Antimicrobial efficacy of tobramycin polymeric nanoparticles for Pseudomonas aeruginosa infections in cystic fibrosis: formulation, characterisation and functionalisation with dornase alfa (DNase).

Inhaled antibiotics, such as tobramycin, for the treatment of Pseudomonas aeruginosa pulmonary infections are associated with the increase in life expectancy seen in cystic fibrosis (CF) patients over recent years. However, the effectiveness of this aminoglycoside is still limited by its inability to penetrate the thick DNA-rich mucus in the lungs of these patients, leading to low antibiotic exposure to resident bacteria. In this study, we created novel polymeric nanoparticle (NP) delivery vehicles for tobramycin. Using isothermal titration calorimetry, we showed that tobramycin binds with alginate polymer and, by exploiting this interaction, optimised the production of tobramycin alginate/chitosan NPs. It was established that NP antimicrobial activity against P. aeruginosa PA01 was equivalent to unencapsulated tobramycin (minimum inhibitory concentration 0.625 mg/L). Galleria mellonella was employed as an in vivo model for P. aeruginosa infection. Survival rates of 90% were observed following injection of NPs, inferring low NP toxicity. After infection with P. aeruginosa, we showed that a lethal inoculum was effectively cleared by tobramycin NPs in a dose dependent manner. Crucially, a treatment with NPs prior to infection provided a longer window of antibiotic protection, doubling survival rates from 40% with free tobramycin to 80% with NP treatment. Tobramycin NPs were then functionalised with dornase alfa (recombinant human deoxyribonuclease I, DNase), demonstrating DNA degradation and improved NP penetration of CF sputum. Following incubation with CF sputum, tobramycin NPs both with and without DNase functionalisation, exhibited anti-pseudomonal effects. Overall, this work demonstrates the production of effective antimicrobial NPs, which may have clinical utility as mucus-penetrating tobramycin delivery vehicles, combining two widely used CF therapeutics into a single NP formulation. This nano-antibiotic represents a strategy to overcome the mucus barrier, increase local drug concentrations, avoid systemic adverse effects and improve outcomes for pulmonary infections in CF.

Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial

Background: Ataluren was developed to restore functional protein production in genetic disorders caused by nonsense mutations, which are the cause of cystic fibrosis in 10% of patients. This trial was designed to assess the efficacy and safety of ataluren in patients with nonsense-mutation cystic fibrosis. 

Methods: This randomised, double-blind, placebo-controlled, phase 3 study enrolled patients from 36 sites in 11 countries in North America and Europe. Eligible patients with nonsense-mutation cystic fibrosis (aged ≥6 years; abnormal nasal potential difference; sweat chloride >40 mmol/L; forced expiratory volume in 1 s [FEV₁] ≥40% and ≤90%) were randomly assigned by interactive response technology to receive oral ataluren (10 mg/kg in morning, 10 mg/kg midday, and 20 mg/kg in evening) or matching placebo for 48 weeks. Randomisation used a block size of four, stratified by age, chronic inhaled antibiotic use, and percent-predicted FEV₁. The primary endpoint was relative change in percent-predicted FEV₁ from baseline to week 48, analysed in all patients with a post-baseline spirometry measurement. This study is registered with ClinicalTrials.gov, number NCT00803205. 

Findings: Between Sept 8, 2009, and Nov 30, 2010, 238 patients were randomly assigned, of whom 116 in each treatment group had a valid post-baseline spirometry measurement. Relative change from baseline in percent-predicted FEV₁ did not differ significantly between ataluren and placebo at week 48 (-2·5% vs -5·5%; difference 3·0% [95% CI -0·8 to 6·3]; p=0·12). The number of pulmonary exacerbations did not differ significantly between treatment groups (rate ratio 0·77 [95% CI 0·57-1·05]; p=0·0992). However, post-hoc analysis of the subgroup of patients not using chronic inhaled tobramycin showed a 5·7% difference (95% CI 1·5-10·1) in relative change from baseline in percent-predicted FEV₁ between the ataluren and placebo groups at week 48 (-0·7% [-4·0 to 2·1] vs -6·4% [-9·8 to -3·7]; nominal p=0·0082), and fewer pulmonary exacerbations in the ataluern group (1·42 events [0·9-1·9] vs 2·18 events [1·6-2·7]; rate ratio 0·60 [0·42-0·86]; nominal p=0·0061). Safety profiles were generally similar for ataluren and placebo, except for the occurrence of increased creatinine concentrations (ie, acute kidney injury), which occurred in 18 (15%) of 118 patients in the ataluren group compared with one (<1%) of 120 patients in the placebo group. No life-threatening adverse events or deaths were reported in either group. I

nterpretation: Although ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients who received this treatment, it might be beneficial for patients not taking chronic inhaled tobramycin. 

Funding: PTC Therapeutics, Cystic Fibrosis Foundation, US Food and Drug Administration's Office of Orphan Products Development, and the National Institutes of Health. 

Effectiveness of a Multifaceted Intervention for Potentially Inappropriate Prescribing in Older Patients in Primary Care: A Cluster-Randomized Controlled Trial (OPTI-SCRIPT Study)

PURPOSE Potentially inappropriate prescribing (PIP) is common in older people and can result in increased morbidity, adverse drug events, and hospitalizations. The OPTI-SCRIPT study (Optimizing Prescribing for Older People in Primary Care, a cluster-randomized controlled trial) tested the effectiveness of a multifaceted intervention for reducing PIP in primary care.

METHODS We conducted a cluster-randomized controlled trial among 21 general practitioner practices and 196 patients with PIP. Intervention participants received a complex, multifaceted intervention incorporating academic detailing; review of medicines with web-based pharmaceutical treatment algorithms that provide recommended alternative-treatment options; and tailored patient information leaflets. Control practices delivered usual care and received simple, patient-level PIP feedback. Primary outcomes were the proportion of patients with PIP and the mean number of potentially inappropriate prescriptions. We performed intention-to-treat analysis using random-effects regression.

RESULTS All 21 practices and 190 patients were followed. At intervention completion, patients in the intervention group had significantly lower odds of having PIP than patients in the control group (adjusted odds ratio = 0.32; 95% CI, 0.15–0.70; P = .02). The mean number of PIP drugs in the intervention group was 0.70, compared with 1.18 in the control group (P = .02). The intervention group was almost one-third less likely than the control group to have PIP drugs at intervention completion, but this difference was not significant (incidence rate ratio = 0.71; 95% CI, 0.50–1.02; P = .49). The intervention was effective in reducing proton pump inhibitor prescribing (adjusted odds ratio = 0.30; 95% CI, 0.14–0.68; P = .04).

CONCLUSIONS The OPTI-SCRIPT intervention incorporating academic detailing with a pharmacist, and a review of medicines with web-based pharmaceutical treatment algorithms, was effective in reducing PIP, particularly in modifying prescribing of proton pump inhibitors, the most commonly occurring PIP drugs nationally.

Design and physicochemical characterisation of novel dissolving polymeric microneedle arrays for transdermal delivery of high dose, low molecular weight drugs

We describe formulation and evaluation of novel dissolving polymeric microneedle (MN) arrays for the facilitated delivery of low molecular weight, high dose drugs. Ibuprofen sodium was used as the model here and was successfully formulated at approximately 50% w/w in the dry state using the copolymer poly(methylvinylether/maleic acid). These MNs were robust and effectively penetrated skin in vitro, dissolving rapidly to deliver the incorporated drug. The delivery of 1.5mg ibuprofen sodium, the theoretical mass of ibuprofen sodium contained within the dry MN alone, was vastly exceeded, indicating extensive delivery of the drug loaded into the baseplates. Indeed in in vitro transdermal delivery studies, approximately 33mg (90%) of the drug initially loaded into the arrays was delivered over 24h. Iontophoresis produced no meaningful increase in delivery. Biocompatibility studies and in vivo rat skin tolerance experiments raised no concerns. The blood plasma ibuprofen sodium concentrations achieved in rats (263μgml(-1) at the 24h time point) were approximately 20 times greater than the human therapeutic plasma level. By simplistic extrapolation of average weights from rats to humans, a MN patch design of no greater than 10cm(2) could cautiously be estimated to deliver therapeutically-relevant concentrations of ibuprofen sodium in humans. This work, therefore, represents a significant progression in exploitation of MN for successful transdermal delivery of a much wider range of drugs.

Irradiation of bioresorbable biomaterials for controlled surface degradation

Bioresorbable polymers increasingly are the materials of choice for implantable orthopaedic fixation devices. Controlled degradation of these polymers is vital for preservation of mechanical properties during tissue repair and controlled release of incorporated agents such as osteoconductive or anti-microbial additives. The work outlined in this paper investigates the use of low energy electron beam irradiation to surface modify polyhydroxyacid samples incorporating beta tricalcium phosphate (β-TCP). This work uniquely demonstrates that surface modification of bioresorbable polymers through electron beam irradiation allows for the early release of incorporated agents such as bioactive additives. Samples were e-beam irradiated at an energy of 125 keV and doses of either 150 kGy or 500 kGy. Irradiated and non-irradiated samples were degraded in phosphate buffered saline (PBS), to simulate bioresorption, followed by characterisation. The results show that low energy e-beam irradiation enhances surface hydrolytic degradation in comparison to bulk and furthermore allows for earlier release of incorporated calcium via dissolution into the surrounding medium.

Pan-retinal photocoagulation and other forms of laser treatment and drug therapies for non-proliferative diabetic retinopathy: systematic review and economic evaluation

BACKGROUND: Diabetic retinopathy is an important cause of visual loss. Laser photocoagulation preserves vision in diabetic retinopathy but is currently used at the stage of proliferative diabetic retinopathy (PDR).

OBJECTIVES: The primary aim was to assess the clinical effectiveness and cost-effectiveness of pan-retinal photocoagulation (PRP) given at the non-proliferative stage of diabetic retinopathy (NPDR) compared with waiting until the high-risk PDR (HR-PDR) stage was reached. There have been recent advances in laser photocoagulation techniques, and in the use of laser treatments combined with anti-vascular endothelial growth factor (VEGF) drugs or injected steroids. Our secondary questions were: (1) If PRP were to be used in NPDR, which form of laser treatment should be used? and (2) Is adjuvant therapy with intravitreal drugs clinically effective and cost-effective in PRP?

ELIGIBILITY CRITERIA: Randomised controlled trials (RCTs) for efficacy but other designs also used.

REVIEW METHODS: Systematic review and economic modelling.

RESULTS: The Early Treatment Diabetic Retinopathy Study (ETDRS), published in 1991, was the only trial designed to determine the best time to initiate PRP. It randomised one eye of 3711 patients with mild-to-severe NPDR or early PDR to early photocoagulation, and the other to deferral of PRP until HR-PDR developed. The risk of severe visual loss after 5 years for eyes assigned to PRP for NPDR or early PDR compared with deferral of PRP was reduced by 23% (relative risk 0.77, 99% confidence interval 0.56 to 1.06). However, the ETDRS did not provide results separately for NPDR and early PDR. In economic modelling, the base case found that early PRP could be more effective and less costly than deferred PRP. Sensitivity analyses gave similar results, with early PRP continuing to dominate or having low incremental cost-effectiveness ratio. However, there are substantial uncertainties. For our secondary aims we found 12 trials of lasers in DR, with 982 patients in total, ranging from 40 to 150. Most were in PDR but five included some patients with severe NPDR. Three compared multi-spot pattern lasers against argon laser. RCTs comparing laser applied in a lighter manner (less-intensive burns) with conventional methods (more intense burns) reported little difference in efficacy but fewer adverse effects. One RCT suggested that selective laser treatment targeting only ischaemic areas was effective. Observational studies showed that the most important adverse effect of PRP was macular oedema (MO), which can cause visual impairment, usually temporary. Ten trials of laser and anti-VEGF or steroid drug combinations were consistent in reporting a reduction in risk of PRP-induced MO.

LIMITATION: The current evidence is insufficient to recommend PRP for severe NPDR.

CONCLUSIONS: There is, as yet, no convincing evidence that modern laser systems are more effective than the argon laser used in ETDRS, but they appear to have fewer adverse effects. We recommend a trial of PRP for severe NPDR and early PDR compared with deferring PRP till the HR-PDR stage. The trial would use modern laser technologies, and investigate the value adjuvant prophylactic anti-VEGF or steroid drugs.

STUDY REGISTRATION: This study is registered as PROSPERO CRD42013005408.

FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Development of polymeric–cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid–polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA.

Drug induced gingival overgrowth: A role for Protease Activated Receptors?

Background: Protease activated receptors (PAR) belong to a subfamily of G protein coupled receptors. They consist of seven transmembrane domains but are not classical receptors as their agonist is a circulating serine proteinase. This proteinase cleaves an N-terminal extracellular domain of the receptor to reveal a new N-terminal tethered ligand which binds intramolecularly, thus converting an extracellular proteolytic event into a transmembrane signal. Therefore, the cleavage and activation of PARs provide a mechanism whereby proteinases can directly influence the inflammatory response. Gingival hyperplasia or gingival enlargement is a side effect of some drugs such as cyclosporine, a potent immunosuppressant. To date, the potential role of PAR in the inflammation associated with the pathogenesis of gingival overgrowth has not been studied. Objectives: The present study was designed to determine whether proteinases derived from extracts of cyclosporine induced hyperplasia were capable of activating PAR in vitro. Methods: Cell lysates were derived from tissue obtained from gingival overgrowth of patients requiring surgical excision. Cell lines over-expressing PARs were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% foetal calf serum (FCS) in 5% CO2. The cells were treated with gingival overgrowth lysates and agonist stimulated calcium release from the cells was recorded using the Fluo-4-Direct™ Calcium Assay Kit from Invitrogen, according to manufacturer's instructions. Results: Calcium release by activated PAR on tumour cells was detected in those treated with gingival hyperplasia lysates. Samples from healthy gingival fibroblasts did not elicit this response. Conclusions: The identification of mediators of the molecular events central to the inflammatory phenotype elicited by gingival hyperplasia is important. To this end, our experiments show that in vitro, enzymes derived from overgrown gingival tissue are capable of activating PAR and thereby provide evidence for the potential role of PAR in sustaining gingival hyperplasia.

Sustained release of the candidate antiretroviral peptides T-1249 and JNJ54310516-AFP from a rod insert vaginal ring

Administration of biomacromolecular drugs in effective quantities from conventional vaginal rings is hampered by poor drug permeability in the polymers from which rings are commonly constructed. Here, we report the formulation development and testing of rod insert rings for sustained release of the candidate antiretroviral peptides T-1249 and JNJ54310516-AFP (JNJ peptide), both of which have potential as HIV microbicides. Rod inserts were prepared comprising antiviral peptides T-1249 or JNJ peptide in combination with a hydrophilic excipient (sodium chloride, sodium glutamate, lactose or zinc acetate) dispersed at different loadings within a medical grade silicone elastomer. The inserts were tested for weight change and swelling when immersed in simulated vaginal fluid (SVF). Dye migration into the inserts was also assessed visually over 28 days. In vitro release of T-1249 and JNJ peptide from rings containing various insert types was tested. Weight change and degree of swelling of rods immersed in SVF was dependent on the type and concentration of excipient present. The rods displayed the following rank order in terms of weight change: sodium glutamate > zinc acetate ≈ sodium chloride > lactose. The weight change and degree of swelling of the inserts did not correlate with the level of dye uptake observed. In vitro release of T-1249 was improved through addition of lactose, sodium chloride and sodium glutamate, while release of JNJ peptide was improved through addition of sodium chloride or sodium glutamate. Sustained release of hydrophobic peptides can be achieved using a rod insert ring design formulated to include a hydrophilic excipient. Release rates were dependent upon the type of excipient used. The degree of release improvement with different inserts partially reflects their ability to imbibe surrounding fluid and swell in aqueous environments.

Microbicide Vaginal Rings: Technological Challenges and Clinical Development

Vaginal rings (VRs) are flexible, torus-shaped, polymeric devices designed to sustain delivery of pharmaceutical drugs to the vagina for clinical benefit. Following first report in a 1970 patent application, several steroid-releasing VR products have since been marketed for use in hormone replacement therapy and contraception. Since 2002, there has been growing interest in the use of VR technology for delivery of drugs that can reduce the risk of sexual acquisition of human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). Although no vaginally-administered product has yet been approved for HIV reduction/prevention, extensive research efforts are continuing and a number of VR devices offering sustained release of so-called ‘HIV microbicide’ compounds are currently being evaluated in late-stage clinical studies. This review article provides an overview of the published scientific literature within this important field of research, focusing primarily on articles published within peer-reviewed journal publications. Many important aspects of microbicide-releasing VR technology are discussed, with a particular emphasis on the technological, manufacturing and clinical challenges that have emerged in recent years.

Biomarker responses to folic acid intervention in healthy adults: a meta-analysis of randomized controlled trials

BACKGROUND: The task of revising dietary folate recommendations for optimal health is complicated by a lack of data quantifying the biomarker response that reliably reflects a given folate intake.

OBJECTIVE: We conducted a dose-response meta-analysis in healthy adults to quantify the typical response of recognized folate biomarkers to a change in folic acid intake.

DESIGN: Electronic and bibliographic searches identified 19 randomized controlled trials that supplemented with folic acid and measured folate biomarkers before and after the intervention in apparently healthy adults aged ≥18 y. For each biomarker response, the regression coefficient (β) for individual studies and the overall pooled β were calculated by using random-effects meta-analysis.

RESULTS: Folate biomarkers (serum/plasma and red blood cell folate) increased in response to folic acid in a dose-response manner only up to an intake of 400 μg/d. Calculation of the overall pooled β for studies in the range of 50 to 400 μg/d indicated that a doubling of folic acid intake resulted in an increase in serum/plasma folate by 63% (71% for microbiological assay; 61% for nonmicrobiological assay) and red blood cell folate by 31% (irrespective of whether microbiological or other assay was used). Studies that used the microbiological assay indicated lower heterogeneity compared with studies using nonmicrobiological assays for determining serum/plasma (I(2) = 13.5% compared with I(2) = 77.2%) and red blood cell (I(2) = 45.9% compared with I(2) = 70.2%) folate.

CONCLUSIONS: Studies administering >400 μg folic acid/d show no dose-response relation and thus will not yield meaningful results for consideration when generating dietary folate recommendations. The calculated folate biomarker response to a given folic acid intake may be more robust with the use of a microbiological assay rather than alternative methods for blood folate measurement.

Effects of Prochlorperazine on Normal Vestibular Ocular and Perceptual Responses: A Randomised, Double-Blind, Crossover, Placebo-Controlled Study

Background: The present study investigated whether prochlorperazine affects vestibular-ocular reflex (VOR) and vestibulo-perceptual function. Methods: We studied 12 healthy naïve subjects 3 hours after a single dose of oral prochlorperazine 5mg in a randomised, placebo-controlled, double-blind, cross-over study in healthy young subjects. Two rotational tests in yaw were used: 1) a Threshold task investigating perceptual motion detection and nystagmic thresholds (acceleration steps of 0.5deg/s/s) and 2) Suprathreshold responses to velocity steps of 90deg/s in which vestibulo-ocular (VO) and vestibulo-perceptual (VP) time constants of decay, as well as VOR gain, were measured. Results: Prochlorperazine had no effect upon any measure of nystagmic or perceptual vestibular function compared to placebo. This lack of effects on vestibular-mediated motion perception suggests that the drug is likely to act more as an antiemetic than as an anti-vertiginous agent.