antigens and the assay is conducted under conditions of antigen excess. Assay parameters have been evaluated and a standardized procedure has been developed. The assay has been compared to a standard extrapolation method and found to give approximately the same result. The test has been used on four different radiolabeled antibodies currently in clinical trials in patients with colorectal cancer. Mean immunoreactive fractions for these radiolabeled antibodies ranged from 35 to 65% and the variability of the immunoreactive fraction ranged from 140 to 240% for different antibodies. We conclude that the quality, defined as the immunoreactive fraction, of radiolabeled antibodies is both low and highly variable, indicating the need for a quality control test of these radiopharmaceuticals in the clinic prior to patient administration.