Dry powder inhalation of macromolecules using novel PEG-co-polyester microparticle carriers

This study investigated optimizing the formulation parameters for encapsulation of a model mucinolytic enzyme, α-chymotrypsin (α-CH), within a novel polymer; poly(ethylene glycol)-co-poly(glycerol adipate-co-ω-pentadecalactone), PEG-co-(PGA-co-PDL) which were then applied to the formulation of DNase I. α-CH or DNase I loaded microparticles were prepared via spray drying from double emulsion (w1/o/w2) utilizing chloroform (CHF) as the organic solvent, l-leucine as a dispersibility enhancer and an internal aqueous phase (w1) containing PEG4500 or Pluronic® F-68 (PLF68). α-CH released from microparticles was investigated for bioactivity using the azocasein assay and the mucinolytic activity was assessed utilizing the degradation of mucin suspension assay. The chemical structure of PEG-co-(PGA-co-PDL) was characterized by 1H NMR and FT-IR with both analyses confirming PEG incorporated into the polymer backbone, and any unreacted units removed. Optimum formulation α-CH-CHF/PLF68, 1% produced the highest bioactivity, enzyme encapsulation (20.08 ± 3.91%), loading (22.31 ± 4.34 μg/mg), FPF (fine particle fraction) (37.63 ± 0.97%); FPD (fine particle dose) (179.88 ± 9.43 μg), MMAD (mass median aerodynamic diameter) (2.95 ± 1.61 μm), and the mucinolytic activity was equal to the native non-encapsulated enzyme up to 5 h. DNase I-CHF/PLF68, 1% resulted in enzyme encapsulation (17.44 ± 3.11%), loading (19.31 ± 3.27 μg/mg) and activity (81.9 ± 2.7%). The results indicate PEG-co-(PGA-co-PDL) can be considered as a potential biodegradable polymer carrier for dry powder inhalation of macromolecules for treatment of local pulmonary diseases

Monitoring the Long-Term Molecular Epidemiology of the Pneumococcus and Detection of Potential ‘Vaccine Escape’ Strains

While the pneumococcal protein conjugate vaccines reduce the incidence in invasive pneumococcal disease (IPD), serotype replacement remains a major concern. Thus, serotype-independent protection with vaccines targeting virulence genes, such as PspA, have been pursued. PspA is comprised of diverse clades that arose through recombination. Therefore, multi-locus sequence typing (MLST)-defined clones could conceivably include strains from multiple PspA clades. As a result, a method is needed which can both monitor the long-term epidemiology of the pneumococcus among a large number of isolates, and analyze vaccine-candidate genes, such as pspA, for mutations and recombination events that could result in 'vaccine escape' strains.We developed a resequencing array consisting of five conserved and six variable genes to characterize 72 pneumococcal strains. The phylogenetic analysis of the 11 concatenated genes was performed with the MrBayes program, the single nucleotide polymorphism (SNP) analysis with the DNA Sequence Polymorphism program (DnaSP), and the recombination event analysis with the recombination detection package (RDP).The phylogenetic analysis correlated with MLST, and identified clonal strains with unique PspA clades. The DnaSP analysis correlated with the serotype-specific diversity detected using MLST. Serotypes associated with more than one ST complex had a larger degree of sequence polymorphism than a serotype associated with one ST complex. The RDP analysis confirmed the high frequency of recombination events in the pspA gene.The phylogenetic tree correlated with MLST, and detected multiple PspA clades among clonal strains. The genetic diversity of the strains and the frequency of recombination events in the mosaic gene, pspA were accurately assessed using the DnaSP and RDP programs, respectively. These data provide proof-of-concept that resequencing arrays could play an important role within research and clinical laboratories in both monitoring the molecular epidemiology of the pneumococcus and detecting 'vaccine escape' strains among vaccine-candidate genes

Neurovascular dysfunction in vascular dementia, Alzheimer’s and atherosclerosis

Efficient blood supply to the brain is of paramount importance to its normal functioning and improper blood flow can result in potentially devastating neurological consequences. Cerebral blood flow in response to neural activity is intrinsically regulated by a complex interplay between various cell types within the brain in a relationship termed neurovascular coupling. The breakdown of neurovascular coupling is evident across a wide variety of both neurological and psychiatric disorders including Alzheimer’s disease. Atherosclerosis is a chronic syndrome affecting the integrity and function of major blood vessels including those that supply the brain, and it is therefore hypothesised that atherosclerosis impairs cerebral blood flow and neurovascular coupling leading to cerebrovascular dysfunction. This review will discuss the mechanisms of neurovascular coupling in health and disease and how atherosclerosis can potentially cause cerebrovascular dysfunction that may lead to cognitive decline as well as stroke. Understanding the mechanisms of neurovascular coupling in health and disease may enable us to develop potential therapies to prevent the breakdown of neurovascular coupling in the treatment of vascular brain diseases including vascular dementia, Alzheimer’s disease and stroke

Development and Validation of a RPLC Method for the Determination of 2-Phenoxyethanol in Senselle Lubricant Formulation

A new and simple reversed-phase liquid chromatographic method has been developed and validated for the determination of 2-phenoxyethanol preservative (0.3%, w/w) in senselle lubricant formulation. The separation was achieved with acetonitrile-tetrahydrofuran-water (21:13:66, v/v/v) as mobile phase, a C8 column, and UV detection at 258 nm. The calibration curve is linear (r2= 0.9999) from 20-140% of the analytical concentration of 0.75 mg/ml. The mean percent relative standard deviation values for intra- and inter-day precision studies are <1%. The recovery of 2-phenoxyethanol ranged between 99.76 and 100.03% from lubricant formulation. The limits of detection and quantitation are determined to be 0.094 and 0.15 mg/ml, respectively. The method was found to be robust and can be successfully and reliably used to determine the 2-phenoxyethanol preservative content of marketed formulations

A New Validated HPLC Method for the Simultaneous Determination of 2-phenoxyethanol, Methylparaben, Ethylparaben and Propylparaben in a Pharmaceutical Gel

A novel reversed-phase HPLC method has been developed and validated for the simultaneous determination of 2-phenoxyethanol, methylparaben, ethylparaben and propylparaben preservatives. The method uses a Lichrosorb C8 (150×4.6 mm, 5 µm) column and isocratic elution. The mobile phase consisted of a mixture of acetonitrile, tetrahydrofuran and water (21:13:66, v/v/v), pumped at a flow rate of 1 ml/min. The UV detection was set at 258 nm. The method was validated with respect to accuracy, precision (repeatability and intermediate precision), specificity, linearity and range. All the parameters examined met the current recommendations for bioanalytical method validation. The developed method was successfully applied to the determination of commercially available pharmaceutical gel products for these preservatives. The procedure describes here is simple, selective and reliable for routine quality control analysis and stability tests

Development and Validation of a Reversed-phase HPLC Method for the Determination of Hydroxybenzene in a Cream Formulation

A rapid, sensitive and specific reversed-phase high performance liquid chromatographic method with diode-array detection has been developed and validated for the determination of hydroxybenzene (0.494%, w/w) in a commercially available cream pharmaceutical formulation. Isocratic chromatography was performed on a C18 column with methanol-water 60:40 (v/v) containing 0.1% phosphoric acid (v/v) as mobile phase at a flow rate of 1.0 ml/min. UV detection was at 254 nm. Linearity of the method was excellent (r2 = 0.9999). The relative standard deviation values for intra- and inter-day precision studies were < 1% and the recovery of hydroxybenzene was >99%. The limit of detection and quantitation for hydroxybenzene was found to be 13.5 η g/ml and 2 μg/ml, respectively. The method was also validated for specificity and robustness. The method was found to be robust and can be reliably used to determine the hydroxybenzene content of marketed formulations