212Pb-radioimmunotherapy potentiates paclitaxel-induced cell killing efficacy by perturbing the mitotic spindle checkpoint.

BACKGROUND: Paclitaxel has recently been reported by this laboratory to potentiate the high-LET radiation therapeutic (212)Pb-TCMC-trastuzumab, which targets HER2. To elucidate mechanisms associated with this therapy, targeted α-particle radiation therapeutic (212)Pb-TCMC-trastuzumab together with paclitaxel was investigated for the treatment of disseminated peritoneal cancers. METHODS: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pre-treated with paclitaxel, followed by treatment with (212)Pb-TCMC-trastuzumab and compared with groups treated with paclitaxel alone, (212)Pb-TCMC-HuIgG, (212)Pb-TCMC-trastuzumab and (212)Pb-TCMC-HuIgG after paclitaxel pre-treatment. RESULTS: (212)Pb-TCMC-trastuzumab with paclitaxel given 24 h earlier induced increased mitotic catastrophe and apoptosis. The combined modality of paclitaxel and (212)Pb-TCMC-trastuzumab markedly reduced DNA content in the S-phase of the cell cycle with a concomitant increase observed in the G2/M-phase. This treatment regimen also diminished phosphorylation of histone H3, accompanied by an increase in multi-micronuclei, or mitotic catastrophe in nuclear profiles and positively stained γH2AX foci. The data suggests, possible effects on the mitotic spindle checkpoint by the paclitaxel and (212)Pb-TCMC-trastuzumab treatment. Consistent with this hypothesis, (212)Pb-TCMC-trastuzumab treatment in response to paclitaxel reduced expression and phosphorylation of BubR1, which is likely attributable to disruption of a functional Aurora B, leading to impairment of the mitotic spindle checkpoint. In addition, the reduction of BubR1 expression may be mediated by the association of a repressive transcription factor, E2F4, on the promoter region of BubR1 gene. CONCLUSION: These findings suggest that the sensitisation to therapy of (212)Pb-TCMC-trastuzumab by paclitaxel may be associated with perturbation of the mitotic spindle checkpoint, leading to increased mitotic catastrophe and cell death.

Preclinical evaluation of a monoclonal antibody targeting the epidermal growth factor receptor as a radioimmunodiagnostic and radioimmunotherapeutic agent.

BACKGROUND AND PURPOSE: The studies described here are the first to evaluate the in vitro and in vivo properties of (111)In-CHX-A''-panitumumab for radioimmunotherapy (alpha- and beta(-)-emitters) and radioimmunoimaging (single photon emission computed tomography and positron emission tomography). EXPERIMENTAL APPROACH: Twenty-seven human carcinoma cell lines were analysed for expression of epidermal growth factor receptors by flow cytometry. Panitumumab was conjugated with CHX-A''-DTPA (diethylenetriamine-pentaacetic acid) and radiolabelled with (111)In. Immunoreactivity of the CHX-A''-DTPA-panitumumab and (111)In-CHX-A''-DTPA-panitumumab was evaluated by radioimmunoassays. Tumour targeting was determined in vivo by direct quantitation of tumour and normal tissues and by gamma-scintigraphy. KEY RESULTS: For 26 of 27 human tumour cell lines, 95% of the cells expressed epidermal growth factor receptors over a range of intensity. Immunoreactivity of panitumumab was retained after modification with CHX-A''-DTPA. Radiolabelling of the immunoconjugate with (111)In was efficient with a specific activity of 19.5 +/- 8.9 mCi.mg(-1) obtained. Immunoreactivity and specificity of binding of the (111)In-panitumumab was shown with A431 cells. Tumour targeting by (111)In-panitumumab was demonstrated in athymic mice bearing A431, HT-29, LS-174T, SHAW or SKOV-3 s.c. xenografts with little uptake observed in normal tissues. The (111)In-panitumumab was also evaluated in non-tumour-bearing mice. Pharmacokinetic studies compared the plasma retention time of the (111)In-panitumumab in both non-tumour-bearing and A431 tumour-bearing mice. Tumour targeting was also visualized by gamma-scintigraphy. CONCLUSIONS AND IMPLICATIONS: Panitumumab can be efficiently radiolabelled with (111)In with high labelling yields. Based on the efficiency in tumour targeting and low normal tissue uptake, panitumumab may be an effective targeting component for radioimmunodiagnostic and radioimmunotherapeutic applications.

Imaging pulmonary emboli and deep venous thrombi with 99mTc-bitistatin, a platelet-binding polypeptide from viper venom.

UNLABELLED: An imaging test that could locate both pulmonary emboli (PE) and their source, active deep venous thrombi (DVT), would be valuable in patient management. Bitistatin, an 83-amino-acid polypeptide isolated from Bitis arietans venom, binds avidly to the glycoprotein IIb/IIIa receptor on platelets. The goal of this study was to label bitistatin with 99mTc and assess its potential for imaging thrombi and emboli in vivo. METHODS: Molecular modeling of bitistatin indicated that its primary amines are located on the opposite side of the molecule from the receptor-binding domain. The primary amines were reacted with succinimidyl-4-hydrazino nicotinate hydrochloride to place 2.4 hydrazino nicotinate (Hn) chelating groups per peptide molecule. Hn-bitistatin was labeled by incubation with 99mTc-glucoheptonate to 96 TBq/mmol and then tested for binding to platelets in vitro and for imaging of 24-h-old DVT and PE in a canine model used previously for other thrombus tracers. RESULTS: 99mTc-Hn-bitistatin bound to stimulated platelets with a dissociation constant (Kd) = 32 nmol/L, similar to that of 125I-bitistatin (Kd = 41 nmol/L). In vivo, focal uptake was observed in planar images as early as 30 min (DVT) and 60 min (PE) after injection. Lesion uptake of 99mTc-Hn-bitistatin at 4 h after injection was calculated in terms of percentage injected dose per gram (%ID/g) of tissue and averaged 0.89 %ID/g PE and 0.79 %ID/g DVT. Lesion-to-background ratios averaged 34:1 (PE-to-lung), 18:1 (DVT-to-blood), and 284:1 (DVT-to-muscle). These values were not significantly different from iodinated bitistatin, but uptakes were higher than other tracers tested in the same model. CONCLUSION: 99mTc-Hn-bitistatin retains the functional activity of the iodinated peptide, has higher DVT and PE uptakes than other thrombus tracers in this standardized model, and has target-to-background characteristics suitable for imaging both PE and DVT in a single test.

High-affinity no-carrier-added 99mTc-labeled chemotactic peptides for studies of inflammation in vivo.

Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys, a chemotactic peptide that binds with high affinity to the chemoattractant receptor on granulocytes and monocytes, was labeled with 99mTc using the diaminedithiol (DADT) chelating system to coordinate the Tc. 99mTc labeling of the DADT-coupled peptide was accomplished in 84% overall yield (room temperature for 10 min) using [99mTc]glucoheptonate as the donor of prereduced Tc. HPLC analysis showed two major 99mTc-labeled peptide peaks, 99mTc-DADT-Pep-I and 99mTc-DADT-Pep-II, were obtained in a ratio of 1:0.85. Using an iodoacetamide-derivatized gel to remove unlabeled peptide from the 99mTc labeling mixtures, essentially no-carrier-added (nca) high-specific activity 99mTc-labeled chemotactic peptides were obtained. The 99Tc analogues of the peptides were synthesized (72% yield) in a similar fashion and correlated with 99mTc complexes I and II by HPLC. In vitro competitive receptor binding assays of the isolated 99Tc analogues were performed against the tritiated chemotactic peptide [3H]N-for-Met-Leu-Phe ([3H]fMLF) using isolated granulocytes. The 99Tc-derivatized peptides showed similar binding affinities to the chemoattractant receptor as the unlabeled Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. The nca 99mTc-labeled peptides gave high contrast images of experimental inflammation in rabbits without causing neutropenia. Thus, it is feasible to attach the Tc-DADT chelate to low-molecular weight receptor binding chemotactic peptides and retain substantial binding to the receptor. Chemotactic peptides labeled with 99mTc via the DADT ligand system have the potential for imaging focal sites of inflammation without toxic effects, an important consideration in the successful utilization of chemotactic peptide agonists.

Design, synthesis, and initial evaluation of high-affinity technetium bombesin analogues.

Potent antagonists of bombesin-like peptides have shown great potential for applications in cancer therapy. A 99mTc-labeled agent capable of identifying patients who could benefit from these emerging therapies would have a great impact on patient management. This study involves the synthesis and initial evaluation of technetium diaminedithiolate analogues derived from the potent bombesin analogue Pyr-Gln-Lys-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (Lys3-bombesin). We coupled two diaminedithiol (DADT) bifunctional chelating agents (BCAs 1 and 2) to the Lys3 residue at the N-terminal region that is not required for binding to the receptor. 99mTc labeling was performed by ligand exchange on addition of [99mTc]glucoheptonate to a solution of the adduct at room temperature. Two products were obtained from each adduct on analysis by HPLC. The major to minor product ratios of the 99mTc-labeled analogues were 3:1 for products from BCA 1 and 9:1 for the products from BCA 2. Macroscopic amounts of the 99Tc analogues were similarly prepared using [99Tc]glucoheptonate. In this case, the major to minor ratios were 2:1 for the products from both BCAs. For initial evaluation of the binding of the Tc-labeled peptides to bombesin receptors, the 99Tc analogues were used in vitro in competitive binding assays in rat brain cortex membranes against [125I-Tyr4]bombesin. Results of the in vitro assays showed that the inhibition constants (Ki) of the major and minor products were 3.5+/-0.7 and 3.9+/-1.5 nM, respectively, for the products from BCA 1; and 7.4+/-2.0 and 5.2+/-1.5 nM for the products derived from BCA 2, respectively. The high affinity exhibited by these technetium analogues is an indication of their potential for use in non-invasive in vivo biochemical characterization of cancers that possess receptors for bombesin.

Validation and application of a solid phase chemistry method for preparation of high effective specific activity technetium-99m radiopharmaceuticals.

Solid phase chemistry with UltraLink Iodoacetyl gel was investigated as a method for removing excess ligand present in preparations of diaminedithiol (DADT) 99mTc radiopharmaceuticals. The process was optimized and tested under 99mTc labeling conditions with 2,2,4,9,9-pentamethyl-4,7-diaza-1,10-decanedithiol (N-methyl-DADT), 1, and 4-(1-(2'-methoxyphenyl) piperazinylbutyl)-2,2,9,9-tetramethyl-4,7-diaza-1,10 -decanedithiol (DADT-4C-MPP), 2, a serotonin receptor-binding ligand. The results illustrated that this gel removed > or = 99.9% excess ligand used in labeling reactions, and was comparable to the level of purification usually obtained by the more labor intensive method of HPLC.

Pulmonary accumulation of neutral diamine dithiol complexes of technetium-99m.

Twenty-two neutral, lipid-soluble 99mTc complexes have been synthesized from diamine dithiol (DADT) ligands which vary in alkyl substitution pattern on nitrogen and carbon. The logarithm of the partition coefficients (log PC), as well as the capacity factor k', of the purified complexes increased linearly with molecular weight. The biodistribution of these complexes was determined in normal mice, and several of the complexes selectively accumulated in the lungs as compared to the liver or other organs. Pulmonary accumulation varied greatly with subtle changes in structure, and a 30-fold range of lung uptake (1-31% of the injected dose/organ) was observed for isomeric technetium complexes which have identical molecular weights and similar log PC. Further, a parabolic relationship between lung uptake and log PC was observed for a subset of the complexes which are derived from a homologous series of tetramethyl-DADT ligands. Neutral and lipophilic radiopharmaceuticals labeled with technetium can therefore be developed which exhibit structurally specific uptake in the lung.

Bifunctional chelator for facile preparation of neutral technetium complexes.

The diaminedithiol (DADT) ligand system has proven to be useful as a carrier of technetium-99m in the preparation of a wide variety of site-specific radiopharmaceuticals. To expand the utility of the ligand system, we have designed and synthesized a bifunctional chelating agent based on the ligand system whose adducts generate a neutral technetium complex core and therefore can penetrate intact membranes. We have evaluated both the coupling of the thiolactone reactive moiety of the bifunctional chelate to benzylamine as a model as well as subsequent labeling with technetium-99m. Reaction with benzylamine was complete at room temperature within 2 h, producing the adduct in 74% isolated yield. On coordination of the benzylamine adduct to technetium-99m, one major product was obtained in high yield (> 90%). The product was stable in serum and physiologic saline at 37 degrees C over a 20-h study period. The partition coefficient of the technetium complex was 101 +/- 6.2, indicating that the complex was lipophilic. Biodistribution studies in mice showed that the brain concentration at 5 min postinjection was 0.91 +/- 0.09% injected dose/g indicating that the complex penetrates the intact blood-brain barrier. This is further evidence that the complex is neutral and lipophilic. This bifunctional chelate should facilitate the incorporation of technetium-99m into molecules of biological interest such as drugs, small peptides, and metabolic substrates.

Novel technetium ligands with affinity for the muscarinic cholinergic receptor.

The synthesis and preliminary biological characterization of two isomeric technetium labeled complexes (2,5,5,9-tetramethyl-4,7-diaza-7-(3' (R)-quinuclidinylcarboxymethyl)-2,9-decanedithiolato oxo 99/99mtechnetium(V), [99/99mTc]-1 and [99/99mTc]-2) designed to exhibit affinity to muscarinic cholinergic receptors are described. In vitro binding assays were conducted in mouse brain homogenates (whole brain-cerebellum) at 37 degrees C by the centrifugation method, where non-specific binding was defined by atropine (1 microM). The measured affinity (KD) of [99Tc]-1 for mAChR was 1.9 +/- 0.5 microM (mean +/- SEM; n = 3) and [99Tc]-2 was 4.5 +/- 0.5 microM (mean +/- SEM; n = 3). Scatchard analysis indicated that Bmax values were 10.6 +/- 0.5 and 16.9 +/- 0.5 pmol/mg tissue, respectively. In competition assays, [99Tc]-1 exhibited an apparent affinity (KI) of 16.5 microM (n = 2) against [125I] iododexetimide, whereas [99Tc]-2 exhibited an affinity (KI) of 105 microM. In vivo, 0.3% of the injected dose of [99mTc]-1 and [99mTc]-2 accumulated in the brain at 5 min after injection. These values indicate technetium analogues of neuroreceptor binding ligands can be synthesized and retain some affinity for the receptor.

Synthesis and in vivo evaluation of a 99m/99Tc-DADT-benzovesamicol: a potential marker for cholinergic neurons.

The diaminodithiol (DADT) ligand has been conjugated to the neuromuscular blocking agent benzovesamicol (BVM) in the 5-position. DADT-BVM 1 was synthesized by coupling of 5-aminomethylbenzovesamicol with a BCA thiolactone reagent. 99mTc radiolabeling of 1 with [99mTc]glucoheptonate gave a 4.7:1 mixture of two 99mTc complexes as determined by HPLC. Biodistribution data of the major [99mTc]-1 complex in CD-1 mice (n = 4-5) showed very little uptake and no regional selectivity in the mouse brain. At all time points examined, the lung and liver showed the highest uptake. For whole brain, the % injected dose values were 0.27, 0.12, 0.04 and 0.01% at t = 1, 5, 30 and 240 min. The major [99mTc]-1 product exhibited a log P = 3.13 +/- 0.06 (SD) with an IC50 = 140-280 nM for the corresponding [99Tc]-1 vs (-)-N-[3H]methyl-5-aminobenzovesamicol. The low brain uptake of [99mTc]-1 vs 5-iodobenzovesamicol is attributed to its higher molecular weight (752) and lower binding affinity.

Structure of a neutral N-alkylated diaminedithiol (dadt) 99TcV complex: syn[99TcO(NEt-tmdadt)].

syn-(4-Ethyl-2,9-dimethyl-4,7-diaza-2,9-decanedithiolato)oxo (99Tc) technetium(V), syn-[99TcO(NEt-tmdadt)], [Tc(O)(C12H25N2S2)], Mr = 375, monoclinic, P2(1)/n, a = 9.638(2), b = 14.371(5), c = 11.893(3) A, beta = 100.79(2) degrees, V = 1618.15(75) A3, Z = 4, Dx = 1.54 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 11.0 cm-1, F(000) = 776, T = 293 K, R = 0.028, wR = 0.042 for 2788 unique observed reflections with I greater than sigma(I). This study is the first which conclusively establishes the structure of the major complex derived from the reaction of an N-substituted diaminedithiol ligand with reduced pertechnetate. An intramolecular repulsion exists between the ethyl substituent and the oxo-metal core, resulting in a larger-than-expected angle for N(2)-Tc-O, 104.7(1) degrees.

Synthesis of a diaminedithiol bifunctional chelating agent for incorporation of technetium-99m into biomolecules.

The synthesis of a bifunctional chelating agent (BCA), 1, based on the diaminedithiol (DADT) ligand system, is described. The six-step synthetic sequence has been accomplished in 16% overall yield, affording 1, which contains a thiolactone as a reactive moiety, which permits direct coupling to nucleophiles without the formation of byproducts. The reactivity of 1 toward benzylamine and subsequent labeling of the ligand with technetium-99m has been evaluated as a model for preparation of various bioconjugates. Both coupling and exchange labeling occur in high yield under mild conditions, and competition reactions with diethylenetriaminepentaacetic acid (DTPA) indicate the superior stability of the technetium-99m-DADT complex. Preparation of BCA 1 thus provides a new avenue into technetium-labeled radiopharmaceuticals.

99mTc labeling of proteins: initial evaluation of a novel diaminedithiol bifunctional chelating agent.

The initial evaluation of a novel thiolactone bifunctional chelating agent, 1, for facile introduction of the diaminedithiol (DADT) ligand system onto proteins for subsequent labeling with 99mTc under mild conditions is presented. For human serum albumin and a monoclonal anti-colon carcinoma antibody (B72.3), time-dependent coupling was achieved at pH 7-9 at room temperature. The coupled proteins could be labeled in a direct fashion (Sn2+/99mTcO4-) and by exchange labeling (with preformed 99mTc-glucoheptonate) in good to excellent yields at pH 7. In contrast, the unmodified proteins could be labeled only by direct methods with maximum yields of 30%. The labeled, coupled proteins were purified by high performance liquid chromatography and found to be stable in vitro over a 20-h period of study with no evidence for the loss of label. This exceptional stability is due to the chelation of 99mTc by the DADT ligand since 66% (3.5 h) and 47% (20 h) of the label were lost from directly labeled unmodified human serum albumin and the unmodified antibody, respectively. Biodistribution studies in normal mice and rabbits confirmed the stability of the 99mTc-DADT-labeled proteins compared to the 99mTc-labeled unmodified proteins in vivo. Thus, the novel thiolactone 1 is a useful bifunctional chelating agent for introducing the DADT ligand system onto a variety of proteins for subsequent incorporation of technetium under mild conditions such that the resultant labeled products are stable in vitro and in vivo.