Application of the crystalline sponge method for metabolite structure elucidation

The identification of metabolites during drug discovery and development can play a crucial role to understand their contribution to efficacy and safety. To investigate the metabolism of new drug candidates in human as early as possible, different in vitro systems from human origin are used. Standard structural elucidation tools like liquid chromatography-mass spectrometry, single-crystal X-ray diffraction or nuclear magnetic resonance spectroscopy are either limited in providing the complete structure of metabolites or require a larger amount of material not available from in vitro incubations. A new technology to overcome these limitations was introduced by Makoto Fujita in 2013 and is commonly known as the crystalline sponge method. The usage of a pre-existing crystal allows the complete crystallographic identification of small molecules at the atomic level by encapsulation of only nanogram amount of analyte material without the need for crystallization of the analyte itself. In this thesis, the structural elucidation of metabolites generated from in vitro incubations using the crystalline sponge method is described. First, the analysis of an active pharmaceutical ingredient and its known metabolites demonstrated, that the crystalline sponge technology is a valuable method to identify the complete structure of trace amounts of analytes isolated from incubation matrices. The crystallographic data provided the exact position of metabolism for phase I and phase II metabolites. The technology was further used to identify various known and unknown metabolites of the anthelmintic drug praziquantel, which has been used for treatment of the parasitic flatworm of the genus Schistosoma for several decades. Moreover, to optimize the soaking process of the crystalline sponge method, which is the key element of the technology, an affinity screening to prioritize the optimal soaking conditions for individual analytes without time-consuming X-ray diffraction measurements, was developed.Die Identifizierung von Metaboliten während der Arzneimittelforschung und -entwicklung spielt eine wichtige Rolle um deren Beteiligung an der Wirksamkeit und Unbedenklichkeit zu verstehen. Um den humanen Metabolismus von neuen Wirkstoffkandidaten so früh wie möglich zu untersuchen, werden verschiedene in vitro Systeme humanen Ursprungs verwendet. Konventionelle Strukturaufklärungsmethoden wie Flüssigchromatographie gekoppelt mit Massenspektrometrie, Einkristall-Röntgenstrukturanalyse oder Kernspinresonanzspektroskopie sind einerseits begrenzt in ihrem Informationsgehalt in Bezug auf die vollständige Metabolitenstruktur oder benötigen eine hohe Menge an Probenmaterial, welches durch in vitro Inkubationen nicht zur Verfügung steht. Eine Möglichkeit zur Überwindung dieser Einschränkungen wurde 2013 von Makoto Fujita vorgestellt und ist allgemein bekannt als Crystalline Sponge Methode. Die Verwendung eines bereits vorhandenen Kristalls erlaubt die kristallografische Bestimmung von kleinen Molekülen auf atomarer Ebene durch die Aufnahme von Nanogramm Mengen an Analytmaterial in das Kristallgitter und vermeidet dadurch die Notwendigkeit eines Einkristalls des Analyten. In der vorliegenden Dissertation wurde die Strukturaufklärung von Metaboliten aus in vitro Inkubationen unter Verwendung der Crystalline Sponge Methode beschrieben. Zunächst wurde anhand eines pharmazeutischen Wirkstoffes und seiner bekannten Metabolite veranschaulicht, dass die Crystalline Sponge Technologie eine nützliche Methode zur Bestimmung der vollständigen Struktur von geringen Mengen isolierter Analyten aus Inkubationsmatrizes darstellt. Die kristallografischen Daten bestimmten die exakte Position des Metabolismus für die vorliegenden Phase I und Phase II Metaboliten. Die Technologie wurde weiterhin zur Strukturaufklärung von bereits bekannten, sowie unbekannten Metaboliten des anthelmintischen Arzneistoffes Praziquantel verwendet, welcher seit vielen Jahrzehnten für die Behandlung des parasitären Plattwurms der Gattung Schistosoma eingesetzt wird. Um den entscheidenden Schritt der Crystalline Sponge Methode zu optimieren, wurde des Weiteren ein Affinitätsscreening zur Priorisierung der optimalen Methodenparameter für individuelle Analyten ohne Einsatz von zeitaufwendigen Röntgenkristallstrukturmessungen entwickelt

Inner ear dysfunction in caspase-3 deficient mice

<p>Abstract</p> <p>Background</p> <p>Caspase-3 is one of the most downstream enzymes activated in the apoptotic pathway. In caspase-3 deficient mice, loss of cochlear hair cells and spiral ganglion cells coincide closely with hearing loss. In contrast with the auditory system, details of the vestibular phenotype have not been characterized. Here we report the vestibular phenotype and inner ear anatomy in the caspase-3 deficient (<it>Casp3</it>^<it>-/-</it>) mouse strain.</p> <p>Results</p> <p>Average ABR thresholds of <it>Casp3</it>^{<it>-/- </it>}mice were significantly elevated (<it>P </it>< 0.05) compared to <it>Casp3</it>^{<it>+/- </it>}mice and <it>Casp3</it>^{<it>+/+ </it>}mice at 3 months of age. In DPOAE testing, distortion product 2F1-F2 was significantly decreased (<it>P </it>< 0.05) in <it>Casp3</it>^{<it>-/- </it>}mice, whereas <it>Casp3</it>^{<it>+/- </it>}and <it>Casp3</it>^{<it>+/+ </it>}mice showed normal and comparable values to each other. <it>Casp3</it>^{<it>-/- </it>}mice were hyperactive and exhibited circling behavior when excited. In lateral canal VOR testing, <it>Casp3</it>^{<it>-/- </it>}mice had minimal response to any of the stimuli tested, whereas <it>Casp3</it>^{<it>+/- </it>}mice had an intermediate response compared to <it>Casp3</it>^{<it>+/+ </it>}mice. Inner ear anatomical and histological analysis revealed gross hypomorphism of the vestibular organs, in which the main site was the anterior semicircular canal. Hair cell numbers in the anterior- and lateral crista, and utricle were significantly smaller in <it>Casp3</it>^{<it>-/- </it>}mice whereas the <it>Casp3</it>^{<it>+/- </it>}and <it>Casp3</it>^{<it>+/+ </it>}mice had normal hair cell numbers.</p> <p>Conclusions</p> <p>These results indicate that caspase-3 is essential for correct functioning of the cochlea as well as normal development and function of the vestibule.</p

Metabolic Profiling of S-praziquantel: Structure Elucidation Using the Crystalline Sponge Method in Combination with Mass Spectrometry and Nuclear Magnetic Resonance

Praziquantel (PZQ) is the drug of choice for treatment of the neglected tropical disease schistosomiasis. Although the drug has been extensively used over several decades and its metabolism well studied (several oxidative metabolites are known from literature), the knowledge of the complete structure of some of its metabolites remains elusive. Conventional techniques, such as nuclear magnetic resonance or liquid chromatography mass spectrometry were used in the past to investigate phase I and phase II metabolites of PZQ. These techniques are either limited to provide the complete molecular structure (liquid chromatography mass spectrometry) or require large amount of sample material (NMR), which are not always available when in vitro systems are used for investigation of the metabolites. In this study, we describe new structures of S-PZQ metabolites generated in vitro from human liver microsomes using the crystalline sponge method. After chromatographic separation and purification of the oxidative metabolites, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis was conducted to narrow down the position of oxidation to a certain part of the molecule. To determine the exact position of hydroxylation, singe-crystal X-ray diffraction analysis of the crystalline sponges and absorbed analyte was used to identify the structure of S-PZQ and its metabolites. The crystalline sponge method allowed for complete structure elucidation of the known metabolites S-trans-4'-hydroxy-PZQ (M1), S-cis-4'-hydroxy-PZQ (M2) and S-/R-11b-hydroxy-PZQ (M6) as well as the unknown metabolites S-9-hydroxy-PZQ (M3) and S-7-hydroxy-S-PZQ (M4). For comparison of structural elucidation techniques, one metabolite (M3) was additionally analyzed using NMR. SIGNIFICANCE STATEMENT: The information content of the metabolic pathway of praziquantel is still limited. The crystalline sponge method allowed the complete structural elucidation of three known and two unknown metabolites of S-praziquantel, using only trace amounts of analyte material, as demonstrated in this study

Revision of the absolute configurations of chelocardin and amidochelocardin

Even with the aid of the available methods, the configurational assignment of natural products can be a challenging task that is prone to errors, and it sometimes needs to be corrected after total synthesis or single-crystal X-ray diffraction (XRD) analysis. Herein, the absolute configuration of amidochelocardin is revised using a combination of XRD, NMR spectroscopy, experimental ECD spectra, and time-dependent density-functional theory (TDDFT)-ECD calculations. As amidochelocardin was obtained via biosynthetic engineering of chelocardin, we propose the same absolute configuration for chelocardin based on the similar biosynthetic origins of the two compounds and result of TDDFT-ECD calculations. The evaluation of spectral data of two closely related analogues, 6-desmethyl-chelocardin and its semisynthetic derivative 1, also supports this conclusion

Dry Sliding-Friction and Wear Behavior of Hot-Extruded Al6061/Si3N4/Cf Hybrid Metal Matrix Composite.

The effects of reinforcement addition and hot extrusion on the microstructures, micro hardness, friction, and wear behavior of aluminium (Al) hybrid composite were investigated. Al6061 dispersed with electroless nickel-coated Si3N4 (6wt.%) and copper-coated carbon fiber (Cf) (1wt.%) hybrid composites was developed through stir casting followed by hot extrusion. Optical micro structural studies confirmed that the size of reinforcements decreased, and their orientations were in the extrusion direction. The decrease in the grain size (29%) of hybrid composites was larger than that in the grain size of matrix alloys under hot-extruded conditions. The synthesized hot-extruded Al6061 hybrid composite exhibited a lower coefficient of friction (51%) and high wear resistance (39%) compared with the hotextruded Al6061base alloy

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P &lt; 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Crystalline sponges as a sensitive and fast method for metabolite identification: Application to gemfibrozil and its phase I and II metabolites

Understanding the metabolism of new drug candidates is important during drug discovery and development, as circulating metabolites may contribute to efficacy or cause safety issues. In the early phase of drug discovery, human in vitro systems are used to investigate human relevant metabolism. Though conventional techniques are limited in their ability to provide complete molecular structures of metabolites (liquid chromatography mass spectrometry) or require a larger amount of material not available from in vitro incubation (nuclear magnetic resonance), we here report for the first time the use of the crystalline sponge method to identify phase I and phase II metabolites generated from in vitro liver microsomes or S9 fractions. Gemfibrozil was used as a test compound. Metabolites generated from incubation with microsomes or S9 fractions, were fractionated using online fraction collection. After chromatographic purification and fractionation of the generated metabolites, single crystal X-ray diffraction of crystalline sponges was used to identify the structure of gemfibrozil metabolites. This technique allowed for complete structure elucidation of 5'-CH2OH gemfibrozil (M1), 4'-OH gemfibrozil (M2), 5'-COOH gemfibrozil (M3), and the acyl glucuronide of gemfibrozil, 1-O-β-glucuronide (M4), the first acyl glucuronide available in the Cambridge Crystallographic Data Centre. Our study shows that when optimal soaking is possible, crystalline sponges technology is a sensitive (nanogram amount) and fast (few days) method that can be applied early in drug discovery to identify the structure of pure metabolites from in vitro incubations. SIGNIFICANCE STATEMENT: Complete structure elucidation of human metabolites plays a critical role in early drug discovery. Low amounts of material (nanogram) are only available at this stage and insufficient for nuclear magnetic resonance analysis. The crystalline sponge method has the potential to close this gap, as demonstrated in this study

Preoperative interleukin-22 values add valuable information for outcome prediction following orthotopic liver transplantation: a preliminary study

BACKGROUND: Recent findings support the idea that interleukin (IL)-22 serum levels are related to disease severity in end-stage liver disease. Existing scoring systems--Model for End-Stage Liver Disease (MELD), Survival Outcomes Following Liver Transplantation (SOFT) and Pre-allocation-SOFT (P-SOFT)--are well-established in appraising survival rates with or without liver transplantation. We tested the hypothesis that IL-22 serum levels at transplantation date correlate with survival and potentially have value as a predictive factor for survival. MATERIAL AND METHODS: MELD, SOFT, and P-SOFT scores were calculated to estimate post-transplantation survival. Serum levels of IL-22, IL-6, IL-10, C-reactive protein (CRP), and procalcitonin (PCT) were collected prior to transplantation in 41 patients. Outcomes were assessed at 3 months, 1 year, and 3 years after transplantation. RESULTS: IL-22 significantly correlated with MELD, P-SOFT, and SOFT scores (Rs 0.35, 0.63, 0.56 respectively, p<0.05) and with the discrimination in post-transplantation survival. IL-6 showed a heterogeneous pattern (Rs 0.40, 0.63, 0.57, respectively, p<0.05); CRP and PCT did not correlate. We therefore added IL-22 serum values to existing scoring systems in a generalized linear model (GLM), resulting in a significantly improved outcome prediction in 58% of the cases for both the P-SOFT (p<0.01) and SOFT scores (p<0.001). CONCLUSIONS: Further studies are needed to address the concept that IL-22 serum values at the time of transplantation provide valuable information about survival rates following orthotopic liver transplantation