Development of PEGylated PLGA nanoparticle for controlled and sustained drug delivery in cystic fibrosis

Background

The mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in CF. The most common mutation, ΔF508-CFTR, is a temperature-sensitive, trafficking mutant with reduced chloride transport and exaggerated immune response. The ΔF508-CFTR is misfolded, ubiquitinated, and prematurely degraded by proteasome mediated- degradation. We recently demonstrated that selective inhibition of proteasomal pathway by the FDA approved drug PS-341 (pyrazylcarbonyl-Phe-Leuboronate, a.k.a. Velcade or bortezomib) ameliorates the inflammatory pathophysiology of CF cells. This proteasomal drug is an extremely potent, stable, reversible and selective inhibitor of chymotryptic threonine protease-activity. The apprehension in considering the proteasome as a therapeutic target is that proteasome inhibitors may affect proteostasis and consecutive processes. The affect on multiple processes can be mitigated by nanoparticle mediated PS-341 lung-delivery resulting in favorable outcome observed in this study.

Results

To overcome this challenge, we developed a nano-based approach that uses drug loaded biodegradable nanoparticle (PLGA-PEG^PS-341) to provide controlled and sustained drug delivery. The in vitro release kinetics of drug from nanoparticle was quantified by proteasomal activity assay from days 1-7 that showed slow drug release from day 2-7 with maximum inhibition at day 7. For in vivo release kinetics and biodistribution, these drug-loaded nanoparticles were fluorescently labeled, and administered to C57BL6 mice by intranasal route. Whole-body optical imaging of the treated live animals demonstrates efficient delivery of particles to murine lungs, 24 hrs post treatment, followed by biodegradation and release over time, day 1-11. The efficacy of drug release in CF mice (Cftr ^-/- ) lungs was determined by quantifying the changes in proteasomal activity (~2 fold decrease) and ability to rescue the Pseudomonas aeruginosa LPS (Pa -LPS) induced inflammation, which demonstrates the rescue of CF lung disease in murine model.

Conclusion

We have developed a novel drug delivery system to provide sustained delivery of CF "correctors" and "anti-inflammatories" to the lungs. Moreover, we demonstrate here the therapeutic efficacy of nano-based proteostasis-modulator to rescue Pa-LPS induced CF lung disease.     

A Ketone Ester Diet Increases Brain Malonyl-CoA and Uncoupling Proteins 4 and 5 while Decreasing Food Intake in the Normal Wistar Rat

Three groups of male Wistar rats were pair fed NIH-31 diets for 14 days to which were added 30% of calories as corn starch, palm oil, or R-3-hydroxybutyrate-R-1,3-butanediol monoester (3HB-BD ester). On the 14th day, animal brains were removed by freeze-blowing, and brain metabolites measured. Animals fed the ketone ester diet had elevated mean blood ketone bodies of 3.5 mm and lowered plasma glucose, insulin, and leptin. Despite the decreased plasma leptin, feeding the ketone ester diet ad lib decreased voluntary food intake 2-fold for 6 days while brain malonyl-CoA was increased by about 25% in ketone-fed group but not in the palm oil fed group. Unlike the acute effects of ketone body metabolism in the perfused working heart, there was no increased reduction in brain free mitochondrial [NAD⁺]/[NADH] ratio nor in the free energy of ATP hydrolysis, which was compatible with the observed 1.5-fold increase in brain uncoupling proteins 4 and 5. Feeding ketone ester or palm oil supplemented diets decreased brain l-glutamate by 15–20% and GABA by about 34% supporting the view that fatty acids as well as ketone bodies can be metabolized by the brain.     

The tailless complex polypeptide-1 ring complex of the heat shock protein 60 family facilitates cross-priming of CD8 responses specific for chaperoned peptides

The tailless complex polypeptide-1 ring complex (TRiC) is a eukaryotic heat shock protein 60 (hsp60) molecule that has been shown to bind N-terminally extended precursors of OVA-derived SIINFEKL in vivo. Binding of peptides to TRiC was shown to be essential for their presentation on MHC class I. We demonstrate in this study that purified TRiC binds antigenic peptides in vitro as well; however, such binding is not restricted to N-terminally extended peptides, suggesting that the results obtained in vivo reflect the availability of peptides in vivo rather than structural constraints of TRiC-peptide binding. Immunization of mice with noncovalent complexes of peptides (derived from OVA or β-galactosidase) and TRiC results in cross-priming of CD8(+) T lymphocytes specific for K(b)/SIINFEKL or L(d)/TPHPARIGL. Mechanistic dissection of this phenomenon shows that TRiC binds APC, and TRiC-chaperoned peptides are processed within the APC and presented on their MHC class I. Immunogenicity of TRiC purified from OVA- or β-galactosidase-expressing cells, that is, of endogenously generated TRiC-peptide complexes, was investigated, and such preparations were observed not to be immunogenic. Consistent with this observation, SIINFEKL or its precursors were not observed to be associated with TRiC purified from cells expressing a fusion GFP-OVA protein. In contrast, immunization with TRiC purified from a tumor elicited specific protection against a challenge with that tumor. These results are interpreted with respect to the cell biological properties of TRiC and suggest that in vivo, TRiC binds a limited proportion of peptides derived from a limited set of intracellular proteins.     

Simultaneous determination and quantification of three pentacyclic triterpenoids—betulinic acid,oleanolic acid,and ursolic acid—in cell cultures of <Emphasis Type="BoldItalic">Lantana camara</Emphasis> L.

A simple procedure has been described for simultaneous determination and improved yield of three pentacyclic triterpenoids—betulinic, oleanolic, and ursolic acids—from callus cultures of Lantana camara. Cell biomass was obtained from leaf disk explants cultured on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with 5 μM 6-benzylaminopurine, 1 μM 2,4-dichlorophenoxyacetic acid, and 1 μM α-naphthaleneacetic acid. Optimum separation of the three compounds was achieved by reverse-phase high-pressure liquid chromatography on a C₁₈ column with 80:20 (v/v) acetonitrile/water as mobile phase. With this route, a yield of 3.1% betulinic acid, 1.88% oleanolic acid, and 4.12% ursolic acid per gram dry weight was obtained from cultures. Leaves from the parent plant, used as control, showed total absence of betulinic acid, and the quantities of oleanolic and ursolic acids present in them were only marginally higher than that found in in vitro-raised cultures. Presence of the three compounds was further confirmed by electrospray ionization mass spectrometry.     

Mapping interactions of gastric inhibitory polypeptide with GIPR N‐terminus using NMR and molecular dynamics simulations

Glucose‐dependent insulinotropic polypeptide (gastric inhibitory polypeptide, or GIP), a 42‐amino acid incretin hormone, modulates insulin secretion in a glucose‐concentration‐dependent manner. Its insulinotropic action is highly dependent on glucose concentration that surmounts the hypoglycemia side effects associated with current therapy. In order to develop a GIP‐based anti‐diabetic therapy, it is essential to establish the 3D structure of the peptide and study its interaction with the GIP receptor (GIPR) in detail. This will give an insight into the GIP‐mediated insulin release process. In this article, we report the solution structure of GIP(1–42, human)NH₂ deduced by NMR and the interaction of the peptide with the N‐terminus of GIPR using molecular modelling methods. The structure of GIP(1–42, human)NH₂ in H₂O has been investigated using 2D‐NMR (DQF‐COSY, TOCSY, NOESY, ¹H‐¹³C HSQC) experiments, and its conformation was built by constrained MD simulations with the NMR data as constraints. The peptide in H₂O exhibits an α‐helical structure between residues Ser8 and Asn39 with some discontinuity at residues Gln29 to Asp35; the helix is bent at Gln29. This bent gives the peptide an ‘L’ shape that becomes more pronounced upon binding to the receptor. The interaction of GIP with the N‐terminus of GIPR was modelled by allowing GIP to interact with the N‐terminus of GIPR under a series of decreasing constraints in a molecular dynamics simulation, culminating with energy minimization without application of any constraints on the system. The canonical ensemble obtained from the simulation was subjected to a detailed energy analysis to identify the peptide–protein interaction patterns at the individual residue level. These interaction energies shed some light on the binding of GIP with the GIPR N‐terminus in a quantitative manner. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Stabilization of anionic and neutral forms of a fluorophoric ligand at the active site of human carbonic anhydrase I

We synthesized a fluorogenic dansylamide derivative (JB2-48), which fills the entire (15 Å deep) active site pocket of human carbonic anhydrase I, and investigated the contributions of sulfonamide and hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic properties of the enzyme–ligand complex. The steady-state and fluorescence lifetime data revealed that the deprotonation of the sulfonamide moiety of the enzyme bound ligand increases the fluorescence emission intensity as well as the lifetime of the fluorophores. This is manifested via the electrostatic interaction between the active site resident Zn²⁺ cofactor and the negatively charged sulfonamide group of the ligand, and such interaction contributes to about 2.2 kcal/mol (ΔΔG°) and 0.89 kcal/mol (ΔΔG^‡) energy in stabilizing the ground and the putative transition states, respectively. We provide evidence that the anionic and neutral forms of JB2-48 are stabilized by the complementary microscopic/conformational states of the enzyme. The implication of the mechanistic studies presented herein in rationale design of carbonic anhydrase inhibitors is discussed.     

Differential modulation of the active site environment of human carbonic anhydrase XII by cationic quantum dots and polylysine

Due to prevalence of negative charges on the protein surface, opposite to the active site pocket of human carbonic anhydrase XII (hCA XII), both positively charged CdTe quantum dots (Qds⁺) and polylysine electrostatically interact with the enzyme, and such interaction does not influence the catalytic activity of the enzyme. However, both these cationic macromolecules differently modulate the active site environment of the enzyme. The steady-state kinetic data revealed that whereas polylysine exhibited no influence on dansylamide (DNSA) dependent inhibition of the enzyme, Qds⁺ overcame such an inhibitory effect, leading to almost 70% restoration of the catalytic activity of the enzyme. We provide evidence that DNSA remains bound to the enzyme upon interaction with both polylysine and Qds⁺. Arguments are presented that the above differential feature of polylysine and Qds⁺ on hCA XII is encoded in the “rigidity” versus “flexibility” of these cationic macromolecules.     

In silico QSAR studies of anilinoquinolines as EGFR inhibitors

Members of the epidermal growth factor receptor (EGFR) family of proteins are frequently overactive in solid tumors. A relatively new therapeutic approach to inhibit the kinase activity is the use of ATP-competitive small molecules. In silico techniques were employed to identify the key interactions between inhibitors and their protein receptors. A series of EGFR inhibitory anilinoquinolines was studied within the framework of hologram quantitative structure activity relationship (HQSAR), density functional theory (DFT)-based QSAR, and three-dimensional (3D) QSAR (CoMFA/CoMSIA). The HQSAR analysis implied that substitutions at certain sites on the inhibitors play an important role in EGFR inhibition. DFT-based QSAR results suggested that steric and electronic interactions contributed significantly to the activity. Ligand-based 3D-QSAR and receptor-guided 3D-QSAR analyses such as CoMFA and CoMSIA techniques were carried out, and the results corroborated the previous two approaches. The 3D QSAR models indicated that steric and hydrophobic interactions are dominant, and that substitution patterns are an important factor in determining activity. Molecular docking was helpful in identifying a bioactive conformer as well as a plausible binding mode. The docked geometry-based CoMFA model with steric and electrostatic fields effect gave q ² = 0.66, r ² = = 0.94 with r ² _predictive = 0.72. Similarly, CoMSIA with hydrophobic field gave q ² = 0.59, r ² = 0.85 with r ² _predictive = 0.63. Bulky groups around site 3 of ring “C”, and hydrophilic and bulky groups at position 6 of ring “A” are desirable, with a hydrophobic and electron-donating group at site 7 of ring “A” being helpful. Accordingly, potential EGFR inhibitors may be designed by modification of known inhibitors.     

白念珠菌SSK1突变株对氟康唑敏感性的初步研究

目的探讨氟康唑作用于白念珠菌双组分信号传导途径SSK1突变株SSK21后药物敏感性的变化。方法采用微量液体稀释法和固醇测定法测定野生株(CAF2-1)和突变株(SSK21)的最小抑菌浓度(MIC);并应用RT-PCR观察氟康唑作用前后,SSK1的表达变化。结果氟康唑对CAF2-1的MIC为16μg/mL,对SSK21为0.032μg/mL。加入氟康唑后,CAF2-1的SSK1表达明显增加,60min时达到最多。结论 SSK21对氟康唑高度敏感,SSK1基因及其相关的重要基因与药物敏感性的关系值得进一步研究,从而为新的抗真菌药物和治疗途径的研发提供参考。