Novel antimicrobial peptide specifically active against Porphyromonas gingivalis.

Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles, lipopolysaccharides, hemolysins and proteinases. Antimicrobial peptides (AMPs) including bacteriocins have been found to inhibit the growth of P. gingivalis; however, these peptides are relatively large molecules. Hence, it is difficult to synthesize them by a scale-up production. Therefore, this study aimed to synthesize a shorter AMP that was still active against P. gingivalis. A peptide that contained three cationic amino acids (Arg, His and Lys), two anionic amino acids (Glu and Asp), hydrophobic amino acids residues (Leu, Ile, Val, Ala and Pro) and hydrophilic residues (Ser and Gly) was obtained and named Pep-7. Its bioactivity and stability were tested after various treatments. The mechanism of action of Pep-7 and its toxicity to human red blood cells were investigated. The Pep-7 inhibited two pathogenic P. gingivalis ATCC 33277 and P. gingivalis ATCC 53978 (wp50) strains at a minimum bactericidal concentration (MBC) of 1.7 µM, but was ineffective against other oral microorganisms (P. intermedia, Tannerella forsythensis, Streptococcus salivarius and Streptococcus sanguinis). From transmission electron microscopy studies, Pep-7 caused pore formation at the poles of the cytoplasmic membranes of P. gingivalis. A concentration of Pep-7 at four times that of its MBC induced some hemolysis but only at 0.3%. The Pep-7 was heat stable under pressure (autoclave at 110 and 121 °C) and possessed activity over a pH range of 6.8-8.5. It was not toxic to periodontal cells over a range of 70.8-4.4 µM and did not induce toxic pro-inflammatory cytokines. The Pep-7 showed selective activity against Porphyromonas sp. by altering the permeability barriers of P. gingivalis. The Pep-7 was not mutagenic in vitro. This work highlighted the potential for the use of this synthetic Pep-7 against P. gingivalis.

Further characterization of a bacteriocin produced by Lactobacillus paracasei HL32.

AIMS: Purification, identification and partial characterization of bacteriocin produced by Lactobacillus paracasei HL32. It has been shown to have activity against Porphyromonas sp. METHODS AND RESULTS: The purification of bacteriocin consisting of gel exclusion followed by anion exchange chromatography produced a single band upon an electrophoresis gel with a molecular weight corresponding to 56 kDa. The isolated protein contained 171 amino acids and the first 151 were sequenced. The bacteriocin contained a high percentage of cationic amino acids near the N-terminus, hydrophobic amino acids in the central region (Leu, Ile, Val, Phe, Trp and Gly) and hydrophilic residues (Ser, Asn and Gln) at the C-terminus. This structure did not match with that of previously reported bacteriocins. The antimicrobial activity of the bacteriocin was determined against some pathogens and normal microbiota (P. gingivalis, P. intermedia, T. forsythensis, S. salivarius and S. sanguinis) found in saliva and crevicular fluid. The bacteriocin was found to inhibit P. gingivalis at the minimum bactericidal concentration (MBC) of 0.14 mmol l(-1), but was found not to inhibit the other oral micro-organisms. The bacteriocin was found from transmission electron microscopy studies to cause pore formation in the cytoplasmic membranes of P. gingivalis at the pole and induce potassium efflux. Bacteriocin concentrations of two to four times of MBC were shown to induce haemolysis. The bacteriocin was heat-stable, surviving at 110 degrees C under pressure and possessed activity over a pH range of 6.8-8.5. Only a small reduction of activity was found to occur after incubation in biological fluids (saliva and crevicular fluid). CONCLUSIONS: A novel bacteriocin has been identified that has selective activity against Porphyromonas sp. associated with periodontal disease. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work gained the knowledge of specific antibacterial activity of bacteriocin against Porphyromonas gingivalis.

Application of spectrofluorometry for evaluation of dry powder inhalers in vitro.

Andersen cascade impactor (ACI) is commonly used for the testing of pharmaceutical aerosols, which has to be coupled with an instrument for quantitative analysis of drug depositing on each stage of the ACI. This procedure consumes much time in operation. Therefore, this study was aimed at speeding up the process of drug analysis in aerosol formulations after obtaining samples from the ACI. From the results obtained, it was proved that the validated spectrofluorometric method was accurate and sensitive. It was capable of giving similar results to those we obtained from HPLC-UV analysis. There was no interference from the amount of lactose carrier incorporated in the formulation in the step of salbutamol analysis indicating specificity of the method. As a result, samples were analyzed without further separation. The detection limit was 0.1 microg/ml. Hence, spectrofluorometry can be used as a substitute method to HPLC-UV in determining the small quantity of salbutamol after aerosolization from dry powder aerosols. The present study suggests that spectrofluometry can be a rapid and efficient method in the pharmaceutical analysis of aerosols.

Stereoselective release behaviors of imprinted bead matrices.

In this work, the stereoselective release behaviors of "low"-swelling molecularly imprinted polymer (MIP) bead matrices in pressed-coat tablet type were studied. Either R-propranolol selective MIP or S-propranolol selective MIP was combined with excipients and racemic propranolol and fabricated into the matrix. Subsequently, the release of different propranolol enantiomers from the matrices was examined. Also, the microscopic structure of the hydrated "low"-swelling MIP matrix was determined using a cryogenic scanning electron microscope in order to compare with that of the hydrated "high"-swelling MIP matrix. In vitro release profiles of the "low"-swelling matrices showed a difference in the release of enantiomers, in that the non-template isomer was released faster than the template isomer. However, in the last phase of dissolution this difference reduced and later reversed, resulting at last in the type of specificity being similar to that obtained previously with "high"-swelling MIP matrices. In summary, MIP beads can be fashioned into matrices and incorporated into different formulations to regulate the resultant stereoselectivity. From the behaviors of stereoselective release observed in MIP matrices, we can conclude that the enantioselective-controlled delivery mechanism of MIPs via formulations depends on the relative affinity of the enantiomer for the template sites, as well as the nature of the polymer, such as hydrophobicity and swellability.

Enantioselective release of controlled delivery granules based on molecularly imprinted polymers.

The objectives of our study were two fold: to examine enantioselective release of controlled delivery granules based on molecularly imprinted polymers (MIPs) for various racemic drugs, including ibuprofen and ketoprofen (NSAIDs) and propranolol (beta-blockers); to evaluate the use of controlled delivery granules containing a combination of different MIPs for the multiple simultaneous enantioselective-controlled delivery of mixed racemic drugs. In this work, the MIP beads selective to S-Ibuprofen, S-ketoprofen, and R-propranolol were prepared using multistep swelling and thermal polymerization method. Afterward, the MIP beads were formulated with racemate of the chiral drugs and a binder and followed by granulation. Then, the enantioselective release of racemic drugs from the prepared MIP granules was investigated by an in vitro dissolution test using a chiral HPLC for assays of enantiomers. The influence of drug/polymer ratio and medium pH on the selective enantiomeric release of MIP granules was explored. Further, the release of the enantiomers of racemic ibuprofen and racemic ketoprofen from the granule containing two MIPs - S-ibuprofen MIP and S-ketoprofen MIP - was examined. The release profiles of both S-ibuprofen MIP granule and R-propranolol MIP granule exhibited differential release of enantiomers. Also, the findings indicated the stereoselective retardation of those controlled delivery granules as well as the influence of MIP formulation on enantioselective release mechanism. The enantioselective release of S-ibuprofen MIP granule and R-propranolol MIP granule appeared to depend on polymer loading and medium pH. In this case, the drug/polymer ratio of 1:25 showed the best enantioselective release with initial enantiomeric excess of 100%. On the other hand, the enantioselectivity of both granules was the greatest in buffer pH 7.4. Furthermore, the efficiency in enantioselective release of the combined MIP granule was higher than its relative single MIP granules, as a result of the cross-reactivities of the MIPs. In our study, controlled delivery granules based on MIPs demonstrated significant enantioselective release for several chiral drugs, and thus it may be developed as a tool to administer chiral pharmaceutical as a single enantiomer.

Evaluation of stereoselective dissolution of racemic salbutamol matrices prepared with commonly used excipients and 1H-NMR study.

The purpose of this work was to examine the in vitro enantioselective dissolution of salbutamol from matrix tablets containing various chiral excipients, such as gamma cyclodextrin (gamma-CD), heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), sulfobutyl-beta-cyclodextrin (SBE-beta-CD), hydroxypropylmethylcellulose (HPMC), and egg albumin. In this study, two types of tablets were prepared; the coated tablet contained the complex of racemic salbutamol and cyclodextrin (gamma-CD, DM-beta-CD, and SBE-beta-CD), and the uncoated tablet was composed of the drug with either HPMC or egg albumin. Subsequently, these formulations were evaluated for enantioselective release. The results revealed that the formulations containing either SBE-beta-CD, HPMC, or egg albumin had no enantioselective release, while the formulation with DM-beta-CD gave slightly different release of the two enantiomers at the end of the dissolution profile. The formulation containing gamma-CD provided significant stereoselectivity throughout the dissolution profile. The release of the eutomer R-salbutamol was higher than that of the distomer S-salbutamol from the gamma-CD tablet. In addition, the enantioselective interaction for the gamma-CD inclusion complex was investigated by 1H-NMR (nuclear magnetic resonance) spectroscopy and gave evidence to support the enantioselectivity obtained on dissolution.

Cyclodextrin as a potential drug carrier in salbutamol dry powder aerosols: the in-vitro deposition and toxicity studies of the complexes.

This research was carried out to develop a new carrier in dry powder aerosols. Two types of cyclodextrin were chosen; gamma cyclodextrin (GCD) and dimethyl-beta-cyclodextrin (DMCD) as carriers in dry powder formulations. Salbutamol was used as a model drug and a control formulation containing lactose and the drug was included. A twin-stage impinger (TSI) was used to evaluate in delivery efficiency of those dry powder formulations. The toxicity of cyclodextrin complexes was investigated in the rat by monitoring blood urea nitrogen (BUN) and urinary creatinine, as well as determining haemolysis of human red blood cells. The release of salbutamol from dry powder formulations was also studied over a period of time. From the results obtained, it was found that the formulation containing GCD-enhanced drug delivery to the lower stage of the TSI (deposition = 65%) much greater than that of both formulations containing DMCD (50%) and the control formulation (40%) (P<0.05). After injecting the GCD complex BUN and creatinine levels in rats were similar to those obtained in the control while those receiving DMCD complex had higher BUN and creatinine. The haemolysis of red blood cells incubated with the DMCD complex was higher than that obtained in the GCD complex. The drug release in both formulations containing GCD and DMCD was fast (over 70% was released in 5 min) and nearly all the drug was released within 30 min. It can be concluded that GCD and DMCD are able to promote salbutamol delivery in dry powder inhaler compared to a formulation containing lactose. In addition, GCD is relatively safe in the rat if the amount of GCD in the formulation is similar to this experiment.

A human oral-throat cast integrated with a twin-stage impinger for evaluation of dry powder inhalers.

The aim of this study was to investigate the applicability of replacing the glass throat from a twin-stage impinger (TSI) with a human oral-throat cast. Monodisperse aerosols were used to calibrate the human oral cast-TSI at 60 L min(-1) and cut-off in particle size was compared with that of the TSI described in the British Pharmacopoeia which employs a glass throat. The amount of salbutamol sulphate (and lactose) delivered by the Cyclohaler depositing on various elements of the in-vitro model were determined. The calibration of the model containing a human oral-throat cast at 60 L min(-1) gave a particle size cut-off of 5.2 microm which was less than that of the TSI (6.3 microm). The oral-throat cast trapped more drug than the glass throat model with a formulation that employed the larger carrier (63-90 microm; P<0.05) while it trapped a lesser amount of drug with those filled with the lower size carrier (Lactochem, micronised lactose). The greater amount of lactose in the formulation that employed the larger-sized carrier (63-90 microm) was deposited closer to the inlet of the oral-throat cast than to the inlet of the glass throat model. Replacement of the glass throat in the TSI by the human oral-throat cast, leads to a change in deposition efficiency, with the cast having a higher filter efficiency and hence more aerosol particles being captured before their entry into the TSI. This should be investigated further to determine whether such a model might provide a more realistic assessment of the in-vivo characteristics of an aerosol in comparison with the TSI currently being employed, which utilises the glass throat as the portal of entry.

Evaluation of matrices containing molecularly imprinted polymers in the enantioselective-controlled delivery of beta-blockers.

Granules and beads of methacrylic acid (MAA) and granules of N-acryloyl-alanine polymer (NAA) were produced using ethylene glycol dimethacrylate as cross-linking monomer either by bulk (in the case of granules) or suspension (in the case of beads) polymerization. Either R- or S-propranolol, were used as an imprint molecule, acting as a template, with a view to conferring enantioselectivity of release upon the polymer. The molecularly imprinted polymers (MIPs) or nonMIPs (control) were formulated with racemic propranolol and other excipients and compressed to form matrix tablets. Enantioselective release of propranolol in vitro was monitored using a stereoselective HPLC assay. The influence of the method of polymer synthesis, drug: polymer ratio, pH and temperature on the release of the two enantiomers was determined. Stereoselectivity of release was identified in tablets containing either MAA or NAA granules or MAA beads, with the latter showing the greatest differences between enantiomers. Release of the enantiomer used as the print was always faster than the release of the nonprint enantiomer. In the case of S-propranolol-MIP bead matrices composed of MAA, greater differences in the release of enantiomers could be promoted by increasing the polymer: drug ratio of the tablet. Differences in the release rate of the two propranolol enantiomers was still apparent as the pH was varied between 3 and 7.4 and when the temperature was decreased from 37 to 25 degrees C. S-Propranolol-MIP bead matrices demonstrated cross-reactivities of stereoselective dissolution for enantiomers of pindolol and oxprenolol, both of which have structural similarities to the imprint molecule. It is concluded that polymers of this type may have great potential in controlling, via means of formulation, the release of drug eutomer whilst enhancing retention of distomer in the dosage form.

A study of drug-carrier interactions in dry powder inhaler formulations using the Andersen cascade impactor, X-ray microanalysis and time of flight aerosol beam spectrometry (TOFABS).

The purpose of this study was to determine the in vitro deposition of both drug (albuterol sulfate) and carrier (lactose) particles in relation to each other from a dry powder inhaler formulation using an Andersen cascade impactor (ACI) and time of flight aerosol beam spectrometry (TOFABS). In addition, scanning electron microscopy (SEM) combined with X-ray microanalysis was employed to distinguish albuterol sulfate from lactose. Drug particles apparently penetrated deeper into the impactor than lactose particles contained in the formulation. In some certain stages of impactor, drug particles were separated from lactose particles. Although the TOFABS cannot distinguish between albuterol sulfate and lactose, the TOF spectra obtained from the Aerosizer would appear to be partly indicative of the interactions which exist between drug and carrier. One symmetrical TOF peak was obtained from drug or lactose alone. The TOF peak of the drug was always lower than the TOF of lactose. The times obtained for each powder between experiments were highly reproducible and typical of material and particle size. The use of SEM-X-ray microanalysis also allowed some qualitative characterization of shape and state of association of the two components.

Chiral determination of various adrenergic drugs by thin-layer chromatography using molecularly imprinted chiral stationary phases prepared with alpha-agonists.

Thin-layer chromatography (TLC) based on molecularly imprinted polymers (MIPs) of alpha-agonists as chiral stationary phases was applied to the determination of enantiomers of various adrenergic drugs including alpha- and beta-agonists and beta-antagonists (beta-blockers). In this study, three MIPs imprinted with (+)-ephedrine, (+)-pseudoephedrine and (+)-norephedrine plus a non-imprinted polymer (non-MIP) were prepared, processed and coated on a glass support as thin layers. then enantiomeric determination of adrenergic drugs was carried out by development of their racemates on the TLC plates, using established conditions. From the results, the racemates of the compounds used as print molecules were well separated into two isomers on the MIP-plates, except on the plate based on MIP of (+)-norephedrine. Most adrenergic drugs structurally related to print molecules were completely resolved into two spots with the MIP plates. In general the retention of (+)-isomers (or 1S-isomers) was greater than that of (-)-isomers (or 1R-isomers), indicating the stereoselectivity of the MIPs with the former isomers. Moreover, the role between the chemical structures of the analytes with chiral recognition of the MIPs has been investigated. The proposed method enables rapid determination of enantiomers and screening of large numbers for optical purity of adrenergic drugs.

Dry powder inhalers: the influence of device resistance and powder formulation on drug and lactose deposition in vitro.

It is a principal in the formulation of a dry powder aerosol that the device should enable a high fine particle fraction (FPF) of drug to be delivered to the lung whilst any carrier, such as lactose, should remain in the upper airways. Both the device and the dry powder formulation itself contribute to the resultant FPF and few studies have considered the deposition of lactose carrier. It was the purpose of this study to determine the effect of the resistance of the device and the influence of powder formulation on the deposition of drug and carrier. Measurement of the pressure drop across the devices investigated in this study showed that the two types of Inhalator Ingelheim had the highest resistance, whilst lower pressure drops were found across the Diskhaler, Cyclohaler and Accuhaler devices. The lowest pressure drops were measured across the Rotahaler and Spinhaler devices. Employing Rotacaps 400 capsules as the formulated salbutamol product, the FPF of drug was greater from the high resistance devices, being in the order Inhalators Ingelheim>Cyclohaler>Rotahaler=Spinhaler. However, the Diskhaler, employing its own developed formulation, produced the highest FPF, approximately twice that from the Accuhaler. There was no statistical difference between the FPF of salbutamol (approximately 20% nominal dose) from the Rotacaps formulation when aerosolised using high resistance devices (Inhalators Ingelheim) operated at 30 l min-1, a medium resistance device (Cyclohaler) operated at 60 l min-1 and low resistance devices (Spinhaler and Rotahaler) operated at a flow-rate of 90 l min-1. The Ventolin Diskhaler using its own formulation operated at 60 l min-1 gave a FPF of 40.33%, but the FPF obtained was sensitive to flow, being only 25.65% of the nominal dose at 30 l min-1. Whereas no lactose was found in the FPF from the Accuhaler operated at 60 l min-1, 100, 400 and 3500 microg were obtained from the Diskhaler, Rotacaps and micronised lactose formulation, respectively, when operated at the same flow-rate. An in-house formulation comprising salbutamol sulphate blended with micronised lactose in a weight ratio of 1:67.5 and aerosolised from a Cyclohaler produced a similar FPF to the Diskhaler at 60 l min-1. When air flow was reduced to 30 l min-1, the FPF from the in-house formulation was reduced considerably less than that from the Diskhaler formulation.