Optimized LC-MS/MS quantification of tuberculosis drug candidate macozinone (PBTZ169), its dearomatized Meisenheimer Complex and other metabolites, in human plasma and urine.

Tuberculosis, and especially multidrug-resistant tuberculosis (MDR-TB), is a major global health threat which emphasizes the need to develop new agents to improve and shorten treatment of this difficult-to-manage infectious disease. Among the new agents, macozinone (PBTZ169) is one of the most promising candidates, showing extraordinary potency in vitro and in murine models against drug-susceptible and drug-resistant Mycobacterium tuberculosis. A previous analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed by our group to support phase I clinical trials of PBTZ169. These plasma sample analyses revealed the presence of several additional metabolites among which the most prominent was H2PBTZ, a reduced species obtained by dearomatization of macozinone, one of the first examples of Meisenheimer Complex (MC) metabolites identified in mammals. Identification of these new metabolites required the optimization of our original method for enhancing the selectivity between isobaric metabolites as well as for ensuring optimal stability for H2PBTZ analyses. Sample preparation methods were also developed for plasma and urine, followed by extensive quantitative validation in accordance with international bioanalytical method recommendations, which include selectivity, linearity, qualitative and quantitative matrix effect, trueness, precision and the establishment of accuracy profiles using ß-expectation tolerance intervals for known and newer analytes. The newly optimized methods have been applied in a subsequent Phase Ib clinical trial conducted in our University Hospital with healthy subjects. H2PBTZ was found to be the most abundant species circulating in plasma, underscoring the importance of measuring accurately and precisely this unprecedented metabolite. Low concentrations were found in urine for all monitored analytes, suggesting extensive metabolism before renal excretion.

Determination of chlorothalonil metabolites in soil and water samples.

Pesticide metabolites are frequently detected in groundwater at concentrations often exceeding those of their parent pesticides. A well-known example is the metabolites of chlorothalonil, a non-systematic, broad spectrum fungicide. Some of the chlorothalonil metabolites occur frequently and at elevated concentrations in groundwater, which is why the use of chlorothalonil was recently banned in the European Union. To estimate the long-term evolution of the concentration of the chlorothalonil metabolites in groundwater after this ban, it is important to know if metabolite residues in soil and unsaturated zone can affect the concentrations in groundwater. We developed and validated a method for the determination of 5 chlorothalonil metabolites in soil (R471811, R417888, SYN507900, SYN548580 and R611968), including those which are frequently detected in groundwater. The developed protocols, based on a solid phase extraction approach (for R471811, R417888, SYN507900, SYN548580) or a QuEChERS approach (for R611968) followed by UHPLC-MS/MS analysis, provided excellent sensitivity (LOQ of 0.5 µg/kg for all metabolites), precision (RSD<10 % at low, medium and high concentrations) and accuracy (84-115 %). In addition, we developed a simple but highly sensitive (LOQ of 5-10 ng/L) direct-injection method for the analysis of these 5 metabolites in water to compare their occurrence in soil and groundwater. The application of these methods to agricultural soil samples and groundwater samples showed that the detection frequency of the 5 chlorothalonil metabolites in soil and groundwater seems to be inversed and dependent on their sorption coefficient. The latter might control the amount of the chlorothalonil metabolites which is retained in the soil or which leaches towards groundwater. Our results provide insights to estimate the retention of the different chlorothalonil metabolites in soil and unsaturated zone and therefore, to assess the influence of the soil and unsaturated zone on the long-term concentration evolution of these metabolites in groundwater.

Development and validation of a multiplex UHPLC-MS/MS method for the determination of the investigational antibiotic against multi-resistant tuberculosis macozinone (PBTZ169) and five active metabolites in human plasma.

The emergence of Mycobacterium tuberculosis strains resistant to current first-line antibiotic regimens constitutes a major global health threat. New treatments against multidrug-resistant tuberculosis (MDR-TB) are thus eagerly needed in particular in countries with a high MDR-TB prevalence. In this context, macozinone (PBTZ169), a promising drug candidate with an unique mode of action and highly potent in vitro tuberculocidal properties against MDR Mycobacterium strains, has now reached the clinical phase and has been notably tested in healthy male volunteers in Switzerland. To that endeavor, a multiplex UHPLC-MS/MS method has been developed for the sensitive and accurate human plasma levels determination of PBTZ169 along with five metabolites retaining in vitro anti-TB activity. Plasma protein precipitation with methanol was carried out as a simplified sample clean-up procedure followed by direct injection of the undiluted supernatant for the bioanalysis of the six analytes within 5 min, using 1.8 µm reversed-phase chromatography coupled to triple quadrupole mass spectrometry employing electrospray ionization in the positive mode. Stable isotopically-labelled PBTZ169 was used as internal standard (ISTD), while metabolites could be reliably quantified using two unlabeled chemical analogues selected as ISTD from a large in-house analogous compounds library. The overall methodology was fully validated according to current recommendations (FDA, EMEA) for bioanalytical methods, which include selectivity, carryover, qualitative and quantitative matrix effect, extraction recovery, process efficiency, trueness, precision, accuracy profiles, method and instrument detection limits, integrity to dilution, anticoagulant comparison and short- and long-term stabilities. Stability studies on the reduced metabolite H2-PBTZ169 have shown no significant impact on the actual PBTZ169 concentrations determined with the proposed assay. This simplified, rapid, sensitive and robust methodology has been applied to the bioanalysis of human plasma samples collected within the frame of a phase I clinical study in healthy volunteers receiving PBTZ169.

Analysis of Riboflavin Compounds in the Rabbit Cornea In Vivo.

PURPOSE: To investigate the composition and concentration of individual riboflavin compounds in the corneal stroma in vivo after soaking with various commercially available riboflavin formulations. METHODS: Experiments were performed in 26 rabbit corneas in vivo: 24 corneas were soaked with riboflavin formulations for 30 minutes or with 0.9% NaCl for control (n = 2). After treatment, corneas were excised and prepared for ultra-high-pressure liquid chromatography (UHPLC) analysis. Additionally, computational chemical analysis of riboflavin compounds and keratan sulfate were performed. RESULTS: The amount of riboflavin and riboflavin phosphate isomers in cornea decreased by a factor of 10 to 100, when compared to the amount in riboflavin formulations. In particular, we found an inverse relationship in the ratio of riboflavin to riboflavin phosphate isomer concentration between formulations and cornea. The electronegativity and ionization potential of riboflavin and phosphate isomers are different. CONCLUSIONS: The inverse relationship observed might be explained by a stronger electronegativity of the phosphate isomers, leading to a stronger repulsion by corneal proteoglycans. Indicating the individual concentration of riboflavin compounds in formulations is more representative than the total riboflavin concentration. Riboflavin formulations and CXL protocols might be improved considering the differences in diffusion and ionization potentials of the different riboflavin compounds.

New prostaglandin analog formulation for glaucoma treatment containing cyclodextrins for improved stability, solubility and ocular tolerance.

Latanoprost is a practically insoluble prostaglandin F2α analog considered a first-line agent for glaucoma treatment. From a pharmaceutical point of view, latanoprost is challenging to be formulated as an eye drop due to its poor water solubility and the presence of an ester bond that needs to be cleaved in vivo but maintained unchanged during storage. Cyclodextrins (CDs) are known to form complexes with hydrophobic drugs, influencing their stability, availability, solubility, and tolerance in a non-predictable manner. A variety of CDs including native α, ß, and γCDs as well as substituted hydroxypropylßCD, hydroxypropylγCD, dimethylßCD, sulphatedßCD, and propylaminoßCD were screened and the most appropriate CD for the formulation of latanoprost for an ocular topical application was selected. Among the tested CDs, propylaminoßCD had the best trade-off between latanoprost stability and availability, which was confirmed by its complex constant value of 3129M(-1). Phase-solubility and NMR investigations demonstrated that the propylaminoßCD effectively formed a complex involving the ester group of latanoprost providing protection to its ester bond, while ensuring proper latanoprost solubilization. Furthermore, in vivo experiments demonstrated that the latanoprost-propylaminoßCD formulation led to lower ocular irritation than the commercial latanoprost formulation used as a reference. The latanoprost-propylaminoßCD formulation was demonstrated to successfully address the main stability, solubility, and tolerance limitations of topical ocular latanoprost therapy for glaucoma.