Liposomal formulation of a new antifungal hybrid compound provides protection against Candida auris in the ex vivo skin colonization model.

The newly emerged pathogen, Candida auris, presents a serious threat to public health worldwide. This multidrug-resistant yeast often colonizes and persists on the skin of patients, can easily spread from person to person, and can cause life-threatening systemic infections. New antifungal therapies are therefore urgently needed to limit and control both superficial and systemic C. auris infections. In this study, we designed a novel antifungal agent, PQA-Az-13, that contains a combination of indazole, pyrrolidine, and arylpiperazine scaffolds substituted with a trifluoromethyl moiety. PQA-Az-13 demonstrated antifungal activity against biofilms of a set of 10 different C. auris clinical isolates, representing all four geographical clades distinguished within this species. This compound showed strong activity, with MIC values between 0.67 and 1.25 µg/mL. Cellular proteomics indicated that PQA-Az-13 partially or completely inhibited numerous enzymatic proteins in C. auris biofilms, particularly those involved in both amino acid biosynthesis and metabolism processes, as well as in general energy-producing processes. Due to its hydrophobic nature and limited aqueous solubility, PQA-Az-13 was encapsulated in cationic liposomes composed of soybean phosphatidylcholine (SPC), 1,2-dioleoyloxy-3-trimethylammonium-propane chloride (DOTAP), and N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt (DSPE-PEG 2000), and characterized by biophysical and spectral techniques. These PQA-Az-13-loaded liposomes displayed a mean size of 76.4 nm, a positive charge of +45.0 mV, a high encapsulation efficiency of 97.2%, excellent stability, and no toxicity to normal human dermal fibroblasts. PQA-Az-13 liposomes demonstrated enhanced antifungal activity levels against both C. auris in in vitro biofilms and ex vivo skin colonization models. These initial results suggest that molecules like PQA-Az-13 warrant further study and development.

In Operando Analysis of Milk-Based Oral Formulations during Digestion Using Synchrotron Small-Angle X-ray Scattering Coupled to Low-Frequency Raman Spectroscopy.

A low-frequency Raman (LFR) probe was coupled to an in-line small-angle X-ray scattering (SAXS) beamline to test the capabilities of a combinatory approach for the determination of lipid and drug behavior during the enzymatic lipolysis of milk-based oral formulations. Cinnarizine was used as the model drug, and its solubilization dynamics as well as its potential impact on the supramolecular structures formed by the digestion products of bovine milk were evaluated from the perspective of both techniques. The SAXS data were superior in distinguishing various liquid crystalline assemblies formed during the digestion process, with LFR providing complementary information regarding the formation of calcium soaps. On the other hand, studying changes in the LFR domain allowed the differentiation of drug solubilization and precipitation; processes that were less clear from the X-ray scattering data. Given the relative simplicity of the combined experimental setup, these results highlight the advantages that the combination of the two techniques can provide for understanding and developing new lipid-based formulations and will help to translate the results obtained at synchrotron facilities to routine analysis procedures in laboratory/industry-based environments.

In Situ Imaging of Subcutaneous Drug Delivery Systems Using Microspatially Offset Low-Frequency Raman Spectroscopy.

The noninvasive in situ monitoring of the status of drug retention and implant integrity of subcutaneous implants would allow optimization of therapy and avoid periods of subtherapeutic delivery kinetics. A proof-of principle study was conducted to determine the use of microspatially offset low-frequency Raman spectroscopy (micro-SOLFRS) for nonintrusive in situ analysis of subcutaneous drug delivery systems. Caffeine was used as the model drug, and it was embedded in a circular-shape Soluplus matrix via vacuum compression molding. For the exploratory analysis, prototype implants were positioned underneath skin tissue samples, and various caffeine concentrations (1-50% w/w) and micro-SOLFRS displacement settings (Δz = 0-8 mm) were tested from the pseudo three-dimensional (3D)-imaging perspective. This format allowed the optimization of real-time micro-SOLFRS analysis of implants through skin tissue that was embedded in an agarose hydrogel. Notably, this analytical approach allowed the temporal and spatial erosion of the implant and solid-state transformations of caffeine to be distinguished. The spectrometric results correlated with complementary high-performance liquid chromatography (HPLC) determination of changes in drug concentration, illustrating drug dissipation/diffusion characteristics. The discovered capability of micro-SOLFRS for in situ measurements of drugs and implants makes it attractive for biomedical diagnostics that, ultimately, could result in development of a new point-of-care technology.

Systematic Investigation of Metabolic Oligosaccharide Engineering Efficiency in Intestinal Cells Using a Dibenzocyclooctyne-Monosaccharide Conjugate.

Metabolic oligosaccharide engineering (MOE) of cells with synthetic monosaccharides can introduce functionality to the glycans of cell membranes. Unnatural sugars (e. g., peracetylated mannose-azide) can be expressed on the cell surface with the azide group in place. After MOE, the azide group can participate in a copper-free click reaction with an alkyne (e. g., dibenzocyclooctyne, DBCO) probe. This allows the metabolic fate of monosaccharides in cells to be understood. However, in a drug delivery context it is desirable to have azide groups on the probe (e. g. a drug delivery particle) and the alkyne (e. g. DBCO) on the cell surface. Consequently, the labelling efficiency of intestinal cell lines (Caco-2 and HT29-MTX-E12) treated with N-dibenzocyclooctyne-tetra-acetylmannosamine, and the concentration- and time-dependent labelling were determined. Furthermore, the labelling of mucus in HT29-MTX-E12 cells with DBCO was shown. This study highlights the potential for using MOE to target azide-functionalised probes to intestinal tissues for drug delivery applications.

Ganaxolone versus Phenobarbital for Neonatal Seizure Management.

OBJECTIVE: Seizures are more common in the neonatal period than at any other stage of life. Phenobarbital is the first-line treatment for neonatal seizures and is at best effective in approximately 50% of babies, but may contribute to neuronal injury. Here, we assessed the efficacy of phenobarbital versus the synthetic neurosteroid, ganaxolone, to moderate seizure activity and neuropathology in neonatal lambs exposed to perinatal asphyxia. METHODS: Asphyxia was induced via umbilical cord occlusion in term lambs at birth. Lambs were treated with ganaxolone (5mg/kg/bolus then 5mg/kg/day for 2 days) or phenobarbital (20mg/kg/bolus then 5mg/kg/day for 2 days) at 6 hours. Abnormal brain activity was classified as stereotypic evolving (SE) seizures, epileptiform discharges (EDs), and epileptiform transients (ETs) using continuous amplitude-integrated electroencephalographic recordings. At 48 hours, lambs were euthanized for brain pathology. RESULTS: Asphyxia caused abnormal brain activity, including SE seizures that peaked at 18 to 20 hours, EDs, and ETs, and induced neuronal degeneration and neuroinflammation. Ganaxolone treatment was associated with an 86.4% reduction in the number of seizures compared to the asphyxia group. The total seizure duration in the asphyxia+ganaxolone group was less than the untreated asphyxia group. There was no difference in the number of SE seizures between the asphyxia and asphyxia+phenobarbital groups or duration of SE seizures. Ganaxolone treatment, but not phenobarbital, reduced neuronal degeneration within hippocampal CA1 and CA3 regions, and cortical neurons, and ganaxolone reduced neuroinflammation within the thalamus. INTERPRETATION: Ganaxolone provided better seizure control than phenobarbital in this perinatal asphyxia model and was neuroprotective for the newborn brain, affording a new therapeutic opportunity for treatment of neonatal seizures. ANN NEUROL 2022;92:1066-1079.

The Influence of Gastrointestinal Biomolecules on Solid-State Transformations in Pharmaceutical Particulates.

Adsorption of gut relevant biomolecules onto particles after oral administration of solid oral dosage forms is expected to form a "gastrointestinal corona", which could influence solution-mediated solid-state transformations on exposure of drug particles to gastrointestinal fluids. Low-frequency Raman (LFR) spectroscopy was used in this study to investigate in situ solid-state phase transformations under biorelevant temperature and pH conditions along with the presence of biomolecules. Melt-quenched amorphous indomethacin was used as a model solid particulate, and its solid-state behavior was evaluated at 37 °C and pH 1.2-6.8 with or without the presence of typical bile salt/phospholipid mixtures emulating fed-state conditions. Overall, a change in the solid-state transformation pathway from amorphous to crystalline drug was observed, where an intermediate ε-form that initially formed at pH 6.8 was suppressed by the addition of endogenous gastrointestinal biomolecules. These solid-state changes were corroborated using time-resolved synchrotron small- and wide-angle X-ray scattering (SAXS/WAXS). Additionally, the bile salt and phospholipid mixture partly prevented the otherwise strong aggregation between drug particles at more acidic conditions (pH ≤ 4.5) and helped to shift the balance against the intrinsic hydrophobicity of indomethacin as well as the plasticization effect brought about by the physiological temperature (i.e., the stickiness arising from the supercooled liquid state at 37 °C). The overall results highlight the importance of evaluating the impact that endogenous biomolecules may have on the solid-state characteristics of drug molecules in dissolution media, where analytical tools such as LFR spectroscopy can serve as an attractive avenue for accessing time-resolved solid-state information on time-scales that are difficult to achieve with other techniques such as X-ray diffraction.

Implications of the Digestion of Milk-Based Formulations for the Solubilization of Lopinavir/Ritonavir in a Combination Therapy.

The development of formulation approaches to coadminister lopinavir and ritonavir antiretroviral drugs to children is necessary to ensure optimal treatment of human immunodeficiency virus (HIV) infection. It was previously shown that milk-based lipid formulations show promise as vehicles to deliver antimalarial drugs by enhancing their solubilization during the digestion of the milk lipids under intestinal conditions. In this study, we investigate the role of digestion of milk and infant formula on the solubilization behavior of lopinavir and ritonavir to understand the fate of drugs in the gastrointestinal (GI) tract after oral administration. Small angle X-ray scattering (SAXS) was used to probe the presence of crystalline drugs in suspension during digestion. In particular, the impact of one drug on the solubilization of the other was elucidated to reveal potential drug-drug interactions in a drug combination therapy. Our results showed that lopinavir and ritonavir affected the solubilization of each other during digestion in lipid-based formulations. While addition of ritonavir to lopinavir improved the overall solubilization of lopinavir, the impact of lopinavir was to reduce ritonavir solubilization as digestion progressed. These findings highlight the importance of assessing the solubilization of individual drugs in a combined matrix in order to dictate the state of drugs available for subsequent absorption and metabolism. Enhancement in the solubilization of lopinavir and ritonavir in a drug combination setting in vitro also supported the potential for food effects on drug exposure.

Impact of pasteurization on the self-assembly of human milk lipids during digestion.

Human milk is critical for the survival and development of infants. This source of nutrition contains components that protect against infections while stimulating immune maturation. In cases where the mother's own milk is unavailable, pasteurized donor milk is the preferred option. Although pasteurization has been shown to have minimal impact on the lipid and FA composition before digestion, no correlation has been made between the impact of pasteurization on the FFA composition and the self-assembly of lipids during digestion, which could act as delivery mechanisms for poorly water-soluble components. Pooled nonpasteurized and pasteurized human milk from a single donor was used in this study. The evolving FFA composition during digestion was determined using GC coupled to a flame ionization detector. In vitro digestion coupled to small-angle X-ray scattering was utilized to investigate the influence of different calcium levels, fat content, and the presence of bile salts on the extent of digestion and structural behavior of human milk lipids. Almost complete digestion was achieved when bile salts were added to the systems containing high calcium to milk fat ratio, with similar structural behavior of lipids during digestion of both types of human milk being apparent. In contrast, differences in the colloidal structures were formed during digestion in the absence of bile salt because of a greater amount of FFAs being released from the nonpasteurized than pasteurized milks. This difference in FFAs released from both types of human milk could result in varying nutritional implications for infants.

Penicillin G concentrations required for prophylaxis against Group A Streptococcus infection evaluated using a hollow fibre model and mathematical modelling.

BACKGROUND: Acute rheumatic fever (ARF), an autoimmune reaction to Group A Streptococcus (Streptococcus pyogenes; Strep A) infection, can cause rheumatic heart disease (RHD). New formulations of long-acting penicillins are being developed for secondary prophylaxis of ARF and RHD. OBJECTIVES: To evaluate the penicillin G concentrations required to suppress growth of Strep A. METHODS: Broth microdilution MIC and MBC for Strep A strains M75611024, M1T15448 and M18MGAS8232 were determined. All strains were studied in a hollow fibre model (initial inoculum 4 log10 cfu/mL). Constant penicillin G concentrations of 0.008, 0.016 and 0.05 mg/L were examined against all strains, plus 0.012 mg/L against M18MGAS8232. Viable counts were determined over 144 h. Subsequently, all penicillin G-treated cartridges were emptied, reinoculated with 5 log10 cfu/mL and counts determined over a further 144 h. Mathematical modelling was performed. RESULTS: MIC and MBC were 0.008 mg/L for all strains; small subpopulations of M75611024 and M1T15448, but not M18MGAS8232, grew at 1× MIC. Following the first inoculation, 0.008 mg/L achieved limited killing and/or stasis against M75611024 and M1T15448, with subsequent growth to ∼6 log10 cfu/mL. Following both inocula, concentrations ≥0.016 mg/L suppressed M75611024 and M1T15448 to <1 log10 cfu/mL from 6 h onwards with eradication. Concentrations ≥0.008 mg/L suppressed M18MGAS8232 to <1 log10 cfu/mL from 24 h onwards with eradication after both inoculations. Mathematical modelling well described all strains using a single set of parameter estimates, except for different maximum bacterial concentrations and proportions of bacteria growing at 1× MIC. CONCLUSIONS: In the absence of validated animal and human challenge models, the study provides guidance on penicillin G target concentrations for development of new penicillin formulations.

TAILOR-MS, a Python Package that Deciphers Complex Triacylglycerol Fatty Acyl Structures: Applications for Bovine Milk and Infant Formulas.

Liquid chromatography tandem mass spectrometry (LC/MS) and other mass spectrometric technologies have been widely applied for triacylglycerol profiling. One challenge for targeted identification of fatty acyl moieties that constitute triacylglycerol species in biological samples is the numerous combinations of 3 fatty acyl groups that can form a triacylglycerol molecule. Manual determination of triacylglycerol structures based on peak intensities and retention time can be highly inefficient and error-prone. To resolve this, we have developed TAILOR-MS, a Python (programming language) package that aims at assisting: (1) the generation of targeted LC/MS methods for triacylglycerol detection and (2) automating triacylglycerol structural determination and prediction. To assess the performance of TAILOR-MS, we conducted LC/MS triacylglycerol profiling of bovine milk and two infant formulas. Our results confirmed dissimilarities between bovine milk and infant formula triacylglycerol composition. Furthermore, we identified 247 triacylglycerol species and predicted the possible existence of another 317 in the bovine milk sample, representing one of the most comprehensive reports on the triacylglycerol composition of bovine milk thus far. Likewise, we presented here a complete infant formula triacylglycerol profile and reported >200 triacylglycerol species. TAILOR-MS dramatically shortened the time required for triacylglycerol structural identification from hours to seconds and performed decent structural predictions in the absence of some triacylglycerol constituent peaks. Taken together, TAILOR-MS is a valuable tool that can greatly save time and improve accuracy for targeted LC/MS triacylglycerol profiling.

The influence of lipid digestion on the fate of orally administered drug delivery vehicles.

This review will focus on orally administered lipid-based drug delivery vehicles and specifically the influence of lipid digestion on the structure of the carrier lipids and their entrained drug cargoes. Digestion of the formulation lipids, which are typically apolar triglycerides, generates amphiphilic monoglycerides and fatty acids that can self-assemble into a diverse array of liquid crystalline structures. Tracking the dynamic changes in self-assembly of the lipid digestion products during digestion has recently been made possible using synchrotron-based small angle X-ray scattering. The influence of lipid chain length and degree of unsaturation on the resulting lipid structuring will be described in the context of the critical packing parameter theory. The chemical and structural transformation of the formulation lipids can also have a dramatic impact on the physical state of drugs co-administered with the formulation. It is often assumed that the best strategy for drug development is to maximise drug solubility in the undigested formulation lipids and to incorporate additives to maintain drug solubility during digestion. However, it is possible to improve drug absorption using lipid digestion in cases where the solubility of the dosed drug or one of its polymorphic forms is greater in the digested lipids. Three different fates for drugs administered with digestible lipid-based formulations will be discussed: (1) where the drug is more soluble in the undigested formulation lipids; (2) where the drug undergoes a polymorphic transformation during lipid digestion; and (3) where the drug is more soluble in the digested formulation lipids.

Chemistry and Geometry of Counterions Used in Hydrophobic Ion Pairing Control Internal Liquid Crystal Phase Behavior and Thereby Drug Release.

The combination of Flash NanoPrecipitation and hydrophobic ion pairing (HIP) is a valuable approach for generating nanocarrier formulations of ionic water-soluble drugs with controllable release properties dictated by liquid crystalline structuring of the ion pairs. However, there are few examples of this in practice in the literature. This work aims to decipher the influence of the nature of the hydrophobic counterion used in HIP and its consequent impact on liquid crystalline structuring and drug release. The hypothesis of this study was that hydrophobic counterions with different head and tail groups used for FNP with HIP would give rise to different liquid crystalline structures, which in turn would result in different drug release behavior. A cationic, water-soluble antibiotic, polymixin B, was complexed with eight different hydrophobic counterions with varying head and tail groups and encapsulated into nanocarriers 100-400 nm in size prepared using FNP. Sixteen formulations were assessed for internal structure by synchrotron small-angle X-ray scattering, and drug release was measured in vitro in physiological conditions. The liquid crystalline phases formed depended on the counterion head group and tail geometry, drug:counterion charge ratio, and the ionic strength and pH of the release medium. Drug release occurred more rapidly when no liquid crystalline phases were present and more slowly when higher-ordered phases existed. Specific findings include that phosphonic acid counterions lead to the formation of lamellar structures that persisted at pH 2.0 but were not present at pH 7.3. In contrast, sulfonic acids lead to lamellar or hexagonal phases that persisted at both pH 7.3 and 2.0, while hydrophobic counterions without alkyl tails did not form internal structures. It was also clear that the lipophilicity of the counterion does not dictate drug release. These findings confirm that the liquid crystalline phase behavior of the drug:counterion complex dictates drug release and significantly improves our understanding of the types of controlled release formulations that are possible using FNP with HIP.

Lipid Compositions in Infant Formulas Affect the Solubilization of Antimalarial Drugs Artefenomel (OZ439) and Ferroquine during Digestion.

Recent studies have shown that the solubilization of two antimalarial drug candidates, artefenomel (OZ439) and ferroquine (FQ), designed to provide a single-dose combination therapy for uncomplicated malaria can be enhanced using milk as a lipid-based formulation. However, milk as an excipient faces significant quality and regulatory hurdles. We therefore have investigated infant formula as a potential alternative formulation approach. The significance of the lipid species present in a formula with different lipid compositions upon the solubilization of OZ439 and FQ during digestion has been investigated. Synchrotron small-angle X-ray scattering was used to measure the diffraction from a dispersed drug during digestion and thereby determine the extent of drug solubilization. High-performance liquid chromatography was used to quantify the amount of drug partitioned into the digested lipid phases. Our results show that both the lipid species and the amount of lipids administered were key determinants for the solubilization of OZ439, while the solubilization of FQ was independent of the lipid composition. Infant formulas could therefore be designed and used as milk substitutes to tailor the desired level of drug solubilization while circumventing the variability of components in naturally derived milk. The enhanced solubilization of OZ439 was achieved during the digestion of medium-chain triacylglycerols (MCT), indicating the potential applicability of MCT-fortified infant formula powder as a lipid-based formulation for the oral delivery of OZ439 and FQ.

Low-Frequency Raman Scattering Spectroscopy as an Accessible Approach to Understand Drug Solubilization in Milk-Based Formulations during Digestion.

Techniques enabling in situ monitoring of drug solubilization and changes in the solid-state of the drug during the digestion of milk and milk-based formulations are valuable for predicting the effectiveness of such formulations in improving the oral bioavailability of poorly water-soluble drugs. We have recently reported the use of low-frequency Raman scattering spectroscopy (region of analysis <200 cm-1) as an analytical approach to probe solubilization of drugs during digestion in milk using ferroquine (SSR97193) as the model compound. This study investigates the wider utilization of this technique to probe the solubilization behavior of other poorly water-soluble drugs (halofantrine, lumefantrine, and clofazimine) in not only milk but also infant formula in the absence or presence of bile salts during in vitro digestion. Multivariate analysis was used to interpret changes to the spectra related to the drug as a function of digestion time, through tracking changes in the principal component (PC) values characteristic to the drug signals. Characteristic low-frequency Raman bands for all of the drugs were evident after dispersing the solid drugs in suspension form in milk and infant formula. The drugs were generally solubilized during the digestion of the formulations as observed previously for ferroquine and correlated with behavior determined using small-angle X-ray scattering (SAXS). A greater extent of drug solubilization was also generally observed in the infant formula compared to milk. However, in the case of the drug clofazimine, the correlation between low-frequency Raman scattering and SAXS was not clear, which may arise due to background interference from clofazimine being an intense red dye, which highlights a potential limitation of this new approach. Overall, the in situ monitoring of drug solubilization in milk and milk-based formulations during digestion can be achieved using low-frequency Raman scattering spectroscopy, and the information obtained from studying this spectral region can provide better insights into drug solubilization compared to the mid-frequency Raman region.

Spontaneous Self-Assembly of Thermoresponsive Vesicles Using a Zwitterionic and an Anionic Surfactant.

Spontaneous formation of vesicles from the self-assembly of two specific surfactants, one zwitterionic (oleyl amidopropyl betaine, OAPB) and the other anionic (Aerosol-OT, AOT), is explored in water using small-angle scattering techniques. Two factors were found to be critical in the formation of vesicles: surfactant ratio, as AOT concentrations less than equimolar with OAPB result in cylindrical micelles or mixtures of micellar structures, and salt concentration, whereby increasing the amount of NaCl promotes vesicle formation by reducing headgroup repulsions. Small-angle neutron scattering measurements reveal that the vesicles are approximately 30-40 nm in diameter, depending on sample composition. Small-angle X-ray scattering measurements suggest preferential partitioning of OAPB molecules on the vesicle inner layer to support vesicular packing. Heating the vesicles to physiological temperature (37 °C) causes them to collapse into smaller ellipsoidal micelles (2-3 nm), with higher salt concentrations (≥10 mM) inhibiting this transition. These aggregates could serve as responsive carriers for loading or unloading of aqueous cargoes such as drugs and pharmaceuticals, with temperature changes serving as a simple release/uptake mechanism.

Correction: Comparison of bulk and microfluidic methods to monitor the phase behaviour of nanoparticles during digestion of lipid-based drug formulations using in situ X-ray scattering.

Correction for 'Comparison of bulk and microfluidic methods to monitor the phase behaviour of nanoparticles during digestion of lipid-based drug formulations using in situ X-ray scattering' by Ben J. Boyd et al., Soft Matter, 2019, 15, 9565-9578.

Solid State Characterization of Ciprofloxacin Liposome Nanocrystals.

Liposomes have been widely researched as drug delivery systems; however, the solid state form of drug inside the liposome, whether it is in solution or in a solid state, is often not studied. The solid state properties of the drug inside the liposomes are important, as they dictate the drug release behavior when the liposomes come into contact with physiological fluid. Recently, a new approach of making liposomal ciprofloxacin nanocrystals was proposed by the use of an additional freeze-thawing step in the liposomal preparation method. This paper aims to determine the solid state properties of ciprofloxacin inside the liposomes after this additional freeze-thawing cycle using cryo-TEM, small-angle X-ray scattering (SAXS), and cross-polarized light microscopy (CPLM). Ciprofloxacin precipitated in the ciprofloxacin hydrate crystal form with a unit cell dimension of 16.7 Å. The nanocrystals also showed a phase transition at 93 °C, which represents dehydration of the hydrate crystals to the anhydrate form of ciprofloxacin, verified by temperature-dependent SAXS measurements. Furthermore, the dependence of the solid state form of the nanocrystals on pH was investigated in situ, and it was shown that the liposomal ciprofloxacin nanocrystals retained their crystalline form at pH 6-10. Understanding the solid state attributes of nanocrystals inside liposomes provides improved understanding of drug dissolution and release as well as opening avenues to new applications where the nanosized crystals can provide a dissolution benefit.

Direct Comparison of Standard Transmission Electron Microscopy and Cryogenic-TEM in Imaging Nanocrystals Inside Liposomes.

The use of electron microscopy techniques in the understanding of shape and size of nanoparticles are commonly applied to drug nanotechnology, but the type of microscopy and suitability for the particles of interest can have a significant impact on the result. The size and shape of the nanoparticles are crucial in clinical applications; however, direct comparison of the results from standard transmission electron microscopy (TEM) and cryo-TEM have rarely been reported. As a useful case for comparison, liposomal drug nanocrystals are studied here. In this study, the effect of thawing temperature on the size and shape of the ciprofloxacin nanocrystals was determined. A quantitative standard TEM assay was developed to allow for high-throughput particle size analysis. These results were compared to size and shape information obtained using the cryo-TEM method. The results showed broad agreement between the two TEM methods and that ciprofloxacin nanocrystals formed shorter and thinner crystals inside the liposomes at higher thawing temperatures. The results provide confidence in the use of standard TEM to determine the size and shape distribution of solid nanoparticles (in this case, encapsulated inside liposomes) from aqueous media without fear of sample preparation altering the conclusions.

Impact of Ferroquine on the Solubilization of Artefenomel (OZ439) during in Vitro Lipolysis in Milk and Implications for Oral Combination Therapy for Malaria.

Milk is an attractive lipid-based formulation for the delivery of poorly water-soluble drugs to pediatric populations. We recently observed that solubilization of artefenomel (OZ439) during in vitro intestinal lipolysis was driven by digestion of triglycerides in full-cream bovine milk, reflecting the ability of milk to act as an enabling formulation in the clinic. However, when OZ439 was co-administered with a second antimalarial drug, ferroquine (FQ) the exposure of OZ439 was reduced. The current study therefore aimed to understand the impact of the presence of FQ on the solubilization of OZ439 in milk during in vitro intestinal digestion. Synchrotron small-angle X-ray scattering was used for in situ monitoring of drug solubilization (inferred via decreases in the intensity of drug diffraction peaks) and polymorphic transformations that occurred during the course of digestion. Quantification of the amount of each drug solubilized over time and analysis of their distributions across the separated phases of digested milk were determined using high-performance liquid chromatography. The results show that FQ reduced the solubilization of OZ439 during milk digestion, which may be due to competitive binding of FQ to the digested milk products. Interactions between the protonated FQ-H+ and ionized liberated free fatty acids resulted in the formation of amorphous salts, which removes the low-energy crystalline state as a barrier to dissolution of FQ, while inhibiting the solubilization of OZ439. We conclude that although milk could enhance the solubilization of poorly water-soluble OZ439 during in vitro digestion principally due to the formation of fatty acids, the solubilization efficiency was reduced by the presence of FQ by competition for the available fatty acids. Assessment of the solubilization of both drugs during digestion of fixed-dose combination lipid formulations (such as milk) is important and may rationalize changes in bioavailability when compared to that of the individual drugs in the same formulation.

Solid-State Behavior and Solubilization of Flash Nanoprecipitated Clofazimine Particles during the Dispersion and Digestion of Milk-Based Formulations.

Clofazimine, a drug previously used to treat leprosy, has recently been identified as a potential new drug for the treatment for cryptosporidiosis: a diarrheal disease that contributes to 500 000 infant deaths a year in developing countries. Rapid dissolution and local availability of the drug in the small intestine is considered key to the treatment of the infection. However, the commercially available clofazimine formulation (Lamprene) is not well-suited to pediatric use, and therefore reformulation of clofazimine is desirable. Development of clofazimine nanoparticles through the process of flash nanoprecipitation (FNP) has been previously shown to provide fast and improved drug dissolution rates compared to clofazimine crystals and Lamprene. In this study, we investigate the effects of milk-based formulations (as possible pediatric-friendly vehicles) on the in vitro solubilization of clofazimine formulated as either lecithin- or zein/casein-stabilized nanoparticles. Milk and infant formula were used as the lipid vehicles, and time-resolved synchrotron X-ray scattering was used to monitor the presence of crystalline clofazimine in suspension during in vitro lipolysis under intestinal conditions. The study confirmed faster dissolution of clofazimine from all the FNP formulations after the digestion of infant formula was initiated, and a reduced quantity of fat was required to achieve similar levels of drug solubilization compared to the reference drug material and the commercial formulation. These attributes highlight not only the potential benefits of the FNP approach to prepare drug particles but also the fact that enhanced dissolution rates can be complemented by considering the amount of co-administered fat in lipid-based formulations to drive the solubilization of poorly soluble drugs.