Strategies for improving the solubility and metabolic stability of griseofulvin analogues.

We report two types of modifications to the natural product griseofulvin as strategies to improve solubility and metabolic stability: the conversion of aryl methyl ethers into aryl difluoromethyl ethers at metabolic hotspots and the conversion of the C-ring ketone into polar oximes. The syntheses of the analogues are described together with their solubility, metabolic half-life in vitro and antiproliferative effect in two cancer cell lines. We conclude that on balance, the formation of polar oximes is the most promising strategy for improving the properties of the analogues.

Adverse effects associated with high-dose olanzapine therapy in patients admitted to inpatient psychiatric care.

CONTEXT: In 2012, Danish psychiatrist raised concerns regarding the use of high-dose olanzapine in the treatment of patients. The present study was part of an audit carried out by the Mental Health Services of the Capitol Region of Denmark regarding this topic. Objective. To assess the potential risks associated with high-dose olanzapine treatment (> 40 mg daily) in inpatient psychiatric units. METHODS: The study was an observational case series based on review of patient charts. The main inclusion criterion was treatment with at least one daily dose > 40 mg olanzapine during the index admission in the period between 1st of January and 15th of March 2012. Six additional criteria were applied in order to target the subgroup of patients most likely to have experienced an adverse event due to treatment with olanzapine. The physician order entry system and the central patient register containing patient specific information about diagnoses and treatments were used for identification of study population. RESULTS: The 91 patients included in the study received maximum daily doses of olanzapine ranging from 45 to 160 mg and in 25% of patients, the total antipsychotic load exceeded 2000 mg of chlorpromazine equivalents. Extrapyramidal symptoms and sedation were the most frequent adverse events with frequencies of 27% and 25%, respectively. Furthermore, other well-known adverse events such as weight gain (14%), hypotension (2%), neuroleptic malignant syndrome (2%) and corrected QT-interval (QTc) prolongation (1%) were also observed in some patients. Five patients died and in two of these cases, olanzapine was concluded to be a possible contributing cause of death. CONCLUSION: Increased frequency of extrapyramidal symptoms and sedation as well as severe toxicity was observed in patients treated with up to 160 mg olanzapine per day. In order to prevent harmful outcomes, the clinicians should be ready to act appropriately if toxic effects of olanzapine occur. Treatment cessation should be immediate if serious adverse events such as neuroleptic malignant syndrome arise.

Lixisenatide for the treatment of type 2 diabetes.

Lixisenatide (trade name Lyxumia®), a short-acting glucagon-like peptide 1 receptor (GLP-1R) agonist, was approved for the treatment of type 2 diabetes by the European Medicines Agency in early 2013. In preclinical investigations, acceptable toxicity and carcinogenicity profiles were demonstrated, as well as pancreatic beta cell-preserving actions and favorable effects on glycemic control. Following subcutaneous administration in humans, lixisenatide displays linear pharmacokinetics and an absorption-dependent elimination half-life of 2-3 hours. In clinical trials of up to 1 year duration in patients with type 2 diabetes, treatment with lixisenatide alone and in combination with insulin and various oral antidiabetics conferred significant reductions in HbA1c, fasting and postprandial plasma glucose. In direct comparison with the other GLP-1R agonists on the market (exenatide and liraglutide), lixisenatide appears to be less efficient, or at best non-inferior in terms of reducing HbA1c, fasting plasma glucose and body weight. Nevertheless, lixisenatide confers fewer adverse events than the other currently marketed GLP-1R agonists, while exhibiting a clinically valuable effect on postprandial hyperglycemia.

Phylogeny and resistance profiles of HIV-1 POL sequences from rectal biopsies and blood.

The phylogeny and resistance profiles of human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) sequences were compared among six patients with HIV-1 who had received numerous treatments. RNA and DNA fractions were obtained from concurrent blood and rectal biopsy samples. Phylogenetic trees and resistance profiles showed that the rectal mucosa and the peripheral blood mononuclear cells (PBMCs) harbored different HIV-1 strains. The resistance-associated mutations found in each strain corresponded to the treatment history of the patients. The resistance mutations acquired during earlier treatment regimens were detected in the sequences obtained from the rectal samples and in the PBMCs in several of the patients. Also, differences in the resistance profiles were observed between anatomical sites and between RNA and DNA fractions. Thus, a single sample probably will not be representative of the HIV-1 archived in different sites. Both the resistance profile and phylogeny of HIV-1 often differed in sequences obtained from RNA and DNA from the same site. These findings suggest that additional information regarding the antiviral resistance profile of the patient might be obtained by testing different anatomical sites.

Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of IgE antibodies to species-specific major allergens Api m1 and Ves v5.

BACKGROUND: In patients with hymenoptera venom allergy diagnostic tests are often positive with honey bee and Vespula venom causing problems in selection of venoms for immunotherapy. METHODS: 100 patients each with allergic reactions to Vespula or honey bee stings and positive i.e. skin tests to the respective venom, were analysed for serum IgE to bee venom, Vespula venom and crossreacting carbohydrate determinants (CCDs) by UNICAP (CAP) and ADVIA Centaur (ADVIA). IgE-antibodies to species specific recombinant major allergens (SSMA) Api m1 for bee venom and Ves v5 for Vespula venom, were determined by ADVIA. 30 history and skin test negative patients served as controls. RESULTS: By CAP sensitivity was 1.0 for bee and 0.91 for Vespula venom, by ADVIA 0.99 for bee and 0.91 for Vespula venom. None of the controls were positive with either test. Double positivity was observed in 59% of allergic patients by CAP, in 32% by ADVIA. slgE to Api m1 was detected in 97% of bee and 17% of Vespula venom allergic patients, slgE to Ves v5 in 87% of Vespula and 17% of bee venom allergic patients. slgE to CCDs were present in 37% of all allergic patients and in 56% of those with double positivity and were more frequent in bee than in Vespula venom allergic patients. CONCLUSIONS: Double positivity of IgE to bee and Vespula venom is often caused by crossreactions, especially to CCDs. IgE to both Api m1 and Ves v5 indicates true double sensitization and immunotherapy with both venoms.

Differences in activation of G2/M checkpoint in keratinocytes after genotoxic stress induced by hydrogen peroxide and ultraviolet A radiation.

Long-wave ultraviolet radiation (UVA) may cause extensive DNA damage via reactive oxygen species (ROS). In this study we examined whether UVA- and H2O2-mediated DNA damage have equivalent effects on the induction of G2/M phase checkpoint and cell cycle progression in a transformed keratinocyte cell line HaCaT. By employing single cell gel electrophoresis (comet assay) we determined the equipotent doses of UVA and H2O2 with respect to the induction of alkali-labile sites (an indicator of oxidative DNA decay). However, in contrast to H2O2 which caused a pronounced G2/M cell cycle arrest 24 h after treatment, UVA irradiation did not affect cell cycle progression. Increasing UVA doses up to 150 kJ/m2 did not affect cell cycle and proliferation whereas increasing H2O2 concentrations caused a cell cycle block or cell death. Cytometric analysis revealed that G2/M cell cycle arrest took place beyond the cyclin B1 restriction point. We conclude that the DNA damage induced by UVA is easily repaired and does not perturb cell growth, whereas the H2O2-induced damage leads ultimately to cell cycle arrest or cell death.

Laser scanning cytometry for comet assay analysis.

BACKGROUND: The comet assay (single-cell gel electrophoresis) is a sensitive method for evaluating nuclear DNA damage. Previously used evaluation methods for the comet assay are time consuming and have an inherent risk of biased selection of comets due to manual selection and categorization of comet images. Laser scanning cytometry (LSC), the principle of which is equivalent to flow cytometry, enables quantification of fluorescence emitted from the cells on a microscope slide. In the present study, we explored whether LSC could be used to determine the degree of DNA damage demonstrated by the comet assay. METHODS: DNA damage was induced by ultraviolet A irradiation of keratinocytes and visualized by the comet assay. The evaluation included (a) LSC determination of DNA-specific fluorescence in 1,000 comet heads (undamaged DNA), (b) image acquisition of comets by rescanning of the microscope slide, and (c) digital image analysis and computation of tail moment and DNA content in the comet tails. RESULTS: Cells with damaged DNA were observed in a sub-G(1) area because the comet head loses DNA to the tail. We found a strong inverse correlation between tail moment and DNA content per nucleus. CONCLUSIONS: LSC enables an automated method for cell recognition and evaluation of the comets, thus providing quantitative information about nuclear DNA damage without subjective selection of analyzed comets.

Hydrogen peroxide is responsible for UVA-induced DNA damage measured by alkaline comet assay in HaCaT keratinocytes.

We investigated the role of different reactive oxygen species (ROS) in ultraviolet A (UVA)-induced DNA damage in a human keratinocyte cell line, HaCaT. UVA irradiation increased the intracellular levels of hydrogen peroxide (H2O2), detected by a fluorescent probe carboxydichlorodihydrofluorescein, and caused oxidative DNA damage, single strand-breaks and alkali-labile sites, measured by alkaline single cell gel electrophoresis (comet assay). Superoxide anion (O2*-) was a likely substrate for H2O2 production since diethyldithiocarbamate (DDC), a superoxide dismutase blocker, decreased the level of intracellular H2O2. Hydrogen peroxide was shown to play a central role in DNA damage. Increasing the intracellular levels of H2O2 with aminotriazole (AT) (a catalase blocker) and buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis) potentiated the UVA-induced DNA damage. Exogenous H2O2 was also able to induce DNA damage. Since H2O2 alone is not able to damage DNA directly, we investigated the significance of the H2O2-derived hydroxyl radical (*OH). Addition of FeSO4, that stimulates *OH formation from H2O2 (Fenton reaction) resulted in a twofold increase of DNA-damage. Desferrioxamine, an iron chelator that blocks the Fenton reaction, prevented UVA-induced DNA damage. We also employed a panel of less specific antioxidants and enzyme modulators. Sodium selenite (Na-Se) present in glutathione peroxidase and thioredoxin reductase and addition of glutathione (GSH) prevented DNA-damage. Tocopherol potently prevented UVA-and H2O2-induced DNA damage and reduced intracellular H2O2 -levels. Ascorbic acid reduced H2O2 production, but only partly prevented DNA damage. Singlet oxygen (1O2) did not seem to play an important role in the UVA-induced DNA-damage since the specific 1O2 scavenger sodium azide (NaN3) and the less specific 1O2 scavenger beta-carotene did not markedly prevent either DNA-damage or H2O2 production. In conclusion the conversion of H2O2 to *OH appears to be the most important step in UVA-induced generation of strand breaks and alkali-labile sites and the bulk H2O2 appears to originate from O2*- generated by UVA irradiation.