Preclinical Biodistribution Studies Research Articles

Automated radiosynthesis and preclinical evaluation of two new PSMA-617 derivatives radiolabelled via [18F]AlF2+ method

BackgroundIn the last decade the development of new PSMA-ligand based radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research. The most promising derivative in terms of interaction with the antigen and clinical properties has been found to be “PSMA-617”, and its lutetium-177 radiolabelled version has recently been approved by EU and USA regulatory agencies for therapeutic purposes. For the above reasons, the development of new derivatives of PSMA-617 radiolabelled with fluorine-18 may still be of great interest. This paper proposes the comparison of two different PSMA-617 derivatives functionalized with NODA and RESCA chelators, respectively, radiolabelled via [18F]AlF2+ complexation.ResultsThe organic synthesis of two PSMA-617 derivatives and their radiolabelling via [18F]AlF2+ complexation resulted to proceed efficiently and successfully. Moreover, stability in solution and in plasma has been evaluated. The whole radiosynthesis procedure has been fully automated, and the final products have been obtained with radiochemical yield and purity potentially suitable for clinical studies. The biodistribution of the two derivatives was performed both in prostate cancer and glioma tumour models. Compared with the reference [18F]F-PSMA-1007 and [18F]F-PSMA-617-RESCA, [18F]F-PSMA-617-NODA derivative showed a higher uptake in both tumors, faster clearance in non-target organs, and lower uptake in salivary glands.ConclusionPSMA-617 NODA and RESCA derivatives were radiolabelled successfully via [18F]AlF2+ chelation, the former being more stable in solution and human plasma. Moreover, preclinical biodistribution studies showed that [18F]F-PSMA-617-NODA might be of potential interest for clinical applications.

Efficacy and biomarker analysis of phase 2 (P2) and window-of-opportunity (WoO) cohorts of patients with recurrent glioblastoma (rGBM) treated with ST101, an inhibitor of the transcription factor C/EBPβ.

2011 Background: CCAAT/enhancer-binding protein β (C/EBPβ) is an important transcription factor in GBM cell maintenance and is associated with worse outcomes and resistance to chemotherapy through transcription of genes associated with cell cycle and DNA repair. C/EBPβ is also required for maintenance of immunosuppressive tumor-associated macrophages. ST101 is a first-in-class antagonist of C/EBPβ that promotes selective GBM cell death without impacting normal cell viability (Darvishi, 2022), and repolarization of macrophages toward an immunostimulatory program. In an all comers P1 study, ST101 was well-tolerated, showed significant tumor uptake, ~40 hr plasma half-life, and signal of efficacy (1PR +8SD). With strong rationale targeting C/EBPβ in GBM and evidence that ST101 crosses the BBB in preclinical biodistribution studies, cohorts in rGBM were explored. Methods: Pts with ≤2 recurrences of GBM were enrolled in a P2 cohort (n=30) and treated with 500mg QW. A WoO cohort of 6 pts with rGBM received 2-4 doses of ST101 500mg QW prior to surgery and resumed ST101 after surgery until progression (NCT04478279). Endpoints for the two cohorts included response, PFS and OS while the WoO cohort also included tissue analyses (GBM subtypes, tumor microenvironment (TME) characterization, ST101 tumor penetration, and C/EBPβ target engagement). Clinical data is available for both cohorts and tissue analysis is available for the WoO cohort. Results: Data is a snapshot as of February 1, 2024. ST101 was safe and well-tolerated in both cohorts with no discontinuations due to AEs. Pts in the P2 study show 30% DCR (1PR + 8SD), 20% 6-mo PFS and 37% OS at 1 year. Median duration of treatment is 3.4 mos with 2 pts stable and still on study for 10+ and 16+ mos. In the WoO study, efficacy from post-surgery MRI showed 67% DCR (2 PR + 2 SD) and ~50% 6-mo PFS. Median OS cannot be assessed at this early stage. The median duration of treatment is 6.02 mos with 2 pts still on study for 7+ and 10+ mos. Tissue compared before and after ST101 exposure shows ST101 uptake and C/EBPβ target engagement by IHC, and pathologic evidence of treatment effect, such as geographic necrosis, in 5/6 pts. Evaluation of tumor and TME characteristics in post-ST101 treatment tissue will be presented. Conclusions: Monotherapy ST101 in pts with rGBM has an excellent safety profile and results in comparable outcomes to current 2nd line treatments. ST101 crosses the BBB, engages its target, and potentially induces treatment-related necrosis. These encouraging clinical activity, safety, and tissue findings, as well as preclinical data showing additive activity in combination with lomustine, support advancing ST101 to a randomized, placebo-controlled study comparing ST101 + lomustine vs. lomustine + placebo in rGBM. We aim to initiate this study in Q3 2024. Clinical trial information: NCT04478279 .

Amelioration of Tumor Targeting andIn VivoBiodistribution of99mTc-Methotrexate-Gold Nanoparticles (99mTc-Mex-AuNPs).

Gold nanoparticles (AuNPs) represent very attractive and promising drug delivery carriers due to their unique dimensions, adjustable surface functions, and controllable drug release. Therefore, AuNPs are used to overcome the limitations of conventional chemotherapy, for example methotrexate (Mex), one of the first-generation chemotherapy drugs for cancer treatment, whose usefulness has been restricted due to drug resistance and dose-dependent side effects. In the present study, the AuNPs drug delivery system was synthesized and loaded with technetium-99m radiolabeled Methotrexate (99mTc-Mex) to produce new potential nanoradiopharmaceutical for tumor targeting and further imaging. The Methotrexate loaded gold nanoparticles (Mex-AuNPs) successfully prepared in small spherical particle size (20.3nm), polydispersity index PDI (< 0.5) and a zeta potential (-17.6mV) with loading efficiency% (93±1.2%) of methotrexate at 30min as an optimum stirring time and showed strong absorption peak for Mex-AuNPs at λmax, 525nm. The in vitro release profile of Mex-AuNPs showed high release percent of methotrexate at pH 5; the Q0.5h and Q8h were 21.2±1.5% and 92.9±3.4%, respectively. The in vitro cytotoxicity was investigated at different concentrations (0.024-50 μl/100 μl) of Mex-AuNPs (1mg/ml) against MCF-7 (Michigan Cancer Foundation-7) breast cancer cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay technique. Mex-AuNPs showed higher anticancer activity with low inhibitory concentration (IC50=0.098 μl/100 μl) that was three times lower than the inhibitory concentration (IC50) of methotrexate (IC50=0.3 μl/100 μl). 99mTc-Mex complex prepared by direct reduction method at maximum radiochemical yield (RCY)% ̴ 98.3±1.09% was loaded in AuNPs to form 99mTc-Mex-AuNPs with loading efficiency% (93±1.2%) at 30min of stirring time. 99mTc-Mex-AuNPs showed convenient in vitro stability in mice serum up to 24h with RCY% > 90%. The preclinical biodistribution studies of 99mTc-Mex-AuNPs were performed in 3 experimental groups A (intravenous (I.V.) injected normal mice), B and C (I.V. and intratumor (I.T.) injected tumor bearing mice, respectively). The 99mTc-Mex-AuNPs achieved highest tumor uptake (93±0.39%ID/g) and highest Target/NonTarget (T/NT) ratio (58.1±0.91) with high Tumor/Blood (T/B) ratio (25.8±0.11) at 10min post I.T. injection and retained high tumor uptake (79±0.65%ID/g) up to 60min post I.T. injection before escaping into blood stream. Consequently, 99mTc-Mex-AuNPs can be considered as new potential nanoradiopharmaceutical in tumor diagnosis.

Perfusion fixation methods for preclinical biodistribution studies: A comparative assessment using automated image processing

Studying the biodistribution of novel therapeutics and biomaterials in vivo requires effective and consistent perfusion and fixation of major organs. Standard methods for removing red blood cells (RBCs) and fixing tissue often involve transcardial perfusion, such as brain-targeted perfusion (via the left ventricle) or lung-targeted perfusion (via the right ventricle). Using autofluorescence measurements and a bespoke ImageJ macro to quantify RBC content from histology, we compared the efficacy and consistency of three whole-body perfusion techniques. We show that lung-targeted perfusion evacuates more blood from the lung vasculature than brain-targeted perfusion (20 ± 54% fewer RBCs), and that our novel approach of ‘dual-targeted’ perfusion (via the right and left ventricles sequentially) had even higher efficacy (30 ± 6% fewer RBCs). Furthermore, by combining aspects of brain- and lung-targeted methods, dual-targeted perfusion achieved the highest consistency in autofluorescence emissions from major organs (64% and 65% lower variance than brain- and lung-targeted perfusion respectively). Since RBC content and autofluorescence can be confounding factors in biodistribution studies using fluorescent probes, our findings and proposed novel approach offer insight into perfusion fixation techniques for pre-clinical studies.

Development of a bioanalytical method for circulating human T cells in animals using Arthrobacter luteus-based quantitative polymerase chain reaction and its application in preclinical biodistribution studies.

IntroductionIn the development of cell therapy products for human use, studies on the biodistribution of transplanted cells in animals are important for assessing the safety and efficacy of these products. Although a few reports have described the biodistribution of human cells in animals using Arthrobacter luteus-based-polymerase chain reaction (Alu-PCR), most have used genomic DNA or synthetic oligonucleotide as calibrators, as opposed to actual cells. In addition, bioanalytical variability in the quantification of cells with respect to specificity, selectivity, accuracy, and precision, has not been evaluated. Accordingly, in this study, we validated the utility of this bioanalytical method for human T cells in mice to establish assay performance using cells as a calibrator.MethodsA standard curve was constructed for the addition of cell lysates to mouse tissues and blood, and DNA was extracted. Alu-PCR was applied for the quantification of human peripheral blood CD8+ T cells in mice. To determine assay performance, we evaluated accuracy, precision, selectivity, specificity, and stability. In vivo cell kinetics and biodistribution were investigated based on intravenous administration of human T cells to mice.ResultsAlu-PCR enabled us to specifically detect human T cells in mouse blood and tissues. The lower detection limit of Alu-PCR was 10 cells/15 mg tissue (7.5 mg for spleen and lung) or cells/50 μL blood. Given that PCR threshold cycle (Cq) values among mouse samples (blood, liver spleen, lung, heart, and kidney) show slight variation, calibration curves should be generated using the same tissue as used for the assay. Most coefficients of variation in the assay were within 30%. The cell kinetics of administered human T cells in mice were successfully evaluated using the established Alu-qPCR.ConclusionsThe Alu-PCR technique developed in this study showed sufficient specificity and sensitivity in detecting human peripheral blood CD8+ T cells in mice. This technique, which targets the primate-specific Alu gene, is applicable for quantifying transplanted human cells in animals without the necessity of cell labeling. The data presented herein will be useful for standardizing bioanalytical approaches in biodistribution studies of cell therapy products.

Highly specific, quantitative polymerase chain reaction probe for the quantification of human cells in cynomolgus monkeys

Quantification of human cells may be performed using quantitative polymerase chain reaction (qPCR). In preclinical studies, the human Alu sequence is widely used as biomarker for human DNA. However, because the Alu gene is shared by primates, its use is limited to non-primate studies. The biodistribution of human cells in primates is also necessary for translational studies. Therefore, we aimed to design a novel, human-specific primer/probe that enables the quantification of human cells in primates and other animal models. A novel primer/probe set was successfully designed based on highly repetitive LINE1 sequences. qPCR efficiency (94.95–99.21%) and linearity of calibration curves (r2 = 0.996–0.999) were confirmed in tissue homogenates of cynomolgus monkey. The lower limit of detection was 10 cells per 15-mg tissue sample, a sensitivity that is equivalent to existing Alu primers/probes. The set was also effective in other animal models such as mice, rabbits, pigs, and common marmosets.To our knowledge, this is the first study describing the successful design of a human-specific qPCR primer/probe for human cell quantification in various animals, including non-human primates, using LINE1 sequence. The excellent selectivity, sensitivity, and versatility of the LINE1 primers/probes make it a promising quantification tool in preclinical biodistribution studies.

Biodistribution studies: understanding international expectations

Biodistribution studies: understanding international expectations

2015 4thTERMIS World CongressBoston, MassachusettsSeptember 8–11, 2015

2015 4^thTERMIS World CongressBoston, MassachusettsSeptember 8–11, 2015

Single vial kit formulation of DOTATATE for preparation of (177) Lu-labeled therapeutic radiopharmaceutical at hospital radiopharmacy.

The clinical applications of radiolabeled somatostatin analogue (177) Lu-DOTA-Tyr(3) -Thr(8) -Octreotide ((177) Lu-DOTATATE) constitute a promising treatment option for patients with disseminated and inoperable neuroendocrine (NET) tumors. Formulation of (177) Lu-DOTATATE in hospital radiopharmacy under aseptic conditions in a safe and reliable manner is a major constraint for its extensive use. The present work was intended to develop a kit for the safe preparation of the therapeutic radiopharmaceutical, viz. (177) Lu-DOTATATE of high quality that can be easily adapted at conventional hospital radiopharmacies. Single vial kits of DOTATATE were formulated and evaluated for suitability for radiolabeling as well as stability on its storage. Patient dose of (177) Lu-DOTATATE (7.4 GBq) could be successfully prepared using semi-automated in-house setup that assures safe handling and high yields of product of pharmaceutical purity suitable for clinical use. Fast clearance of activity via renal route was observed in preclinical biodistribution studies of (177) Lu-DOTATATE carried out in normal Swiss mice. Deployment of in-house produced (177) LuCl3 , cold kits and easy adaptability of synthesis setup at hospital radiopharmacy for preparation is likely to expand applications of peptide receptor radionuclide therapy.

Abstract 716: ASPH_0047: a potent and selective antisense oligonucleotide-targeting transforming growth factor beta 2 (TGF-β2)

Abstract Transforming growth factor beta (TGF-β) is a key member of a large family of cytokines, including bone morphogenetic protein (BMP), nodals, activins and others, which play critical, pleiotropic roles in the pathophysiology of various human diseases, such as cancer, inflammation, autoimmune disease, and cirrhosis/fibrosis. In particular, the different TGF-β isoforms (TGF-β1, -β2, and -β3, encoded by different genes but sharing high sequence and structure homology) are overexpressed in many human tumors. Correlations between TGF-β expression, cancer stage and clinical parameters have been reported and linked to poor clinical outcome. TGF-β has been associated with a wide range of tumor-promoting processes, including tumor cell invasion and migration, angiogenesis, immunosuppression, as well as tumor stem cell maintenance and protection. Therefore, blocking the TGF-β signaling pathway via inhibition of TGF-β expression appears as an attractive therapeutic intervention in Oncology. We have previously reported the rational design and preliminary outcome of an extensive discovery program for identification of antisense oligodeoxynucleotides targeting the various TGF-β isoforms. We present here ASPH_0047, a 17-mer LNA-modified gapmer based on the sequence of the human TGF-β2 mRNA, as novel preclinical development candidate. ASPH_0047 shows potent and selective target downregulation (mRNA and protein) in various cell-based assays. Preliminary preclinical biodistribution studies in rodent point at tissue penetration in expected target organs (e.g., liver, kidney, spleen) and accumulation in tumor tissue consistent with observed target downregulation and efficacy in relevant in vivo experimental antitumor models. Key pharmacology properties and preclinical features of ASPH_0047 supporting rapid advancement to clinical development will be presented. Citation Format: Katja Wosikowski, Frank Jaschinski, Hanna Kohonen, Stephan Braun, Eugen Leo, Michel Janicot. ASPH_0047: a potent and selective antisense oligonucleotide-targeting transforming growth factor beta 2 (TGF-β2). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 716. doi:10.1158/1538-7445.AM2014-716

Enhanced Tumor Retention of a Radiohalogen Label for Site-Specific Modification of Antibodies

A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical radioimmunotherapies.

Preclinical biodistribution studies of 68Ga-labeled anti-miR15b molecules

Preclinical biodistribution studies of 68Ga-labeled anti-miR15b molecules

INGN 007, an oncolytic adenovirus vector, replicates in Syrian hamsters but not mice: comparison of biodistribution studies

Preclinical biodistribution studies with INGN 007, an oncolytic adenovirus (Ad) vector, supporting an early stage clinical trial were conducted in Syrian hamsters, which are permissive for Ad replication, and mice, which are a standard model for assessing toxicity and biodistribution of replication-defective (RD) Ad vectors. Vector dissemination and pharmacokinetics following intravenous administration were examined by real-time PCR in nine tissues and blood at five time points spanning 1 year. Select organs were also examined for the presence of infectious vector/virus. INGN 007 (VRX-007), wild-type Ad5 and AdCMVpA (an RD vector) were compared in the hamster model, whereas only INGN 007 was examined in mice. DNA of all vectors was widely disseminated early after injection, but decayed rapidly in most organs. In the hamster model, DNA of INGN 007 and Ad5 was more abundant than that of the RD vector AdCMVpA at early times after injection, but similar levels were seen later. An increased level of INGN 007 and Ad5 DNA but not AdCMVpA DNA in certain organs early after injection, and the presence of infectious INGN 007 and Ad5 in lung and liver samples at early times after injection, strongly suggests that replication of INGN 007 and Ad5 occurred in several Syrian hamster organs. There was no evidence of INGN 007 replication in mice. In addition to providing important information about INGN 007, the results underscore the utility of the Syrian hamster as a permissive immunocompetent model for Ad5 pathogenesis and oncolytic Ad vectors.

The production, recovery, and purification of 172Hf for utilization in nuclear medicine as the generator of 172Lu

172Hf is of potential value in nuclear medicine research as the generator of its daughter nuclide, 172Lu. The latter isotope, a heavy rare-earth tracer, could have applications in compound labelling, pre-clinical biodistribution studies, and other areas of investigation. 172Hf is produced by inducing spallation reactions in Ta targets with medium-energy protons at LAMPF, and a chemical procedure has been developed for the separation of radiohafnium from the bulk target material and various spallogenic impurities. Nuclide identification was accomplished by gamma-ray spectrometry, and radiochemical yields of nine different elements in the analytical scheme were measured. 172Hf is thus recovered quantitatively (93% chemical yield) and is sufficiently decontaminated for use in generator development.