Radioimmunotherapy with alpha-particle-emitting immunoconjugates.

Alpha particles are energetic short-range ions whose higher linear energy transfer produces extreme cytotoxicity. An alpha-particle-emitting radioimmunoconjugate consisting of a bismuth-212-labeled monoclonal immunoglobulin M specific for the murine T cell/neuroectodermal surface antigen Thy 1.2 was prepared. Analysis in vitro showed that the radioimmunoconjugate was selectively cytotoxic to a Thy 1.2+ EL-4 murine tumor cell line. Approximately three bismuth-212-labeled immunoconjugates per target cell reduced the uptake of [3H]thymidine by the EL-4 target cells to background levels. Mice inoculated intraperitoneally with EL-4 cells were cured of their ascites after intraperitoneal injection of 150 microcuries of the antigen-specific radioimmunoconjugate, suggesting a possible role for such conjugates in intracavitary cancer therapy.

First human treatment of resistant neoplastic meningitis by intrathecal administration of MTX plus (125)IUdR.

PURPOSE: Neoplastic meningitis is often the final outcome of disseminated cancer and is rapidly lethal. Its limited treatment relies on systemic or intrathecal chemotherapy with methotrexate (MTX) or thiotepa. When 5-iodo-2'-deoxyuridine labeled with (125)I ((125)IUdR) is incorporated into the DNA of mitotic tumor cells, the Auger electrons emitted during iodine decay are highly cytotoxic. The radiotherapeutic efficacy of (125)IUdR administered intrathecally has also been established in animals bearing spinal cord tumors, and MTX is known to potentiate the response. This approach has not been tested in the clinic. METHODS: A 44-year-old woman, with locally advanced pancreatic cancer, was treated for three years with complete systemic remission, but then relapsed with cytologically proven neoplastic meningitis. The patient was given four successive intrathecal injections of MTX (10 mg) every 12 h and, with the fourth dose, 1850 MBq (125)IUdR, followed by four additional MTX doses. The response was monitored by cytology and CA19.9 (carbohydrate antigen 19.9) levels in the cerebrospinal fluid (CSF) as well as by clinical status of the patient. RESULTS: The follow-up of cytology and CA19.9 levels in the CSF showed dramatic improvement within 26 days followed by a biological relapse on Day +36. There was no evidence of local central nervous system toxicity. Three months later, neoplastic meningitis recurred and meningeal tumor infiltration was observed on magnetic resonance imaging. Six months after MTX-(125)IUdR treatment, the patient died. CONCLUSION: (125)IUdR treatment proved to be feasible without acute neurological toxicity and seemed to have produced a biological response. This attempt provides the basis for designing prospective clinical trials.

Absence of preferential uptake of [125I]iododihydrorhodamine 123 by four human tumor xenografts.

The biodistribution of [125I]iododihydrorhodamine 123 has been studied over a 96-h period in four human tumor xenograft models: HT-29 colon adenocarcinoma, PC-3 prostate carcinoma, HT-1080 fibrosarcoma, and PaCa-2 pancreatic carcinoma. Elimination of radioactivity in the tumor-bearing nude mice was rapid during the first 24 h and slow thereafter. The lack of uptake in the thyroid indicated there was little, if any, deiodination of the molecule. Activity was found mainly in the liver and spleen. Accumulation of radioactivity was low in all four tumors examined. At 4 h postinjection, as well as at 24 and 48 h, however, the total radioactive content in each of the four tumors was directly proportional to the weight of the tumor sample. This correlation was independent of tumor type, route of injection (i.v./i.p.) or dose (1.2-6 microCi/mouse). This was not true for any of the normal tissues, suggesting that this accumulation may be governed by certain intrinsic characteristics of the cancers tested.

Specific uptake of the auger electron-emitting thymidine analogue 5-[123I/125I]iodo-2'-deoxyuridine in rat brain tumors: diagnostic and therapeutic implications in humans.

Glial neoplasms of the human central nervous system are malignancies that have defied treatment. Part of the problem lies in the limitations of current diagnostic techniques which are unable to identify small collections of neoplastic glia within normal parenchyma and in the difficulty of sterilizing these tumors because of limited selectivity of the cytotoxic agents available. The thymidine analogue 5-iodo-2'-deoxyuridine (IdUrd) radiolabeled with 123I and 125I was injected directly into an intracerebral rat 9L gliosarcoma and found to be a sensitive and specific agent for the detection of this neoplasm in rats. External gamma camera imaging (123I) visualized tumors as small as 0.5 mm in diameter. Autoradiography (125I) indicated that IdUrd was incorporated into the DNA of neoplastic glia only. Since 123I emits gamma-photons suitable for scintigraphy, [123I]IdUrd holds promise for the diagnosis of brain tumors in humans as well. Furthermore, since 123I and 125I are Auger electron emitters that have demonstrated antineoplastic effects, direct administration of [123I]IdUrd or [125I]IdUrd into tumors may also have potential for the treatment of central nervous system malignancies.

Fluoresceinated phosphoethanolamine for flow-cytometric measurement of lipid peroxidation.

A new lipophilic fluorescein probe (fluor-DHPE) has been identified that can assay lipid peroxidation in mammalian cells on a cell-by-cell or selected-cell-subpopulation basis by flow cytometry. Application of this approach requires that the fluorescent probe be nonexchangeable among cells. Fluorescein is an appropriate fluorophore, since its fluorescence matches the specifications of common flow cytometers and the compound loses its fluorescence upon reaction with peroxyl radicals. Upon examination of four lipophilic derivatives of fluorescein, fluor-DHPE was found to be the only probe that was nonexchangeable among labeled and unlabeled rat RBC for at least 24 h. The exposure of fluor-DHPE-labeled RBC to benzoyl peroxide followed by mixing the sample with RBC unexposed to peroxide led to a decrease in fluorescence. Furthermore, the flow cytometer could clearly select the subpopulation of cells undergoing lipid peroxidation from those cells that were not. Fluor-DHPE-labeled-RBC obtained from rats and exposed to cumene hydroperoxide also displayed a gradual decrease in fluorescence. This decrease was preventable by either regulation of the vitamin E content in the animal diet or in vitro supplementation of cells with vitamin E. We conclude that fluor-DHPE is a stable and nonexchangeable probe for monitoring lipid peroxidation in cell subpopulations by flow cytometry.

A fluorimetric method for the detection of copper-mediated hydroxyl free radicals in the immediate proximity of DNA.

An optical method to detect copper-mediated hydroxyl free radicals generated close to DNA and other biomolecules has been developed. Low-molecular-weight polylysines were labeled with SECCA, a derivative of coumarin that generates the fluorescent 7-OH-SECCA following its interaction with hydroxyl free radicals in aqueous solution. These polylysines were then complexed with DNA to place the detector molecule SECCA in the vicinity of the nucleic acid. Following addition of copper sulfate (0-10 mumol dm-3), free radicals were generated by incubation with ascorbic acid (0-1 mmol dm-3) and hydrogen peroxide (0-1 mmol dm-3). A rapid increase in the induced fluorescence was observed corresponding to the formation of the fluorescent 7-OH-SECCA in the polylysine-nucleic acid complex. This fluorescence was not decreased significantly by addition of high concentrations of hydroxyl free-radical scavengers (DMSO, methanol, ethanol and tert-butanol), but was diminished by addition of relatively low concentrations of EDTA (0.1 mmol dm-3), histidine (0.1 mmol dm-3) or catalase (8.3 x 10(-5) mmol dm-3). On the other hand, when such reaction mixtures were incubated with SECCA molecules that were free in solution or SECCA-labeled polylysine in the absence of DNA, the induced fluorescence was diminished by all hydroxyl free-radical scavengers. The efficiency by which the scavengers reduce the fluorescence increases as their hydroxyl rate constant increases. The data indicate that the detector molecule SECCA can be used to detect copper-mediated hydroxyl free radicals generated close to DNA.

Novel fluorescein-based flow-cytometric method for detection of lipid peroxidation.

The novel property of fluorescein to detect peroxyl radicals is demonstrated. On the basis of this observation, a fluorescein-based, flow-cytometric method to directly and continuously detect free radicals generated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein-labeled polylysine lose their fluorescence gradually upon addition of a peroxyl-radical-generating system (thermal decomposition of 2,2'-azobis(2-amidinopropane) [AAPH]). 5-/6-CF retains its fluorescence when exposed to AAPH in the presence of the peroxyl radical scavenger Trolox. When 5-/6-CF free in solution is incubated with red blood cells exposed to cumene hydroperoxide (CH), a similar loss of fluorescence occurs due to lipid peroxidation on RBC membranes, which is preventable by pretreatment of the cells with Trolox or vitamin E. Undecylamine-fluorescein (C11-fluor), a lipophilic fluorescein conjugate, has been incorporated into the membranes of RBC. Upon addition of CH, a decrease in fluorescence is fluorometrically observed that is proportional to the amount of hydroperoxide added and inhibited by preincubation with Trolox or vitamin E. Flow-cytometric studies are then performed to demonstrate that C11-fluor can monitor free radicals generated during lipid peroxidation on a cell-by-cell basis. When exposed to CH, a time-dependent shift of the flow-cytometric profile toward lower values is observed that is inhibited by Trolox or vitamin E. This approach in conjunction with multiparametric flow cytometry may allow examination of the biologic significance of lipid peroxidation by correlation to other cellular end points on single cells.

Measurement of hydroxyl radicals catalyzed in the immediate vicinity of DNA by metal-bleomycin complexes.

A recently developed sensitive fluorimetric assay has been used to examine whether free hydroxyl radicals (HO.) are generated in the immediate vicinity of DNA by Fe(II)-bleomycin. When aqueous solutions of SECCA (the succinimidyl ester of coumarin-3-carboxylic acid) are irradiated with gamma rays or incubated with Fe(II)-bleomycin or Fe (II)-EDTA in the presence of ascorbate and H2O2, 7-hydroxy-SECCA, a fluorescent product of the interaction of HO. with SECCA, is generated. Studies with catalase and several HO. scavengers indicate that the fluorescence induction is mediated by HO. On the contrary, Cu(II)-bleomycin complexes under similar conditions fail to induce 7-hydroxy-SECCA fluorescence. When SECCA is conjugated to DNA via SECCA-polylysine-DNA complexes and incubated in the same iron-containing systems, the relative ability of the scavengers to reduce the fluorescence again demonstrates the generation of 7-hydroxy-SECCA by HO. However, while the fluorescence is practically eliminated by high concentrations of DMSO (100 mumols dm-3) in the systems with Fe(II) or Fe(II)-EDTA, it is not possible to reduce it similarly in the case of Fe(II)- bleomycin. These data demonstrate the generation of HO. by Fe(II)-bleomycin in the immediate vicinity of DNA. Because the experiments simulate the lifetime of HO. expected in cells, these data suggest that, if such DNA-associated HO. radicals are also produced in vivo by bleomycin, these would not be scavengable by intracellular scavengers and they could interact with chromatin.

HPLC analysis of the dissociation and recombination of rabbit immunoglobulin G.

Rabbit immunoglobulin G (RIgG) was reduced with dithioerythritol and analyzed by high performance liquid chromatography. A quantitative method for determining the percentage of reduced half-molecules in the mixture was developed. An acetic acid concentration-dependent rate of dissociation of reduced half-molecules was observed. The specific optical absorptivity was determined for whole molecules and half-molecules and found to be significantly greater for the half-molecules. Purified half-molecules were reconstituted into RIgG with a yield greater than 90% following a 16 h incubation at pH 8.0 and room temperature.

Biodistribution studies of anti-Thy 1.2 IgM immunoconjugates: implications for radioimmunotherapy.

We have prepared 111In radioimmunoconjugates (RICs) of the IgM isotype with specificity for the murine T cell/neuroectodermal surface antigen, Thy 1.2. Using gamma camera immunoscintigraphy, we have analyzed the biodistribution patterns of the RICs after intravenous and intraperitoneal injection into normal Thy 1.2+ and Thy 1.2- mice. Both routes of administration show antigen-specific uptake by the splenic T lymphocyte population. A high degree of nonspecific uptake by the reticuloendothelial system is also observed. Analysis of the specific activity of various segments of spleens from RIC-injected animals shows inhomogeneous uptake of the RIC not readily apparent by immunoscintigraphy. Animals injected with the RIC and then given high dose total body irradiation showed rapid shifts in radionuclide distribution away from the target cell population and into the general reticuloendothelial system, suggesting that death of the target cell can alter RIC biodistribution. Analyses of RIC biodistribution patterns will contribute to optimization of treatment by radioimmunotherapy.

5-[123I]iodo-2'-deoxyuridine in the radiotherapy of an early ascites tumor model.

The extreme biological toxicity of Auger emitters is caused by the decay-associated, highly localized deposition of energy. The antineoplastic capability of an Auger-electron emitter, iodine-123, incorporated into the thymidine analog, 5-iodo-2'-deoxyuridine (IUdR) was evaluated in an intraperitoneal (i.p.) murine ovarian tumor (MOT) in female C3HeB/FeJ mice. Total doses of 0.37 to 8.88 MBq (10-240 microCi) 123IUdR were administered i.p. in five equally divided fractions at 24, 28, 32, 36, and 40 hr after the i.p. inoculation of 0.5 to 1.6 x 10(6) tumor cells per mouse. Control tumor-bearing animals were injected with identical volumes of saline at 4-hr intervals. Biodistribution studies demonstrated a distinct and localized uptake of 123IUdR in the MOT cells (1% of the injected dose was associated with MOT cells 24 hr after the last injection), whereas in animals without tumor there was no radioactivity associated with the peritoneal cells. Analogous results were obtained from scintigraphic images where the focal area of abdominal activity persisted only in MOT-bearing mice while it cleared from the abdomen of the controls. The 50% survival (median survival) of the control group was 19 days for an inoculum of 1.6 x 10(6) MOT cells per animal, whereas the median survival of MOT-bearing animals treated with 123IUdR increased by 11 days for the highest administered dose (8.88 MBq, 240 microCi) and resulted in a 20% absolute survival at 7 weeks. Statistically significant absolute survival prolongation was found with all of the total administered doses. The prolongation of both median and absolute survival time of the tumor-bearing animals treated with 123IUdR conclusively indicates the substantial antineoplastic activity of the Auger-electron emitter iodine-123.

[125I/127I/131I]Iodorhodamine: synthesis, cellular localization, and biodistribution in athymic mice bearing human tumor xenografts and comparison with [99mTc]hexakis(2-methoxyisobutylisonitrile).

The synthesis of halogenated rhodamine (Rh) derivatives was carried out by controlling the stoichiometry of the halogenating agents, bromine and iodine monochloride. In the no-carrier-added synthesis of radioiodinated rhodamine 123, direct labeling of rhodamine 123 (Rh 123) with Na125I/Na131I required the presence of the oxidant peracetic acid. 125I/131I-Rh 123 was synthesized in modest yields (40-45%). HPLC purification separated Rh 123 from its mono- and diiodo derivatives. Monohalogenation of Rh 123 did not alter the compound's ability to permeate viable cells and localize in mitochondria. 125I/131I-Rh 123 was stable in serum in vitro but rapidly metabolized after intravenous injection into mice. Consequently, scintigraphy and biodistribution data reveal poor targeting of subcutaneously growing human tumor xenografts. The results are compared to those obtained following the administration of [99mTc]hexakis(2-methoxyisobutylisonitrile) which also did not image human tumor xenografts in nude mice.

Synthesis and biological studies of iodinated (127/125I) derivatives of rhodamine 123.

Rhodamine 123, a mitochondrial stain that preferentially accumulates in certain cancer cells, has been reduced and iodinated by using NaI in the presence of N-chlorosuccinimide. The various mono-, di-, and triiodo derivatives have been isolated and characterized. These nonfluorescent compounds are taken up by mammalian cells, become fluorescent within the cytoplasm (presumably following oxidation), and show the same pattern of localization as the parent compound. Iodination with no-carrier-added Na125I yields the same mixture of compounds. All 125I derivatives accumulate preferentially in PC3 adenocarcinoma cells compared with V79 lung fibroblasts, with the differential being greatest for the monoiodo compound, followed by the di- and triiodo derivatives.

[125I/127I]iodoHoechst 33342: synthesis, DNA binding, and biodistribution.

An iodinated analog of the DNA-minor-groove-binding agent Hoechst 33342 has been synthesized and evaluated for DNA binding and tumor targeting. The bis-benzimidazole ring system of the title compound was constructed from the piperazinyl terminus via a Pinner-type cyclization followed by oxidative cyclization of the diamine Schiff base. To synthesize radioiodoHoechst 33342, (trimethylstannyl)Hoechst 33342 was prepared by the same strategy and subjected to mild radioiododestannylation in the presence of lactoperoxidase. After purification by HPLC, the radiochemical was separated in carrier-free form with > 85% radiochemical yield and > 99% chemical and radiochemical purity. Fluorescence spectrometric analysis of the binding of iodoHoechst 33342 to calf thymus DNA gave an equilibrium association constant (Ka) of 2.57 x 10(7) M-1 comparable to the Ka value of Hoechst 33342. Fluorescence microscopy of viable V79 cells demonstrated that the iodinated dye stained the nuclei with avidity similar to that of the noniodinated dye. The biodistribution of [125I]-iodoHoechst 33342 in LS174T tumor-bearing athymic mice 4 h postadministration showed a tumor uptake of 3-4% injected dose per gram (ID/g), tumor/blood ratio of 6-8, and tumor/ nontumor ratios above unity for most organs. A low thyroid uptake (approximately 2% ID/g) indicated that the radiochemical did not deiodinate and was stable in vivo.

A rapid and reproducible method for the separation of cells from radioactive media.

A simple method is described for the rapid separation of cells, containing radioactive material, from their suspending medium. Cells, in aqueous medium, are layered on fetal bovine serum in microfuge tubes. The tubes are immediately spun for 1 min, frozen in ethanol-dry-ice, and their tips sliced and assayed. This method is both faster and more convenient than currently employed techniques.

Chemotoxicity of indium-111 oxine in mammalian cells.

We have studied the uptake and toxicity of [111In]oxine in Chinese hamster V79 lung fibroblasts. The incorporation of the radionuclide into these cells reached a plateau within 2 hr. Uptake was proportional to the extracellular radioactive concentration. Both radioactive and "decayed" [111In]oxine exhibited similar toxicities, indicating that the observed toxicity was chemical in nature. These results are discussed in terms of the present status of this radiolabeling agent.

Preclinical animal studies with radioiododeoxyuridine.

UNLABELLED: We examined the diagnostic and therapeutic potential of 5-iodo-2'- deoxyuridine (IUdR) radiolabeled with the Auger electron emitters 123I and 125I in several animal tumor models. METHODS: Experiments were conducted using mice bearing an intraperitoneal ovarian tumor (*IUdR by intracavitary injection), and rats bearing either bladder cancer (*IUdR by intravesical injection), brain gliosarcomas (intratumoral injection), or intrathecal gliosarcomas (intrathecal injection). RESULTS: After locoregional administration, [123I]IUdR and [125I]IUdR localize within tumor, clear rapidly from the rest of the body and are therapeutically effective. CONCLUSION: When labeled with the Auger electron emitter 123I or 125I, IUdR demonstrates therapeutic efficacy. In addition, [123I]IUdR has a potential role in the scintigraphic detection of cancer.

Limitations of conventional internal dosimetry at the cellular level.

A theoretic examination of the validity at the cellular level of assumptions used in classic internal dosimetry has been undertaken. An alternate dosimetric model accounting for the consequences of selective uptake of a radiolabeled compound by specific cells in a multicellular cluster of hexagonal geometry has been developed. At the cellular level, derived dose estimates for electrons have been compared to dose estimates obtained employing the assumptions of conventional internal dosimetry. The study has been performed for all electron energies and then applied specifically to electrons emitted by 99mTc, 201Tl, 111In, and 123I. The dosimetric consequences of altering (a) the intracellular-to-extracellular radionuclide concentration, (b) the labeled cell density, and (c) the cell size have been examined for the labeled and nonlabeled cells in a cell cluster, and the conditions in which conventional dosimetry underestimates or overestimates the dose to individual cells have been indicated. It is shown that when selective intracellular uptake of a radiolabeled compound occurs in specific cells within a cell cluster, conventional dosimetry underestimates the radiation dose delivered to the labeled cells by twofold to more than 25-fold if the emitted electrons have ranges of a few micrometers or less, i.e., energies smaller than approximately 10 keV. Under the same conditions, conventional dosimetry overestimates slightly (20% to 50%) the electron radiation dose to the nonlabeled cells of the cell cluster. It is shown that inclusion of photons in the calculation of the total dose to individual cells does not alter significantly the conclusions of the present investigation.

Intracellular distribution and radiotoxicity of chromium-51 in mammalian cells: Auger-electron dosimetry.

The kinetics of uptake and of radiotoxicity of chromium-51, an Auger-electron emitter, have been studied in V79 lung fibroblasts of the Chinese hamster. Intracellular radioactivity was directly proportional to the incubation period and to the extracellular concentration of the Cr-51. About 14% of the cellular activity was associated with the nucleus, whereas approximately 2% was guanidine-precipitable and therefore bound to DNA. The growth rate of V79 cells was slowed following intracellular incorporation of Cr-51. The cell-survival curve, in terms of colony-forming ability, was of the low-LET type, with a D37 of 6.2 pCi/cell. Theoretical dosimetric estimates indicate that, under the given experimental conditions, the mean lethal dose to the cell nucleus was 870 rad. Although this value is somewhat larger than the x-ray D37 dose of 580 rad for this cell line, it is more realistic than the gross underestimate obtained by classical MIRD calculations (2-3 rad/cell).

Kit formulation for the preparation of radiolabeled iododeoxyuridine by demetallation.

UNLABELLED: The fastest and most reliable preparation of radiolabeled 5-iodo-2'- deoxyuridine (*IUdR) is accomplished by iododemetallation. METHODS: We describe a kit formulation for the preparation of *IUdR by demercuration whereby [123I/125I/131I]IUdR is synthesized virtually instantaneously following the incubation of an aqueous solution of the chloromercuric precursor with Na123I/125I/131I in the presence of lodogen. We also report the conditions for the radiosynthesis of IUdR by destannylation of the tributylstannyl precursor using hydrogen peroxide as the oxidant. RESULTS: In each case, the total procedure is completed in 5 min. HPLC indicates the total transformation of iodide into IUdR with no detectable UV-absorbing by-products. The metal content of the sample is low. The product, therefore, does not require purification. CONCLUSION: *IUdR can be prepared instantly, by either demercuration of ClHgUdR or destannylation of Bu3SnUdR. The use of a mercuric precursor favors a kit formulation since the metallic derivative is stable when kept in aqueous solution, aliquoted in vials coated with the oxidant, for up to 3 mo.