Cyclodextrin complexes of a globular protein and a lipophilic oligopeptide: the effect of structure and physicochemical properties.

Cyclosporine A (CyA) is a lipophilic oligopeptide that has a very limited solubility in water of only 0.008 mg/ml at ambient temperature. It has the ability to form inclusion complexes with cyclodextrin (CD) whose complexes can self-assemble to form aggregates. We have previously developed eye drops with CyA/CD aggregates. Our aim was to study cyclodextrin complexes of lysozyme, a small polar globular protein, and to compare the results with those obtained for CyA. We also wanted to test the stabilizing effect of CDs on lysozyme. Phase-solubility studies of various CDs were performed with CyA and lysozyme. Complexation and particle size measurements were made with dynamic light scattering (DLS) and UV. Solid drug fractions were determined. Thermal and chemical stability studies were performed on lysozyme in the presence of various CDs. Recovery of lysozyme activity in the presence of various CDs after a heat shock was determined. Both CyA and lysozyme are able to form non-inclusion complexes with CD and those complexes can self-assemble and form micro sized aggregates. In case of lysozyme the forces involved are relativity weak and the lysozyme/CD complexes dissociate upon centrifuging, however for CyA the aggregates are stronger and do not dissociate upon centrifuging. CyA is therefore suitable for eye drop preparations containing CDs for sustained drug release whereas lysozyme is not. This is mainly due to the fact that CyA forms inclusion complexes with CDs, whereas lysozyme is not able to do so due to its polar surface. The lysozyme/CD non-inclusion complexes can offer some protection against lysozymes chemical and thermal denaturation. CD can, however, form complexes with unfolded lysozyme and hamper refolding of the protein after heat shock.

TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein.

One approach to investigate gene function, by silencing the activity of certain proteins, is the usage of double stranded decoy oligodeoxynucleotides (ds decoy ODNs). Decoy, in this sense, is ds ODNs bearing the consensus binding sequence for a DNA-binding protein. This can be used in clinical settings to attenuate the effect of overexpressed transcription factors in tumor cells. We here choose to target the oncogenic protein Myc. Since oligonucleotides are poorly internalized to cells, a cell-penetrating peptide, TP10, was coupled to the Myc decoy, using two different strategies. Either TP10 was simply mixed with ds decoy ODNs forming complexes through non-covalent electrostatic interactions, or by having a nona-nucleotide overhang in one of the decoy strands, and adding a complementary PNA sequence coupled to an NLS sequence and TP10, which could hybridize to the Myc decoy. By using these strategies, uptake was significantly enhanced, especially with the co-incubation approach. Interestingly, various endocytosis inhibitors had no effect on the uptake pattern, suggesting that uptake of these complexes is not mediated via endocytosis. Finally, a decreased proliferative capacity was observed when treating the neuroblastoma cell line N2a with TP10-PNA conjugate hybridized to Myc decoy compared to naked Myc decoy and untreated cells. A dose-dependent decrease in proliferation was also observed in MCF-7 cells, when using both strategies. These results suggest an alternative way to efficiently deliver ds ODNs into cells using the cell-penetrating peptide TP10 and prevent tumor growth by targeting the oncogenic protein Myc.

Experimental study of the virulence of Streptococcus pneumoniae with reduced susceptibility to penicillin.

Streptococcus pneumoniae is a major cause of morbidity and mortality in all age groups. In a few years, penicillin non-susceptible pneumococci (PNSP) have emerged worldwide as a new threat. In order to better understand the mechanisms behind the rapid expansion of these strains, the virulence of 10 clinical and two transformed PNSP strains were compared with the virulence of three fully susceptible strains in a mouse model of bacteremia and a rat model of acute otitis media. Serotype, antibiotic susceptibility, and to some extent also genetic profile and growth rate of the strains were investigated before inoculation. The animals were monitored for up to 7 days after challenge by clinical examinations/otomicroscopy and cultures from middle ears and blood. The results of the study demonstrated that the PNSP strains had a significantly reduced ability to persist at the infectious site, and to some extent also to induce infections, compared with fully susceptible strains. The reduction was most evident for strains isolated from sources other than blood. It is therefore possible that other factors than virulence factors are of importance for the ability of PNSP strains to expand.

Effects on the ciliated epithelium of protein D-producing and -nonproducing nontypeable Haemophilus influenzae in nasopharyngeal tissue cultures.

A pair of isogenic, nontypeable Haemophilus influenzae strains, one expressing protein D and the other protein D-negative, was compared in their ability to cause damage in a human nasopharyngeal tissue culture model. Damage was assessed by measuring the ciliary beat frequency (CBF) of tissue specimens at 12 h intervals. Cultures inoculated with H. influenzae manifested a decrease in CBF beginning after 12 h, with a maximum decrease after 36 h. The impairment of ciliary function by the protein D-expressing strain was significantly greater than that caused by the protein D-negative mutant (P<.01). Tissue specimens examined by scanning and transmission electron microscopy after 24 h appeared normal. After 48 h of incubation, the protein D-expressing strain caused a significant loss of cilia. These findings suggest that protein D is involved in the pathogenesis of upper respiratory tract infections due to nontypeable H. influenzae, probably by enhancing functional and morphological damage to cilia.