Efficacy of a HER2-Targeted Thorium-227 Conjugate in a HER2-Positive Breast Cancer Bone Metastasis Model.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-30% of breast cancers but has low expression in normal tissue, making it attractive for targeted alpha therapy (TAT). HER2-positive breast cancer typically metastasizes to bone, resulting in incurable disease and significant morbidity and mortality. Therefore, new strategies for HER2-targeting therapy are needed. Here, we present the preclinical in vitro and in vivo characterization of the HER2-targeted thorium-227 conjugate (HER2-TTC) TAT in various HER2-positive cancer models. In vitro, HER2-TTC showed potent cytotoxicity in various HER2-expressing cancer cell lines and increased DNA double strand break formation and the induction of cell cycle arrest in BT-474 cells. In vivo, HER2-TTC demonstrated dose-dependent antitumor efficacy in subcutaneous xenograft models. Notably, HER2-TTC also inhibited intratibial tumor growth and tumor-induced abnormal bone formation in an intratibial BT-474 mouse model that mimics breast cancer metastasized to bone. Furthermore, a match in HER2 expression levels between primary breast tumor and matched bone metastases samples from breast cancer patients was observed. These results demonstrate proof-of-concept for TAT in the treatment of patients with HER2-positive breast cancer, including cases where the tumor has metastasized to bone.

Mesothelin-Targeted Thorium-227 Conjugate (MSLN-TTC): Preclinical Evaluation of a New Targeted Alpha Therapy for Mesothelin-Positive Cancers.

PURPOSE: Targeted thorium-227 conjugates (TTC) represent a new class of molecules for targeted alpha therapy (TAT). Covalent attachment of a 3,2-HOPO chelator to an antibody enables specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. Because of the high energy and short penetration range, TAT efficiently induces double-strand DNA breaks (DSB) preferentially in the tumor cell with limited damage to the surrounding tissue. We present herein the preclinical evaluation of a mesothelin (MSLN)-targeted thorium-227 conjugate, BAY 2287411. MSLN is a GPI-anchored membrane glycoprotein overexpressed in mesothelioma, ovarian, pancreatic, lung, and breast cancers with limited expression in healthy tissue. EXPERIMENTAL DESIGN: The binding activity and radiostability of BAY 2287411 were confirmed bioanalytically. The mode-of-action and antitumor potency of BAY 2287411 were investigated in vitro and in vivo in cell line and patient-derived xenograft models of breast, colorectal, lung, ovarian, and pancreatic cancer. RESULTS: BAY 2287411 induced DSBs, apoptotic markers, and oxidative stress, leading to reduced cellular viability. Furthermore, upregulation of immunogenic cell death markers was observed. BAY 2287411 was well-tolerated and demonstrated significant antitumor efficacy when administered via single or multiple dosing regimens in vivo. In addition, significant survival benefit was observed in a disseminated lung cancer model. Biodistribution studies showed specific uptake and retention of BAY 2287411 in tumors and enabled the development of a mechanistic pharmacokinetic/pharmacodynamic model to describe the preclinical data. CONCLUSIONS: These promising preclinical results supported the transition of BAY 2287411 into a clinical phase I program in mesothelioma and ovarian cancer patients (NCT03507452).

Targeted alpha therapy using a novel CD70 targeted thorium-227 conjugate in in vitro and in vivo models of renal cell carcinoma.

The cell surface receptor CD70 has been previously reported as a promising target for B-cell lymphomas and several solid cancers including renal cell carcinoma. We describe herein the characterization and efficacy of a novel CD70 targeted thorium-227 conjugate (CD70-TTC) comprising the combination of the three components, a CD70 targeting antibody, a chelator moiety and the short-range, high-energy alpha-emitting radionuclide thorium-227 (227Th). In vitro analysis demonstrated that the CD70-TTC retained binding affinity to its target and displayed potent and specific cytotoxicity compared to an isotype control-TTC. A biodistribution study in subcutaneous tumor-bearing nude mice using the human renal cell carcinoma cell line 786-O demonstrated significant uptake and retention with 122 ± 42% of the injected dose of 227Th per gram (% ID/g) remaining in the tumor seven days post dose administration compared to only 3% ID/g for the isotype control-TTC. Tumor accumulation correlated with a dose dependent and statistically significant inhibition in tumor growth compared to vehicle and isotype control-TTC groups at radioactivity doses as low as 50 kBq/kg. The CD70-TTC was well tolerated as evidenced by only modest changes in hematology and normal gain in body weight of the mice. To our knowledge, this is the first report describing molecular targeting of CD70 expressing tumors using a targeted alpha-therapy (TAT).

The fate of Gd and chelate following intravenous injection of gadodiamide in rats.

OBJECTIVE: The biodistribution of gadolinium (Gd) and chelate was studied in rats injected intravenously with a commercially available gadodiamide magnetic resonance contrast agent spiked with trace amounts of (14)C-labelled GdDTPA-BMA. METHODS: Biodistribution of the (14)C-labelled ligand in whole animals was visualised using quantitative whole-body autoradiography, and quantified in individual tissue samples by analysing for radioactivity using beta-counting. Biodistribution of Gd was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and inductively coupled plasma sector field mass spectrometry (ICP-SF-MS). RESULTS: The injected dose was rapidly excreted, with only 1.0% remaining in the body at 24 h. The radioactivity thereafter was mainly associated with kidney cortex, liver, lung, muscle and skin, with a similar rate of clearance for both ligand and Gd from these tissues. The ratio between (14)C-labelled substance and Gd was not significantly different from that of the injected substance in most tissue samples up to 24 h after injection; the ratio then slowly decreased. CONCLUSIONS: The data clearly show that measurements of Gd concentration alone in tissue samples from animals injected with Gd-based contrast agents (GBCAs) cannot be used as a measure of Gd released from the ligand. To our knowledge, such measurements comparing Gd and ligand concentrations and distribution in tissue samples have not been published previously for any of the commercial GBCAs.

Pharmacokinetics of perfluorobutane following intravenous bolus injection and continuous infusion of sonazoid in healthy volunteers and in patients with reduced pulmonary diffusing capacity.

The ultrasound contrast agent Sonazoidtrade mark was administered as an i.v. bolus injection of 0.6 microL microbubbles/kg body weight or as a continuous infusion over 30 min at a rate of 1.2 microL microbubbles/kg body weight to healthy volunteers and patients with reduced pulmonary diffusing capacity. Expired air and blood samples were collected from 32 subjects and perfluorobutane (PFB) gas was analyzed using validated gas chromatography mass spectrometry methods. Blood concentrations of PFB declined biphasicly with a distribution half-life (t(0.5 to 15)) of 2 to 3 min and an elimination half-life (t(15 to 120)) of 30 to 45 min. Area under the curve (AUC) values in patients with impaired gas diffusion were significantly larger than those in healthy volunteers. The exhalation kinetics were somewhat variable with a PFB elimination half-life (t(15 to 120)) of 28 to 111 min. Clearance of PFB was independent of study population and mode of administration. There were no deaths and no serious adverse events that resulted in the withdrawal of a subject from the study. With the exception that arthralgia predominated in healthy volunteers, healthy volunteers and diseased subjects did not show a different adverse event profile whether Sonazoid was administered as a bolus injection or as an infusion. Assessment of laboratory parameters (serum biochemistry, haematology and urinalysis), vital signs, oxygen saturation and electrocardiograms (ECGs) showed no changes which caused safety concern. (E-mail: Kristin.Landmark@ge.com).

Quantification of 13C pyruvate and 13C lactate in dog blood by reversed-phase liquid chromatography-electrospray ionization mass spectrometry after derivatization with 3-nitrophenylhydrazine.

Injection of hyperpolarized (13)C-labelled pyruvate ((13)C pyruvate) is under evaluation as an agent for medical metabolic imaging by measuring formation of (13)C lactate using magnetic resonance spectroscopy of the (13)C nuclei. A quantitative method for analysis of these (13)C-labelled substances in dog blood was needed as part of the development of this agent and we here describe a liquid chromatography-mass spectrometry method for that purpose. Immediately after blood collection, the blood proteins were precipitated using methanol added internal standard ([U-(13)C]pyruvate and [U-(13)C]lactate). Prior to analysis, the compounds were derivatized using 3-nitrophenylhydrazine. Following separation on a Supelco Discovery HS C18 column, (13)C pyruvate and (13)C lactate were detected using negative electrospray ionization mass spectrometry. Calibration standards (4.5-4500 microM (13)C pyruvate and 9-9000 microM (13)C lactate) and added internal standard were used to make the calibration curves, which were fitted to a non-linear equation y=a+bx+cx(2) and weighted with a weighting factor of 1/y(2). The analytical lower limit of quantification of (13)C pyruvate and (13)C lactate was 4.5 and 9 microM, respectively. The total precision of the method was below 9.2% for (13)C pyruvate and below 5.8% for (13)C lactate. The accuracy of the method showed a relative error less than 2.4% for (13)C pyruvate and less than 6.3% for (13)C lactate. The recoveries were in the range 93-115% for (13)C pyruvate and 70-111% for (13)C lactate. Both substances were stable in protein-free supernatant when stored for up to 3 weeks in a -20 degrees C freezer, during three freeze/thaw cycles, and when stored in an autosampler for at least 30 h.

Quantification of phosphatidylserine, phosphatidic acid and free fatty acids in an ultrasound contrast agent by normal-phase high-performance liquid chromatography with evaporative light scattering detection.

Sonazoid is a new contrast agent for ultrasound imaging. The product is an aqueous suspension of perfluorobutane microbubbles coated with phospholipids obtained from hydrogenated egg phosphatidylserine (H-EPS). A normal-phase high-performance liquid chromatographic (HPLC) method with evaporative light scattering detection was developed for quantification of free fatty acids, phosphatidylserine and phosphatidic acid in H-EPS and Sonazoid. Separation of the lipids was carried out on an HPLC diol column and a gradient of chloroform and methanol with 0.2% formic acid titrated to pH 7.5 with ammonia. The calibration standards contained stearic acid, distearoyl-phosphatidic acid (DSPA) and distearoyl-phosphatidylserine (DSPS) in the concentration range of 0.016-1.0mg/ml (0.4-25microg injected). The method was validated with a limit of quantification of the three lipids set to 0.4microg (approximately 20-60microM). The best fit of the three calibration curves were obtained when the logarithmic transformed theoretical lipid concentration was plotted against the logarithmic transformed area under the peak and fitted to a second order polynomial equation. Stearic acid, DSPA and DSPS were analysed with an intermediate precision ranging from 4.4% to 5.3% R.S.D. and they were extracted from an aqueous suspension with a recovery ranging from 103.3% to 113.3%. The sum of total phospholipid concentration determined in H-EPS ranged from 96.4% to 103.2% of the theoretical values. The lipids in the ultrasound product were quantitated with a repeatability ranging from 6.2% to 11.7% R.S.D.

Disposition of perfluorobutane in rats after intravenous injection of Sonazoid.

The new ultrasound contrast agent Sonazoid was injected IV in rats at doses of 0.8 and 8 muL perfluorobutane (PFB)-containing microbubbles/kg body weight. Samples were obtained from blood, liver, spleen, fat, kidney, muscle, heart, lung and brain from both males and females and the PFB gas was analyzed using validated gas chromatography mass spectrometry methods. No differences were observed between genders or doses for any of the pharmacokinetic parameters. For all tissues, the highest concentrations were observed at the first time point (i.e., 5 min postinjection) (51% of injected dose in liver; total recovery of 69%). The highest concentrations of PFB in tissue were observed in spleen > liver > lung > kidney >> other tissues. At 24 h after dosing, the total amount of PFB remaining in the tissues was 1.9%. These data fit well with the finding that after a Sonazoid dose of 8 microL microbubbles/kg to male rats, more than 50% of the injected PFB was recovered in exhaled air by 20 min after dosing. During the first 24 h after administration, more than 96% of the PFB dose was recovered in exhaled air.

Nc100668, a new tracer for imaging of venous thromboembolism: disposition and metabolism in rats.

The 99mTc-complex of NC100668 [Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194] is being evaluated for nuclear medical imaging of venous thromboembolism. NC100668 is a 13-amino acid peptide with a Tc-binding chelator [NC100194; -NH-CH2-CH2-N(CH2-CH2-NH-C(CH3)2-C(CH3)=N-OH)2] linked to the C-terminal end. Following injection in rats of [Asn-U-14C]NC100668 (labeling of the N-terminal amino acid), approximately 70% of the radioactivity was recovered in urine within 3 days. Following injection of [Lys-U-14C]NC100668 (labeling close to the C-terminal amino acid), radioactivity was cleared more slowly, with only 8% recovered in urine and approximately 80% of the radioactivity present in the body after 3 days. The highest concentration of radioactivity in the body following injection of [Lys-U-14C]NC100668 was observed in the kidney inner cortex; this probably represents 14C-labeled Lys, which is reabsorbed in the kidney tubules and incorporated into protein metabolism. Metabolite profiling by high-performance liquid chromatography with radiochemical detection revealed that following injection of [Asn-U-14C]NC100668, there is a rapid appearance in blood of one peak containing radioactive metabolite(s) originating from the N-terminal part of the molecule. In urine samples, only this radioactive peak was observed with no intact NC100668 remaining; this very hydrophilic N-terminal metabolite was probably either the N-terminal amino acid or a very short peptide. Liquid chromatography-mass spectrometry analyses of rat urine samples obtained after injection of nonlabeled NC100668 confirmed the identity of two metabolites generated from the C-terminal end of the molecule; Gly-NC100194 was identified as the major of these metabolites and NC100194 as a minor metabolite present at approximately one-tenth the amount of Gly-NC100194. No other metabolites were identified.

A respiration-metabolism chamber system and a GC-MS method developed for studying exhalation of perfluorobutane in rats after intravenous injection of the ultrasound contrast agent Sonazoid.

Sonazoid is a new contrast agent for ultrasound imaging comprising an aqueous suspension of lipid-stabilised perfluorobutane (PFB) gas microbubbles. A respiration-metabolism chamber system was developed to collect exhaled air following intravenous administration of Sonazoid to rats. Analysis of PFB in the exhaled rat air was performed using a modified version of an earlier published method for blood samples, i.e. an automatic headspace gas chromatographic mass spectrometric (GC-MS) method using electron impact ionisation. The calibration standards were PFB diluted in air (2.5-1800 pg/ml). Perfluoropentane (PFP) was used as an internal standard and the MS detector was set to single ion monitoring of the base fragment ions of PFB (m/z 69 and 119) and PFP (m/z 69). The calibration curve, made by plotting the peak area ratios of PFB (m/z 69) to PFP (m/z 69) against the theoretical concentration of PFB, was fitted to a linear equation with weighting 1/y2 and found to be reproducible. The lower limit of quantification (LLOQ) was 2.5 pg PFB/ml. The between-day variation of the method was below 2.6% relative standard deviation (R.S.D.) and the within-day variation of the method was below 6.4% R.S.D. The accuracy of the method was evaluated and showed a relative error less than 5.2%. PFB was found to be stable for 14 days when stored in Tedlar sample bags at room temperature. An even lower detection limit may be obtained by using the more time-consuming process of solid-phase micro extraction; thus, by concentrating PFB on carboxen-PDMS fibres an LLOQ of 0.5 pg PFB/ml was obtained. When five rats were given an i.v. bolus injection of Sonazoid at a dose of 8 microl microbubbles/kg a mean recovery of 96% (range, 81-110%) was found during 24 h; more than 50% was exhaled during the first 30 min after injection.