Cerastecin Inhibition of the Lipooligosaccharide Transporter MsbA to Combat Acinetobacter baumannii: From Screening Impurity to In Vivo Efficacy.

Acinetobacter baumannii, a commonly multidrug-resistant Gram-negative bacterium responsible for large numbers of bloodstream and lung infections worldwide, is increasingly difficult to treat and constitutes a growing threat to human health. Structurally novel antibacterial chemical matter that can evade existing resistance mechanisms is essential for addressing this critical medical need. Herein, we describe our efforts to inhibit the essential A. baumannii lipooligosaccharide (LOS) ATP-binding cassette (ABC) transporter MsbA. An unexpected impurity from a phenotypic screening was optimized as a series of dimeric compounds, culminating with 1 (cerastecin D), which exhibited antibacterial activity in the presence of human serum and a pharmacokinetic profile sufficient to achieve efficacy against A. baumannii in murine septicemia and lung infection models.

Generation of Complex Protein Structures by Coinfection with High-Quality Recombinant Baculovirus.

The baculovirus expression vector system (BEVS) is a powerful platform for protein expression in insect cells. A prevalent application is the expression of complex protein structures consisting of multiple, interacting proteins. Coinfection with multiple baculoviruses allows for production of complex structures, facilitating structure-function studies, allowing augmentation of insect cell functionality, and production of clinically relevant products such as virus-like particles (VLPs) and adeno-associated viral vectors (AAV). Successful coinfections require the generation of robust and well-quantified recombinant baculovirus stocks. Virus production through homologous recombination, combined with rigorous quantification of viral titers, allows for synchronous coinfections producing high end-product titers. In this chapter, we describe the streamlined workflow for generation and quantification of high-quality recombinant baculovirus stocks and successful coinfection as defined by a preponderance of dually infected cells in the insect cell culture.

Cerastecins inhibit membrane lipooligosaccharide transport in drug-resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.

Risk Factors Associated with Incident Vertebral Fractures in Steroid-treated Males with Duchenne Muscular Dystrophy.

PURPOSE: Prevention of fractures is an unmet need in glucocorticoid (GC)-treated Duchenne muscular dystrophy. This study explored factors associated with incident vertebral fractures (VFs) to inform future fracture prevention efforts. METHODS: VFs were evaluated prospectively at study baseline and 12 months on lateral spine radiographs in participants aged 4 to 25 years with Duchenne muscular dystrophy. Clinical factors were analyzed for their association with the change in Spinal Deformity Index (sum of the Genant-defined VF grades from T4 to L4) between baseline and 12 months. RESULTS: Thirty-eight males were evaluated (mean ± SD age at baseline 11.0 ± 3.6 years; mean ± SD GC duration at baseline 4.1 ± 3.1 years; 74% ambulatory). Nine of 38 participants (24%) had 17 incident VFs, of which 3/17 VFs (18%) were moderate/severe. Participants with 12-month incident VF had lower mean ± SD baseline lumbar spine areal bone mineral density Z-scores (-2.9 ± 1.0 vs -1.9 ± 1.1; P = .049) and lower total body less head areal bone mineral density Z-scores (-3.1 ± 1.2 vs -1.6 ± 1.7; P = .036). Multivariable linear regression showed that at least 1 VF at baseline (P < .001), a higher number of antecedent non-VF (P < .001), and greater bone age delay at baseline (P = .027) were significant predictors of an increase in the Spinal Deformity Index from baseline to 12 months. CONCLUSION: The observation that ≥ 1 prevalent VF and/or non-VF were the strongest predictors of incident VFs at 12 months supports the need for prevention of first fractures in this high-risk setting. Bone age delay, a marker of GC exposure, may assist in the prioritization of patients in efforts to prevent first fractures.

Chemical Stability of Epinephrine 10 mcg/mL Diluted in 0.9% Sodium Chloride and Stored in Polypropylene Syringes at 4 degrees C and 25 degrees C.

Studies have evaluated epinephrine stability in higher concentrations and shorter durations than we require. The objective of this study was to evaluate the chemical stability of epinephrine in syringes at concentrations of 10 mcg/mL in 0.9% sodium chloride at 4°C and 25°C. Solutions of 10 mcg/mL epinephrine in 0.9% sodium chloride were prepared and stored in 10-mL Becton, Dickinson and Company syringes. Three units of each container were stored at 4°C and 25°C. Concentration analysis was completed on study days 0, 2, 7, 14, 21, 28, 42, 56, 72, and 91 using a validated stability-indicating liquid chromatographic method with ultraviolet detection. Chemical stability was based on the intersection of the lower limit of the 95% confidence interval of the observed degradation rate and the time to achieve 90% of the initial concentration (T-90). The analytical method separated degradation products from epinephrine to measure concentration specifically, accurately, and reproducibly. During the study period, all solutions at 4°C retained more than 89.62% of the initial concentration for 91 days. Solutions stored at 25°C retained more than 90% for 21 days. Multiple linear regression revealed significant differences in percent remaining due to study day (P<0.001) and temperature (P=0.002). The calculated T-90, with 95% confidence, was 71.40 days for solutions stored at 4°C but only 12.77 days for solutions stored at 25°C. We conclude that 10 mcg/mL epinephrine solution diluted in 0.9% sodium chloride stored at 4°C is chemically and physically stable for 64 days, with 95% confidence. The syringe may be held at room temperature for up to 24 hours during this period and still retain more than 90% of the initial concentration.

CDC20 Is Regulated by the Histone Methyltransferase, KMT5A, in Castration-Resistant Prostate Cancer.

The methyltransferase KMT5A has been proposed as an oncogene in prostate cancer and therefore represents a putative therapeutic target. To confirm this hypothesis, we have performed a microarray study on a prostate cancer cell line model of androgen independence following KMT5A knockdown in the presence of the transcriptionally active androgen receptor (AR) to understand which genes and cellular processes are regulated by KMT5A in the presence of an active AR. We observed that 301 genes were down-regulated whilst 408 were up-regulated when KMT5A expression was reduced. KEGG pathway and gene ontology analysis revealed that apoptosis and DNA damage signalling were up-regulated in response to KMT5A knockdown whilst protein folding and RNA splicing were down-regulated. Under these conditions, the top non-AR regulated gene was found to be CDC20, a key regulator of the spindle assembly checkpoint with an oncogenic role in several cancer types. Further investigation revealed that KMT5A regulates CDC20 in a methyltransferase-dependent manner to modulate histone H4K20 methylation within its promoter region and indirectly via the p53 signalling pathway. A positive correlation between KMT5A and CDC20 expression was also observed in clinical prostate cancer samples, further supporting this association. Therefore, we conclude that KMT5A is a valid therapeutic target for the treatment of prostate cancer and CDC20 could potentially be utilised as a biomarker for effective therapeutic targeting.

Mitigating the Denosumab-Induced Rebound Phenomenon with Alternating Short- and Long-Acting Anti-resorptive Therapy in a Young Boy with Severe OI Type VI.

Osteogenesis imperfecta (OI) type VI, a recessively inherited form of OI caused by mutations in SERPINF1, is a severe form distinguished by osteomalacia on bone histomorphometry. We describe a boy with severe OI type VI who was initially treated with intravenous (IV) zoledronic acid (ZA) at 1.4 years of age; however, a year later he transitioned to denosumab 1 mg/kg sub-cutaneously every three months in an effort to decrease fracture rates. After two years on denosumab, he presented with symptomatic hypercalcemia due to the denosumab-induced, hyper-resorptive rebound phenomenon. Laboratory parameters at the time of the rebound were as follows: elevated serum ionized calcium (1.62 mmol/L, N 1.16-1.36), elevated serum creatinine due to hypercalcemia-induced muscle catabolism (83 µmol/L, N 9-55), and suppressed parathyroid hormone (PTH) (< 0.7 pmol/L, N 1.3-5.8). The hypercalcemia was responsive to low-dose IV pamidronate, with a rapid decline in serum ionized calcium, and otherwise normalization of the aforementioned parameters within 10 days. To benefit from the powerful, albeit short-term, anti-resorptive effect of denosumab without further rebound episodes, he was treated thereafter with denosumab 1 mg/kg alternating every three months with IV ZA 0.025 mg/kg. Five years later, he remained on dual alternating anti-resorptive therapy without further rebound episodes, and an overall improvement in his clinical status. This novel pharmacological approach of alternating short- and long-term anti-resorptive therapy every three months has not previously been described. Our report suggests this strategy may be an effective method for prevention of the rebound phenomenon in select children for whom denosumab may be beneficial.

Functional Diversity of Gram-Negative Permeability Barriers Reflected in Antibacterial Activities and Intracellular Accumulation of Antibiotics.

Gram-negative bacteria are notoriously more resistant to antibiotics than Gram-positive bacteria, primarily due to the presence of the outer membrane and a plethora of active efflux pumps. However, the potency of antibiotics also varies dramatically between different Gram-negative pathogens, suggesting major mechanistic differences in how antibiotics penetrate permeability barriers. Two approaches are used broadly to analyze how permeability barriers affect intracellular accumulation of antibiotics. One compares the antibacterial activities of compounds, while the other measures the total intracellular concentrations of compounds in nongrowing cells, with both approaches using strains harboring wild-type or genetically modified efflux systems and permeability barriers. Whether the two assays provide similar mechanistic insights remains unclear. In this study, we analyzed the intracellular accumulation and antibacterial activities of antibiotics representative of major clinical classes in three Gram-negative pathogens of high clinical importance, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii. We found that both assays are informative about properties of permeability barriers, but there is no quantitative agreement between the assays. Our results show that the three pathogens differ dramatically in their permeability barriers, with the outer membrane playing the dominant role in E. coli and P. aeruginosa but efflux dominating in A. baumannii. However, even compounds of the same chemotype may use different permeation pathways depending on small chemical modifications. Accordingly, a classification analysis revealed limited conservation of molecular properties that define compound penetration into the three bacteria.

Pandemic-Era Digital Education: Insights from an Undergraduate Medical Programme.

The undergraduate medical programme at Newcastle University (NU) includes a fundamental 'Essentials of Medical Practice' (EOMP) phase comprising the first 2 years of study. This period is designed to support entrants in their transition from further education into the advanced study and practice of clinical medicine. The anatomical sciences of gross anatomy, histology and embryology, and life sciences including physiology, pharmacology and genetics are key disciplines taught within the integrated case-based EOMP curriculum. Learners apply basic science knowledge to clinical scenarios during training in practical examination, communication and reasoning skills. Within the modern pedagogic landscape, the development and introduction of technology-enhanced learning strategies have enhanced the provision of remote learning resources in pre-clinical education. However, the emergence of COVID-19 has resulted in widespread technological challenges for educators and learners, and has raised pedagogic, logistical and ethical concerns. Nonetheless, the pandemic has produced favourable conditions for the creation of valuable digital visualisation strategies for learning and teaching, and for developing and modernising universal approaches to remote education. Here, we describe our technology-enhanced adaptations to COVID-19 across the domains of teaching, learning and academic support for pre-clinical learners studying basic life sciences and clinical skills. Moreover, we outline research-informed digital visualisation solutions to pandemic-era challenges and reflect upon experiences gained within our own educational context. In doing so, we provide insights into the impacts and successes of our interventions. While providing a record of unprecedented contemporary circumstances, we also aim to utilise our observations and experiences of COVID-19 pedagogy when developing ongoing strategies for delivering curricula and futureproofing educational practice.

Risk factors associated with prevalent vertebral fractures in Duchenne muscular dystrophy.

Patients with Duchenne muscular dystrophy (DMD) have a high fracture burden due to progressive myopathy and steroid-induced osteoporosis. This study in males with DMD showed that markers of systemic glucocorticoid exposure including shorter stature, greater bone age delay, and lower lumbar spine bone mineral density were associated with spine fragility. INTRODUCTION: Fragility  fractures are frequent in DMD. The purpose of this study was to identify clinical factors associated with prevalent vertebral fractures (VF) in boys, teens/young adults with Duchenne muscular dystrophy (DMD). METHODS: This was a cross-sectional study of males aged 4-25 years with DMD. VF were evaluated using the modified Genant semi-quantitative method on T4-L4 lateral spine radiographs. Areal bone mineral density (aBMD) was measured at the lumbar spine (LS) and used to estimate volumetric BMD (vBMD). Clinical factors were analyzed for their association with the Spinal Deformity Index (SDI, the sum of the Genant grades). RESULTS: Sixty participants were enrolled (mean age 11.5 years, range 5.4-19.5). Nineteen participants (32%) had a total of 67 VF; 23/67 VF (34%) were moderate or severe. Participants with VF were shorter (mean height Z-score ± standard deviation: - 3.1 ± 1.4 vs. - 1.8 ± 1.4, p = 0.001), had longer glucocorticoid exposure (mean duration 6.0 ± 3.3 vs. 3.9 ± 3.3 years, p = 0.027), greater bone age (BA) delay (mean BA to chronological age difference - 3.2 ± 3.4 vs. - 1.3 ± 1.2 years, p = 0.035), and lower LSaBMD Z-scores (mean - 3.0 ± 1.0 vs. - 2.2 ± 1.2, p = 0.023). There was no difference in LSvBMD Z-scores. Multivariable Poisson regression showed that every 0.1 mg/kg/day increment in average glucocorticoid daily dose was associated with a 1.4-fold SDI increase (95% confidence interval: 1.1-1.7, p = 0.013). Greater BA delay (p < 0.001), higher weight Z-score (p = 0.004), decreased height Z-score (p = 0.025), and lower LSvBMD Z-score (p = 0.025) were also associated with SDI increase. CONCLUSION: Readily measurable clinical variables were associated with prevalent VF in males with glucocorticoid-treated DMD. These variables may be useful to identify candidates for primary osteoporosis prevention after glucocorticoid initiation.

The Impact of Extreme Low Temperatures on Raman Spectra of Amino Acids Relevant for the Search for Life on Europa.

Raman spectroscopy, an emerging technology for in situ space exploration, has been suggested for life detection for the Europa Lander Mission. However, obtaining spectra of samples from the europan icy shell requires measurements at temperatures down to -233°C, which will affect the Raman spectra of any potential biosignatures. In this study, we obtained Raman spectra of amino acids using a 785 nm Raman system at temperatures ranging down to -196°C, analogous to Europa's surface and near subsurface. Significant Raman band width narrowing and decreasing variance were observed at lower temperatures leading to higher-precision Raman measurements, which required higher spectral resolution that could be as high as 2 cm-1 for full identification of amino acids. Such spectral resolution is much higher than the resolution of contemporary Raman instruments for planetary exploration and may be particularly problematic for miniaturized instruments. Shifting of Raman bands to both higher and lower frequencies by as much as ∼25 cm-1 together with changes in the Raman band intensity were recorded. The emergence of new bands and diminishing of the original bands also occurred for some amino acids. A significantly increased fluorescence background was observed in spectra of fluorescent molecules (i.e., tryptophan). A link between the type of vibrational modes associated with Raman bands and the change in their Raman shift at extreme low temperatures was identified and described. This link offers an exciting new method of molecule identification solely based on the comparison of spectra collected at two different temperatures and could greatly improve the identification capabilities in Raman spectroscopy for a wide array of applications.

Cryogenically induced signal enhancement of Raman spectra of porphyrin molecules.

Raman spectroscopy is a powerful analytical technique in contemporary medicine and biomedical research due to its exceptional ability to provide an unambiguous spectroscopic signature of the molecular chemical composition, structure and atom arrangements. Among other applications, investigations of the Raman spectra of porphyrins and their derivatives have been critical in the study of ligand binding mechanisms and drug interactions with healthy and diseased blood cells, as well as for the analysis of blood, hemoproteins and the oxygenation process of human erythrocyte. However, obtaining Raman spectra with satisfactory definition of porphyrin-based molecules can be challenging due to their inherent photo- and thermal sensitivity which leads to laser damage even at low laser power. This severely affects the Raman spectra of porphyrins and limits the Raman signal strength and spectra quality. In this study, we examine two important porphyrins, hemin and protoporphyrin IX, at cryogenic temperatures down to 77 K using a 532 nm excitation Raman instrument in order to study the Raman signal strength and spectral quality dependence on the sample temperature at these extreme low temperatures. We report a significant Raman signal enhancement of up to 310% in the spectra at cryogenic temperatures compared to room temperature measurements. This provides a remarkable improvement of the quality and definition within the spectra and demonstrates that cryogenic Raman measurements can be used as an exceptionally effective method of enhancing the Raman signal and spectra quality for investigations of porphyrins and their derivatives regardless of the excitation wavelength selection. This can greatly improve the effectiveness of Raman spectroscopy in biomedical research, especially in the field of drug design and development, medical diagnostics and disease monitoring and analysis.

Comparison of Preoperative MRI With Intraoperative Findings for Peroneal Tendon Pathologies.

Magnetic resonance imaging (MRI) is commonly used to evaluate soft tissue pathology of the foot and ankle. Prior investigations have reported limitations of this modality, however, in evaluation of pathologies related to the peroneal tendons. This article investigates the correlation of pre-operative MRI studies with intraoperative findings. Five board-certified radiologists interpreted MRIs of 80 ankles that subsequently underwent surgical procedures performed by one board-certified foot and ankle surgeon, after which comparison was made between their findings. Statistically significant disagreement was found between radiologist and surgeon findings of a normal peroneus brevis (PB), PB and peroneus longus (PL) tendinosis, PB and PL hypertrophy, PB and PL partial linear tears, PB and PL flattening, PB longitudinal split tears, and the PB attritional spectrum (combined analysis of flattening, partial linear tearing, and longitudinal split tears). These results suggest that given the disconcordance between MRI and intraoperative findings, surgeons should remain cautious in their reliance upon this imaging modality when evaluating this anatomic region.

Stability of Extemporaneously Compounded Suspensions of Trimethoprim and Sulfamethoxazole in Amber Plastic Bottles and Amber Plastic Syringes.

BACKGROUND: Trimethoprim (TMP) and sulfamethoxazole (SMX) are widely used, in combination, to treat or prevent various infections. Unfortunately, no liquid oral formulation is currently available in Canada for patients who are unable to swallow tablets. OBJECTIVE: To evaluate the stability of suspensions of TMP and SMX (8 and 40 mg/mL, respectively) prepared in Oral Mix or Oral Mix SF vehicle (Medisca Pharmaceutique Inc) and stored for up to 90 days in amber plastic bottles or amber plastic syringes at 5°C or 25°C. METHODS: Suspensions were prepared from bulk powder and from tablets in Oral Mix and Oral Mix SF vehicles, then transferred to amber plastic (polyethylene terephthalate glycol) bottles and plastic oral syringes and stored at 5°C and 25°C. Samples were collected on predetermined study days (0, 7, 14, 23, 45, 60, 75, and 90 days) and analyzed using a validated high-performance liquid chromatography - ultraviolet detection method. A suspension was considered stable if it maintained at least 90% of its initial concentration with 95% confidence. Observations of organoleptic characteristics such as colour and odour, as well as pH, were used to assess physical stability. RESULTS: Suspensions prepared from bulk powder maintained concentrations of TMP and SMX of at least 97% of the initial concentration over the 90-day study period. No obvious changes in colour, odour, or pH were observed. However, acceptable suspensions could not be prepared from the commercial tablets. A persistent foam that developed at the surface of all suspensions prepared from tablets could result in inconsistent dosing. CONCLUSIONS: Extemporaneously compounded oral suspensions of TMP and SMX (8 and 40 mg/mL, respectively) prepared from bulk powder in Oral Mix and Oral Mix SF vehicles and stored in amber plastic bottles or syringes at 5°C or 25°C remained stable for at least 90 days. Suspensions made from tablets produced unacceptable formulations.

CONTEXTE: Le triméthoprime (TMP) et le sulfaméthoxazole (SMX) sont largement utilisés conjointement pour traiter ou prévenir diverses infections. Malheureusement, aucune formulation liquide orale n'est actuellement disponible au Canada pour les patients incapables d'avaler des comprimés. OBJECTIF: Évaluer la stabilité des suspensions de TMP et de SMX (respectivement 8 et 40 mg/mL) préparées dans un véhicule Oral Mix ou Oral Mix SF (Medisca Pharmaceutique Inc.) et stockées pendant 90 jours dans des flacons ou des seringues en plastique ambré à 5 °C ou 25 °C. MÉTHODES: Les suspensions ont été préparées à partir de poudre en vrac et de comprimés dans les véhicules Oral Mix et Oral Mix SF, puis transférées dans des flacons en plastique ambré (polyéthylène téréphtalate glycol) et dans des seringues orales en plastique et stockées à 5 °C et 25 °C. Des échantillons ont été recueillis à des jours prédéterminés (0, 7, 14, 23, 45, 60, 75 et 90 jours) et analysés à l'aide d'une méthode de détection par ultraviolet validée de chromatographie en phase liquide à haute performance. La suspension était jugée stable si elle préservait au moins 90 % de sa concentration initiale avec un seuil de confiance de 95 %. Les observations des caractéristiques organoleptiques, comme la couleur et l'odeur, ainsi que le pH, ont été faites pour évaluer la stabilité physique. RÉSULTATS: Les suspensions préparées à partir de poudre en vrac préservaient au moins 97 % de la concentration initiale de TMP et de SMX pendant la période d'étude de 90 jours. Aucun changement manifeste de couleur, d'odeur ou de pH n'a été observé. Cependant, les suspensions acceptables n'ont pas pu être préparées à partir des comprimés commerciaux. Une mousse homogène se formait à la surface de ces suspensions, ce qui pourrait entraîner un dosage incohérent. CONCLUSIONS: Les suspensions orales composées extemporanées de TMP et SMX (respectivement 8 et 40 mg/mL) préparées à partir de poudre en vrac dans des véhicules Oral Mix et Oral Mix SF et stockées dans des flacons ou des seringues en plastique ambré à 5 °C ou 25°C sont restées stables pendant au moins 90 jours. Les suspensions préparées à partir de comprimés ont donné des formulations inacceptables.

Stability of Compounded Clozapine 25 mg/mL and 50 mg/mL Suspensions in Plastic Bottles.

BACKGROUND: Clozapine oral suspension is not commercially available in Canada but is required for administration to patients who cannot swallow intact tablets. OBJECTIVE: To evaluate the stability of 25 mg/mL and 50 mg/mL clozapine suspensions prepared in a 50:50 mixture of methylcellulose gel 1% and Oral Syrup (flavoured syrup vehicle, Medisca Pharmaceutique Inc) and stored in amber glycol-modified polyethylene terephthalate (PET-G) bottles over 120 days at 4°C and 25°C. METHODS: This study used a validated reverse-phase stability-indicating liquid chromatographic method capable of quantifying clozapine, 3 known degradation compounds, a known impurity, and an unknown compound. Three separate batches of 25 mg/mL and 50 mg/mL clozapine suspensions were prepared, divided into 100-mL aliquots, and stored in 120-mL PET-G bottles. Half of the bottles from each concentration were stored at room temperature (20°C to 25°C) and the other half were stored in the refrigerator (2°C to 8°C). On study days 0, 28, 60, 90, and 120, concentrations of clozapine, each of the 3 known clozapine degradation products, a known impurity, and an unknown compound were determined. RESULTS: When suspensions were stored in PET-G containers at room temperature or under refrigeration for 120 days, the concentration of clozapine remained above 95% of initial concentration, and the measured concentration of degradation products and impurities did not exceed the 0.5% limits set by regulatory authorities worldwide. The proportion of the initial concentration of clozapine remaining on day 120, based on fastest degradation rate with 95% confidence (1-sided), exceeded 92%, and the only degradation product found (clozapine lactam, 0.2%) and an unknown impurity (0.2%) also did not exceed allowable limits. CONCLUSIONS: Compounded clozapine suspensions of 25 mg/mL and 50 mg/mL can be stored in amber PET-G containers for up to 120 days after preparation with storage at room temperature or under refrigeration.

CONTEXTE: La clozapine en suspension orale n'est pas disponible sur le marché canadien, mais elle est nécessaire pour les patients qui ne peuvent l'avaler sous forme de comprimé intact. OBJECTIF: Évaluer la stabilité des suspensions de clozapine de 25 mg/mL et de 50 mg/mL, préparées dans un mélange 50:50 de gel méthylcellulose à 1 % et de Sirop Oral (véhicule de sirop aromatisé, MEDISCA) et conservées dans des flacons ambrés en polytéréphtalate d'éthylène modifié au glycol (PET-G) pendant 120 jours à des températures de 4°C et 25°C. MÉTHODE: Cette étude a utilisé une méthode validée par chromatographie liquide indicatrice de stabilité en phase inverse pouvant quantifier la clozapine, trois composés de dégradation connus, une impureté connue et un composé inconnu. Trois lots séparés de suspensions de clozapine de 25 mg/mL et de 50 mg/mL ont été préparés, divisés dans des aliquotes de 100-mL et stockés dans des flacons en PET-G de 120-mL. La moitié des flacons de chaque concentration a été conservée à température ambiante (de 20°C à 25°C), et l'autre moitié au réfrigérateur (de 2°C à 8°C). Aux jours 0, 28, 60, 90 et 120 de l'étude, on a déterminé les concentrations de clozapine, celles de chacun des trois produits de dégradation de la clozapine, celles d'une impureté connue et d'un complexe inconnu. RÉSULTATS: Lorsque les suspensions étaient stockées dans des contenants en PET-G à température ambiante et réfrigérées pendant 120 jours, la concentration de clozapine demeurait au-dessus de 95 % de la concentration initiale; la concentration mesurée des produits de dégradation et des impuretés ne dépassait pas la limite de 0,5 % fixée par les autorités de règlementation mondiales. La proportion de concentration initiale de clozapine restante au 120e jour, sur la base du taux de dégradation le plus rapide avec un intervalle de confiance de 95 % (unilatéral), dépassait 92 %, et le seul produit de dégradation trouvé (clozapine lactam, 0,2 %) ainsi qu'une impureté inconnue (0,2 %) ne dépassaient pas non plus les limites autorisées. CONCLUSIONS: Les suspensions de clozapine composées de 25 mg/mL et de 50 mg/mL peuvent être conservées dans des contenants ambrés PET-G jusqu'à 120 jours après leur préparation, soit à température ambiante, soit dans un réfrigérateur.

Stability of Extemporaneously Compounded Nadolol 10-mg/mL Suspension in Oral Mix in Glass and Plastic Bottles and Plastic Syringes.

An oral liquid formulation of nadolol, which is required for administration to patients who cannot swallow intact tablets, is not commercially available. The objective of this study was to evaluate the stability of nadolol 10 mg/mL prepared in Oral Mix vehicle and stored in amber glass, amber polyethylene terephthalate, or amber polyvinyl chloride for 91 days at 4ÆC and 25ÆC; and polypropylene oral plastic syringes at 25ÆC only. Three separate batches of nadolol suspension 10 mg/mL were prepared with Oral Mix. Of the suspension, 50-mL aliquots were stored in 100-mL bottles (amber glass, amber polyethylene terephthalate, or amber polyvinyl chloride). Half of the bottles from each container type were stored at 25ÆC and the other half at 4ÆC. On study days 0, 2, 7, 14, 21, 28, 42, 56, 72, and 91, nadolol concentration was determined using a reverse-phase, stability-indicating liquid chromatographic method from samples drawn from each type of container at each temperature. Oral syringes (3 mL), filled with 2 mL of suspension, were stored at 25ÆC and tested on days 0, 2, 7, 21, 42, and 91. The concentration of nadolol 10 mg/mL in Oral Mix in all study samples from bottles and oral syringes remained within 3.5% of the initial concentration. Based on the fastest degradation rate with 95% confidence, on day 91, between 99% to 100% and 98% to 100% remained in suspensions stored in bottles at 25ÆC and 4ÆC, respectively. Oral syringes at 25ÆC had 94% remaining on day 91. Multiple linear regression analysis demonstrated that the percent remaining was related to study day and container, but not temperature. On day 91, nadolol 10 mg/mL oral suspensions prepared with Oral Mix and stored in all bottle types at 4ÆC will retain more than 98% of the initial concentration compared to 99% at 25ÆC and only 94% when stored in oral syringes.

Are outer-membrane targets the solution for MDR Gram-negative bacteria?

The outer membrane (OM) of Gram-negative bacteria confers a significant barrier to many antibacterial agents targeting periplasmic and cytosolic functions. 'Synergist' approaches to disrupt the OM have been hampered by poor specificity and accompanying toxicities. The OM contains proteins required for optimal growth and pathogenesis, including lipopolysaccharide (LPS) and capsular polysaccharide (CPS) transport, porins for uptake of macromolecules, and transporters for essential elements (such as iron). Does the external proximity of these proteins offer an enhanced potential to identify effective therapies? Here, we review recent experiences in exploiting Gram-negative OM proteins (OMPs) to address the calamity of exploding antimicrobial resistance. Teaser: Multidrug-resistant (MDR) Gram-negative bacteria are a growing crisis. Few new antimicrobial chemotypes or targets have been identified after decades of screening. Are OMP targets a solution to MDR Gram-negative bacteria?

Stability of Generic Formulations of Bortezomib 1.0 and 2.5 mg/mL in Vials and Syringes Stored at 4°C and Room Temperature (23°C or 25°C).

BACKGROUND: The availability of generic versions of bortezomib raises questions about the reliability of extrapolating stability data from one brand to another. OBJECTIVE: To evaluate the stability of bortezomib formulations available from Janssen, Teva Canada, Actavis Pharma, Dr. Reddy's Laboratories, Apotex, and MDA, reconstituted with 0.9% sodium chloride (normal saline) to produce solutions of either 1.0 or 2.5 mg/mL and stored over at least 21 days under refrigeration (4°C) or at room temperature (either 23°C or 25°C) in the manufacturer's original glass vials or in polypropylene syringes. METHODS: On study day 0, solutions with concentration 1.0 mg/mL or 2.5 mg/mL of the Teva, Actavis, Dr. Reddy's, Apotex, and MDA generic formulations were prepared. Three units of each type of container (glass vials and syringes) were stored at 4°C and 3 units at room temperature. Concentration and physical inspection were completed on at least 8 study days (including day 0) over a 21- to 84-day study period. Bortezomib concentrations were determined by a validated stability-indicating liquid chromatographic method with ultraviolet detection. The end point of these studies was the time to reach 90% of the initial concentration (T-90) with 95% confidence, which is expressed as "T-9095%CI", where CI refers to the confidence interval. In addition to estimating the T-9095%CI, differences in stability among products from all manufacturers were compared using multiple linear regression. Previously published data for the Janssen product were included in the overall comparisons. RESULTS: In all of the studies, the analytical method separated degradation products from bortezomib, such that the concentration of bortezomib was measured specifically, accurately (deviations < 2.5%), and reproducibly (average replicate error 2.5%). During all studies, solutions retained more than 94% of the initial concentration at 4°C. The T-9095%CI exceeded the study period for all formulations under all combinations of concentration, container, and temperature, except the 84-day study for the MDA product. Multiple linear regression showed no significant differences among manufacturers (p = 0.57). CONCLUSIONS: In this study, formulations of bortezomib currently marketed in Canada (by Janssen, Teva Canada, Actavis Pharma, Dr. Reddy's Laboratories, Apotex, and MDA) were pharmaceutically equivalent and interchangeable. Given that there was no difference in stability related to manufacturer, nominal concentration, or container, we conclude that these formulations are physically and chemically stable for at least 35 days under refrigeration and at least 25 days at room temperature.

CONTEXTE: La disponibilité de versions génériques de bortezomib soulève des questions relatives à la fiabilité de l'extrapolation des données concernant la stabilité d'une marque à l'autre. OBJECTIF: Évaluer la stabilité des formules de bortezomib de Janssen, de Teva Canada, d'Actavis Pharma, des Laboratoires du Dr Reddy, d'Apotex et de MDA, reconstituées avec 0,9 % de chlorure de sodium (solution saline normale) pour produire des solutions de 1 ou de 2,5 mg/mL et réfrigérées au moins 21 jours à 4 °C ou à température ambiante (23 °C ou 25 °C), dans des fioles en verre du fabricant ou dans des seringues en polypropylène. MÉTHODES: La préparation des solutions avec une concentration de 1 mg/mL ou 2,5 mg/mL des formules génériques de Teva, d'Actavis, du Dr Reddy, d'Apotex et de MDA a eu lieu le jour 0 de l'étude. Trois unités de chaque contenant (fioles en verre et seringues) étaient stockées à 4 °C et 3 unités, à température ambiante. L'inspection de la concentration et l'inspection physique ont été réalisées pendant au moins 8 jours (y compris le jour 0) de l'étude qui a duré de 21 à 84 jours. Les concentrations de bortezomib ont été déterminées par une méthode chromatographique liquide validée, indiquant la stabilité à l'aide d'une détection par rayons ultraviolets. Le point final de ces études était le temps nécessaire pour que le produit atteigne 90 % de la concentration initiale (T-90) avec un seuil de confiance de 95 %, exprimé par T-90IC 95 %, IC indiquant l'intervalle de confiance. En plus de l'estimation du T-90IC 95 %, les différences de stabilité des produits de tous les fabricants ont été comparées à l'aide d'une régression linéaire multiple. Les données publiées précédemment sur le produit Jansen sont incluses dans les comparaisons globales. RÉSULTATS: La méthode analytique de toutes les études qui ont été menées a séparé les produits de dégradation du bortezomib de telle manière que la concentration était mesurée de manière spécifique, précise (déviations < 2,5 %) et reproductible (erreur de réplique 2,5 %). Tout au long des études, les solutions ont retenu plus de 94 % de la concentration initiale à 4 °C. Le T-90IC 95 % de toutes les formules dans toutes les combinaisons de concentration, de contenant et de température, dépassait la durée des études, à l'exception du produit MDA dans l'étude de 84 jours. La régression linéaire multiple n'a indiqué aucune différence importante parmi les fabricants (p = 0,57). CONCLUSIONS: Dans cette étude, les formules de bortezomib actuellement commercialisées au Canada (par Janssen, Teva Canada, Actavis Pharma, les Laboratoires du Dr Reddy, Apotex et MDA) étaient équivalentes et interchangeables d'un point de vue pharmaceutique. Puisqu'aucune différence de stabilité, de concentration nominale ou de contenant liée à l'un ou l'autre des fabricants n'a été révélée, nous concluons que ces formules sont physiquement et chimiquement stables pendant au moins 35 jours sous réfrigération et au moins 25 jours à température ambiante.