Experimental study of a gentamicin aerosol]

The study of gentamicin aerosol showed its relative innocuousness: it did not inhibit the growth and development of young animals, did not induce pathological changes in the upper respiratory tract, kidneys, liver, heart and spleen on its prolonged use. Pathohistological examination revealed slight irritating effect of the gentamicin aerosol in the lungs after its use in a dose of 8 or 25 mg/kg for 6 weeks. A procedure for investigating the effect of the aerosol on the activity of the trachea ciliated epithelium of warm blooded animals was developed. The gentamicin aerosols prepared from solutions of different concentrations (1 to 50 mg/ml) induced ingibition of the ciliated epithelium function at average from 15 to 35 per cent which was associated with the solution acidity (pH 4.54 to 4.82). Such a decrease in the function of the ciliated epithelium due to the antibiotic aerosol use was a factor prolonging the antibiotic retention time in the respiratory organs. It was found that aqueous solutions of drugs used for inhalation, such as ephedrin, euphelin, dimedrol, N-acetyl-L-cystein and others had no effect on the activity of gentamicin and may be used with it in a form of aerosols.     

Tolerance of gentacycol--a local dosage form of gentamicin based on collagen--animal experiments

Gentacycol is a local dosage form of gentamicin based on collagen for implantation to wounds in treatment of patients with infections of soft tissues and prevention of contamination of open injuries of the bones and soft tissues. General toxic and organotropic properties of gentacycol were studied on animals with subcutaneous implantation of the dosage form in doses equivalent to the therapeutic dose for man and exceeding it 2-fold. The study showed that the dosage form had no unfavourable side effects on the animal general state, hearing, the functional state of the liver and kidneys and the peripheral blood. In the doses tested gentacycol did not influence the indices of the cardiovascular system and neuromuscular conduction. Morphological examination of the skin and hypodermic tissues in the implantation site revealed no damaging action of the dosage form on the surrounding tissues.     

Development of a liposome formulation for D-cycloserine local delivery

Multilamellar liposomes loaded with D-cycloserine (D-CS) were prepared by a thin layer evaporation technique, followed by freezing and thawing cycles. Charged components and bioadhesive material, such as distearolylphosphatitylethanolamine covalently coupled with methoxypolyethyleneglycol, were used to prepare liposomes with different physico-chemical and technological properties. Negatively charged liposomes showed higher D-CS encapsulation efficiency (about 37%, w/w) than neutral and positively charged liposomes (about 5 and 17%, w/w, respectively). All formulations showed in vitro, after a burst effect, a prolonged release of the encapsulated drug. Lipid vesicles made of dipalmitoylphosphatidylcholine (DPPC) were used as a biomembrane model to evaluate in vitro the interaction of D-CS with biological membranes. Differential scanning calorimetry was used as a simple and noninvasive technique of analysis. D-CS was distributed in the aqueous compartments of liposomes for interaction with the phospholipid polar head-groups (enhancement of Delta H value). However, due to its high diffusibility the drug was also able to freely permeate through DPPC liposomes, altering during this passage the hydrophobic domains of the bilayers. Stability studies were performed at different temperatures and pH values to assay the integrity of the drug during the liposome production steps. D-CS was rapidly degraded at acidic pH, but no significant hydrolysis was observed at pH 7.4 after 7 days.     

Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease.

Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single-chain recombinant form of the protease has been constructed in which NS4A residues 21-32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3-181) through a tetrapeptide linker. The single-chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size-exclusion chromatography. The single-chain recombinant protease domain shows full proteolytic activity cleaving the NS5A-5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and k(cat) of 20.0 +/- 2.0 microM and 9.6 +/- 2.0 min(-1), respectively; parameters identical to those of the authentic NS3(1-631)/NS4A(1-54) protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392-3401).     

S-nitrosothiols as novel, reversible inhibitors of human rhinovirus 3C protease

Human rhinovirus (HRV) 3C protease was inactivated by a series of S-nitrosothiols. These compounds exhibited different inhibitory activities in a time- and concentration-dependent manner with second-order rate constants (kinact/K(I)) ranging from 131 to 5360 M(-1) min(-1). The inactive enzyme could be re-activated by DTT, GSH and ascorbate, which indicated the inactivation mechanism was through an S-transnitrosylation process.     

Characterisation of a novel murine intestinal serine protease, DISP

A putative novel murine serine protease, DISP, was identified by cDNA indexing and shown to be expressed primarily in distal gut. FISH analysis showed it to be localised to mouse chromosome 17A3. A possible human homologue for DISP has been identified. DISP is a novel member of clan SA/family S1 of the serine proteases, at present of unknown function.     

Efficacy and tolerability of a novel herbal formulation for weight management

Objective:

To evaluate the efficacy of an herbal blend.

Design and Methods:

A randomized, double‐blind, clinical trial in 60 subjects with body mass index (BMI) between 30 and 40 kg/m². Participants were randomized into two groups receiving either 400 mg herbal capsules or 400 mg placebo capsules twice daily. The herbal blend comprises of extracts from Sphaeranthus indicus and Garcinia mangostana. Participants received a standard diet (2,000 kcal per day) and walked 30 min 5 days per week.

Results:

After 8 weeks, significant net reductions in body weight (3.74 kg; P < 0.0001), BMI (1.61 kg/m²; P < 0.0001), and waist circumference (5.44 cm; P < 0.05) were observed in the herbal group compared with placebo. Additionally, a significant increase in serum adiponectin concentration was found in the herbal group versus placebo (P = 0.001). Adverse events were mild and were equally distributed between the two groups. In vitro studies in the 3T3‐L1 adipocyte cell line showed that the herbal extract markedly downregulated the expression of peroxisome proliferator‐activated receptor gamma, adipocyte‐differentiation related protein, and cluster of differentiation 36 but increased adiponectin expression. The herbal extract also reduced the expression and the recruitment of perilipin onto the membrane of lipid droplets.

Conclusion:

Supplementation with the herbal blend resulted in a greater degree of weight loss than placebo over 8 weeks.     

Stability and local toxicity evaluation of a liposomal prilocaine formulation

This study reports a physicochemical stability evaluation of a previously reported liposomal prilocaine (PLC(LUV)) formulation (Cereda et al. J. Pharm. Pharmaceut. Sci. 7:235, 2004) before and after steam sterilization as well as its local toxicity evaluation. Prilocaine (PLC) was encapsulated into extruded unilamellar liposomes (LUVs) composed by egg phosphatidylcholine:cholesterol:alfa-tocopherol (4:3:0.07, mole %). Laser light-scattering analysis (p > 0.05) and thiobarbituric acid reaction (p > 0.05) were used to evaluate the liposomes physical (size) and chemical (oxidation) stability, respectively. The prilocaine chemical stability was followed by (1)H-nuclear magnetic resonance. These tests detected no differences on the physicochemical stability of PLC or PLC(LUV), sterilized or not, up to 30 days after preparation (p > 0.05). Finally, the paw edema test and histological analysis of rat oral mucosa were used to assess the possible inflammatory effects of PLC(LUV). PLC(LUV) did not evoke rat paw edema (p > 0.05), and no significant differences were found in histological analysis, when compared to the control groups (p > 0.05). The present work shows that PLC(LUV) is stable for a 30-day period and did not induce significant inflammatory effects both in the paw edema test and in histological analysis, giving supporting evidence for its safety and possible clinical use in dentistry.     

Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops

Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp. WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region. The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins. However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions. Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution. The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form. After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide. The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 degrees C, and lost activity with a half-life of 30 min at 80 degrees C in the presence of 10 mM CaCl2.     

Basulin,a long-acting formulation of human insulin based on medusa nanoparticles

Conclusion • Basulin? is the only once-a-day human insulin with a proof of efficacy in human clinical trials. • Basulin has demonstrated a good safety and tolerability and confirmed the 24 hours efficacy in Phase IIa during 14 days with 20 patients. • Basulin maintains the full activity of human insulin as a human growth hormone essential to reduce long-term complications associated with the lack of insulin (retinopathy, heart attack, amputation, etc...) compare to other strategies of insulin analogues (insulin glargine, insulin determir). • Basulin? offers all the advantages of the present systems of injection (pen, syringe with 31tw gauge needle). • The insulin market continues to grow significantly. • Basulin? could reach the market in 2009 and is protected by patents until 2024. • Basulin? is available for licensing worldwide.     

Design, synthesis, and conformational analysis of a novel series of HIV protease inhibitors

A combination of structure-based design and both solution, and solid-phase synthesis were utilized to derive a potent (nM) series of HIV-1 protease inhibitors bearing a structurally novel backbone. Detailed structural analysis of several inhibitors prepared in this series has suggested that rigidification of the P₁/P₂ region of this class of molecules may result in compounds with improved potency.     

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35?kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60?°C. It was determined that the enzyme had remained stable at the range of pH 7.0–10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20–80?°C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. K_M and V_max values were calculated as 0.197?mg/mL and 7.29?μmol.mL^?1.min^?1, respectively.     

PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation

Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.     

Characterizing and optimizing protease/peptide inhibitor interactions, a new application for spot synthesis

A new method is presented that uses parallel peptide array synthesis on cellulose membranes to characterize protease/peptide inhibitor interactions. A peptide comprising P5-P4' of the third domain of turkey ovomucoid inhibitor was investigated for both binding to and inhibition of porcine pancreatic elastase. Binding was studied directly on the cellulose membrane, while inhibition was measured by an assay in microtiter plates with punched out peptide spots. The importance of each residue for binding or inhibition was determined by substitutional analyses, exchanging every original amino acid with all other 19 coded amino acids. Seven hundred eighty individual peptides were investigated for binding behavior to porcine pancreatic elastase, and 320 individual peptides were measured in inhibition experiments. The results provide new insights into the interaction between the ovomucoid derived peptide and subsites in the active site of elastase. Combining these data with length analysis we designed new peptides in a step-wise fashion which in the end not only inhibited elastase 400 times more strongly than the original peptide, but are highly specific for the enzyme. In addition, the optimized inhibitor peptide was protected against exopeptidase attack by substituting D-amino acids at both termini.     

Purification, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus

A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the K(m) value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca(2+) binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.     

Inhibition of Candida albicans secreted aspartic protease by a novel series of peptidomimetics,also active on the HIV-1 protease

Nineteen reduced amide, monohydroxy- or dihydroxyethylene-based transition-state peptidomimetics, known to be good inhibitors of the aspartic protease of HIV-1, were tested against a secreted aspartic protease (Sap2), purified from the culture medium of a virulent strain of Candida albicans. Ten of these compounds exhibited IC(50)s against Sap2 lower than 15 microM; the best inhibitor, Kyn-Val-Phe-Psi[OH-OH]-Phe-Val-Kyn, when added to the C. albicans culture, repressed the hydrolysis of bovine serum albumin (BSA), contained in the culture medium, and inhibited the growth of the fungus.