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Computational methods including machine learning and molecular dynamics simulations have strong potential to characterize, understand, and ultimately predict the properties of proteins relevant to their stability and function as therapeutics. Such methods would streamline the development pathway by minimizing the current experimental testing required for many protein variants and formulations. The molecular understanding of thermostability and aggregation propensity has advanced significantly along with predictive algorithms based on the sequence-level or structural-level information on a protein. However, these approaches focus largely on a comparison of protein sequence variations to correlate the properties of proteins to their stability, solubility, and aggregation propensity. For therapeutic protein development, it is of equal importance to take into account the impact of the formulation conditions to elucidate and predict the stability of the antibody drugs. At the macroscopic level, changing temperature, pH, ionic strength, and the addition of excipients can significantly alter the kinetics of protein aggregation. The mechanisms controlling aggregation kinetics have been traced back to a combination of molecular features, including conformational stability, partial unfolding to aggregation-prone states, and the colloidal stability governed by surface charges and hydrophobicity. However, very little has been done to evaluate these features in the context of protein dynamics in different formulations. In this work, we have combined a range of molecular features calculated from the Fab A33 protein sequence and molecular dynamics simulations. Using the power of advanced, yet interpretable, statistical tools, it has been possible to uncover greater insights into the mechanisms behind protein stability, validating previous findings, and also develop models that can predict the aggregation kinetics within a range of 49 different solution conditions.

Holliday 4-way junctions are key to important biological DNA processes (insertion, recombination, and repair) and are dynamic structures that adopt either open or closed conformations, the open conformation being the biologically active form. Tetracationic metallo-supramolecular pillarplexes display aryl faces about a cylindrical core, an ideal structure to interact with open DNA junction cavities. Combining experimental studies and MD simulations, we show that an Au pillarplex can bind DNA 4-way (Holliday) junctions in their open form, a binding mode not accessed by synthetic agents before. Pillarplexes can bind 3-way junctions too, but their large size leads them to open up and expand that junction, disrupting the base pairing, which manifests in an increased hydrodynamic size and lower junction thermal stability. At high loading, they rearrange both 4-way and 3-way junctions into Y-shaped forks to increase the available junction-like binding sites. Isostructural Ag pillarplexes show similar DNA junction binding behavior but lower solution stability. This pillarplex binding contrasts with (but complements) that of metallo-supramolecular cylinders, which prefer 3-way junctions and can rearrange 4-way junctions into 3-way junction structures. The pillarplexes' ability to bind open 4-way junctions creates exciting possibilities to modulate and switch such structures in biology, as well as in synthetic nucleic acid nanostructures. In human cells, the pillarplexes do reach the nucleus, with antiproliferative activity at levels similar to those of cisplatin. The findings provide a new roadmap for targeting higher-order junction structures using a metallo-supramolecular approach, as well as expanding the toolbox available to design bioactive junction binders into organometallic chemistry.

Purpose: The use of ionic liquids (ILs) in drug delivery has focused attention on non-toxic IL counterions. Cationic lipids can be used to form ILs with weakly acidic drugs to enhance drug loading in lipid-based formulations (LBFs). However, cationic lipids are typically toxic. Here we explore the use of lipoaminoacids (LAAs) as cationic IL counterions that degrade or digest in vivo to non-toxic components., Methods: LAAs were synthesised via esterification of amino acids with fatty alcohols to produce potentially digestible cationic LAAs. The LAAs were employed to form ILs with tolfenamic acid (Tol) and the Tol ILs loaded into LBF and examined in vitro and in vivo., Results: Cationic LAAs complexed with Tol to generate lipophilic Tol ILs with high drug loading in LBFs. Assessment of the LAA under simulated digestion conditions revealed that they were susceptible to enzymatic degradation under intestinal conditions, forming biocompatible FAs and amino acids. In vitro dispersion and digestion studies of Tol ILs revealed that formulations containing digestible Tol ILs were able to maintain drug dispersion and solubilisation whilst the LAA were breaking down under digesting conditions. Finally, in vivo oral bioavailability studies demonstrated that oral delivery of a LBF containing a Tol IL comprising a digestible cationic lipid counterion was able to successfully support effective oral delivery of Tol., Conclusions: Digestible LAA cationic lipids are potential IL counterions for weakly acidic drug molecules and digest in situ to form non-toxic breakdown products., (© 2022. The Author(s).)

Type III lipid-based formulations (LBFs) combine poorly water-soluble drugs with oils, surfactants, and cosolvents to deliver the drugs into the systemic circulation. However, the solubility of the drug can be influenced by the colloidal phases formed in the gastrointestinal tract as the formulation is dispersed and makes contact with bile and other materials present within the GI tract. Thus, an understanding of the phase behavior of LBFs in the gut is critical for designing efficient LBFs. Molecular dynamics (MD) simulation is a powerful tool for the study of colloidal systems. In this study, we modeled the internal structures of five type III LBFs of loratadine containing poly(ethylene oxide) nonionic surfactants polysorbate 80 and polyoxyl hydrogenated castor oil (Kolliphor RH40) using long-timescale MD simulations (0.4-1.7 μs). We also conducted experimental investigations (dilution of formulations with water) including commercial Claritin liquid softgel capsules. The simulations show that LBFs form continuous phase, water-swollen reverse micelles, and bicontinuous and phase-separated systems at different dilutions, which correlate with the experimental observations. This study supports the use of MD simulation as a predictive tool to determine the fate of LBFs composed of medium-chain lipids, polyethylene oxide surfactants, and polymers.

Objective: Molecular dynamics (MD) simulations provide an in silico method to study the structure of lipid-based formulations (LBFs) and the incorporation of poorly water-soluble drugs within such formulations. In order to validate the ability of MD to effectively model the properties of LBFs, this work investigates the well-known cyclosporine A formulations, Sandimmune® and Neoral®. Sandimmune® exhibits poor dispersibility and its absorption from the gastrointestinal tract is enhanced when administered after food, whereas Neoral® disperses comparatively well and shows no food effect., Methods: MD simulations were performed of both LBFs to investigate the differences observed in fasted and fed conditions. These conditions were also tested using an in vitro experimental model of dispersion and digestion., Results: These MD simulations were able to show that the food effect observed for Sandimmune® can be explained by large changes in drug solubilization on addition of bile. In contrast, Neoral® is well dispersed in water or in simulated fasted conditions, and this dispersion is relatively unchanged on moving to fed conditions. These differences were confirmed using dispersion and digestion in vitro experimental model., Conclusions: The current data suggests that MD simulations are a potential method to model the fate of LBFs in the gastrointestinal tract, predict their dispersion and digestion, investigate behaviour of APIs within the formulations, and provide insights into the clinical performance of LBFs., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Successful oral peptide delivery faces two major hurdles: low enzymatic stability in the gastro-intestinal lumen and poor intestinal membrane permeability. While lipid-based formulations (LBF) have the potential to overcome these barriers, effective formulation of peptides remains challenging. Lipophilic salt (LS) technology can increase the apparent lipophilicity of peptides, making them more suitable for LBF., Methods: As a model therapeutic peptide, octreotide (OCT) was converted to the docusate LS (OCT.DoS 2 ), and compared to the commercial acetate salt (OCT.OAc 2 ) in oral absorption studies and related in vitro studies, including parallel artificial membrane permeability assay (PAMPA), Caco-2, in situ intestine perfusion, and simulated digestion in vitro models. The in vivo oral absorption of OCT.DoS 2 and OCT.OAc 2 formulated in self-emulsifying drug delivery systems (SEDDS) was studied in rats., Results: LS formulation improved the solubility and loading of OCT in LBF excipients and OCT.DoS 2 in combination with SEDDS showed higher OCT absorption than the acetate comparator in the in vivo studies in rats. The Caco-2 and in situ intestine perfusion models indicated no increases in permeability for OCT.DoS 2 . However, the in vitro digestion studies showed reduced enzymatic degradation of OCT.DoS 2 when formulated in the SEDDS formulations. Further in vitro dissociation and release studies suggest that the enhanced bioavailability of OCT from SEDDS-incorporating OCT.DoS 2 is likely a result of higher partitioning into and prolonged retention within lipid colloid structures., Conclusion: The combination of LS and LBF enhanced the in vivo oral absorption of OCT primarily via the protective effect of LBF sheltering the peptide from gastrointestinal degradation.

Lipid based formulations (LBFs) can enhance oral bioavailability, however, their utility may be restricted by low drug loading in the formulation. Converting drugs to drug-ionic liquids (drug-ILs) or lipophilic salts can significantly increase lipid solubility but this approach is complicated in some cases by salt disproportionation, leading to a reduction in solubility and physical instability. Here we explore the physical stability of the weakly basic model drug, cinnarizine (CIN), when paired with a decanoate counterion (Dec) to form the drug-IL, cinnarizine decanoate (CIN.Dec). Consistent with published studies of salt disproportionation in aqueous solution, weakly acidic counterions such as Dec lead to the generation of drug-IL lipid solutions with pHs below pH max . This leads to CIN deprotonation to the less soluble free base and precipitation. Subsequent studies however, show that these effects can be reversed by acidification of the formulation (either with excess decanoic acid or other lipid soluble acids), stimulating a pH shift to the salt plateau of CIN.Dec and the formation of stable lipid solutions of CIN.Dec. Altering formulation pH to more acidic conditions, therefore stabilises drug-ILs formed using weakly acidic lipophilic counterions, and is a viable method to promote formulation stability via inhibition of disproportionation of some drug-ILs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role. IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host's immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system., (Copyright © 2020 Farrant et al.)

Lipid based formulations (LBFs) are extensively utilised as an enabling technology in drug delivery. The use of ionic liquids (ILs) or lipophilic salts (LS) in drug delivery has also garnered considerable interest due to unique solubility properties. Conversion of active pharmaceutical ingredients (API) to ILs by pairing with an appropriately lipophilic counterion has been shown to decrease melting point of the salt complex and improve solubility in LBFs. However, the relationship between the structure of the counterion, the physicochemical properties of the resulting salts and solubility in LBFs has not been systematically explored. This study investigates the relationship between alkyl sulfate counterion structure and melting temperature ( T m or T g ) in addition to LBF solubility, utilizing cinnarizine and lumefantrine as model weakly basic APIs. Three series of structurally diverse alkyl sulfate counterions were chosen to probe this relationship. Pairing cinnarizine and lumefantrine with a majority of these alkyl sulfate counterions resulted in a reduction in melting temperature and enhanced solubility in model medium chain and long chain LBFs. The chain length of the alkyl sulfate plays a crucial role in performance, and consistently branched alkyl sulfate counterions perform better than straight chain alkyl sulfate counterions, as predicted. Most interestingly, trends in counterion performance were found to be consistent across two APIs with disparate chemical structures. The findings from this study will facilitate the design of counterions which enhance solubility of ionisable drugs and unlock the potential to develop compounds previously restrained by poor solubility., Competing Interests: This article describes intellectual property in the use of ionic liquids/lipophilic salts in drug delivery that has been assigned to Lonza. Authors L. F., H. D. W. and H. B. are from Lonza., (This journal is © The Royal Society of Chemistry.)

Lipid based formulations (LBFs) are commonly employed to enhance the absorption of highly lipophilic, poorly water-soluble drugs. However, the utility of LBFs can be limited by low drug solubility in the formulation. Isolation of ionizable drugs as low melting, lipophilic salts or ionic liquids (ILs) provides one means to enhance drug solubility in LBFs. However, whether different ILs benefit from formulation in different LBFs is largely unknown. In the current studies, lumefantrine was isolated as a number of different lipophilic salt/ionic liquid forms and performance was assessed after formulation in a range of LBFs. The solubility of lumefantrine in LBF was enhanced 2- to 80-fold by isolation as the lumefantrine docusate IL when compared to lumefantrine free base. The increase in drug loading subsequently enhanced concentrations in the aqueous phase of model intestinal fluids during in vitro dispersion and digestion testing of the LBF. To assess in vivo performance, the systemic exposure of lumefantrine docusate after administration in Type II-MCF, IIIB-MCF, IIIB-LCF, and IV formulations was evaluated after oral administration to rats. In vivo exposure was compared to control lipid and aqueous suspension formulations of lumefantrine free base. Lumefantrine docusate in the Type IIIB-LCF showed significantly higher plasma exposure compared to all other formulations (up to 35-fold higher). The data suggest that isolation of a lipid-soluble IL, coupled with an appropriate formulation, is a viable means to increase drug dose in an oral formulation and to enhance exposure of lumefantrine in vivo ., Competing Interests: This work describes intellectual property in the use of ionic liquids/lipophilic salts in drug delivery that has been assigned to Lonza from Monash University. H.D.W., H.B. and L.F. are all employees of Lonza and participated in the conduct or write up of this study.

Lipid-based formulations (LBF) are widely used by industry and accepted by the regulatory authorities for oral drug delivery in the pharmaceutical and consumer healthcare market. Innovation in the LBF field is however needed in order to meet the demands of modern drugs, their more challenging problem statements and growing needs for achieving optimal pharmacokinetics (i.e., no food-effects, low variability) on approval. This review describes a new lipophilic salt / ionic liquid approach in combination with LBF, and how this salt strategy can be used to better tailor the properties of a drug to LBFs. The potential advantages of lipophilic salts are discussed in the context of dose escalation studies during toxicological evaluation, reducing the pill burden, increasing drug absorption of new drugs and in life-cycle management. Commentary on lipophilic salt synthesis, scale-up, LBF design and the regulatory aspects are also provided. These topics are discussed in the broad context of bringing the widely recognized advantages of LBFs to a broader spectrum of drugs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Antimicrobial peptides (AMP), part of the innate immune system, are well studied for their ability to kill pathogenic microorganisms. However, many also possess important immunomodulatory effects, and this area has potential for the development of novel therapies to supplement traditional methods such as the use of antibiotics. Here, we characterise the microbicidal and immunomodulatory potential of the proline-rich bovine AMP, Bactenecin 5 (Bac5). We demonstrate broad antimicrobial activity, including against some mycobacterial species, which are important pathogens of fish, cattle and humans. Bac5 is able to activate macrophage-like THP-1 cells and can synergistically trigger the upregulation of tnf-α when co-stimulated with M. marinum. Furthermore, Bac5 sensitises A549 epithelial cells to stimulation with TNF-α. For the first time, we characterise the activity of Bac5 in vivo, and show it to be a potent chemokine for macrophages in the zebrafish (Danio rerio) embryo model of infection. Bac5 also supports the early recruitment of neutrophils in the presence of M. marinum. In the absence of host adaptive immunity, exogenous injected Bac5 is able to slow, although not prevent, infection of zebrafish with M. marinum., Competing Interests: The authors declare that they have no competing interests.

The absolute bioavailability of many small molecule kinase inhibitors (smKIs) is low. The reasons for low bioavailability are multifaceted and include constraints due to first pass metabolism and poor absorption. For smKIs where absorption limits oral bioavailability, low aqueous solubility and high lipophilicity, often in combination with high-dose requirements have been implicated in low and variable absorption, food-effects, and absorption-related drug-drug interactions. The current study has evaluated whether preparation of smKIs as lipophilic salts/ionic liquids in combination with coadministration with lipid-based formulations is able to enhance absorption for examples of this compound class. Lipophilic (docusate) salt forms of erlotinib, gefitinib, ceritinib, and cabozantinib (as example smKIs demonstrating low aqueous solubility and high lipophilicity) were prepared and isolated as workable powder solids. In each case, the lipophilic salt exhibited high and significantly enhanced solubility in lipidic excipients (>100 mg/g) when compared to the free base or commercial salt form. Isolation as the lipophilic salt facilitated smKI loading in model lipid-based formulations at high concentration, increased in vitro solubilization at gastric and intestinal pH and in some cases increased oral absorption (∼2-fold for cabozantinib formulations in rats). Application of a lipophilic salt approach can therefore facilitate the use of lipid-based formulations for examples of the smKI compound class where low solubility limits absorption and is a risk factor for increased variability due to food-effects.

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus However, the exact role of this molecule has not been identified. dacA , the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus ., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Higher lipid solubility of lipophilic salt forms creates new product development opportunities for high-dose liquid-filled capsules. The purpose of this study is to determine if lipophilic salts of Biopharmaceutical Classification System (BCS) Class I amlodipine and BCS Class III fexofenadine, ranitidine, and metformin were better lipid formulation candidates than existing commercial salts. Lipophilic salts were prepared from lipophilic anions and commercial HCl or besylate salt forms, as verified by 1 H-NMR. Thermal properties were assessed by differential scanning calorimetry and hot-stage microscopy. X-ray diffraction and polarized light microscopy were used to confirm the salt's physical form. All lipophilic salt forms were substantially more lipid-soluble (typically >10-fold) when compared to commercial salts. For example, amlodipine concentrations in lipidic excipients were limited to <5-10 mg/g when using the besylate salt but could be increased to >100 mg/g when using the docusate salt. Higher lipid solubility of the lipophilic salts of each drug translated to higher drug loadings in lipid formulations. In vitro tests showed that lipophilic salts solubilized in a lipid formulation resulted in dispersion behavior that was at least as rapid as the dissolution rates of conventional salts. This study confirmed the applicability of forming lipophilic salts of BCS I and III drugs to promote the utility of lipid-based delivery systems., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

This study aimed to transform weakly acidic poorly water-soluble drugs (PWSD) into ionic liquids (ILs) to promote solubility in, and the utility of, lipid-based formulations. Ionic liquids (ILs) were formed directly from tolfenamic acid (Tolf), meclofenamic acid, diclofenac, and ibuprofen by pairing with lipophilic counterions. The drug-ILs were obtained as liquids or low melting solids and were significantly more soluble (either completely miscible or highly soluble) in lipid based, self-emulsifying drug delivery systems (SEDDS) when compared to the equivalent free acid. In vivo assessment of a SEDDS lipid solution formulation of Tolf didecyldimethylammonium salt and the same formulation of Tolf free acid at low dose (18 mg/kg, where the free acid was soluble in the SEDDS), resulted in similar absorption profiles and overall exposure. At high dose (100 mg/kg), solution SEDDS formulations of the Tolf ILs (didecyldimethylammonium, butyldodecyldimethylammonium or didecylmethylammonium salts) were possible, but the lower lipid solubility of Tolf free acid dictated that administration of the free acid was only possible as a suspension in the SEDDS formulation or as an aqueous suspension. Under these conditions, total drug plasma exposure was similar for the IL formulations and the free acid, but the plasma profiles were markedly different, resulting in flatter, more prolonged exposure profiles and reduced C max for the IL formulations. Isolation of a weakly acidic drug as an IL may therefore provide advantage as it allows formulation as a solution SEDDS rather than a lipid suspension, and in some cases may provide a means of slowing or sustaining absorption. The current studies compliment previous studies with weakly basic PWSD and demonstrate that transformation into highly lipophilic ILs is also possible for weakly acidic compounds.

Pseudomonas aeruginosa opportunistically infects the airways of patients with cystic fibrosis and causes significant morbidity and mortality. Initial infection can often be eradicated though requires prompt detection and adequate treatment. Intermittent and then chronic infection occurs in the majority of patients. Better detection of P. aeruginosa infection using biomarkers may enable more successful eradication before chronic infection is established. In chronic infection P. aeruginosa adapts to avoid immune clearance and resist antibiotics via efflux pumps, β-lactamase expression, reduced porins and switching to a biofilm lifestyle. The optimal treatment strategies for P. aeruginosa infection are still being established, and new antibiotic formulations such as liposomal amikacin, fosfomycin in combination with tobramycin and inhaled levofloxacin are being explored. Novel agents such as the alginate oligosaccharide OligoG, cysteamine, bacteriophage, nitric oxide, garlic oil and gallium may be useful as anti-pseudomonal strategies, and immunotherapy to prevent infection may have a role in the future. New treatments that target the primary defect in cystic fibrosis, recently licensed for use, have been associated with a fall in P. aeruginosa infection prevalence. Understanding the mechanisms for this could add further strategies for treating P. aeruginosa in future., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Lipid based formulations (LBF) provide well proven opportunities to enhance the oral absorption of drugs and drug candidates that sit close to, or beyond, the boundaries of Lipinski's 'rule-of-five' chemical space. Advantages in permeability, efflux and presystemic metabolism are evident; however, the primary benefit is in increases in dissolution and apparent intestinal solubility for lipophilic, poorly water soluble drugs. This review firstly details the inherent advantages of LBF, their general properties and classification, and provides a brief retrospective assessment of the development of LBF over the past fifty years. More detailed analysis of the ability of LBF to promote intestinal solubilisation, supersaturation and absorption is then provided alongside review of the methods employed to assess formulation performance. Critical review of the ability of simple dispersion and more complex in vitro digestion methods to predict formulation performance subsequently reveals marked differences in the correlative ability of in vitro tests, depending on the properties of the drug involved. Notably, for highly permeable low melting drugs e.g. fenofibrate, LBF appear to provide significant benefit in all cases, and sustained ongoing solubilisation may not be required. In other cases, and particularly for higher melting point drugs such as danazol, where re-dissolution of crystalline precipitate drug is likely to be slow, correlations with ongoing solubilisation and supersaturation are more evident. In spite of their potential benefits, one limitation to broader use of LBF is low drug solubility in the excipients employed to generate formulations. Techniques to increase drug lipophilicity and lipid solubility are therefore explored, and in particular those methods that provide for temporary enhancement including lipophilic ionic liquid and prodrug technologies. The transient nature of these lipophilicity increases enhances lipid solubility and LBF viability, but precludes enduring effects on receptor promiscuity and off target toxicity. Finally, recent efforts to generate solid LBF are briefly described as a means to circumvent the need to encapsulate in soft or hard gelatin capsules, although the latter remain popular with consumers and a proven means of LBF delivery., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development.

Purpose: In vitro lipid digestion models are commonly used to screen lipid-based formulations (LBF), but in vitro-in vivo correlations are in some cases unsuccessful. Here we enhance the scope of the lipid digestion test by incorporating an absorption 'sink' into the experimental model., Methods: An in vitro model of lipid digestion was coupled directly to a single pass in situ intestinal perfusion experiment in an anaesthetised rat. The model allowed simultaneous real-time analysis of the digestion and absorption of LBFs of fenofibrate and was employed to evaluate the influence of formulation digestion, supersaturation and precipitation on drug absorption., Results: Formulations containing higher quantities of co-solvent and surfactant resulted in higher supersaturation and more rapid drug precipitation in vitro when compared to those containing higher quantities of lipid. In contrast, when the same formulations were examined using the coupled in vitro lipid digestion - in vivo absorption model, drug flux into the mesenteric vein was similar regardless of in vitro formulation performance., Conclusion: For some drugs, simple in vitro lipid digestion models may underestimate the potential for absorption from LBFs. Consistent with recent in vivo studies, drug absorption for rapidly absorbed drugs such as fenofibrate may occur even when drug precipitation is apparent during in vitro digestion.

The Crc protein, together with the Hfq protein, participates in catabolite repression in pseudomonads, helping to coordinate metabolism. Little is known about how Crc affects the hierarchy of metabolite assimilation from complex mixtures. Using proton Nuclear Magnetic Resonance (NMR) spectroscopy, we carried out comprehensive metabolite profiling of culture supernatants (metabolic footprinting) over the course of growth of both Pseudomonas putida and P. aeruginosa, and compared the wild-type strains with deletion mutants for crc. A complex metabolite consumption hierarchy was observed, which was broadly similar between the two species, although with some important differences, for example in sugar utilization. The order of metabolite utilization changed upon inactivation of the crc gene, but even in the Crc-null strains some compounds were completely consumed before late metabolites were taken up. This suggests the presence of additional regulatory elements that determine the time and order of consumption of compounds. Unexpectedly, the loss of Crc led both species to excrete acetate and pyruvate as a result of unbalanced growth during exponential phase, compounds that were later consumed in stationary phase. This loss of carbon during growth helps to explain the contribution of the Crc/Hfq regulatory system to evolutionary fitness of pseudomonads., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Absorption after oral administration is a requirement for almost all drug products but is a challenge for drugs with intrinsically low water solubility. Here, the weakly basic, poorly water-soluble drugs (PWSDs) itraconazole, cinnarizine, and halofantrine were converted into lipophilic ionic liquids to facilitate incorporation into lipid-based formulations and integration into lipid absorption pathways. Ionic liquids were formed via metathesis reactions of the hydrochloride salt of the PWSDs with a range of lipophilic counterions. The resultant active pharmaceutical ingredient-ionic liquids (API-ILs) were liquids or low melting point solids and either completely miscible or highly soluble in lipid based, self-emulsifying drug delivery systems (SEDDS) comprising mixtures of long or medium chain glycerides, surfactants such as Kolliphor-EL and cosolvents such as ethanol. They also readily incorporated into the colloids formed in intestinal fluids during lipid digestion. Itraconazole docusate or cinnarizine decylsulfate API-ILs were subsequently dissolved in long chain lipid SEDDS at high concentration, administered to rats and in vivo exposure assessed. The data were compared to control formulations based on the same SEDDS formulations containing the same concentrations of drug as the free base, but in this case as a suspension (since the solubility of the free base in the SEDDS was much lower than the API-ILs). For itraconazole, comparison was also made to a physical mixture of itraconazole free base and sodium docusate in the same SEDDS formulation. For both drugs plasma exposure was significantly higher for the API-IL containing formulations (2-fold for cinnarizine and 20-fold for itraconazole), when compared to the suspension formulations (or the physical mixture in the case of itraconazole) at the same dose. The liquid SEDDS formulations, made possible by the use of the API-ILs, also provide advantages in dose uniformity, capsule filling, and stability compared to similar suspension formulations. The data suggest that the formation of lipophilic ionic liquids provides a means of increasing dissolved-drug loading in lipid based formulations and thereby promoting the exposure of poorly water-soluble drugs after oral administration.

Purpose: Lipid-based formulations (LBF) are substrates for digestive lipases and digestion can significantly alter their properties and potential to support drug absorption. LBFs have been widely examined for their behaviour in the presence of pancreatic enzymes. Here, the impact of gastric lipase on the digestion of representative formulations from the Lipid Formulation Classification System has been investigated., Methods: The pHstat technique was used to measure the lipolysis by recombinant dog gastric lipase (rDGL) of eight LBFs containing either medium (MC) or long (LC) chain triglycerides and a range of surfactants, at various pH values [1.5 to 7] representative of gastric and small intestine contents under both fasting and fed conditions., Results: All LBFs were hydrolyzed by rDGL. The highest specific activities were measured at pH 4 with the type II and IIIA MC formulations that contained Tween®85 or Cremophor EL respectively. The maximum activity on LC formulations was recorded at pH 5 for the type IIIA-LC formulation. Direct measurement of LBF lipolysis using the pHstat, however, was limited by poor LC fatty acid ionization at low pH., Conclusions: Since gastric lipase initiates lipid digestion in the stomach, remains active in the intestine and acts on all representative LBFs, its implementation in future standardized in vitro assays may be beneficial. At this stage, however, routine use remains technically challenging.

The impact of gastrointestinal (GI) processing and first pass metabolism on danazol oral bioavailability (BA) was evaluated after administration of self-emulsifying drug delivery systems (SEDDS) in the rat. Danazol absolute BA was determined following oral and intraduodenal (ID) administration of LFCS class IIIA medium chain (MC) formulations at high (SEDDSH-III) and low (SEDDSL-III) drug loading and a lipid free LFCS class IV formulation (SEDDS-IV). Experiments were conducted in the presence and absence of ABT (1-aminobenzotriazole) to evaluate the effect of first pass metabolism. A series of modified in vitro lipolysis tests were developed to better understand the in vivo processing of SEDDS in the rat. Danazol BA was low (<13%) following oral and ID administration of either SEDDS. Increasing drug loading, ID rather than oral administration, and administration of SEDDS-IV rather than SEDDS-III led to higher oral BA. After pretreatment with ABT, however, danazol oral BA significantly increased (e.g., 60% compared to 2% after administration of SEDDSL-III), no effect was observed on increasing drug loading, and differences between SEDDS-III and -IV were minimal. In vitro digestion models based on the lower enzyme activity and lower dilution conditions expected in the rat resulted in significantly reduced danazol precipitation from SEDDS-III or SEDDS-IV on initiation of digestion. At the doses administered here (4-8 mg/kg), the primary limitation to danazol oral BA in the rat was first pass metabolism, and the fraction absorbed was >45% after oral administration of SEDDS-III or SEDDS-IV. In contrast, previous studies in dogs suggest that danazol BA is less dependent on first pass metabolism and more sensitive to changes in formulation processing. In vitro digestion models based on likely rat GI conditions suggest less drug precipitation on formulation digestion when compared to equivalent dog models, consistent with the increases in in vivo exposure (fraction absorbed) seen here in ABT-pretreated rats.

The impact of pancreatin and calcium addition on a wide array of lipid-based formulations (LBFs) during in vitro lipolysis, with regard to digestion rates and distribution of the model drug danazol, was investigated. Pancreatin primarily affected the extent of digestion, leaving drug distribution somewhat unaffected. Calcium only affected the extent of digestion slightly but had a major influence on drug distribution, with more drug precipitating at higher calcium levels. This is likely to be caused by a combination of removal of lipolysis products from solution by the formation of calcium soaps and calcium precipitating with bile acids, events known to reduce the solubilizing capacity of LBFs dispersed in biorelevant media. Further, during the digestion of hydrophilic LBFs, like IIIA-LC, the un-ionized-ionized ratio of free fatty acids (FFA) remained unchanged at physiological calcium levels. This makes the titration curves at pH 6.5 representable for digestion. However, caution should be taken when interpreting lipolysis curves of lipophilic LBFs, like I-LC, at pH 6.5, at physiological levels of calcium (1.4 mM); un-ionized-ionized ratio of FFA might change during digestion, rendering the lipolysis curve at pH 6.5 non-representable for the total digestion. The ratio of un-ionized-ionized FFAs can be maintained during digestion by applying non-physiological levels of calcium, resulting in a modified drug distribution with increased drug precipitation. However, as the main objective of the in vitro digestion model is to evaluate drug distribution, which is believed to have an impact on bioavailability in vivo, a physiological level (1.4 mM) of calcium is preferred.

We have previously reported cyanide at concentrations of up to 150 μM in the sputum of cystic fibrosis patients infected with Pseudomonas aeruginosa and a negative correlation with lung function. Our aim was to investigate possible mechanisms for this association, focusing on the effect of pathophysiologically relevant cyanide levels on human respiratory cell function. Ciliary beat frequency measurements were performed on nasal brushings and nasal air-liquid interface (ALI) cultures obtained from healthy volunteers and cystic fibrosis patients. Potassium cyanide decreased ciliary beat frequency in healthy nasal brushings (n = 6) after 60 min (150 μM: 47% fall, p<0.0012; 75 μM: 32% fall, p<0.0001). Samples from cystic fibrosis patients (n = 3) showed similar results (150 μM: 55% fall, p = 0.001). Ciliary beat frequency inhibition was not due to loss of cell viability and was reversible. The inhibitory mechanism was independent of ATP levels. KCN also significantly inhibited ciliary beat frequency in ALI cultures, albeit to a lesser extent. Ciliary beat frequency measurements on ALI cultures treated with culture supernatants from P. aeruginosa mutants defective in virulence factor production implicated cyanide as a key component inhibiting the ciliary beat frequency. If cyanide production similarly impairs mucocilliary clearance in vivo, it could explain the link with increased disease severity observed in cystic fibrosis patients with detectable cyanide in their airway., (©ERS 2014.)

For over 20years, stealth drug delivery has been synonymous with nanoparticulate formulations and intravenous dosing. The putative determinants of stealth in these applications are the molecular weight and packing density of a hydrophilic polymer (commonly poly(ethylene glycol) (PEG)) that forms a steric barrier at the surface of the nanoparticle. The current study examined the potential translation of the concepts learned from stealth technology after intravenous administration to oral drug delivery and specifically, to enhance drug exposure after administration of oral lipid-based formulations (LBFs) containing medium-chain triglycerides (MCT). MCT LBFs are rapidly digested in the gastrointestinal tract, typically resulting in losses in solubilisation capacity, supersaturation and drug precipitation. Here, non-ionic surfactants containing stealth PEG headgroups were incorporated into MCT LBFs in an attempt to attenuate digestion, reduce precipitation risk and enhance drug exposure. Stealth capabilities were assessed by measuring the degree of digestion inhibition that resulted from steric hindrance of enzyme access to the oil-water interface. Drug-loaded LBFs were assessed for maintenance of solubilising capacity during in vitro digestion and evaluated in vivo in rats. The data suggest that the structural determinants of stealth LBFs mirror those of parenteral formulations, i.e., the key factors are the molecular weight of the PEG in the surfactant headgroup and the packing density of the PEG chains at the interface. Interestingly, the data also show that the presence of labile ester bonds within a PEGylated surfactant also impact on the stealth properties of LBFs, with digestible surfactants requiring a PEG Mw of ~1800g/mol and non-digestible ether-based surfactants ~800g/mol to shield the lipidic cargo. In vitro evaluation of drug solubilisation during digestion showed stealth LBFs maintained drug solubilisation at or above 80% of drug load and reduced supersaturation in comparison to digestible counterparts. This trend was also reflected in vivo, where the relative bioavailability of drug after administration in two stealth LBFs increased to 120% and 182% in comparison to analogous digestible (non-stealth) formulations. The results of the current study indicate that self-assembled "stealth" LBFs have potential as a novel means of improving LBF performance., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The analysis of protein-protein interactions in Mycobacterium tuberculosis has the potential to shed light on the functions of the large number of predicted open-reading frames annotated as conserved hypothetical proteins. We have developed a formaldehyde crosslinking system to detect in vivo interactions in mycobacteria. Our Gateway-adapted vector system uses three promoter strengths, including constitutive and regulatable versions, for the expression of target proteins with either an N- or C-terminal His-Strep-Strep tag. Tandem affinity purification using the His- and Strep-tags is well-suited to the isolation of protein complexes with a high purity and no detectable background. We have validated this approach using the well-described pyruvate dehydrogenase complex., (Copyright © 2014. Published by Elsevier B.V.)

Bile components play a significant role in the absorption of dietary fat, by solubilizing the products of fat digestion. The absorption of poorly water-soluble drugs from the gastrointestinal tract is often enhanced by interaction with the pathways of fat digestion and absorption. These processes can enhance drug absorption. Thus, the phase behavior of bile components and digested lipids is of great interest to pharmaceutical scientists who seek to optimize drug solubilization in the gut lumen. This can be achieved by dosing drugs after food or preferably by formulating the drug in a lipid-based delivery system. Phase diagrams of bile salts, lecithin, and water have been available for many years, but here we investigate the association structures that occur in dilute aqueous solution, in concentrations that are present in the gut lumen. More importantly, we have compared these structures with those that would be expected to be present in the intestine soon after secretion of bile. Phosphatidylcholines are rapidly hydrolyzed by pancreatic enzymes to yield equimolar mixtures of their monoacyl equivalents and fatty acids. We constructed phase diagrams that model the association structures formed by the products of digestion of biliary phospholipids. The micelle-vesicle phase boundary was clearly identifiable by dynamic light scattering and nephelometry. These data indicate that a significantly higher molar ratio of lipid to bile salt is required to cause a transition to lamellar phase (i.e., liposomes in dilute solution). Mixed micelles of digested bile have a higher capacity for solubilization of lipids and fat digestion products and can be expected to have a different capacity to solubilize lipophilic drugs. We suggest that mixtures of lysolecithin, fatty acid, and bile salts are a better model of molecular associations in the gut lumen, and such mixtures could be used to better understand the interaction of drugs with the fat digestion and absorption pathway.

The Lipid Formulation Classification System Consortium looks to develop standardized in vitro tests and to generate much-needed performance criteria for lipid-based formulations (LBFs). This article highlights the value of performing a second, more stressful digestion test to identify LBFs near a performance threshold and to facilitate lead formulation selection in instances where several LBF prototypes perform adequately under standard digestion conditions (but where further discrimination is necessary). Stressed digestion tests can be designed based on an understanding of the factors that affect LBF performance, including the degree of supersaturation generated on dispersion/digestion. Stresses evaluated included decreasing LBF concentration (↓LBF), increasing bile salt, and decreasing pH. Their capacity to stress LBFs was dependent on LBF composition and drug type: ↓LBF was a stressor to medium-chain glyceride-rich LBFs, but not more hydrophilic surfactant-rich LBFs, whereas decreasing pH stressed tolfenamic acid LBFs, but not fenofibrate LBFs. Lastly, a new Performance Classification System, that is, LBF composition independent, is proposed to promote standardized LBF comparisons, encourage robust LBF development, and facilitate dialogue with the regulatory authorities. This classification system is based on the concept that performance evaluations across three in vitro tests, designed to subject a LBF to progressively more challenging conditions, will enable effective LBF discrimination and performance grading., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The current study determined the extent to which the desorption of lipid-based formulations (LBFs) from a mesoporous magnesium aluminometasilicate (Neusilin®-US2) carrier is governed by drug properties, LBF composition, and LBF-to-adsorbent ratio. A secondary objective was to evaluate the impact of testing parameters (medium composition, pH, dilution, and agitation) on in vitro LBF performance. Two self-emulsifying LBFs, with high/low lipid-surfactant ratios were studied in detail using danazol, fenofibrate, cinnarizine, and mefenamic acid as model drugs. A wider range of 38 different danazol-containing LBF were also evaluated, where desorption was evaluated immediately after preparation and after 1 month of storage. The results revealed that incomplete desorption from Neusilin® was a feature of all drugs and LBFs tested. Desorption was insensitive to agitation but increased under conditions where ionizable drugs were charged. In addition, formulations containing a higher proportion (>30%) of hydrophilic surfactant consistently exhibited higher desorption, and were least susceptible to decreased desorption on storage. In summary, although Neusilin® is an effective vehicle for LBF solidification, its use is accompanied by a risk of incomplete desorption of the vehicle from the carrier, irrespective of the drug. Lipid Formulation Classification System (LFCS)Type IIIB LBFs comprising higher quantities of hydrophilic surfactants appear to desorb most from Neusilin®. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Purpose: To explore the possibility that age-related changes in physiology may result in differences in drug bioavailability after oral administration of lipid based formulations of danazol., Methods: Danazol absorption from lipid formulations with increasing drug load was examined in younger (9 months) and older (8 years) beagles. Age related changes to hepatic function were assessed via changes to systemic clearance and serum bile acid concentrations. Changes to lipolytic enzyme activity and intestinal bile salt concentration were evaluated using in vitro lipolysis., Results: Drug exposure increased linearly with dose in younger animals. In older animals, bioavailability increased with increasing dose to a tipping point, beyond which bioavailability reduced (consistent with initiation of precipitation). No differences in hepatic function were apparent across cohorts. Changes to enzyme concentrations in lipolysis studies had little impact on drug precipitation/solubilisation. In contrast, higher bile salt concentrations better supported supersaturation at higher drug loads., Conclusions: Differences in animal cohort can have a significant impact on drug absorption from lipid based formulation. For danazol, bioavailability was enhanced under some circumstances in older animals. In vitro experiments suggest that this was unlikely to reflect changes to metabolism or lipolysis, but might be explained by increases in luminal bile salt/phospholipid concentrations in older animals.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the airways of Cystic Fibrosis (CF) patients during which it adapts and undergoes clonal expansion within the lung. It commonly acquires inactivating mutations of the anti-sigma factor MucA leading to a mucoid phenotype, caused by excessive production of the extracellular polysaccharide alginate that is associated with a decline in lung function. Alginate production is believed to be the key benefit of mucA mutations to the bacterium in the CF lung. A phenotypic and gene expression characterisation of the stationary phase physiology of mucA22 mutants demonstrated complex and subtle changes in virulence factor production, including cyanide and pyocyanin, that results in their down-regulation upon entry into stationary phase but, (and in contrast to wildtype strains) continued production in prolonged stationary phase. These findings may have consequences for chronic infection if mucoid P. aeruginosa were to continue to make virulence factors under non-growing conditions during infection. These changes resulted in part from a severe down-regulation of both AHL-and AQ (PQS)-dependent quorum sensing systems. In trans expression of the cAMP-dependent transcription factor Vfr restored both quorum sensing defects and virulence factor production in early stationary phase. Our findings have implications for understanding the evolution of P. aeruginosa during CF lung infection and it demonstrates that mucA22 mutation provides a second mechanism, in addition to the commonly occurring lasR mutations, of down-regulating quorum sensing during chronic infection this may provide a selection pressure for the mucoid switch in the CF lung.

The performance of self-emulsifying drug delivery systems (SEDDS) is influenced by their tendency to generate supersaturated systems during dispersion and digestion in the gastrointestinal tract. This study investigated the effect of drug loading on supersaturation during digestion of fenofibrate or danazol SEDDS, each formulated using long-chain lipids and a range of nonionic surfactants. Supersaturation was described by the maximum supersaturation ratio (SR(M) ) produced by in vitro digestion. This parameter was calculated as the ratio of the total concentration of drug present in the digestion vessel versus the drug solubility in the colloidal phases formed by digestion of the SEDDS. SR(M) proved to be a remarkable indicator of performance across a range of lipid-based formulations. SEDDS containing danazol showed little evidence of precipitation on digestion, even at drug loads approaching saturation in the formulation. In contrast, fenofibrate crystallized extensively on digestion of the corresponding series of SEDDS, depending on the drug loading. The difference was explained by the generation of higher SR(M) values by fenofibrate formulations. A threshold SR(M) of 2.5-2.6 was identified in six of the seven SEDDS. This is not a definitive threshold for precipitation, but in general when SR(M) is greater than 3, fenofibrate supersaturation could not be maintained., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Ionic liquids (ILs) have been exploited to improve the absorption of poorly water-soluble drugs. Custom-made ILs solubilized very high quantities of the poorly water-soluble drugs, danazol and itraconazole, and maintained drug solubilization under simulated gastro-intestinal conditions. A danazol-containing self-emulsifying IL formulation gave rise to 4.3-fold higher exposure than the crystalline drug and prolonged exposure compared with a lipid formulation.

Pseudomonas aeruginosa produces the toxic secondary metabolite hydrogen cyanide (HCN) at high cell population densities and low aeration. Here, we investigated the impact of HCN as a signal in cell-cell communication by comparing the transcriptome of the wild-type strain PAO1 to that of an HCN-negative mutant under cyanogenic conditions. HCN repressed four genes and induced 12 genes. While the individual functions of these genes are unknown, with one exception (i.e. a ferredoxin-dependent reductase), a highly inducible six-gene cluster (PA4129-PA4134) was found to be crucial for protection of P. aeruginosa from external HCN intoxication. A double mutant deleted for PA4129-PA4134 and cioAB (encoding cyanide-insensitive oxidase) did not grow with 100 μM KCN, whereas the corresponding single mutants were essentially unaffected, suggesting a synergistic action of the PA4129-PA4134 gene products and cyanide-insensitive oxidase., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Uptake and excretion of nutrients is an integral part of a cell's physiology. Using analytical chemistry techniques, metabolite uptake and excretion from the culture medium can be quantified. As cellular metabolism changes throughout growth, additional information is available if transient and growth phase-dependent changes are monitored. Here, we describe time-resolved metabolic footprinting (TReF), a technique which employs nuclear magnetic resonance spectroscopy and nonlinear curve fitting to understand and visualize metabolite utilization of P. aeruginosa.

Cyanide is produced by a few bacterial species, including Pseudomonas aeruginosa, and it has a role in the opportunistic infections of this bacterium including in cystic fibrosis lung infections. We describe two methods for determining cyanide in culture and patient sputum samples. One uses an ion-selective electrode to provide a convenient, rapid method of cyanide quantitation in culture or sputum, and the second is a semiquantitative method using Feigl-Anger paper that is useful for screening large numbers of bacterial strains for cyanide production.

Drugs with low aqueous solubility commonly show low and erratic absorption after oral administration. Myriad approaches have therefore been developed to promote drug solubilization in the gastrointestinal (GI) fluids. Here, we offer insight into the unique manner by which lipid-based formulations (LBFs) may enhance the absorption of poorly water-soluble drugs via co-stimulation of solubilization and supersaturation. Supersaturation provides an opportunity to generate drug concentrations in the GI tract that are in excess of the equilibrium crystalline solubility and therefore higher than that achievable with traditional formulations. Incorporation of LBF into lipid digestion and absorption pathways provides multiple drivers of supersaturation generation and the potential to enhance thermodynamic activity and absorption. These drivers include 1) formulation dispersion, 2) lipid digestion, 3) interaction with bile and 4) lipid absorption. However, high supersaturation ratios may also stimulate drug precipitation and reduce exposure where re-dissolution limits absorption. The most effective formulations are likely to be those that generate moderate supersaturation and do so close to the site of absorption. LBFs are particularly well suited to these criteria since solubilization protects against high supersaturation ratios, and supersaturation initiation typically occurs in the small intestine, at the absorptive membrane.

Purpose: Recent studies have shown that digestion of lipid-based formulations (LBFs) can stimulate both supersaturation and precipitation. The current study has evaluated the drug, formulation and dose-dependence of the supersaturation - precipitation balance for a range of LBFs., Methods: Type I, II, IIIA/B LBFs containing medium-chain (MC) or long-chain (LC) lipids, and lipid-free Type IV LBF incorporating different doses of fenofibrate or tolfenamic acid were digested in vitro in a simulated intestinal medium. The degree of supersaturation was assessed through comparison of drug concentrations in aqueous digestion phases (APDIGEST) during LBF digestion and the equilibrium drug solubility in the same phases., Results: Increasing fenofibrate or tolfenamic acid drug loads (i.e., dose) had negligible effects on LC LBF performance during digestion, but promoted drug crystallization (confirmed by XRPD) from MC and Type IV LBF. Drug crystallization was only evident in instances when the calculated maximum supersaturation ratio (SR(M)) was >3. This threshold SR(M) value was remarkably consistent across all LBF and was also consistent with previous studies with danazol., Conclusions: The maximum supersaturation ratio (SR(M)) provides an indication of the supersaturation 'pressure' exerted by formulation digestion and is strongly predictive of the likelihood of drug precipitation in vitro. This may also prove effective in discriminating the in vivo performance of LBFs.

The solubilizing properties of lipid-based formulations (LBFs) can change dramatically following dispersion and digestion of the formulation components. This study investigated the performance of self-emulsifying LBFs consisting of four different long-chain (LC)/medium-chain (MC) lipid blends formulated with the lipophilic drug fenofibrate and either a water-insoluble surfactant polysorbate 85 (Tween 85) or its more hydrophilic relative, polysorbate 80 (Tween 80). These components allowed closely related Type II and IIIA LBFs of fenofibrate to be evaluated during in vitro dispersion and in vitro digestion testing. Initial assessment of the solvent capacity of drug-free LBFs during dispersion and digestion revealed that the solubility of fenofibrate was more dependent on the surfactant type rather than lipid composition. Type II LBFs in the dispersed state were generally better at solubilizing fenofibrate than equivalent Type IIIA LBFs, regardless of lipid composition. However, even when high drug loadings were used, supersaturation/drug precipitation after dispersion of Type II or Type IIIA LBFs was only moderate. In contrast, digestion of both Type II and IIIA LBFs led to much higher levels of drug supersaturation, and this resulted in drug precipitation. After digestion the ability of each LBF to maintain drug in a solubilized state was highly dependent on lipid composition as well as the choice of surfactant. Notably, MC lipids exhibited very good solubilizing properties in the dispersed state, but resulted in a higher degree of supersaturation on digestion, leading to higher susceptibility to drug precipitation. This study showed that replacing LC lipids with MC lipids in Type II and IIIA LBF, in the proportions used here has little effect on fenofibrate solubilization during dispersion, but is likely to promote supersaturation on digestion. Without careful consideration of drug loading and choice of surfactant in Type II/IIIA MC lipid formulations, there is a high risk of precipitation of drug in the intestine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles. E.g. for two-component system mutants, the cognate response regulator and sensor kinase genes clustered tightly together. Some strains had metabolic phenotypes (metabotypes) that could be related to the known gene function. E.g. pyruvate dehydrogenase mutants accumulated large amounts of pyruvate in the medium. In other cases, the metabolic phenotypes were not easily interpretable. The rpoN mutant, which lacks the alternative σ factor RpoN (σ(54)), accumulated high levels of gluconate in the medium. In addition, endometabolome profiling of intracellular metabolites identified a number of systemic metabolic changes. We linked this to indirect regulation of the catabolite repression protein Crc via the non-coding RNA crcZ and found that a crcZ (but not crc) mutant also shared the high-gluconate phenotype. We profiled an additional set of relevant metabolic enzymes and transporters, including Crc targets, and showed that the Crc-regulated edd mutant (gluconate-6-phosphate dehydratase) had similar gluconate levels as the rpoN mutant. Finally, a set of clinical isolates showed patient- and random amplification of polymorphic DNA (RAPD) type-specific differences in gluconate production, which were associated significantly with resistance across four antibiotics (tobramycin, ciprofloxacin, aztreonam, and imipenem), indicating that this has potential clinical relevance.

We grew Pseudomonas aeruginosa in LB and artificial sputum medium (ASM) (filtered and unfiltered) and quantified metabolite utilization and excretion by nuclear magnetic resonance (NMR) spectroscopy (metabolic footprinting or extracellular metabolomics). Utilization rates were similar between media, but there were differences in excretion-e.g., acetate was produced only in unfiltered ASM.

Approximately 80% of adult patients with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa and consequently require antibiotic therapy at intervals throughout their lives. Achieving lethal concentrations of antibiotics in the lung remains a challenge. Recent evidence from Escherichia coli and Staphylococcus aureus suggests that the generation of hydroxyl radicals by sublethal concentrations of antibiotics may induce mutagenesis and confer bacteria with resistance to a wide range of antimicrobials. As Ps. aeruginosa can persist for many years following colonization of the airways and during this time it is repeatedly exposed to bactericidal antibiotics, we tested whether its exposure to sublethal levels increases mutation frequency. We demonstrate that sublethal levels of three classes of bactericidal antibiotics commonly used against Ps. aeruginosa infections, β-lactams, aminoglycosides and quinolones lead to an increase in mutation frequency, varying between c. threefold increase with aminoglycosides and a c. 14-fold increase in mutation frequency with β-lactam antibiotics. These findings could be clinically significant because exposure to sublethal concentrations of antibiotics during chronic infection leading to increased mutation frequency may facilitate adaptive radiation of pathogenic bacteria in the heterogeneous environment of the CF lung., (© 2012 The Society for Applied Microbiology.)