Adverse events related to the new psychoactive substance 3-fluorophenmetrazine – results from the Swedish STRIDA project

Background: New psychoactive substances (NPS) are often poorly pharmacologically documented and the production is unregulated, implying high risks for toxic side effects. This report from the STRIDA project describes analytically confirmed non-fatal intoxications involving the phenmetrazine analogue 3-fluorophenmetrazine (3-FPM).

Study design and methods: Observational case series of patients with suspected acute NPS exposure requiring hospital care. Blood and urine samples were collected from patients presenting in emergency departments (ED) or intensive care units (ICU), after consultation with the Swedish Poisons Information Centre (PIC). Laboratory analysis was performed by multi-component liquid chromatography–mass spectrometry. Clinical data were collected during PIC consultations and retrieved from medical records.

Results: From November 2014 to October 2015, eight cases were registered as 3-FPM or “phenmetrazine” intoxications at the PIC after consultation. During the same period, analysis of STRIDA project samples confirmed 3-FPM use in a total of 19 patients (84% men) aged 22–54 (median 30) years. 3-FPM was detected in 15 out of 19 serum (2.7–1416?ng/mL) and in 14 out of 14 urine (1.0–6857?μg/mmol creatinine) samples. All patients were also tested positive for other psychoactive substances, with benzodiazepines being most common (57% of the cases). Ten patients were monitored in the ED for <4?h, while six needed ICU monitoring of which five were graded as severe intoxications (Poisoning Severity Score 3). Prominent clinical signs were tachycardia (47%), depressed consciousness (42%), agitation/anxiety (37%), delirium (37%), dilated pupils (26%), and seizures (16%). All patients survived.

Conclusion: In 19 patients testing positive for 3-FPM, a high incidence of severe clinical features was demonstrated. However, as all patients had also used other psychoactive substances, it was difficult to identify a unique toxidrome for 3-FPM. The results further showed that many 3-FPM intoxications would have been missed, if relying solely on information from PIC consultations. These results emphasize the importance of performing bioanalytical investigation in cases of suspected NPS intoxication.     

Novel membrane frizzled‐related protein gene mutation as cause of posterior microphthalmia resulting in high hyperopia with macular folds

Purpose: We present a genetic and clinical analysis of two sisters, 3 and 4 years of age, with nanophthalmos and macular folds. Methods: Ophthalmological examination, general paediatric examination and molecular genetic analysis of the MFRP gene were performed in both affected siblings. Results: Clinical analysis showed high hyperopia (+11 D and +12 D), short axial lengths (15 mm) and the presence of macular folds and optic nerve head drusen. Autofluorescence of the retina was generally normal with subtle macular abnormalities. Sequence analysis showed compound heterozygosity for severe MFRP mutations in both sisters: a previously reported p.Asn167fs (c.498dupC) and a novel stop codon mutation p.Gln91X (c.271C>T). Conclusion: These are the youngest nanophthalmos patients in the literature identified with severe loss of MFRP function, showing already the known structural abnormalities for this disease. Adult patients affected by homozygous or compound heterozygous MFRP mutations generally show signs of retinal dystrophy, with ERG disturbances and RPE abnormalities on autofluorescence imaging. ERG examination could not be performed in these children, but extensive RPE abnormalities were not seen at this young age.     

The host defense peptide LL‐37 is detected in human parotid and submandibular/sublingual saliva and expressed in glandular neutrophils

The human host defense peptide, LL‐37, is an important player in the first line of defense against invading microorganisms. LL‐37 and its precursor, hCAP18, have been detected in unstimulated whole saliva but no reports showing hCAP18/LL‐37 in isolated, parotid, and/or submandibular/sublingual saliva have been presented. Here, we measured the levels of hCAP18/LL‐37 in human parotid and submandibular/sublingual saliva and investigated the expression of hCAP18/LL‐37 in parotid and submandibular gland tissue. Parotid and submandibular/sublingual saliva was collected from healthy volunteers, and the levels of hCAP18/LL‐37 in saliva were analyzed by dot blot, ELISA, and western blotting. Cellular expression of hCAP18/LL‐37 in human parotid and submandibular glands was investigated by immunohistochemistry. Immunoreactivity for hCAP18/LL‐37 was detected in both parotid and submandibular/sublingual saliva of all individuals. The concentration of hCAP18/LL‐37 was similar in parotid and submandibular/sublingual saliva, and was determined by densitometric scanning of each dot and normalization to the total protein concentration of each sample, and by ELISA. Double immunohistochemistry revealed that intravascular neutrophils of both parotid and submandibular glands express hCAP18/LL‐37. For the first time, we demonstrate hCAP18/LL‐37 in isolated human parotid and submandibular/sublingual saliva and expression of hCAP18/LL‐37 in glandular intravascular neutrophils, indicating that neutrophils of the major salivary glands contribute to the LL‐37 content of whole saliva.     

Caspase-mediated degradation of human 5-lipoxygenase in B lymphocytic cells

5-Lipoxygenase (5-LO) is a tightly regulated enzyme in the synthesis of bioactive lipids from arachidonic acid. Here, we demonstrate that 5-LO is regulated by caspases, which are signaling molecules that control critical biological processes by means of specific limited proteolysis. Cell splitting of the Epstein-Barr virus-transformed B lymphocytic cell line BL41-E95-A caused a pronounced, but transient, reduction of functional 5-LO protein, accompanied by the appearance of a 62-kDa 5-LO cleavage product. In parallel, splitting of BL41-E95-A cells induced activation of caspase-6 (casp-6) and casp-8. Caspase activation and 5-LO degradation were blocked by the protein-synthesis inhibitor cycloheximide, and cell-permeable peptide inhibitors of casp-6 and casp-8 prevented 5-LO cleavage. Activation of casp-6 and casp-8 was connected to subsequent enhancement of cell proliferation, whereas selective caspase inhibition blocked cell growth. Last, isolated human 5-LO was cleaved by recombinant casp-6 in vitro to a 58-kDa fragment. Based on site-directed mutagenesis studies, 5-LO is cleaved by casp-6 after Asp-170, which in a homology-based 3D model of 5-LO is located on the enzyme periphery. We suggest that splitting of BL41-E95-A cells induces de novo synthesis of a protein involved in the activation of casp-6, which cleaves 5-LO.     

An evaluation of a high‐pressure 11CO carbonylation apparatus

[¹¹C]Carbon monoxide (¹¹CO) is a versatile building block for the synthesis of Positron Emission Tomography (PET) radioligands. However, the difficulty of trapping ¹¹CO in a small solvent volume has limited its utility. We here report an evaluation of a simple, fully automated high‐pressure synthesizer prototype for the use in ¹¹C‐carbonylation reactions. [¹¹C]Carbon monoxide was easily prepared by online reduction of [¹¹C]carbon dioxide using either Mo(s) or Zn(s) as the reducing agent. The conversion yield of ¹¹CO was >99% when zinc was used as the reducing agent, and the corresponding value for Mo was approximately 71%. When the Zn or Mo column was constantly kept under inert atmosphere, no significant decrease in reducing properties was observed for more than 100 ¹¹CO productions. However, in our hands, Mo reductant was much easier to service. A total of nine functional groups were successfully radiolabeled using the ¹¹CO synthesizer prototype. All measured radiochemical yields exceeded 37%, and the ¹¹CO trapping efficiency was generally above 90%, except for the Suzuki coupling where the trapping efficiency was 80%. This high‐pressure synthesizer using [¹¹C]carbon monoxide as the labeling precursor is easy to operate allowing for ¹¹C‐carbonylation reactions to be performed in a high yield and in a routinely fashion.     

Jejunal secretion of secretory immunoglobulins and gliadin antibodies in celiac disease

The aim of this study was to determine the secretion of secretory immunoglobulins and gliadin antibodies in the small bowel in celiac disease. Twenty-four patients were investigated by perfusion of a defined jejunal segment. Four of the patients studied had a serum IgA deficiency and had no measurable amounts of secretory IgA in the perfusion fluid. The other patients demonstrated a significant increase in the jejunal concentration of secretory IgA (median 28.5 mg/liter) compared with healthy controls (median 16 mg/liter,N=16) and of IgM, celiac (median 12.3 mg/liter) compared to healthy controls (median 6.8 mg/liter,N=16). Jejunal IgA gliadin antibodies were detected in all patients except those with an IgA deficiency. All patients had jejunal IgM gliadin antibodies, but none of the patients had measurable jejunal IgG gliadin antibodies. A positive correlation was detected between serum and jejunal IgA gliadin antibody levels in the celiac patients, (P<0.01). Calculation of the ratio between gliadin antibodies and total levels of IgA and IgM in serum and jejunal perfusate demonstrated that the jejunal synthesis of gliadin antibodies of IgA and IgM type is both more pronounced and persistent than the systemic humoral immune response to gliadin.This work was supported by grants from the Swedish Medical Research Council, the Swedish Life Insurance Companies' Trust for Medical Research, and Th.C. Berghs Foundation and Pharmacia AB, Sweden.