Detection of Experimental Colorectal Peritoneal Metastases by a Novel PDGFRß-Targeting Nanobody.

Peritoneal metastases in colorectal cancer (CRC) belong to Consensus Molecular Subtype 4 (CMS4) and are associated with poor prognosis. Conventional imaging modalities, such as Computed Tomography (CT) and Fluorodeoxyglucose-Positron Emission Tomography (FDG-PET), perform very poorly in the detection of peritoneal metastases. However, the stroma-rich nature of these lesions provides a basis for developing molecular imaging strategies. In this study, conducted from 2019 to 2021, we aimed to generate a Platelet-Derived Growth Factor Receptor beta (PDGFRB)-binding molecular imaging tracer for the detection of CMS4 CRC, including peritoneal metastases. The expression of PDGFRB mRNA discriminated CMS4 from CMS1-3 (AUROC = 0.86 (95% CI 0.85-0.88)) and was associated with poor relapse-free survival. PDGFRB mRNA and protein levels were very high in all human peritoneal metastases examined (n = 66). Therefore, we generated a PDGFRB-targeting llama nanobody (VHH1E12). Biotin-labelled VHH1E12 bound to immobilized human and mouse PDGFRB with high affinity (EC50 human PDGFRB = 7 nM; EC50 murine PDGFRB = 0.8 nM), and to PDGFRB-expressing HEK293 cells grown in vitro. A pharmacokinetic analysis of IRDye-800CW-conjugated VHH1E12 in mice showed that the plasma half-life was 6 min. IRDye-800CW-conjugated VHH1E12 specifically accumulated in experimentally induced colorectal cancer peritoneal metastases in mice. A tissue analysis subsequently demonstrated co-localization of the nanobody with PDGFRB expression in the tumour stroma. Our results demonstrate the potential value of PDGFRB-targeted molecular imaging as a novel strategy for the non-invasive detection of CMS4 CRC, in particular, peritoneal metastases.

GMP production of [68Ga]Ga-BOT5035 for imaging of liver fibrosis in microdosing phase 0 study.

INTRODUCTION: Early detection of liver fibrosis and monitoring response to treatment crucial for the management of patients are currently not feasible in clinical practice. Platelet derived growth factor receptor ß (PDGFR-ß) expression is regarded as a potential biomarker to determine the stages of fibrotic diseases including liver fibrosis. [68Ga]Ga-BOT5035 comprising a bicyclic peptide was developed for specific targeting of PDGFR-ß overexpressed in pathological fibrosis. The realization of microdosing phase 0 study using [68Ga]Ga-BOT5035 positron emission tomography required automated good manufacturing practice (GMP) compliant production of [68Ga]Ga-BOT5035 presented herein. Moreover, the investigation of radiation dosimetry was conducted to ensure possibility of multiple annual examinations for disease monitoring in clinical setup. METHODS: The active pharmaceutical ingredient starting material BOT5035 (GMP grade) was provided by BiOrion Technologies BV. The 68Ga-labelling process was developed and automated using synthesis platform (Modular-Lab PharmTrace, Eckert & Ziegler), disposable cassettes for 68Ga-labelling, and pharmaceutical grade 68Ge/68Ga generator (GalliaPharm®) purchased from Eckert & Ziegler. Radiolysis sensitive BOT5035 required development and systematic optimization of the labelling synthesis parameters such as time, temperature, precursor concentration, radical scavenger, buffer concentration and pH. The validation process was conducted with regard to the product quality and quantity, as well as production reproducibility. Human organ equivalent doses and total body effective doses were calculated using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1), based on ex vivo organ distribution in Sprague-Dawley rats. RESULTS: The GMP compliant automated production of [68Ga]Ga-BOT5035 with on-line documentation demonstrated high reproducibility. The time for the labelling synthesis and quality control was approximately 60 min. The non-decay corrected radiochemical yield and radiochemical purity of the radiopharmaceutical were 43.7 ±â€¯7.6% (n = 3, process validation) and 97.7 ±â€¯0.4% (n = 3, process validation), respectively. Predefined acceptance criteria were met for the sterility, endotoxins level, radionuclidic purity and residual solvent content. The stability at ambient temperature was controlled for 120 min with approved results. Ex vivo organ distribution data revealed fast blood clearance and washout from most of the organs. The dose-limiting organs were kidney and bone marrow. The total effective dose as limiting parameter would allow for up to 3-4 PET scans per annum. CONCLUSION: The fully automated and GMP compliant production of [68Ga]Ga-BOT5035 was developed and thoroughly validated. The radiopharmaceutical was approved by Swedish Medicinal Products Agency and the Ethical Review Authority for the Phase 0 clinical study of the quantitative imaging of liver fibrosis. Human dosimetry calculations extrapolated from animal experiment indicated possibility of 3-4 PET examinations per year.

Double-blind study of pardoprunox, a new partial dopamine agonist, in early Parkinson's disease.

This study examined the efficacy and safety of the partial dopamine agonist, pardoprunox (SLV308), in the treatment of patients with early Parkinson's disease (PD). Patients were randomized to receive pardoprunox (n = 69) or placebo (n = 70). Pardoprunox was titrated to each patient's optimal dose (9-45 mg/d) over 2 to 6 weeks and then maintained at this dose for a further 3 weeks. Concomitant anti-Parkinson treatment was not permitted. In the primary analysis, Unified PD Rating Scale (UPDRS)-Motor score was improved in pardoprunox-treated patients (overall mean dose 23.8 mg/d; -7.3 points), as compared with placebo (-3.0 points; P = 0.0001), from baseline to end point. At end point, there were more responders (> or = 30% reduction in UPDRS-Motor score) in the pardoprunox group (50.7%) than in the placebo group (15.7%; P < 0.0001). In other secondary analyses, UPDRS-activities of daily living (ADL) and -ADL+Motor scores were also significantly more improved in the pardoprunox group. Nausea was reported by 32 of 68 (47.1%) pardoprunox-treated patients (vs. 3/70, 4.3%, placebo-treated patients), with dizziness, somnolence, headache, and asthenia also reported by > or = 10 patients. In this exploratory proof-of-concept study, pardoprunox significantly improved motor function in patients with early PD. The efficacy and safety profile of pardoprunox justifies its further investigation in PD.

In vitro characterization of SLV308 (7-[4-methyl-1-piperazinyl]-2(3H)-benzoxazolone, monohydrochloride): a novel partial dopamine D2 and D3 receptor agonist and serotonin 5-HT1A receptor agonist.

Present Parkinson's disease treatment strategies are far from ideal for a variety of reasons; it has therefore been suggested that partial dopamine receptor agonism might be a potential therapeutic approach with potentially fewer side effects. In the present study, we describe the in vitro characterization of the nonergot ligand SLV308 (7-[4-methyl-1-piperazinyl]-2(3H)-benzoxazolonemonohydrochloride). SLV308 binds to dopamine D(2), D(3), and D(4) receptors and 5-HT(1) (A) receptors and is a partial agonist at dopamine D(2) and D(3) receptors and a full agonist at serotonin 5-HT(1) (A) receptors. At cloned human dopamine D(2,L) receptors, SLV308 acted as a potent but partial D(2) receptor agonist (pEC(50) = 8.0 and pA(2) = 8.4) with an efficacy of 50% on forskolin stimulated cAMP accumulation. At human recombinant dopamine D(3) receptors, SLV308 acted as a partial agonist in the induction of [(35)S]GTPgammaS binding (intrinsic activity of 67%; pEC(50) = 9.2) and antagonized the dopamine induction of [(35)S]GTPgammaS binding (pA(2) = 9.0). SLV308 acted as a full 5-HT(1) (A) receptor agonist on forskolin induced cAMP accumulation at cloned human 5-HT(1) (A) receptors but with low potency (pEC(50) = 6.3). In rat striatal slices SLV308 concentration-dependently attenuated forskolin stimulated accumulation of cAMP, as expected for a dopamine D(2) and D(3) receptor agonist. SLV308 antagonized the inhibitory effect of quinpirole on K(+)-stimulated [(3)H]-dopamine release from rat striatal slices (pA(2) = 8.5). In the same paradigm, SLV308 had antagonist properties in the presence of quinpirole (pA(2) = 8.5), but the partial D(2) agonist terguride had much stronger antagonistic properties. In conclusion, SLV308 combines high potency partial agonism at dopamine D(2) and D(3) receptors with full efficacy low potency serotonin 5-HT(1) (A) receptor agonism and is worthy of profiling in in vivo models of Parkinson's disease.

Piperazinyl oxime ethers as NK-1 receptor antagonists.

The synthesis and structure-activity relations for a new class of centrally active NK-1 receptor antagonists are described. The new compounds are based on piperazine 2 and contain an oxime ether functionality. Several new compounds have high affinity for the NK-1 receptor and show good antagonistic activity in the gerbil foot-tapping assay.

Tonic inhibition by orphanin FQ/nociceptin of noradrenaline neurotransmission in the amygdala.

The present microdialysis study investigated whether nociceptin/orphanin FQ exerts a tonic inhibition of the release of noradrenaline in the basolateral nucleus of the amygdala in awake rats. The non-peptide competitive nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist J-113397 (20 mg/kg i.p.) induced an increase in the release of noradrenaline to about 150-200%. The increase was strongly suppressed by local infusion of an endogenous N/OFQ peptide receptor agonist, nociceptin/orphanin FQ (1 microM) via retrograde microdialysis, into the basolateral nucleus of the amygdala. Local infusion of nociceptin/orphanin FQ (1 microM) itself reduced noradrenaline release in the basolateral nucleus of the amygdala to about 70% of basal levels. These results indicate that a large part of basal release of noradrenaline in the basolateral nucleus of the amygdala is under tonic inhibitory control by endogenous nociceptin/orphanin FQ through the N/OFQ peptide receptors localized within the basolateral nucleus of the amygdala.