Design and Preclinical Evaluation of a Novel Prostate-Specific Membrane Antigen Radioligand Modified with a Transthyretin Binder.

Transthyretin binders have previously been used to improve the pharmacokinetic properties of small-molecule drug conjugates and could, thus, be utilized for radiopharmaceuticals as an alternative to the widely explored "albumin binder concept". In this study, a novel PSMA ligand modified with a transthyretin-binding entity (TB-01) was synthesized and labeled with lutetium-177 to obtain [177Lu]Lu-PSMA-TB-01. A high and specific uptake of [177Lu]Lu-PSMA-TB-01 was found in PSMA-positive PC-3 PIP cells (69 ± 3% after 4 h incubation), while uptake in PSMA-negative PC-3 flu cells was negligible (<1%). In vitro binding studies showed a 174-fold stronger affinity of [177Lu]Lu-PSMA-TB-01 to transthyretin than to human serum albumin. Biodistribution studies in PC-3 PIP/flu tumor-bearing mice confirmed the enhanced blood retention of [177Lu]Lu-PSMA-TB-01 (16 ± 1% IA/g at 1 h p.i.), which translated to a high tumor uptake (69 ± 13% IA/g at 4 h p.i.) with only slow wash-out over time (31 ± 8% IA/g at 96 h p.i.), while accumulation in the PC-3 flu tumor and non-targeted normal tissue was reasonably low. Further optimization of the radioligand design would be necessary to fine-tune the biodistribution and enable its use for therapeutic purposes. This study was the first of this kind and could motivate the use of the "transthyretin binder concept" for the development of future radiopharmaceuticals.

Investigations Using Albumin Binders to Modify the Tissue Distribution Profile of Radiopharmaceuticals Exemplified with Folate Radioconjugates.

Introducing an albumin-binding entity into otherwise short-lived radiopharmaceuticals can be an effective means to improve their pharmacokinetic properties due to enhanced blood residence time. In the current study, DOTA-derivatized albumin binders based on 4-(p-iodophenyl)butanoate (DOTA-ALB-1 and DOTA-ALB-3) and 5-(p-iodophenyl)pentanoate entities (DOTA-ALB-24 and DOTA-ALB-25) without and with a hydrophobic 4-(aminomethyl)benzoic acid (AMBA) linker unit, respectively, were synthesized and labeled with lutetium-177 for in vitro and in vivo comparison. Overall, [177Lu]Lu-DOTA-ALB-1 demonstrated ~3-fold stronger in vitro albumin-binding affinity and a longer blood residence time (T50%IA ~8 h) than [177Lu]Lu-DOTA-ALB-24 (T50%IA ~0.8 h). Introducing an AMBA linker enhanced the albumin-binding affinity, resulting in a T50%IA of ~24 h for [177Lu]Lu-DOTA-ALB-3 and ~2 h for [177Lu]Lu-DOTA-ALB-25. The same albumin binders without or with the AMBA linker were incorporated into 6R- and 6S-5-methyltetrahydrofolate-based DOTA-conjugates (177Lu-RedFols). Biodistribution studies in mice performed with both diastereoisomers of [177Lu]Lu-RedFol-1 and [177Lu]Lu-RedFol-3, which comprised the 4-(p-iodophenyl)butanoate moiety, demonstrated a slower accumulation in KB tumors than those of [177Lu]Lu-RedFol-24 and [177Lu]Lu-RedFol-25 with the 5-(p-iodophenyl)pentanoate entity. In all cases, the tumor uptake was high (30-45% IA/g) 24 h after injection. Both diastereoisomers of [177Lu]Lu-RedFol-1 and [177Lu]Lu-RedFol-3 demonstrated high blood retention (3.8-8.7% IA/g, 24 h p.i.) and a 2- to 4-fold lower kidney uptake than the corresponding diastereoisomers of [177Lu]Lu-RedFol-24 and [177Lu]Lu-RedFol-25, which were more rapidly cleared from the blood (<0.2% IA/g, 24 h after injection). Kidney retention of the 6S-diastereoisomers of all 177Lu-RedFols was consistently higher than that of the respective 6R-diastereoisomers, irrespective of the albumin binder and linker unit used. It was demonstrated that the blood clearance data obtained with 177Lu-DOTA-ALBs had predictive value for the blood retention times of the respective folate radioconjugates. The use of these albumin-binding entities without or with an AMBA linker may serve for fine-tuning the blood retention of folate radioconjugates and also other radiopharmaceuticals and, hence, optimize their tissue distribution profiles. Dosimetry estimations based on patient data obtained with one of the most promising folate radioconjugates will be crucial to identify the dose-limiting organ, which will allow for selecting the most suitable folate radioconjugate for therapeutic purposes.

Dual Action of Curcumin as an Anti- and Pro-Oxidant from a Biophysical Perspective.

Curcumin, a natural polyphenol widely used as a spice, colorant and food additive, has been shown to have therapeutic effects against different disorders, mostly due to its anti-oxidant properties. Curcumin also reduces the efficiency of melanin synthesis and affects cell membranes. However, curcumin can act as a pro-oxidant when blue light is applied, since upon illumination it can generate singlet oxygen. Our review aims to describe this dual role of curcumin from a biophysical perspective, bearing in mind its concentration, bioavailability-enhancing modifications and membrane interactions, as well as environmental conditions such as light. In low concentrations and without irradiation, curcumin shows positive effects and can be recommended as a beneficial food supplement. On the other hand, when used in excess or irradiated, curcumin can be toxic. Therefore, numerous attempts have been undertaken to test curcumin as a potential photosensitizer in photodynamic therapy (PDT). At that point, we underline that curcumin-based PDT is limited to the treatment of superficial tumors or skin and oral infections due to the weak penetration of blue light. Additionally, we conclude that an increase in curcumin bioavailability through the using nanocarriers, and therefore its concentration, as well as its topical use if skin is exposed to light, may be dangerous.

Establishment of vestibular schwannoma primary cell cultures obtained from cavitron ultrasonic surgical aspirator tissue material.

BACKGROUND: Vestibular schwannoma (VS) is a benign tumor arising from the Schwann cells of the eighth cranial nerve. The complexity in treatment is associated with unpredictable progression of this tumor. Some of the VS do not alter for years, while others rapidly increase in size. The mechanisms behind size progression are not well studied. Furthermore, despite several studies, there is no pharmacological treatment available for sporadic VS. Therefore, in vitro models are essential tools to study the cellular and molecular processes of VS. In addition, patient-derived cell cultures are important for substance screening to investigate pharmacological approaches in vitro. NEW METHOD: This study presents a simple and fast method for culturing VS cells from patient tissue material obtained using a cavitron ultrasonic surgical aspirator (CUSA). In addition, the cells were characterized based on the expression of schwannoma markers, growth properties and screened for fibroblast contamination. RESULT: We could show that CUSA obtained material is a suitable resource for isolation of VS primary cultures and enables real time analysis on living cells. COMPARISON WITH EXISTING METHODS: To date, only a few protocols are available for culturing VS cells from patient tissue material. A disadvantage of these methods is the relatively large amount of tissue needed to obtain the primary cells, which can be difficult, especially in small VS. By obtaining the cells from the CUSA, there is the possibility to establish a primary culture even with limited material. CONCLUSION: This approach could be particularly useful for testing substances that represent candidates for drug therapy of vestibular schwannoma.

Cr(vi) permanently binds to the lipid bilayer in an inverted hexagonal phase throughout the reduction process.

Cr(vi) is a harmful, carcinogenic agent with a high permeability rate throughout the lipid membranes. In an intracellular environment and during interactions with cellular membranes, it undergoes an instant reduction to lower oxidation states throughout radical states, recognized as the most dangerous factor for cells. The cellular membrane is the most visible cellular organelle in the interior and exterior of a cell. In this study, liposomes and non-lamellar inverted hexagonal phase lipid structures based on phosphoethanolamine (PE) were used as model cellular bilayers because of their simple composition, preparation procedure, and the many other properties of natural systems. The lipid membranes were subjected to 0.075 mM Cr(vi) for 15 min, after which the Cr content was removed via dialysis. This way, the remaining Cr content could be studied qualitatively and quantitatively. Using the combined XRF/XAS/EPR approach, we revealed that some Cr content (Cr(iii) and Cr(vi)) was still present in the samples even after long-term dialysis at a temperature significantly above the phase transition for the chosen liposome. The amount of bound Cr increased with increasing PE and -C[double bond, length as m-dash]C- bond content in lipid mixtures. Internal membrane order decreased in less fluid membranes, while in more liquified ones, internal order was only slightly changed after subjecting them to the Cr(vi) agent. The results suggest that the inverted hexagonal phase of lipid structures is much more sensitive to oxidation than the lamellar lipid phase, which can play an important role in the strong cytotoxicity of Cr(vi).

Neuropsychological Assessment of Cognitive Impairment in Kidney Transplantation (NAsKiT) and its related risk factors: a study protocol.

BACKGROUND: Association of cognitive impairment with chronic kidney disease has been reported over the last decade. Individuals show better cognitive performance after kidney transplantation than individuals on dialysis but are more likely to be affected by cognitive impairment than age-matched comparison groups. Better knowledge of the prevalence as well as course and profile of cognitive impairment is important for the design of future studies assessing the clinical impact of cognitive impairment and developing management strategies. The goal of our study is to examine the extent of cognitive impairment before and after transplantation and to derive a distinct profile of cognitive function using standard neurocognitive tests. Furthermore, we aim to assess whether transplantation per se leads to an improvement in cognitive performance. METHODS: We are conducting a prospective single-center cohort study involving 100 kidney transplant individuals. Individuals who are wait-listed to receive a kidney transplantation or have already received one will be included in this study. Individuals will undergo a battery of detailed neurocognitive tests at baseline (in part before surgery), and then 3 and 12 months afterwards. Furthermore, the enrolled patients will complete a validated German version of the Cognitive Failure Questionnaire for self-assessment (s-CFQ) as well as the Hospital Anxiety and Depression Scale -Deutsche (HADS-D), a self-report screening instrument with two scales that capture anxiety and depression. In addition, a hair sample will be taken at each measurement time point for the determination of hair cortisol levels as a parameter for the cumulative hypothalamic-pituitary-adrenocortical axis activity over the previous three months. The primary outcome measure will be (a) the effect of kidney transplantation on the cognitive performance up to 12 months after transplantation and (b) the course of cognitive performance following kidney transplantation over time. DISCUSSION: The results of our study have potentially important implications for the prevention and treatment of cognitive impairment in kidney transplant individuals. By increasing our knowledge of the neurocognitive profile and assigning the corresponding deficits, it might be possible to create an individualized training program to positively impact cognitive deficits in kidney transplant patients.

Preclinical Investigations to Explore the Difference between the Diastereomers [177Lu]Lu-SibuDAB and [177Lu]Lu-RibuDAB toward Prostate Cancer Therapy.

[177Lu]Lu-Ibu-DAB-PSMA, a radioligand modified with ibuprofen as the albumin binder, showed higher accumulation in PSMA-positive tumors of mice than the clinically used [177Lu]Lu-PSMA-617 but lower retention in non-targeted tissues than previously developed albumin-binding PSMA radioligands. The aim of this study was to investigate whether the stereochemistry of the incorporated ibuprofen affects the radioligand's in vitro and in vivo properties and to select the more favorable radioligand for further development. For this purpose, SibuDAB and RibuDAB containing (S)- and (R)-ibuprofen, respectively, were synthesized and labeled with lutetium-177. In vitro, the two isomers had similar properties; however, [177Lu]Lu-SibuDAB showed increased binding to mouse and human plasma proteins (91 ± 1 and 88 ± 2%, respectively) compared to [177Lu]Lu-RibuDAB (75 ± 2 and 79 ± 2%, respectively). In vivo, [177Lu]Lu-SibuDAB was metabolically more stable than [177Lu]Lu-RibuDAB with ∼90 vs ∼67% intact radioligand detected in the blood at 4 h post injection (p.i.). In line with the lower albumin-binding affinity, the blood clearance of [177Lu]Lu-RibuDAB in mice was considerably faster [27% of injected activity (% IA), 1 h p.i.] than for [177Lu]Lu-SibuDAB (50% IA, 1 h p.i.). Time-dependent biodistribution studies performed in tumor-bearing athymic nude mice showed high PSMA-specific tumor uptake for both isomers. A twofold increased area under the curve (AUC0→8d) of the blood retention was determined for [177Lu]Lu-SibuDAB as compared to [177Lu]Lu-RibuDAB, whereas the kidney AUC0→8d value of [177Lu]Lu-SibuDAB was only half as high as for [177Lu]Lu-RibuDAB. As a result, a more favorable tumor-to-kidney AUC0→8d ratio was obtained for [177Lu]Lu-SibuDAB, which was also visualized on SPECT/CT images. Based on its improved kidney clearance and higher metabolic stability, [177Lu]Lu-SibuDAB was selected as the more favorable radioligand. Therapy studies performed with [177Lu]Lu-SibuDAB (5 MBq/mouse) demonstrated the anticipated therapeutic superiority over the current gold-standard [177Lu]Lu-PSMA-617 (5 MBq/mouse). The significantly increased survival time of mice treated with [177Lu]Lu-SibuDAB as compared to those injected with [177Lu]Lu-PSMA-617 justifies further development of this novel radioligand toward clinical application.

Mitochondrial Impairment by MitoBloCK-6 Inhibits Liver Cancer Cell Proliferation.

Augmenter of liver regeneration (ALR) is a critical multi-isoform protein with its longer isoform, located in the mitochondrial intermembrane space, being part of the mitochondrial disulfide relay system (DRS). Upregulation of ALR was observed in multiple forms of cancer, among them hepatocellular carcinoma (HCC). To shed light into ALR function in HCC, we used MitoBloCK-6 to pharmacologically inhibit ALR, resulting in profound mitochondrial impairment and cancer cell proliferation deficits. These effects were mostly reversed by supplementation with bioavailable hemin b, linking ALR function to mitochondrial iron homeostasis. Since many tumor cells are known for their increased iron demand and since increased iron levels in cancer are associated with poor clinical outcome, these results help to further advance the intricate relation between iron and mitochondrial homeostasis in liver cancer.

Novel Synthetic Strategies Enable the Efficient Development of Folate Conjugates for Cancer Radiotheranostics.

The folate receptor (FR) is an interesting target for radiotheranostics due to its overexpression in several tumor types. The progress in developing novel folate radioconjugates is, however, slow due to the synthetic challenges that folate chemistry presents. The goal of this study was, thus, to establish versatile solid-phase synthetic strategies for a convenient preparation of novel folate conjugates. Two approaches were established based on an orthogonal fluorenylmethyloxycarbonyl (Fmoc)-protection strategy to enable a modular buildup of an albumin-binding DOTA conjugate (known as OxFol-1) using folic acid (oxidized folate version) as a targeting agent. The main difference between the two approaches was the sequence of conjugating the single structural units. The approach that introduced the folate entity as the last unit appeared particularly useful for the preparation of conjugates based on 6R- or 6S-5-methyltetrahydrofolic acid (5-MTHF; a reduced folate version) as targeting entity. Three types of folate conjugates were synthesized either with a p-iodophenyl-based albumin binder (OxFol-1, 6R-RedFol-1, and 6S-RedFol-1) or without an albumin-binding entity (OxFol-14, 6R-RedFol-14, and 6S-RedFol-14). All six conjugates were obtained with high chemical purity (>98%) after 9-13 synthesis steps and a single final HPLC purification. Radiolabeling with lutetium-177 was feasible at high molar activity, and the resulting radioconjugates were stable over at least 24 h. Biodistribution and SPECT/CT imaging studies confirmed the favorable effect of an albumin-binding entity to increase the tumor uptake and reduce kidney retention of folate radioconjugates. The increased tumor-to-kidney ratios obtained with [177Lu]Lu-6R-RedFol-1 and [177Lu]Lu-6S-RedFol-1 as compared to [177Lu]Lu-OxFol-1 indicated that 5-MTHF is the preferred FR-targeting agent for albumin-binding radioconjugates. This was, however, not the case for folate radioconjugates without an albumin binder. Thanks to the established synthesis strategy, the preparation of further folate radioconjugates will be facilitated, potentially enabling the optimization of the tissue distribution characteristics even more.

Comparison of Serum and Urinary 5-Hydroxyindoleacetic Acid as Biomarker for Neuroendocrine Neoplasms.

CONTEXT: Patients with serotonin-secreting neuroendocrine neoplasms (NENs) have increased serum 5-hydroxyindoleacetic acid (5HIAA) concentrations. Serum 5HIAA thus serves as a biomarker in NEN. OBJECTIVE: To evaluate an improved tandem mass spectrometric serum 5HIAA assay for diagnosis and follow-up of NEN in a clinical cohort. DESIGN: A retrospective study during 2016-2018 at the Diagnostic Center and Department of Endocrinology at Helsinki University Hospital, Finland. METHODS: Detailed patient data was obtained from 116 patients. Serum 5HIAA was analyzed by 2 different liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays with samples prepared either by protein precipitation or solid phase extraction. Twenty-four-hour urine 5HIAA samples (n = 33) were analyzed by amperometric LC, and the results were compared. Specificity and sensitivity were calculated by receiver operating characteristic (ROC) analysis. RESULTS: We achieved 5 to10 000 nmol/L linearity and ≤2.5% variation with our new serum 5HIAA assay. In ROC analysis, the area under curve was 85% by serum assays [upper reference limit (URL) value 123 nmol/L] and 88% by the 24-h urine 5HIAA assay (URL value of 47.1 µmol), respectively. A difference (P < 0.001) between patients with active NEN and patients in remission was found by all 5HIAA assays. CONCLUSION: Serum 5HIAA by LC-MS/MS after protein precipitation performs equally well for the diagnosis of NEN as urinary 5HIAA LC assay. The outcome and sensitivity for serum and 24-h urine assays are convergent. Due to much more reliable and convenient sampling, we recommend serum instead of 24-h urine 5HIAA for diagnosis and follow-up of NEN patients.

Stability of viral haemorrhagic septicaemia virus, infectious hematopoietic necrosis virus and cyprinid herpesvirus 3 in various water samples.

Rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) are the two most common species in traditional fish farming in Germany. Their aquaculture is threatened upon others by viruses that can cause a high mortality. Therefore, this work focuses on three viruses-viral haemorrhagic septicaemia virus, infectious hematopoietic necrosis virus and cyprinid herpesvirus 3 (CyHV-3)-that endanger these species. To prevent their spread and contain further outbreaks, it is essential to know how long they can outlast in environmental waters and what affects their infectivity outside the host. Hence, the stability of the target viruses in various water matrices was examined and compared in this work. In general, all three viruses were quite stable within sterile water samples (showing mostly ≤1 log reduction after 96 hr) but were inactivated faster and to a higher extent (up to five log steps within 96 hr) in unsterile environmental water samples. The inactivation of the viruses correlated well with the increasing bacterial load of the samples, suggesting that bacteria had the greatest effect on their stability in the examined samples. In comparison, CyHV-3 seemed to be the most sensitive and maintained its infectivity for the shortest period.

Effects of Curcumin on Lipid Membranes: an EPR Spin-label Study.

Curcumin is a yellow-orange dye widely used as a spice, food coloring and food preservative. It also exhibits a broad range of therapeutic effects against different disorders such as cancer, diabetes, or neurodegenerative diseases. As a compound insoluble in water curcumin accumulates in cell membranes and due to this location it may indirectly lead to the observed effects by structurally altering the membrane environment. To exert strong structural effects on membrane curcumin needs to adopt a transbilayer orientation. However, there is no agreement in literature as to curcumin's orientation and its structural effects on membranes. Here, we investigated the effects of curcumin on lipid order, lipid phase transition, and local polarity in a model liposome membranes made of DMPC or DSPC using electron paramagnetic resonance (EPR) spin labeling technique. Curcumin affected lipid order at different depths within the membrane: it slightly increased the phospholipid polar headgroup mobility as monitored by spectral parameters of T-PC, while along the acyl chain the ordering effect was observed in terms of order parameter S. Also, rotational correlation times τ2B and τ2C of 16-PC in the membrane center were increased by curcumin. Polarity measurements performed in frozen suspensions of liposomes revealed enhancement of water penetration by curcumin in the membrane center (16-PC) and in the polar headgroup region (T-PC) while the intermediate positions along the acyl chain (5-PC and 10-PC) were not significantly affected. Curcumin at a lower concentration (5 mol%) shifted the temperature of the DMPC main phase transition to lower values and increased the transition width, and at a higher concentration (10 mol%) abolished the transition completely. The observed effects suggest that curcumin adopts a transbilayer orientation within the membrane and most probably form oligomers of two molecules, each of them spanning the opposite bilayer leaflets. The effects are also discussed in terms of curcumin's protective activity and compared with those imposed on membranes by other natural dyes known for their protective role, namely polar carotenoids, lutein and zeaxanthin.

Response to Agomelatine Treatment is Independent of Smoking Status and Dosage: Results From the AGOPSYCH Study.

INTRODUCTION: Cigarette smoking influences response to antidepressant treatment. It accelerates the metabolism of several cytochrome P450 (CYP) subtypes, including CYP1A2, and therefore bears the risk of pharmacokinetic interactions with psychotropic drugs using that pathway. Agomelatine is a substrate of CYP1A2; the association between nicotine use and agomelatine dosage, however, has never been studied before. METHODS: Smoking habits were correlated with agomelatine doses and treatment outcomes in a sample of 27 patients with lifetime diagnoses within the schizophrenia spectrum who received agomelatine treatment in addition to their stable antipsychotic treatment regimen because of depressive symptoms. RESULTS: No interactions were found between smoking status and agomelatine dosage, and treatment outcomes did not differ between smokers and nonsmokers. DISCUSSION: Agomelatine efficacy appears to be independent of dosage and smoking status, pointing toward mechanisms beyond mere dose-response relationships. Further research will be necessary to validate these findings.

Health-related quality of life in patients with nasal prosthesis.

OBJECTIVE: To evaluate the health-related quality of life (QOL) in patients with nasal prosthesis after tumour resection with partial or total rhinectomy. MATERIAL AND METHODS: Patients with nasal prosthesis were asked to complete a quality of life questionnaire. 43 patients with prosthesis after partial or total rhinectomy completed the University of Washington quality of life questionnaire. Answers were systematically analysed. RESULTS: 27 patients were male, 16 were female. Mean age was 62 years, and the mean interval between tumour resection and date of study inclusion was 45.3 months. There are gender-specific differences for the QOL domain 'recreation' and age-specific differences for the domains 'appearance' and 'saliva'. The fields 'activity', 'mood' and 'appearance' are the most severely affected QOL domains in patients with nasal prosthesis. CONCLUSION: Similar to other head and neck cancers there is a need and a deficiency in psycho-oncological support after partial or total rhinectomy, the influence on QOL however was much lower than initially expected. This should be considered with regard to the oncological benefits of this surgical procedure. Compared to the results of other head and neck malignancies the 'appearance' is only slightly affected. A stable prosthesis is needed to enable the maintenance of the accustomed activities.