Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

ABSTRACT

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by ¹²⁵I) and subsequent accumulation of an ¹¹¹In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of ¹¹¹In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using ¹¹¹In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.     

Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22

 LL2 is an anti-CD22 pan-B-cell monoclonal antibody which, when radiolabeled, has a high sensitivity for detecting B-cell, non-Hodgkin’s lymphoma (NHL), as well as an antitumor efficacy in therapeutic applications. The aim of this study was to determine whether intracellularly retained radiolabels have an advantage in the diagnosis and therapy of lymphoma with LL2. In vitro studies showed that iodinated LL2 is intracellularly catabolized, with a rapid release of the radioiodine from the cell. In contrast, residualizing radiolabels, such as radioactive metals, are retained intracellularly for substantially longer. In vivo studies were performed using LL2-labeled with radioiodine by a non-residualizing (chloramine-T) or a residualizing method (dilactitol-tyramine, DLT), or with a radioactive metal (¹¹¹In). The biodistribution of a mixture of ¹²⁵I (non-residualizing chloramine-T compared to residualizing DLT), ¹¹¹In-labeled LL2 murine IgG2a or its fragments [F(ab′)₂, Fab′], as well as its humanized, CDR-grafted form, was studied in nude mice bearing the RL human B-cell NHL cell line. Radiation doses were calculated from the biodistribution data according to the Medical International Radiation Dose scheme to assess the potential advantage for therapeutic applications. At all assay times, tumor uptake was higher with the residualizing labels (i.e., ¹¹¹In and DLT-¹²⁵I) than with the non-residualizing iodine label. For example, tumor/blood ratios of ¹¹¹In-labeled IgG were 3.2-, 3.5- and 2.8-fold higher than for non-residualizing iodinated IgG on days 3, 7 and 14, respectively. Similar results were obtained for DLT-labeled IgG and fragments with residualized radiolabels. Tumor/organ ratios also were higher with residualizing labels. No significant differences in tumor, blood and organ uptake were observed between murine and humanized LL2. The conventionally iodinated anti-CD20 antibody, 1F5, had tumor uptake values comparable to those of iodinated LL2, the uptake of both antibodies being strongly dependent on tumor size. These data suggest that, with internalizing antibodies such as LL2, labeling with intracellularly retained isotopes has an advantage over released ones, which justifies further clinical trials with residualizing ¹¹¹In-labeled LL2 for diagnosis, and residualizing ¹³¹I and ⁹⁰Y labels for therapy. Received: 23 August 1996 / Accepted: 7 February 1997     

Studies on the metabolic fate of 111In-labeled antibodies

Indium-111-labeled antibodies, though providing superior photon flux to iodine-labeled antibodies, can exhibit high levels of accumulation in some non-target organs. In an effort to understand the nature of this non-target uptake we have evaluated the molecular weight of ¹¹¹In species retained in several tissues by radio-FPLC (sizing chromatography) following injection of [¹¹¹In]DTPA 5G6.4, a murine monoclonal antibody, into normal mice. Blood, liver and kidneys were removed, and liver and kidneys were homogenized at several time points after antibody injection. The proportion of ¹¹¹In-containing species was found to vary with the tissue and with time. Analysis of blood showed only radiolabeled antibody. In the liver, several ¹¹¹In species were identified with molecular weights compatible with intact antibody, [¹¹¹In]transferrin, and low molecular weight complexes, with an increase in the proportion of [¹¹¹In]transferrin and low molecular weight species occurring over time. While the same molecular weight species were also identified in the kidneys, the kidneys contained the largest percentage of low molecular weight species which increased over time. When ¹²⁵I-labeled 5G6.4 was injected and the tissues similarly analyzed, only radioactive material with the molecular weight of intact antibody was detected. Comparison of two methods of purification of [¹¹¹In]labeled antibody after labeling revealed a significant difference in the organ uptake of radiolabeled products for ¹¹¹In. Although dialysis was sufficient for the removal of labile ¹¹¹In, as determined by TLC, subsequent sizing chromatography on Bio-Gel P-60 dramatically dropped the hepatic and renal uptake of ¹¹¹In relative to blood and diminished the proportion of the low molecular weight species present on sizing FPLC of extracts from tissues. These data indicate that low and intermediate molecular weight ¹¹¹In compounds are accreted in the liver and kidneys following the i.p. injection of ¹¹¹In-labeled monoclonal antibodies and that their uptake can be diminished by more stringent radioantibody purification. This knowledge may be valuable in developing methods for reducing non-target ¹¹¹In uptake.     

Site-specifically modified 111In labelled antibodies give low liver backgrounds and improved radioimmunoscintigraphy

Site-specific modification of monoclonal antibodies at their oligosaccharide had previously been demonstrated to produce excellent ¹¹¹In imaging in a xenograft model using a Brown Norway (BN) rat lymphoma and a rat anti-BN MHC monoclonal antibody [Lee C. et al. Fed. Proc. Abstr. 43 3014 (1984)]. These results are due, in part, to lack of liver uptake, so we wanted to evaluate the extent of hepatic uptake observed with different monoclonal antibodies in normal mice. Biodistribution data were obtained for four monoclonal antibodies by first modifying each antibody at its carbohydrate with a diethylenetriaminepentaacetic acid (DTPA) derivative. The antibodies were then labelled with ¹¹¹In and injected into normal mice. Images were obtained 24 h post-injection, and at 48 h the mice were dissected and the tissue-to-blood (T:B) ratios determined. T:B ratios were approximately 1 (or less) for every organ evaluated, indicating minimal non-specific uptake into these organs. Data is also presented for the BN-rat system which shows excellent localization into the tumor xenograft and low non-specific organ uptake. These data indicate that modification of antibodies site-specifically at their oligosaccharide results in minimal non-specific uptake into non-target tissues and enhanced localization into the tumor target, and that this may represent a preferred method for production of ¹¹¹In labelled antibodies.     

Monoclonal antibodies as reversible equilibrium carriers of radiopharmaceuticals

We have prepared monoclonal antibodies (MoAbs) with the specific ability to bind metal chelates such as ¹¹¹In benzyl EDTA. One, 10, 50 and 100 μg MoAb CHA255 K_b = 4 × 10E9 was complexed with ¹¹¹In BLEDTA II, BLEDTA IV, and benzyl EDTA and injected i.v. in Balb/c mice with KHJJ tumor. The biological half-life by whole body counting was profoundly altered for all three compounds; from minutes to hours with 10 μg; to days with 100 μg. Tumor uptake increased 50 fold at 24 h with increasing MoAb but satisfactory tumor concentrations (3% per g) and tumor/blood ratios (1.8:1) were obtained with an amount equivalent to 7 mg for a human. Blood level and whole body activity were decreased 30–50% within 3 h or i.v. injection of a “flushing” dose of unlabeled indium benzyl EDTA, increasing tumor/blood ratios to 50:1.     

Discovery of potent and selective bicyclic A2B adenosine receptor antagonists via bioisosteric amide replacement

Several new potent and selective A_2B adenosine receptor antagonists have been prepared in which the aryl–amide moiety of the lead series, exemplified by 1a, has been replaced by bioisosteric bicyclic moieties. Although the majority of compounds had generally improved microsomal stability as compared to 1a, this was not translated into overall improvements in the pharmacokinetic profiles of a representative set of compounds.     

Evaluation of two new bifunctional chelates for radiolabeling a parathyroid-specific monoclonal antibody with In-111

BB5-G1, a monoclonal antibody specific for human parathyroid cell membrane antigen was conjugated with two new ligands, BrMe₂HBED and BrφHBED and radiolabeled with ¹¹¹In. We have compared the biodistribution of ¹¹¹In-labeled BBS using the new ligands to conventionally labeled (¹²⁵I-labeled and ¹¹¹In-DTPA-labeled) BBS in a nude mouse model. Both ¹¹¹In-BrMe₂HBED-BB5 and ¹¹¹In-BrφHBED-BB5 attained high parathyroid-to-blood and parathyroid-to-muscle ratios by 72–96 h. ¹¹¹In-BrφHBED-BB5 showed lower %ID/g than ¹¹¹In-BrMe₂HBED-BB5 in the clearance organs, the liver and kidney; renal activity had cleared significantly by 120 h. This work suggests that ¹¹¹In-BrφHBED-BB5 offers improved in vivo behavior and may be useful as a radiopharmaceutical for localizing parathyroid tissue.     

Detection of hepatic metastases from colorectal carcinoma using indium-111 (111In) labeled monoclonal antibody (mAB): MSKCC experience with mAb 111In-C110

Sixteen patients with colorectal carcinoma and a rising serum carcinoembryonic antigen (CEA) level and no evidence of extra-abdominal disease were administered 5 mg of an anti-CEA monoclonal antibody (mAb), C110, labeled with approx. 5 mCi of ¹¹¹In. All patients subsequently underwent exploratory laparotomy, and samples of tumor and normal tissue were obtained. Hepatic lesions (confirmed by histopathology) were visualized as areas of increased radiotracer uptake in 13 of 16 patients. Single photon emission computed tomography (SPECT) considerably aided detection, being positive in two patients with normal planar images. Ten of the 16 patients had positive x-ray computed tomographic (CT) images. The radioimmunodiagnostic study was falsely negative in 3 of 16 patients with subsequently proven hepatic disease, in one of whom CT was also normal. The antibody study was positive in 80% of lesions, thus being, in this small series, significantly more sensitive (P < 0.01) than CT. ¹¹¹In-C110 is a promising monoclonal antibody for the detection of hepatic metastases from colorectal carcinoma; this is the first study to show consistently greater concentration of ¹¹¹In-labeled antibody in hepatic lesions than in surrounding normal hepatic parenchyma.     

Synthesis and SAR studies of novel benzodiazepinedione-based inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB)

Synthesis and structure-activity relationships (SAR) of a novel series of benzodiazepinedione-based inhibitors of Clostridium difficile toxin B (TcdB) are described. Compounds demonstrating low nanomolar affinity for TcdB, and which possess improved stability in mouse plasma vs. earlier compounds from this series, have been identified. Optimized compound 11d demonstrates a good pharmacokinetic (PK) profile in mouse and hamster and is efficacious in a hamster survival model of Clostridium difficile infection.     

Direct 99mTc labeling of monoclonal antibodies: Radiolabeling and in vitro stability

Direct labeling involves ^99mTc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind ^99mTc. The direct ^99mTc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability.Stable direct antibody labeling with ^99mTc requires the generation of sulfhydryl groups, which show high affinity binding sites for ^99mTc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the ^99mTc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution.Very good ^99mTc-antibody stability is obtained with activated IgG (IgGa) and Fab′ fragment, which makes these substances possible candidates for immunoscintigraphy use.     

Optimization of pyrimidinol antioxidants as mitochondrial protective agents: ATP production and metabolic stability

Previously we described a novel series of pyrimidinol antioxidants and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Our initial lead compound was a potent antioxidant in vitro, but was subsequently found to exhibit poor stability to oxidative metabolism. The current study focused on balancing potency with metabolic stability through structural modification, and involved modifications at positions 2 and 4 of the pyrimidinol redox core, likely sites of oxidative metabolism. Eight new analogues have been prepared and their ability to suppress lipid peroxidation and reactive oxygen species (ROS), and to preserve mitochondrial membrane potential (Δψ_m) and support ATP production, has been investigated. The metabolic stability of the prepared compounds was also assessed in vitro using bovine liver microsomes to obtain preliminary insight on this class of compounds. This study revealed the complexity of balancing reasonable metabolic stability with efficient antioxidant properties. While a few analogues appear promising, especially in terms of metabolic stability, a 4-isopropoxy derivative conserved the favorable biological activity and exhibited good metabolic stability. The favorable metabolic stability conferred by the combination of the azetidine and isopropoxy moieties in analogue 6 makes this compound an excellent candidate for further evaluation.     

Targeting properties of an anti-CD16/anti-CD30 bispecific antibody in an in vivo system

Bispecific antibodies are currently being used in clinical trials in increasing numbers in the areas of breast cancer, prostate cancer, non-Hodgkin's lymphoma and Hodgkin's lymphoma. We have previously performed two clinical trials in patients with Hodgkin's disease with an anti-CD30/anti-CD16 bispecific antibody and demonstrated a 30% response rate in a cohort of patients otherwise resistant to standard therapeutic modalities. However, no surrogate marker could be defined in these trials indicative of optimal antibody dosing/scheduling or predictive for favorable response. In order to evaluate accurately the potential biodistribution properties of bispecific antibody in patients, we have performed a detailed analysis of the binding properties and animal model in vivo characteristics of these constructs. For this purpose, the parental antibodies (anti-CD30 and anti-CD16) and the bispecific antibody (anti-CD30/anti-CD16) were radiolabeled with either ¹²⁵I or ¹¹¹In. Antibody integrity and binding properties after labeling were confirmed by Scatchard plot and Lindmo analysis. ¹¹¹In-labeled antibodies revealed superior targeting properties in a standard SCID mouse tumor model. Both the bivalent parental anti-CD30 monoclonal antibody and the monovalent anti-CD30/anti-CD16 bispecific antibody showed excellent uptake in CD30⁺ tumors which did not differ significantly between the two (maximum uptake 16.5% ± 4.2% vs. 18.4% ± 3.8% injected dose/gram tissue). The equivalent targeting properties of the bispecific antibody compared with the parental anti-CD30 antibody encourages the further clinical development of this bispecific antibody, and might help to explain the clinical responses seen with this antibody so far in patients suffering from Hodgkin's disease. Received: 26 October 2000 / Accepted: 15 December 2000     

Facile synthesis and evaluation of C-functionalized benzyl-1-oxa-4,7,10-triazacyclododecane-N,N',N″-triacetic acid as chelating agent for ¹¹¹In-labeled polypeptides

A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N,N′,N″-triacetic acid (ODTA), has been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetraacetic acid (DOTA). However, neither synthetic procedure for a C-functionalized ODTA (C-ODTA) nor its chelating ability with a trace amount of radioactive indium-111 (¹¹¹In) has been elucidated. We herein present a facile synthetic procedure for C-ODTA, and estimated its ability as a chelating agent for radiolabeling peptides and proteins with ¹¹¹In. The synthetic procedure involves the synthesis of a linear precursor using a para-substituted phenylalanine derivative as a starting material. The following intramolecular cyclization reaction was best performed (>73% yield) when Boc-protected linear compound and the condensation reagent, HATU, were simultaneously added to the reaction vessel at the same flow rate. The cyclic compound was then reduced with BH₃ and alkylated with tert-butyl bromoacetate. The synthetic procedure was straightforward and some optimization would be required. However, most of the intermediate compounds were obtained easily in good yields, suggesting that the present synthetic procedure would be useful to synthesize C-ODTA derivatives. The intramolecular cyclization reaction might also be applicable to synthesize polyazamacrocycles of different ring sizes and cyclic peptides. In ¹¹¹In radiolabeling reactions, C-ODTA provided ¹¹¹In chelates in higher radiochemical yields at low ligand concentrations when compared with C-DOTA. The ¹¹¹In-labeled C-ODTA remained unchanged in the presence of apo-transferrin. The biodistribution studies also showed that the ¹¹¹In-labeled compound was mainly excreted into urine as intact. These findings indicate that C-ODTA would be useful to prepare ¹¹¹In-labeled peptides of high specific activities in high radiochemical yields.     

Altered biodistribution of indium-111-labeled monoclonal antibody 96.5 to tumors and normal tissues of nude mice bearing human melanoma xenografts in visceral organs

Summary Human melanoma xenografts were produced in the subcutis, kidney, cecum and liver of different nude mice. An¹¹¹In-labeled anti-(human melanoma) monoclonal antibody (96.5) or an¹¹¹In-labeled nonspecific control monoclonal antibody (ZCE-025) was injected intravenously in separate groups of mice. Radioactive antibody accumulation was measured in tumor, blood, viscera, and carcasses. mAb 96.5 targeted specifically to tumor tissue regardless of site of growth. Tumors in the liver exhibited significantly (P <0.05) higher tumor-to-blood ratios (45±6, mean ±SEM) than xenografts at other visceral organs, the lowest value being found for subcutaneous melanoma (2.6±0.5). The differences in tumor-to-blood ratio were due to significant alterations of antibody biodistribution, since the actual antibody concentration in the different tumor sites was similar. The percentage of recovered anti-melanoma antibody per milliliter of blood in mice with visceral lesions (4.6±1.1%/ml) was significantly lower than that found in mice with subcutaneous tumors (9.5±1.4%/ml,P <0.05). Moreover, significantly higher levels (18.2±3.2%/g, 31.0±5.1%/g, respectively) of the melanoma mAb 96.5 were found in normal liver and spleen tissue recovered from mice with visceral tumors as compared to tissue from mice with subcutaneous tumors (9.2±0.9%/g, 13.5±1.9%/g, respectively;P <0.05). These results demonstrate that the presence of visceral tumor can significantly affect tumor-to-blood ratios, blood levels, and biodistribution of¹¹¹In-labeled mAb 96.5.This work was supported in part by funds from the National Institutes of Health, National Cancer Institute, Grant R35-CA42107 and Core Grant CA16672     

Synthesis and anticandidal evaluation of new benzothiazole derivatives with hydrazone moiety

In this study, we have performed the synthesis of new N′-(arylidene)-4-[(benzothiazol-2-yl)thio]butanoylhydrazide derivatives (3a–s) bearing azole moiety and hydrazone group in a lipophilic structural framework. The target compounds were prepared by a three step synthetic procedure starting from 2-mercaptobenzothiazole. The structures of the target compounds were elucidated by IR, ¹H NMR, ¹³C NMR spectra and elemental analysis. The antifungal activity of the obtained compounds has been determined against a number of clinic and fluconazole-resistant Candida strains by using microdilution method. Compounds (3a–3s) exhibited anticandidal activity in different ratios varying between the range of MIC: 50 and 200?µg/mL.     

Synthesis of aminobenzyltriethylenetetraaminohexaacetic acid: conjugation of the chelator to protein by an alkylamine linkage

The conjugation of a chelating agent to an antibody as an anchoring site for a radionuclide is the first step in the successful preparation of a radiolabeled antibody for a diagnostic and therapeutic application. The high affinity of the protein bound chelator towards radionuclide ensures a higher selectivity in the delivery of the radionuclide to the targeted tissue. 4-Aminobenzylderivativetriethlenetetraaminohexaacetic acid (TTHA), a hexadentate chelating agent has been now prepared for conjugation with proteins in view of the higher affinity of TTHA metal ions as compared to DTPA. The latent crosslinking potential of -hydroxy aldehydes has been used to conjugate the new chelating agent to proteins through an alkylamine linkage. On incubation of amino benzyl TTHA with glycoladehyde at neutral pH and room temperature, the reagent is converted to oxoethyl amino benzyl TTHA. On addition of albumin to this reaction mixture, the oxo ethylamino benzyl TTHA generates reversible schiff base adducts with the amino groups of albumin. The reduction of the Schiff base adducts of the chelator with the protein by sodium cyanoborohydride stabilizes the schiff base adducts as stable alkylamine linkages. 4-Thiocyanatobenzyl TTHA has also been prepared and conjugated to albumin through a thiocarbamoyl linkage. Both preparations of TTHA conjugated albumin complexed with ^99mTc and ¹¹¹In, with high affinity and no decomposition of the complex was noticed for at least up to 6 hrs after the preparation. The radiolabels complexed with these TTHA -albumin conjugates could not be chased out by free DTPA. A comparison of the biodistribution of ¹¹¹In, bound to the TTHA conjugated through an alkylamine and a thiocarbamoyl linkage showed that ¹¹¹In complexed with alkylamine linked TTHA was retained in blood to a level nearly 17% higher compared to that seen with thicarbamoyl linked TTHA, one hr after the injection into mice. Thus, the alkylamine linkage appears to be more stable under the in vivo conditions. The glycolaldehyde mediated alkylation procedure offers a mild, simple and rapid method for preparation of drug-protein (antibody) conjugates.     

A potential copper radiopharmaceutical for imaging the heart and brain: Copper-labeled pyruvaldehyde bis(N4-methylthiosemicarbazone)

An investigation of the biodistribution of lipophilic copper-64 (half-life = 12.7 h) compounds has been initiated in order to screen potential tracers that could be used to measure regional cerebral and/or myocardial blood flow when labeled with generator-produced ⁶²Cu. The ⁶⁴Cu complex of pyruvaldehyde bis(N⁴-methylthiosemicarbazone), [⁶⁴Cu]Cu-PTSM, was prepared and found to be lipophlic (octanol/saline partition coefficient, log p = 1.97 ± 0.03). Biodistribution studies following i.v. injection of [⁶⁴Cu]Cu-PTSM into rats show tracer uptake by the brain and heart. At 1, 5, and 15 min post-injection 3.3, 3.0 and 2.7% of the injected dose was found in the brain. Corresponding brain to blood ratios (per gram) were 3.2, 3.6 and 4.0 respectively. Heart to blood ratios of 7.6, 7.6 and 7.3 were observed at these same time points.     

Synthesis and biological evaluation of Esaprazole analogues showing σ1 binding and neuroprotective properties in vitro

Esaprazole, a molecule previously acknowledged to protect against stomach and intestinal ulcers was surprisingly discovered to have neuroprotective activities and σ₁ binding in vitro. A highly diverse set of Esaprazole analogues 2–5 was prepared in order to increase blood–brain barrier penetration. The analogues showed a structure–activity relationship at the σ₁ receptor closely matching already published pharmacophores. Many of the analogues were shown to have neuroprotective properties in two assays using primary cultures of cortical neurons exposed to glutamate and hydrogen peroxide. However, no apparent SAR for these two assays could be developed. Metabolic stability of the analogues were also investigated and the structure of R¹ had a significant bearing on the ADME properties of the compound resulting in two series of compounds. Compounds in which R¹ was a H or acyl group had good metabolic stability in RLM but poor BBB penetration, whereas compounds where R¹ was a cyclo- or bicyclo-alkyl group had poor metabolic stability but good BBB penetration.     

Serum stability of 67Cu chelates: Comparison with 111In and 57Co

Simple chelates and chelate conjugated monoclonal antibodies labeled with ¹¹¹In, ⁵⁷Co and ⁶⁷Cu demonstrate marked differences in stability when exposed to a serum environment. Among these radiometals, on DTPA, the order of stability is ¹¹¹In ⁵⁷Co ⪢ ⁶⁷Cu. On benzyl-EDTA, the order of stability is ¹¹¹In ⋍ ⁵⁷Co ⪢ ⁶⁷Cu. Among those investigated, the only serum stable ⁶⁷Cu chelate found was ⁶⁷Cu-TETA. The order of stability observed for ⁵⁷Co vs ⁶⁷Cu is contrary to published equilibrium constants. These in vitro studies suggest that the in vivo behavior of metal chelates exposed to a complex molecular environment may not be predicted by classically determined equilibrium constants.     

Target tissue uptake selectivity of three fluorine-substituted progestins: Potential imaging agents for receptor-positive breast tumors

We have studied three new fluorine-substituted progestins (1–3) as potential imaging agents for progesterone receptor (PgR)-positive human breast tumors. Two of these are fluorine-substituted analogs of the potent progestin R5020 (promegestone), derived from (21S)-hydroxy R 5020 (RU 27987) and (21R)-hydroxy R 5020 (RU 27988), known metabolites of R 5020, which have affinities for PgR that are 116 and 4%, respectively (relative to R 5020 = 100%). These precursors were protected as their 3,3-dioxolane derivatives and converted to the 21-trifluoromethanesulfonate derivatives. Fluoride ion displacement, followed by acid-catalyzed deprotection, furnished in good yield the epimeric fluoroanalogs, (21S)- and (21R)-fluoro R 5020 (1 and 2, affinities for PgR, 11 and 45%, respectively). These compounds were also prepared in ¹⁸F labeled form by the same route, in 14–32% overall radiochemical yield (decay corrected; synthesis time 90 min; sp. act. 370–1060 Ci/mmol). In tissue distribution studies in estrogen-primed immature rats, uterus-to-muscle ratios were 4.3 at 1 h for the 21S-epimer and 1.1 for the 21R-epimer, paralleling their relative binding affinities. Considerable metabolic defluorination was observed. The third fluorine-substituted progestin, DU 41165, has a novel retroprogesterone (9β, 10α) structure, substituted with fluorine at C-6; its binding affinity is 145% relative to R 5020, and it was prepared in tritium-labeled form by acetylation of DU 41231, the 17α-hydroxy precursor, with [³H]acetic anhydride. In estrogen-primed immature rats, this compound shows uterus-to-muscle ratios of 15 at 1 h, and 18–71 between 2 and 6 h, suggesting that compounds in this retroprogesterone series may be very promising candidates for selective imaging of PgR-positive tissues and tumors.