A survey of women who continue to smoke during pregnancy in Slovenia.

INTRODUCTION: Smoking in pregnancy is unhealthy and can also have negative effects on the foetus. However, there are still some women who do not quit smoking during pregnancy. The aim of the study was to identify Slovenian women at risk who smoke during pregnancy. METHODS: A quantative approach using an online survey was adopted. The study was conducted in May and June 2017, in Slovenia. A snowball sample was used. Participation in the survey was anonymous and voluntary, and 118 women who identified themselves as smokers participated in the study. Descriptive statistics was used to analyse the data. RESULTS: More than half (66%) did not quit smoking during pregnancy. Women who continued to smoke during pregnancy were usually younger, less educated with a partner who smoked. Women who smoked more cigarettes per day before conception were less likely to refrain from smoking during pregnancy. The most common reason for not quitting smoking during pregnancy was failure in attempts to quit (37%). CONCLUSIONS: Women who smoke need support to stop smoking before pregnancy or, at least, in the first trimester. Special groups for smoking cessation need to be created. Further and more extensive research is warranted in Slovenia to assess this issue.

Cysteine-specific PEGylation of rhG-CSF via selenylsulfide bond.

A new PEGylation reagent enabling selective modification of free thiol groups is described in this article. The reagent was synthesized by attaching linear polyethylene glycol (PEG) N-hydroxysuccinimide to selenocystamine. The reaction was very fast, resulting in over 95% conversion yield. The active group of this new PEG-Se reagent is a diselenide, reacting with thiols via thiol/diselenide exchange reaction. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) with an unpaired cysteine at the position 18 (Cys18) was used as a model protein. It was comparatively PEGylated with the new PEG-Se reagent, as well as with commercially available maleimide (PEG-Mal) and ortho-pyridyl disulfide (PEG-OPSS) PEG reagents. The highest PEGylation yield was obtained with PEG-Mal, followed by PEG-OPSS and PEG-Se. The reaction rates of PEG-Mal and PEG-Se were comparable, while the reaction rate of PEG-OPSS was lower. Purified monoPEGylated rhG-CSF conjugates were characterized and compared. Differences in activity, stability, and in vivo performance were observed, although all conjugates contained a 20 kDa PEG attached to the Cys18. Minor conformational changes were observed in the conjugate prepared with PEG-Mal. These changes were also reflected in low in vitro biological activity and aggregate formation of the maleimide conjugate. The conjugate prepared with PEG-Se had the highest in vitro biological activity, while the conjugate prepared with PEG-OPSS had the best in vivo performance.

PEGylation of antibody fragments for half-life extension.

Antibody fragments (Fab's) represent important structure for creating new therapeutics. Compared to full antibodies Fab' fragments possess certain advantages, including higher mobility and tissue penetration, ability to bind antigen monovalently and lack of fragment crystallizable (Fc) region-mediated functions such as antibody-dependent cell mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The main drawback for the use of Fab's in clinical applications is associated with their short half-life in vivo, which is a consequence of no longer having the Fc region. To exert meaningful clinical effects, the half-life of Fab's need to be extended, which has been achieved by postproduction chemical attachment of polyethylene glycol (PEG) chain to protein using PEGylation technology. The most suitable approach employs PEG-maleimide attachment to cysteines, either to the free hinge cysteine or to C-terminal cysteines involved in interchain disulfide linkage of the heavy and light chain. Hence, protocols for mono-PEGylation of Fab via free cysteine in the hinge region and di-PEGylation of Fab via interchain disulfide bridge are provided in this chapter.

The Characterization and Potential use of G-CSF Dimers and their Pegylated Conjugates.

G-CSF successfully prevents chemotherapy-induced neutropenia. Two second-generation drugs with improved therapeutic properties are already available and the development of new forms is still ongoing. For an efficient receptor dimerization two G-CSF molecules have to bind. Development of G-CSF dimers acting as receptor dimerizers was explored and their potential use evaluated. The in vitro biological activities of the prepared dimers were lower than G-CSF monomer activity, presumably due to non-optimal spatial orientation of the molecules. Most likely two dimers had to bind to trigger receptor dimerization instead of one dimer acting as a dimerizer. Although significantly lower in the residual in vitro biological activity, the diPEG-Fdim conjugate exhibited pharmacokinetical (PK) and pharmacodynamical (PD) properties comparable to pegfilgrastim or even better. An interesting PD profile with the second maximum in absolute neutrophil count (ANC) and a balanced elevated ANC profile over the longer time interval was namely observed.

Correlations between in vitro potency of polyethylene glycol-protein conjugates and their chromatographic behavior.

Pegylation is the most widely used and accepted methodology for half-life extension of biopharmaceutical drugs that also improves physicochemical and biological characteristics of proteins considerably. Most of the positive pharmacological effects of pegylated proteins are believed to be related to an increased hydrodynamic volume and molecular size. To explore the size impact of polyethylene glycol (PEG) on in vitro potency, a series of well-defined conjugates of interferon alpha-2b (IFN) were prepared with PEGs of different lengths and shapes specifically attached to the N-terminal amino group of the protein. Specificity of the attachment was confirmed by peptide mapping and mass spectroscopy. When potency values determined by reporter gene assay were correlated with methods for molecular weight and size characterization, such as size exclusion chromatography and dynamic light scattering, rough parallels were found. Unexpectedly, the retention times on cation exchange chromatography showed much higher correlation with experimentally determined in vitro potency. It appears that in a series of N-terminally pegylated IFNs, their in vitro potency could be predicted from the retention times on the cation exchange chromatography columns, probably because both methods reflect not only the influence of molecular size but also the impact of protein masking exerted by attached PEG moiety.