Evaluation of a bromine-76-labeled progestin 16alpha,17alpha-dioxolane for breast tumor imaging and radiotherapy: in vivo biodistribution and metabolic stability studies.

INTRODUCTION: Progesterone receptors (PRs) are present in many breast tumors, and their levels are increased by certain endocrine therapies. They can be used as targets for diagnostic imaging and radiotherapy. METHOD: 16alpha,17alpha-[(R)-1'-alpha-(5-[(76)Br]Bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione ([(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione (3)), a PR ligand with relative binding affinity (RBA)=65 and log P(o/w)=5.09+/-0.84, was synthesized via a two-step reaction, and its tissue biodistribution and metabolic stability were evaluated in estrogen-primed immature female Sprague-Dawley rats. RESULTS: [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 was synthesized in 5% overall yield with specific activity being 200-1250 Ci/mmol. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 demonstrated high PR-mediated uptake in the target tissue uterus (8.72+/-1.84 %ID/g at 1 h) that was reduced by a blocking dose of unlabeled progestin R5020, but the nonspecific uptake in blood and muscle (2.11+/-0.14 and 0.89+/-0.16 %ID/g at 1 h, respectively) was relatively high. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 was stable in whole rat blood in vitro, but it was not stable in vivo due to the fast metabolism that occurred in the liver, resulting in the formation of a more polar radioactive metabolite and free [(76)Br]bromide. The level of free [(76)Br]bromide in blood remained high during the experiment (2.11+/-0.14 %ID/g at 1 h and 1.52+/-0.24 %ID/g at 24 h). The tissue distribution of [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 at 1 and 3 h was compared with that of the (18)F analogs, [(18)F]FFNP fluoro furanyl norprogesterone (FFNP) 1 and ketal 2. CONCLUSION: [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 may have potential for imaging PR-positive breast tumors at early time points, but it is not suitable for imaging at later times or for radiotherapy.

Hybridisation of [76Br]-labelled antisense oligonucleotides to Chromogranin A mRNA verified by RT-PCR.

Methods have been developed to label oligonucleotides (ODNs) in the 5'-position with (76)Br via a prosthetic group on a hexylamino-linker. The purpose of the study was to explore whether the labelling procedure would prevent specific hybridisation by using reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing of the PCR product. Antisense ODNs (30 mer, specific for rat Chromogranin A [CgA] mRNA) with phosphodiester (O-ODN) or phosphothioate (S-ODN) backbone, either unlabelled or labelled with (76)Br, served as one of the primers in individual PCR reactions. Using O-ODN as a primer, irrespective of being labelled or not, a selected 225-bp PCR fragment was successfully amplified. However, no amplification was obtained using S-ODN as a primer. The proper PCR products were only detected in the sample prepared from the adrenal gland, but not in that from the heart, liver or kidney. Autoradiographic recording of the gel, after gel electrophoresis, revealed radioactive signals corresponding to the amplified PCR products. The sequence of the PCR product matched the rat CgA mRNA sequence obtained from the EMBL database. RT-PCR is an attractive method to identify the selective binding of modified ODNs to target mRNA. This method confirmed that the labelling with (76)Br did not change the hybridisation ability of antisense O-ODN.

Positron emission tomography and radioimmunotargeting--general aspects.

To optimize radioimmunotherapy, in vivo information on individual patients, such as radionuclide uptake, kinetics, metabolic patterns and optimal administration methods, is important. An overriding problem is to determine accurately the absorbed dose in the target organ as well as critical organs. Positron Emission Tomography (PET) is a superior technique to quantify regional kinetics in vivo with a spatial resolution better than 1 cm3 and a temporal resolution better than 10 s. However, target molecules often have distribution times of several hours to days. Conventional PET nuclides are not applicable and alternative positron-emitting nuclides with matching half-lives and with suitable labelling properties are thus necessary. Over many years we have systematically developed convenient production methods and labelling techniques of suitable positron nuclides, such as 110In(T(1/2) = 1.15 h), 86Y(T(1/2) = 14 h), 76Br(T(1/2) = 16 h) and 124I(T(1/2) = 4 days). 'Dose planning' can be done, for example, with 86Y- or 124I-labelled ligands before therapy, and 90Y- and 131I-labelled analogues and double-labelling, e.g. with a 86Y/90Y-labelled ligand, can be used to determine the true radioactivity integral from a pure beta-emitting nuclide. The usefulness of these techniques was demonstrated in animal and patient studies by halogen-labelled MAbs and EGF-dextran conjugates and peptides chelated with metal ions.

Tissue dose estimates following the selective uptake of 125IUdR and other radiolabelled thymidine precursors in resistant tumours.

For the purposes of evaluation of the therapeutic potential of the radiohalogenated thymidine analogue 125IUdR, estimation of the radiation dose to the tumour cells and normal tissues is important. To determine the dose to any tissue from the radionuclide 125I is not simple, since the major emissions are very short-range Auger electrons. The cytotoxicity of 125I is strongly dependent on the position of the decay relative to the DNA, the principal target for cell sterilization. Estimates of the cytotoxicity of 125I based on the traditional MIRD recommended formulation (ICRU Report 32, 1979) may produce gross underestimates if it is incorporated into the DNA via the thymidine precursor 125IUdR. In this work, tissue count and autoradiography (ARG) data from studies by Bagshawe et al were used to estimate tissue doses following the administration of 125IUdR to LS174/T (a colorectal carcinoma) and CC3 (a choriocarcinoma) tumour-bearing animals, after a hydroxyurea block of the normal tissue turnover. The tumour cell toxicity is estimated from ARG data on the degree of 125I incorporation into the cell nucleus. Major drawbacks with 125I for this type of therapy are the long 60-day half-life, leading to radiological and waste disposal problems and the extreme short range of the radiotoxic effects. Possible alternative radiohalogens, 13I, 77Br, 131I and 211At, are suggested in place of 125I in the thymidine analog iododeoxyuridine. Dose calculations are performed and cytotoxicities estimated on the assumption that their biological retention characteristics are the same as for 125IUdR.

Production of no carrier added 80mBr for investigation of Auger electron toxicity.

80mBr (half-life = 4.43 h) is an Auger electron emitting nuclide with convenient properties for investigating Auger electron cytotoxicity and with potential for labeling in vivo radiotherapeutic agents. We have investigated three cyclotron target systems capable of generating 80mBr of sufficiently high specific radioactivity (no carrier added) for biomedical experiments. A 83Kr gas target irradiated with 21.5 MeV deuterons made 80mBr at a production yield of 1.6 +/- 0.2 mCi/muAh at saturation. A five-fold increase in 80mBr yield was obtained from 15 MeV proton irradiation of thin elemental Se enriched in 80Se targets although technical improvements are expected to further raise this production yield. This route is therefore superior for current medical cyclotrons. Irradiation of a reusable 80Se copper selenide target also yielded multi-millicurie amounts of 80mBr, and recovery of radiobromine by dry distillation is faster and more convenient than in the elemental Se target, but an optimum copper selenide target for 80mBr production has not yet been built.

Bromine-80m-labeled estrogens: Auger electron-emitting, estrogen receptor-directed ligands with potential for therapy of estrogen receptor-positive cancers.

To assess their possible use for estrogen receptor (ER)-directed radiotherapy of estrogen receptor-containing cancers, two estrogens were synthesized with the Auger electron-emitting nuclide bromine-80m and administered to immature female rats. Both the triphenylethylene-based estrogen, [80mBr]-2-bromo-1,1-bis(4-hydroxyphenyl)phenylethylene (Br-BHPE) and the steroidal estrogen [80mBr]17 alpha-bromovinylestradiol, showed substantial diethylstilbestrol-inhibitable localization only in the estrogen target tissues, the uterus, pituitary, ovaries, and vagina and, except for the liver and intestines, generally lower concentrations in all other tissues at both 0.5 and 2 h. The [80mBr]Br-BHPE (specific activity, 8700 Ci/mmol), was shown to bind specifically to the low salt extractable ER of the rat uterus. Comparing i.p., i.v., and s.c. administration of [80mBr]BHPE the i.p. route was found to be particularly advantageous to effect maximum, DES-inhibitable concentrations of radiobromine in the ER-rich target organs in the peritoneal cavity. When the tissue distribution of the [80mBr]Br-BHPE was compared with that of sodium bromide-80m, it was apparent that no substantial amounts of radiobromine were released from the bromoestrogen prior to its target tissue localization. The substantial concentration of these bromine-80m-labeled estrogens in ER-rich tissues, combined with previously reported evidence for the effective radiotoxicity of Auger electron-emitting nuclides within cell nuclei suggest a good potential for such ligands for therapy of ER positive cancers.