Development of an automated production process of [64Cu][Cu (ATSM)] for positron emission tomography imaging and theranostic applications

Cyclotron-produced copper-64 radioisotope tracers offer the possibility to perform both diagnostic investigation by positron emission tomography (PET) and radiotherapy by a theranostic approach with bifunctional chelators. The versatile chemical properties of copper add to the importance of this isotope in medicinal investigation. [⁶⁴Cu][Cu (ATSM)] has shown to be a viable candidate for imaging of tumor hypoxia; a critical tumor microenvironment characteristic that typically signifies tumor progression and resistance to chemo-radiotherapy. Various production and radiosynthesis methods of [⁶⁴Cu][Cu (ATSM)] exist in labs, but usually involved non-standardized equipment with varying production qualities and may not be easily implemented in wider hospital settings. [⁶⁴Cu][Cu (ATSM)] was synthesized on a modified GE TRACERlab FXN automated synthesis module. End-of-synthesis (EOS) molar activity of [⁶⁴Cu][Cu (ATSM)] was 2.2–5.5 Ci/μmol (HPLC), 2.2–2.6 Ci/μmol (ATSM-titration), and 3.0–4.4 Ci/μmol (ICP-MS). Radiochemical purity was determined to be >99% based on radio-HPLC. The final product maintained radiochemical purity after 20 h. We demonstrated a simple and feasible process development and quality control protocols for automated cyclotron production and synthesis of [⁶⁴Cu][Cu (ATSM)] based on commercially distributed standardized synthesis modules suitable for PET imaging and theranostic studies.     

Low-dose hydrocortisone in patients with COVID-19 and severe hypoxia (COVID STEROID) trial—Protocol and statistical analysis plan

Introduction

Severe acute respiratory syndrome coronavirus-2 has caused a pandemic of coronavirus disease (COVID-19) with many patients developing hypoxic respiratory failure. Corticosteroids reduce the time on mechanical ventilation, length of stay in the intensive care unit and potentially also mortality in similar patient populations. However, corticosteroids have undesirable effects, including longer time to viral clearance. Clinical equipoise on the use of corticosteroids for COVID-19 exists.

Methods

The COVID STEROID trial is an international, randomised, stratified, blinded clinical trial. We will allocate 1000 adult patients with COVID-19 receiving ≥10 L/min of oxygen or on mechanical ventilation to intravenous hydrocortisone 200 mg daily vs placebo (0.9% saline) for 7 days. The primary outcome is days alive without life support (ie mechanical ventilation, circulatory support, and renal replacement therapy) at day 28. Secondary outcomes are serious adverse reactions at day 14; days alive without life support at day 90; days alive and out of hospital at day 90; all-cause mortality at day 28, day 90, and 1 year; and health-related quality of life at 1 year. We will conduct the statistical analyses according to this protocol, including interim analyses for every 250 patients followed for 28 days. The primary outcome will be compared using the Kryger Jensen and Lange test in the intention to treat population and reported as differences in means and medians with 95% confidence intervals.

Discussion

The COVID STEROID trial will provide important evidence to guide the use of corticosteroids in COVID-19 and severe hypoxia.

     

Effects of growth hormone on glucose and fat metabolism in human subjects.

This article focuses on in vivo data from tests performed in normal subjects and in patients who had abnormal growth hormone (GH) status. Experimental data in human subjects demonstrate that GH acutely inhibits glucose disposal in skeletal muscle. At the same time GH stimulates the turnover and oxidation of free fatty acid (FFA), and experimental evidence suggests a causal link between elevated FFA levels and insulin resistance in skeletal muscle. Observational data in GH-deficient adults do not indicate that GH replacement is associated with significant impairment of glucose tolerance, but it is recommended that overdosing be avoided and glycemic control be monitored.     

Lung function development in the first 2 yr of life is independent of allergic diseases by 2 yr

The aim of the study was to assess if lung function at birth predicts lung function at 2 yr and secondly, if lung function development was influenced by the common phenotypes of recurrent bronchial obstruction (rBO) or atopic eczema (AE) by 2 yr. Lung function was assessed at birth (n = 802) and at 2 yr within the prospective birth cohort study 'the Environment and Childhood Asthma Study' in Oslo. The 135 children with lung function measured at birth by tidal flow volume (TFV) loops and passive respiratory mechanics, who were included in a nested case-control study were reinvestigated at 2 yr with clinical examination, TFV loops (n = 90) (mean age 26.6 (3.7 s.d.) months), skin prick test and parental interview. Children were categorized into quartiles (lower, middle two, upper) according to time to peak tidal expiratory flow/total expiratory time (t(PTEF)/t(E)) at birth as well as clinical phenotype based on the presence of rBO and/or AE (ever) by 2 yr. The observed reduction in mean t(PTEF)/t(E) from birth to 2 yr within the quartiles, were not significantly different after controlling for 'regression to the mean'. t(PTEF)/t(E) at birth correlated significantly with t(PTEF)/t(E) at 2 yr, (r = 0.475, p < 0.001). Children with both rBO and AE by 2 yr had significantly lower t(PTEF)/t(E) at 2 yr (p = 0.002) and at birth (p = 0.027), compared with children with no rBO or AE. Clinical phenotype at 2 yr did not influence the change in t(PTEF)/t(E) from birth to 2 yr. This study demonstrates a clear tracking of lung function from birth, not influenced by rBO or AE by 2 yr.     

Clinical symptoms and signs in sore throat patients with large colony variant β-haemolytic streptococci groups C or G versus group A

BACKGROUND: The role of large colony streptococci groups C or G as pathogen agents in sore throat has been questioned. AIM: To analyse clinical features of patients with large colony streptococci groups C or G compared with patients with group A streptococci (GAS) and with negative cultures. DESIGN OF STUDY: Prospective study of patients with sore throat. SETTING: Two Norwegian general practices in Stokke and Kongsberg communities with 6500 patients.METHOD: Frequency of clinical features in the three patient categories including the four Centor criteria (fever, anterior cervical lymphadenopathy, tonsillar exudates, and lack of cough), degree of pain on swallowing, pharyngeal rubor, C-reactive protein (CRP) values, patient age between 3 and 14 years, and duration of symptoms before seeing the doctor. A logistic regression analysis to find independent predictors was performed. RESULTS: Out of 306 patients with a sore throat, 244 were adults and 62 were children under 10 years old; 40% were men. One hundred and twenty-seven had GAS, 33 had streptococci groups C or G, and 146 had negative throat cultures. Forty-eight per cent of the GAS patients and 45% of the C or G patients met three or four of the Centor criteria. The logistic regression revealed that in patients with GAS considerable pain on swallowing, an age of 3-14 years and a duration of symptoms of < or =3 days or less were significantly associated with GAS infection in addition to the Centor criteria. The same results were found when all streptococci were analysed together, in addition elevated CRP was significant. In patients with streptococci group C or G an elevated CRP-value was significantly associated. CONCLUSION: Patients with tonsillitis caused by streptococcus groups C or G have, to a large extent, the same clinical picture as patients with GAS. Large colony streptococci groups C and G should be considered as throat pathogens in line with GAS.     

Effects of dietary broccoli on human in vivo drug metabolizing enzymes: evaluation of caffeine, oestrone and chlorzoxazone metabolism

Ingestion of cruciferous vegetables may prevent chemically inducedcarcinogenesis by their influence on specific cytochrome P450enzymes (CYP) and phase II drug metabolizing enzymes in humansand rodents. Thus CYP enzymes are involved in transformationof procarcinogens, mutagens, steroid hormones and a large varietyof other endogenous and exogenous components. In order to learnmore about the influence of cruciferous vegetables on drug metabolizingenzymes in man two CYP enzymespreviously suggested to be inducedby vegetables were selected in an in vivo experiment in humans.Sixteen healthy non-smoking subjects, two females and 14 males,were exposed to three different types of diets and afterwardsassayed for CYP1A2 catalysed caffeine metabolites and for CYP2E1catalysed 6-hydroxylation of chlorzoxazone. Further, 2-hydroxyoestrone:16     

HRAS1-selected, chromosome-mediated transformants vary in phenotype in vitro and tumorigenic potential in vivo

Transfection of mouse C127 cells with mitotic chromosomes isolated from a human EJ bladder carcinoma cell line gave rise, at high frequency, to foci of transformed cells. Independent, HRAS1-selected chromosome-mediated transformants displayed distinctive cellular morphologies in monolayer culture and colony-forming abilities in low-melting-point agarose. Subcutaneous inoculation of neonatally thymectomized, Ara-C-protected, total-body-irradiated CBA mice was used to compare the tumorigenic potential of each transformant. Significant quantitative and qualitative differences in tumorigenicity were found between transformants which correlated with differences in malignant phenotype observed in vitro. The sensitivity of the tumorigenicity assay is such that rare transformation events can be selected directly in vivo.     

Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption.

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. INTRODUCTION: Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. MATERIALS AND METHODS: Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1. RESULTS: In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. CONCLUSIONS: We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.