Search

DNA-minor-groove-binding ligands are potent antineoplastic molecules. The antibiotic distamycin A is the prototype of one class of these DNA-interfering molecules that have been largely used in vitro. The affinity of distamycin A for DNA is well known, and the structural details of the complexes with some B-DNA and G-quadruplex-forming DNA sequences have been already elucidated. Here, we show that distamycin A binds S100β, a protein involved in the regulation of several cellular processes. The reported affinity of distamycin A for the calcium(II)-loaded S100β reinforces the idea that some biological activities of the DNA-minor-groove-binding ligands arise from the binding to cellular proteins. Copyright © 2015 John Wiley & Sons, Ltd.

Background Escherichia coli asparaginase is an enzyme that depletes serum levels of asparagine. It is used to treat acute lymphoblastic leukaemia and related forms of non-Hodgkin’s lymphoma. Polyethylene glycosylated–asparaginase (pegaspargase), obtained by covalently attaching polyethylene glycol to the native enzyme, has been shown to sustain similar reductions in serum asparagine concentrations compared with the native enzyme. In addition, pegaspargase has a decreased immunogenicity and a prolonged half-life. The summary of product characteristics (Oncaspar) indicates that the intravascular infusion should be given over a period of 1–2 h but nothing is known on the long term stability and activity of the enzyme after dilution. Purpose Evaluation of the biological activity of pegaspargase diluted to 16 UI/mL in NaCl 0.9% and stored up to 48 h at 4°C and at room temperature. A study of drug degradation was also carried out. Material and methods Samples of pegaspargase solution diluted in NaCl 0.9% were stored refrigerated at 4°C and at room temperature and protected from light. The biological activity of the two solutions was determined by measuring hydrolysis of L-asparagine, and the ammonia released by the enzyme was quantified with Nessler’s reagent. The absence of degradation products or aggregates in the two solutions was verified using size exclusion fast protein liquid chromatography (SEC-FPLC) under the following condition: Superdex 200 10/300 column; Tris buffer pH=8.6; 0.5 mL/min flow rate; 280 nm UV detection; 100 µL injection volume. Results In the samples stored both at 4°C and at room temperature, enzymatic activity was preserved over a period of 48 h. No degradation or aggregation was observed in these samples over the same period. Conclusion The variation in enzymatic activity of the diluted pegaspargase solutions compared with the fresh solution was less than 5% after 48 h, with no significant differences between storage at 4°C or at room temperature. Preservation of the enzymatic activity and the stability of the solutions evaluated will allow us to store pegaspargase for up to 48 h with costs savings and an improvement in patient compliance. A microbiological study is in progress to validate the aseptic manufacturing process in order to guarantee the sterility of the stored solutions. No conflict of interest.

Background Vernal Keratoconjunctivitis (VKC) is an allergic conjunctivitis, often not easily diagnosed and properly treated. The disease is very debilitating for patients, may be complicated by corneal lesions and can evolve to keratoconus. Purpose The exponential increase in VKC patients led us to start a close collaboration between pharmacists and allergists, ophthalmologists, and chemists. The goal was to address and solve problems caused by the lack of adequate knowledge of VKC, in order to find a diagnostic-therapeutic course, improve patient compliance and provide high-quality products as an alternative to conventional treatments. Material and methods After discussions with allergists and ophthalmologists, pharmacists formulated 3 different kinds of eye drops as treatment: ciclosporin 1% and tacrolimus 0.1%, both in methylcellulose 0.15%, and ciclosporin 2% in sunflower oil. The stability of such formulations was demonstrated by using liquid chromatography coupled to a triple quadrupole mass spectrometer. The pharmacist now prepares a weekly supply of eye drops, after allergists pass on the number of children who will undergo eye examinations. Then, the pharmacist proceeds, after allergist confirmation, to arrange for eye drops to be sent directly to patients’ homes in the whole country. Results The LC/MS/MS and sterility analysis results allowed the pharmacist to declare that formulations in methylcellulose 0.15% and in sunflower oil were safe for up to 45 days. Such formulations were chosen considering also patient compliance. Indeed, one of the results of the team collaboration has been the development of the formulation in sunflower oil, which can be stored at room temperature; thus leading to huge advantages in terms of patient compliance. Conclusion The preparation of a galenical formulation of such quality has contributed to the efficacy of the treatment. Moreover, the sharing of information between medical doctors, pharmacists and nurses has led to personalised assistance that is highly responsive to health needs. Reference Leonardi A. Prog Retin Eye Res 2002;21(3):319–39 No conflict of interest.

The homodimeric structure of human S100A16 in the apo state has been obtained both in the solid state and in solution, resulting in good agreement between the structures with the exception of two loop regions. The homodimeric solution structure of human S100A16 was also calculated in the calcium(II)-bound form. Differently from most S100 proteins, the conformational rearrangement upon calcium binding is minor. This characteristic is likely to be related to the weak binding affinity of the protein for the calcium(II) ions. In turn, this is ascribed to the lack of the glutamate residue at the end of the S100-specific N-domain binding site, which in most S100 proteins provides two important side chain oxygen atoms as calcium(II) ligands. Furthermore, the presence of hydrophobic interactions stronger than for other S100 proteins, present in the closed form of S100A16 between the third and fourth helices, likely make the closed structure of the second EF-hand particularly stable, so even upon calcium(II) binding such a conformation is not disrupted.