DNA-PKcs inhibitors sensitize neuroendocrine tumor cells to peptide receptor radionuclide therapy <i>in vitro</i> and <i>in vivo</i>

Background: Peptide receptor radionuclide therapy (PRRT) increases progression-free survival and quality of life of neuroendocrine tumor (NET) patients, however complete cures are rare and dose-limiting toxicity has been reported. PRRT induces DNA damage of which DNA double strand breaks (DSBs) are the most cytotoxic. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key player in DSB repair and its inhibition therefore is a potential way to enhance PRRT efficacy without increasing the dosage. Methods: We analyzed effects of combining PRRT and DNA-PKcs inhibitor AZD7648 on viability, cell death and clonogenic survival on SSTR2-expressing cell lines BON1-SSTR2, GOT1 and NCI-H69. Therapy-induced DNA damage response was assessed by analyzing DSB foci levels and cell cycle distributions. In vivo efficacy was investigated in BON1-SSTR2 and NCI-H69 xenografted mice and hematologic and renal toxicity were monitored by blood counts, creatinine levels and analyzing renal morphology. Results: Combining PRRT and AZD7648 significantly decreased viability of BON1-SSTR2, GOT1 and NCI-H69 cells and induced cell death in GOT1 and BON1-SSTR2 cells. A strong effect of AZD7648 on PRRT-induced DSB repair was found. In GOT1 cells, this was accompanied by induction of cell cycle blocks. However, BON1-SSTR2 cells were unable to fully arrest their cell cycle and polyploid cells with high DNA damage levels were detected. In vivo, AZD7648 significantly sensitized BON1-SSTR2 and NCI-H69 xenograft models to PRRT. In addition, combination therapy did not induce significant changes in body weight, blood composition, plasma creatinine levels and renal morphology, indicating the absence of severe acute hematologic and renal toxicity. Conclusion: These results highlight that the potentiation of the therapeutic effect of PRRT by DNA-PKcs inhibition is a highly effective and well-Tolerated therapeutic strategy. Based on our findings, we recommend initiation of phase I/II studies in patients to find a safe and effective combination regimen.</p

Investigation of Factors Determining the Enhanced Permeability and Retention Effect in Subcutaneous Xenografts

Liposomal chemotherapy offers several advantages over conventional therapies, including high intratumoral drug delivery, reduced side effects, prolonged circulation time and the possibility to dose higher. The efficient delivery of liposomal chemotherapeutics relies however on the enhanced permeability and retention (EPR) effect, which refers to the ability of macromolecules to extravasate leaky tumor vessels and accumulate in the tumor tissue. Using a panel of human xenograft tumors, we evaluated the influence of the EPR effect on liposomal distribution in vivo by injection of pegylated liposomes radiolabeled with 111In. Liposomal accumulation in tumors and organs was followed over time by SPECT/CT imaging. We observed that fast growing xenografts, which may be less representative of tumor development in patients, showed higher liposomal accumulation as compared to slow growing xenografts. Additionally, several other parameters determining the EPR effect were evaluated, such as blood and lymphatic vessel density, intratumoral hypoxia, and the presence of macrophages. The investigation of various parameters showed a few correlations. Although hypoxia, proliferation and macrophage presence were associated with tumor growth, no hard conclusions or predictions could be made regarding the EPR effect or liposomal uptake. However liposomal uptake was

Measurement of reaction kinetics of [177Lu]Lu-DOTA-TATE using a microfluidic system

Microfluidic synthesis techniques can offer improvement over batch syntheses which are currently used for radiopharmaceutical production. These improvements are, for example, better mixing of reactants, more efficient energy transfer, less radiolysis, faster reaction optimization, and overall improved reaction control. However, scale-up challenges hinder the routine clinical use, so the main advantage is currently the ability to optimize reactions rapidly and with low reactant consumption. Translating those results to clinical systems could be done based on calculations, if kinetic constants and diffusion coefficients were known. This study describes a microfluidic system with which it was possible to determine the kinetic association rate constants for the formation of [177Lu]Lu-DOTA-TATE under conditions currently used for clinical production. The kinetic rate constants showed a temperature dependence that followed the Arrhenius equation, allowing the determination of Arrhenius parameters for a Lu-DOTA conjugate (A = 1.24 ± 0.05 × 1019 M-1 s-1, EA = 109.5 ± 0.1 × 103 J mol-1) for the first time. The required reaction time for the formation of [177Lu]Lu-DOTA-TATE (99% yield) at 80 °C was 44 s in a microfluidic channel (100 μm). Simulations done with COMSOL Multiphysics® indicated that processing clinical amounts (3 mL reaction solution) in less than 12 min is possible in a micro- or milli-fluidic system, if the diameter of the reaction channel is increased to over 500 μm. These results show that a continuous, microfluidic system can become a viable alternative to the conventional, batch-wise radiolabelling technique

Influence of tumour size on the efficacy of targeted alpha therapy with 213Bi-[DOTA0,Tyr3]-octreotate

BACKGROUND: Targeted alpha therapy has been postulated to have great potential for the treatment of small clusters of tumour cells as well as small metastases. (213)Bismuth, an α-emitter with a half-life of 46 min, has shown to be effective in preclinical as well as in clinical applications. In this study, we evaluated whether (213)Bi-[DOTA(0), Tyr(3)]-octreotate ((213)Bi-DOTATATE), a (213)Bi-labelled somatostatin analogue with high affinity for somatostatin receptor subtype 2 (SSTR(2)), is suitable for the treatment of larger neuroendocrine tumours overexpressing SSTR(2) in comparison to its effectiveness for smaller tumours. We performed a preclinical targeted radionuclide therapy study with (213)Bi-DOTATATE in animals bearing tumours of different sizes (50 and 200 mm(3)) using two tumour models: H69 (human small cell lung carcinoma) and CA20948 (rat pancreatic tumour). METHODS: Pharmacokinetics was determined for calculation of dosimetry in organs and tumours. H69- or CA20948-xenografted mice with tumour volumes of approximately 120 mm(3) were euthanized at 10, 30, 60 and 120 min post injection of a single dose of (213)Bi-DOTATATE (1.5–4.8 MBq). To investigate the therapeutic efficacy of (213)Bi-DOTATATE, xenografted H69 and CA20948 tumour-bearing mice with tumour sizes of 50 and 200 mm(3) were administered daily with a therapeutic dose of (213)Bi-DOTATATE (0.3 nmol, 2–4 MBq) for three consecutive days. The animals were followed for 90 days after treatment. At day 90, mice were injected with 25 MBq (99m)Tc-DMSA and imaged by SPECT/CT to investigate possible renal dysfunction due to (213)Bi-DOTATATE treatment. RESULTS: Higher tumour uptakes were found in CA20948 tumour-bearing animals compared to those in H69 tumour-bearing mice with the highest tumour uptake of 19.6 ± 6.6 %IA/g in CA20948 tumour-bearing animals, while for H69 tumour-bearing mice, the highest tumour uptake was found to be 9.8 ± 2.4 %IA/g. Nevertheless, as the anti-tumour effect was more pronounced in H69 tumour-bearing mice, the survival rate was higher. Furthermore, in the small tumour groups, no regrowth of tumour was found in two H69 tumour-bearing mice and in one of the CA20948 tumour-bearing mice. No renal dysfunction was observed in (213)Bi-DOTATATE-treated mice after the doses were applied. CONCLUSIONS: (213)Bi-DOTATATE demonstrated a great therapeutic effect in both small and larger tumour lesions. Higher probability for stable disease was found in animals with small tumours. (213)Bi-DOTATATE was effective in different neuroendocrine (H69 and CA20948) tumour models with overexpression of SSTR(2) in mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13550-016-0162-2) contains supplementary material, which is available to authorized users

Posture Does Not Matter! Paw Usage and Grasping Paw Preference in a Small-Bodied Rooting Quadrupedal Mammal

BACKGROUND: Recent results in birds, marsupials, rodents and nonhuman primates suggest that phylogeny and ecological factors such as body size, diet and postural habit of a species influence limb usage and the direction and strength of limb laterality. To examine to which extent these findings can be generalised to small-bodied rooting quadrupedal mammals, we studied trees shrews (Tupaia belangeri). METHODOLOGY/PRINCIPAL FINDINGS: We established a behavioural test battery for examining paw usage comparable to small-bodied primates and tested 36 Tupaia belangeri. We studied paw usage in a natural foraging situation (simple food grasping task) and measured the influence of varying postural demands (triped, biped, cling, sit) on paw preferences by applying a forced-food grasping task similar to other small-bodied primates. Our findings suggest that rooting tree shrews prefer mouth over paw usage to catch food in a natural foraging situation. Moreover, we demonstrated that despite differences in postural demand, tree shrews show a strong and consistent individual paw preference for grasping across different tasks, but no paw preference at a population level. CONCLUSIONS/SIGNIFICANCE: Tree shrews showed less paw usage than small-bodied quadrupedal and arboreal primates, but the same paw preference. Our results confirm that individual paw preferences remain constant irrespective of postural demand in some small-bodied quadrupedal non primate and primate mammals which do not require fine motoric control for manipulating food items. Our findings suggest that the lack of paw/hand preference for grasping food at a population level is a universal pattern among those species and that the influence of postural demand on manual lateralisation in quadrupeds may have evolved in large-bodied species specialised in fine manipulations of food items

Gastrin-releasing peptide receptor-based targeting using bombesin analogues is superior to metabolism-based targeting using choline for in vivo imaging of human prostate cancer xenografts

Purpose: Prostate cancer (PC) is a major health problem. Overexpression of the gastrin-releasing peptide receptor (GRPR) in PC, but not in the hyperplastic prostate, provides a promising target for staging and monitoring of PC. Based on the assumption that cancer cells have increased metabolic activity, metabolism-based tracers are also being used for PC imaging. We compared GRPR-based targeting using the68Ga-labelled bombesin analogue AMBA with metabolism-based tar

Comparing dogs and great apes in their ability to visually track object transpositions

Knowing that objects continue to exist after disappearing from sight and tracking invisible object displacements are two basic elements of spatial cognition. The current study compares dogs and apes in an invisible transposition task. Food was hidden under one of two cups in full view of the subject. After that both cups were displaced, systematically varying two main factors, whether cups were crossed during displacement and whether the cups were substituted by the other cup or instead cups were moved to new locations. While the apes were successful in all conditions, the dogs had a strong preference to approach the location where they last saw the reward, especially if this location remained filled. In addition, dogs seem to have especial difficulties to track the reward when both containers crossed their path during displacement. These results confirm the substantial difference that exists between great apes and dogs with regard to mental representation abilities required to track the invisible displacements of objects

Litsea Deccanensis Ameliorates Myocardial Infarction in Wistar Rats: Evidence from Biochemical and Histological Studies

The present study was designed to evaluate the cardioprotective effects of methanolic extract of Litsea deccanensis (MELD) against isoproterenol-induced myocardial infarction in rats by studying cardiac markers, lipid peroxidation, lipid profile, and histological changes. Male Wistar rats were treated orally with MELD (100 and 200 mg/kg) daily for a period of 21 days. After 21 days of pretreatment, isoproterenol (100 mg/kg) was injected subcutaneously to rats at an interval of 24 h for 2 days to induce myocardial infarction. Isoproterenol-induced rats showed significant (P < 0.05) increase in the levels of serum creatine kinase, lactate dehydrogenase, thiobarbituric acid reactive substances, and lipid hydro peroxides. The serum lipid levels were altered in the isoproterenol-induced myocardial infarcted rats. The histopathological findings of the myocardial tissue evidenced myocardial damage in isoproterenol-induced rats. The oral pretreatment with MELD restored the pathological alterations in the isoproterenol-induced myocardial infarcted rats. The MELD pretreatment significantly reduced the levels of biochemical markers, lipid peroxidation and regulated the lipid profile of the antioxidant system in the isoproterenol-induced rats. An inhibited myocardial necrosis was evidenced by the histopathological findings in MELD pretreated isoproterenol-induced rats. Our study shows that oral pretreatment with MELD prevents isoproterenol-induced oxidative stress in myocardial infarction. The presence of phenolic acid and flavonoid contents were confirmed by preliminary phytochemical tests. The reducing power and free radical scavenging activities of the MELD may be the possible reason for it pharmacological actions

Mass extinctions drove increased global faunal cosmopolitanism on the supercontinent Pangaea

Mass extinctions have profoundly impacted the evolution of life through not only reducing taxonomic diversity but also reshaping ecosystems and biogeographic patterns. In particular, they are considered to have driven increased biogeographic cosmopolitanism, but quantitative tests of this hypothesis are rare and have not explicitly incorporated information on evolutionary relationships. Here we quantify faunal cosmopolitanism using a phylogenetic network approach for 891 terrestrial vertebrate species spanning the late Permian through Early Jurassic. This key interval witnessed the Permian–Triassic and Triassic–Jurassic mass extinctions, the onset of fragmentation of the supercontinent Pangaea, and the origins of dinosaurs and many modern vertebrate groups. Our results recover significant increases in global faunal cosmopolitanism following both mass extinctions, driven mainly by new, widespread taxa, leading to homogenous ‘disaster faunas’. Cosmopolitanism subsequently declines in post-recovery communities. These shared patterns in both biotic crises suggest that mass extinctions have predictable influences on animal distribution and may shed light on biodiversity loss in extant ecosystems