Pharmacokinetic Effects of Different Models of Nonalcoholic Fatty Liver Disease in Transgenic Humanized OATP1B Mice.

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 (collectively, OATP1B) transporters encoded by the solute carrier organic anion transporter (SLCO) genes mediate uptake of multiple pharmaceutical compounds. Nonalcoholic steatohepatitis (NASH), a severe form of nonalcoholic fatty liver disease (NAFLD), decreases OATP1B abundance. This research characterized the pathologic and pharmacokinetics effects of three diet- and one chemical-induced NAFLD model in male and female humanized OATP1B mice, which comprises knock-out of rodent Oatp orthologs and insertion of human SLCO1B1 and SLCO1B3. Histopathology scoring demonstrated elevated steatosis and inflammation scores for all NAFLD-treatment groups. Female mice had minor changes in SLCO1B1 expression in two of the four NAFLD treatment groups, and pitavastatin (PIT) area under the concentration-time curve (AUC) increased in female mice in only one of the diet-induced models. OATP1B3 expression decreased in male and female mice in the chemical-induced NAFLD model, with a coinciding increase in PIT AUC, indicating the chemical-induced model may better replicate changes in OATP1B3 expression and OATP substrate disposition observed in NASH patients. This research also tested a reported multifactorial pharmacokinetic interaction between NAFLD and silymarin, an extract from milk thistle seeds with notable OATP-inhibitory effects. Males showed no change in PIT AUC, whereas female PIT AUC increased 1.55-fold from the diet alone and the 1.88-fold from the combination of diet with silymarin, suggesting that female mice are more sensitive to pharmacokinetic changes than male mice. Overall, the humanized OATP1B model should be used with caution for modeling NAFLD and multifactorial pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Advanced stages of NAFLD cause decreased hepatic OATP1B abundance and increase systemic exposure to OATP substrates in human patients. The humanized OATP1B mouse strain may provide a clinically relevant model to recapitulate these observations and predict pharmacokinetic interactions in NAFLD. This research characterized three diet-induced and one drug-induced NAFLD model in a humanized OATP1B mouse model. Additionally, a multifactorial pharmacokinetic interaction was observed between silymarin and NAFLD.

Rifampin- and Silymarin-Mediated Pharmacokinetic Interactions of Exogenous and Endogenous Substrates in a Transgenic OATP1B Mouse Model.

Organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3, encoded by the SLCO gene family of the solute carrier superfamily, are involved in the disposition of many exogenous and endogenous compounds. Preclinical rodent models help assess risks of pharmacokinetic interactions, but interspecies differences in transporter orthologs and expression limit direct clinical translation. An OATP1B transgenic mouse model comprising a rodent Slco1a/1b gene cluster knockout and human SLCO1B1 and SLCO1B3 gene insertions provides a potential physiologically relevant preclinical tool to predict pharmacokinetic interactions. Pharmacokinetics of exogenous probe substrates, pitavastatin and pravastatin, and endogenous OATP1B biomarkers, coproporphyrin-I and coproporphyrin-III, were determined in the presence and absence of known OATP/Oatp inhibitors, rifampin or silymarin (an extract of milk thistle [Silybum marianum]), in wild-type FVB mice and humanized OATP1B mice. Rifampin increased exposure of pitavastatin (4.6- and 2.8-fold), pravastatin (3.6- and 2.2-fold), and coproporphyrin-III (1.6- and 2.1-fold) in FVB and OATP1B mice, respectively, but increased coproporphyrin-I AUC0-24h only (1.8-fold) in the OATP1B mice. Silymarin did not significantly affect substrate AUC, likely because the silymarin flavonolignan concentrations were at or below their reported IC50 values for the relevant OATPs/Oatps. Silymarin increased the Cmax of pitavastatin 2.7-fold and pravastatin 1.9-fold in the OATP1B mice. The data of the OATP1B mice were similar to those of the pitavastatin and pravastatin clinical data; however, the FVB mice data more closely recapitulated pitavastatin clinical data than the data of the OATP1B mice, suggesting that the OATP1B mice are a reasonable, though costly, preclinical strain for predicting pharmacokinetic interactions when doses are optimized to achieve clinically relevant plasma concentrations.

Green Tea Catechins Decrease Solubility of Raloxifene In Vitro and Its Systemic Exposure in Mice.

PURPOSE: Green tea is a widely consumed beverage. A recent clinical study reported green tea decreased systemic exposure of raloxifene and its glucuronide metabolites by 34-43%. However, the underlying mechanism(s) remains unknown. This study investigated a change in raloxifene's solubility as the responsible mechanism. METHODS: The effects of green tea extract, (-)-epigallocatechin gallate (EGCG), and (-)-epigallocatechin (EGC) on raloxifene's solubility were assessed in fasted state simulated intestinal fluids (FaSSIF) and fed state simulated intestinal fluids (FeSSIF). EGCG and EGC represent green tea's main bioactive constituents, flavan-3-gallate and flavan-3-ol catechins respectively, and the tested concentrations (mM) match the µg/mg of each compound in the extract. Our mouse study (n = 5/time point) evaluated the effect of green tea extract and EGCG on the systemic exposure of raloxifene. RESULTS: EGCG (1 mM) and EGC (1.27 mM) decreased raloxifene's solubility in FaSSIF by 78% and 13%, respectively. Micelle size in FaSSIF increased with increasing EGCG concentrations (> 1000% at 1 mM), whereas EGC (1.27 mM) did not change micelle size. We observed 3.4-fold higher raloxifene solubility in FeSSIF compared to FaSSIF, and neither green tea extract nor EGCG significantly affected raloxifene solubility or micelle size in FeSSIF. The mice study showed that green tea extract significantly decreased raloxifene Cmax by 44%, whereas EGCG had no effect. Green tea extract and EGCG did not affect the AUC0-24 h of raloxifene or the metabolite-to-parent AUC ratio. CONCLUSIONS: This study demonstrated flavan-3-gallate catechins may decrease solubility of poorly water-soluble drugs such as raloxifene, particularly in the fasted state.

Semisynthesis, Characterization, and Biological Evaluation of Fluorinated Analogues of the Spirobisnaphthalene, Diepoxin-η.

Diepoxin-η (1) is a cytotoxic fungal metabolite belonging to the spirobisnaphthalene structural class. In this study, four mono fluorinated analogues (2-5) of diepoxin-η (1) were semisynthesized in a single-step by selectively fluorinating the naphthalene moiety with Selectfluor. The structures of 2-5 were elucidated using a set of spectroscopic and spectrometric techniques and were further confirmed by means of TDDFT-ECD and isotropic shielding tensors calculations. Compounds 2-5 showed equipotent cytotoxic activity to 1 when tested against OVCAR3 (ovarian) and MDA-MB-435 (melanoma) cancer cell lines with IC50 values that range from 5.7-8.2 µM.

Goldenseal-Mediated Inhibition of Intestinal Uptake Transporters Decreases Metformin Systemic Exposure in Mice.

Goldenseal is a perennial plant native to eastern North America. A recent clinical study reported goldenseal decreased metformin Cmax and area under the blood concentration versus time curve (AUC) by 27% and 23%, respectively, but half-life and renal clearance were unchanged. These observations suggested goldenseal altered processes involved in metformin absorption. The underlying mechanism(s) remain(s) unknown. One mechanism for the decreased metformin systemic exposure is inhibition by goldenseal of intestinal uptake transporters involved in metformin absorption. Goldenseal extract and three goldenseal alkaloids (berberine, (-)-ß-hydrastine, hydrastinine) were tested as inhibitors of organic cation transporter (OCT) 3, plasma membrane monoamine transporter (PMAT), and thiamine transporter (THTR) 2 using human embryonic kidney 293 cells overexpressing each transporter. The goldenseal extract, normalized to berberine content, was the strongest inhibitor of each transporter (IC50: 4.9, 13.1, and 5.8 µM for OCT3, PMAT, and THTR2, respectively). A pharmacokinetic study in mice compared the effects of berberine, (-)-ß-hydrastine, goldenseal extract, and imatinib (OCT inhibitor) on orally administered metformin. Goldenseal extract and imatinib significantly decreased metformin Cmax by 31% and 25%, respectively, and had no effect on half-life. Berberine and (-)-ß-hydrastine had no effect on metformin pharmacokinetics, indicating neither alkaloid alone precipitated the interaction in vivo. A follow-up murine study involving intravenous metformin and oral inhibitors examined the contributions of basolateral enteric/hepatic uptake transporters to the goldenseal-metformin interaction. Goldenseal extract and imatinib had no effect on metformin AUC and half-life, suggesting lack of inhibition of basolateral enteric/hepatic uptake transporters. Results may have implications for patients taking goldenseal with drugs that are substrates for OCT3 and THTR2. SIGNIFICANCE STATEMENT: Goldenseal is used to self-treat respiratory infections and digestive disorders. We investigated potential mechanisms for the clinical pharmacokinetic interaction observed between goldenseal and metformin, specifically inhibition by goldenseal of intestinal uptake transporters (OCT3, PMAT, THTR2) involved in metformin absorption. Goldenseal extract inhibited all three transporters in vitro and decreased metformin systemic exposure in mice. These data may have broader implications for patients co-consuming goldenseal with other drugs that are substrates for these transporters.

Evaluating the Antioxidant Properties of the Ancient-Crop Tef (Eragrostis tef) Grain Extracts in THP-1 Monocytes.

Tef (Eragrostis tef) is an orphan crop that is widely grown in East Africa, primarily in Ethiopia as a staple crop. It is becoming popular in the Western world owing to its nutritious and gluten-free grains and the forage quality of its biomass. Tef is also considered to have a high antioxidant capacity based on cell-free studies. However, the antioxidant activity of tef has never been validated using a physiologically relevant cell model. The purpose of this study was to investigate the antioxidant capacity of tef grain extracts using a mammalian cell model. We hypothesized that the tef grain extracts are capable of modulating the cellular antioxidant response via the modulation of glutathione (GSH) biosynthetic pathways. Therefore, we evaluated the antioxidant activity of purified tef grain extracts in the human acute monocytic leukemia (THP-1) cell line. Our findings revealed that the organic fraction of grain extracts increased the cellular GSH level, which was more evident for brown-colored tef than the ivory variety. Moreover, a brown-tef fraction increased the expressions of GSH-pathway genes, including γ-glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits and glutathione reductase (GR), an enzyme that plays a key role in GSH biosynthesis, suggesting that tef extracts may modulate GSH metabolism. Several compounds were uniquely identified via mass spectrometry (MS) in GSH-modulating brown-tef samples, including 4-oxo-ß-apo-13-carotenone, γ-linolenic acid (methyl ester), 4,4'-(2,3-dimethyl-1,4-butanediyl)bis-phenol (also referred to as 8,8'-lignan-4,4'-diol), and (3ß)-3-[[2-[4-(Acetylamino)phenoxy]acetyl]oxy]olean-12-en-28-oic acid. Tef possesses antioxidant activity due to the presence of phytochemicals that can act as direct antioxidants, as well as modulators of antioxidant-response genes, indicating its potential role in alleviating diseases triggered by oxidative stresses. To the best of our knowledge, this is the first report revealing the antioxidant ability of tef extracts in a physiologically relevant human cell model.

What was old is new again: Phenotypic screening of a unique fungal library yields pyridoxatin, a promising lead against extensively resistant Acinetobacter baumannii (AB5075).

Due to the emergence of resistance, the World Health Organization considers Gram-negative pathogen Acinetobacter baumannii a top priority for therapeutic development. Using this priority pathogen and a phenotypic, agar plate-based assay, a unique library of extracts from 2,500 diverse fungi was screened for antimicrobial activity against a highly virulent, drug-resistant strain of A. baumannii (AB5075). The most potent hit from this screen was an extract from the fungus Tolypocladium sp., which was found to produce pyridoxatin. Another active extract from the fungi Trichoderma deliquescens was characterized and yielded trichokonin VII and trichokonin VIII. Evaluation of pyridoxatin against A. baumannii (AB5075) in a broth microdilution assay revealed a minimum inhibitory concentration (MIC) of 38 µM, compared to the known antibiotic levofloxacin with MIC of 28 µM. Mass spectrometry, Marfey's analysis and nuclear magnetic resonance spectroscopy analyses confirmed the structures of trichokonins VII and VIII to be consistent with previous reports. In an in vivo Galleria mellonella model, pyridoxatin tested at 150 mg/kg exhibited minimal toxicity (90% survival) and promising antimicrobial efficacy (50% survival) after 5 days. Trichokonins VII and VIII tested at 150 mg/kg were toxic to G. mellonella, with 20% survival and 40% survival after 5 days, respectively. The findings of this project suggest that pyridoxatin may serve as a lead compound for the development of antimicrobials against A. baumannii. They also demonstrate the value of the phenotypic screening approach employed herein.

A Widely Distributed Biosynthetic Cassette Is Responsible for Diverse Plant Side Chain Cross-Linked Cyclopeptides.

Cyclopeptide alkaloids are an abundant class of plant cyclopeptides with over 200 analogs described and bioactivities ranging from analgesic to antiviral. While these natural products have been known for decades, their biosynthetic basis remains unclear. Using a transcriptome-mining approach, we link the cyclopeptide alkaloids from Ceanothus americanus to dedicated RiPP precursor peptides and identify new, widely distributed split BURP peptide cyclase containing gene clusters. Guided by our bioinformatic analysis, we identify and isolate new cyclopeptides from Coffea arabica, which we named arabipeptins. Reconstitution of the enzyme activity for the BURP found in the biosynthesis of arabipeptin A validates the activity of the newly discovered split BURP peptide cyclases. These results expand our understanding of the biosynthetic pathways responsible for diverse cyclic plant peptides and suggest that these side chain cross-link modifications are widely distributed in eudicots.

Hepatic organic anion transporting polypeptides mediate disposition of milk thistle flavonolignans and pharmacokinetic silymarin-drug interactions.

Silybum marianum (L.) Gaertn. (Asteraceae), commonly known as milk thistle, is a botanical natural product used to self-treat multiple diseases such as Type 2 diabetes mellitus and nonalcoholic steatohepatitis (NASH). An extract from milk thistle seeds (achenes), termed silymarin, is comprised primarily of several flavonolignans. Systemic concentrations of these flavonolignans can influence the potential biologic effects of silymarin and the risk for pharmacokinetic silymarin-drug interactions. The aims of this research were to determine the roles of organic anion transporting polypeptides (OATPs/Oatps) in silymarin flavonolignan disposition and in pharmacokinetic silymarin-drug interactions. The seven major flavonolignans from silymarin were determined to be substrates for OATP1B1, OATP1B3, and OATP2B1. Sprague Dawley rats were fed either a control diet or a NASH-inducing diet and administered pitavastatin (OATP/Oatp probe substrate), followed by silymarin via oral gavage. Decreased protein expression of Oatp1b2 and Oatp1a4 in NASH animals increased flavonolignan area under the plasma concentration-time curve (AUC) and maximum plasma concentration. The combination of silymarin inhibition of Oatps and NASH-associated decrease in Oatp expression caused an additive increase in plasma pitavastatin AUC in the animals. These data indicate that OATPs/Oatps contribute to flavonolignan cellular uptake and mediate the interaction between silymarin and NASH on pitavastatin systemic exposure.

Delivery of eupenifeldin via polymer-coated surgical buttresses prevents local lung cancer recurrence.

Lung cancer is the leading cause of cancer deaths worldwide. Unfortunately, high recurrence rates and poor survival remain despite surgical resection and conventional chemotherapy. Local drug delivery systems are a promising intervention for lung cancer treatment with the potential for improved efficacy with reduced systemic toxicity. Here, we describe the development of a chemotherapy-loaded polymer buttress, to be implanted along the surgical margin at the time of tumor resection, for achieving local and prolonged release of a new anticancer agent, eupenifeldin. We prepared five different formulations of buttresses with varying amounts of eupenifeldin, and additional external empty polymer coating layers (or thicknesses) to modulate drug release. The in vitro eupenifeldin release profile depends on the number of external coating layers with the formulation of the greatest thickness demonstrating a prolonged release approaching 90 days. Similarly, the long-term cytotoxicity of eupenifeldin-loaded buttress formulations against murine Lewis lung carcinoma (LLC) and human lung carcinoma (A549) cell lines mirrors the eupenifeldin release profiles and shows a prolonged cytotoxic effect. Eupenifeldin-loaded buttresses significantly decrease local tumor recurrence in vivo and increase disease-free survival in a lung cancer resection model.

The Chemistry of Kratom [Mitragyna speciosa]: Updated Characterization Data and Methods to Elucidate Indole and Oxindole Alkaloids.

Two separate commercial products of kratom [Mitragyna speciosa (Korth.) Havil. Rubiaceae] were used to generate reference standards of its indole and oxindole alkaloids. While kratom has been studied for over a century, the characterization data in the literature for many of the alkaloids are either incomplete or inconsistent with modern standards. As such, full 1H and 13C NMR spectra, along with HRESIMS and ECD data, are reported for alkaloids 1-19. Of these, four new alkaloids (7, 11, 17, and 18) were characterized using 2D NMR data, and the absolute configurations of 7, 17, and 18 were established by comparison of experimental and calculated ECD spectra. The absolute configuration for the N(4)-oxide (11) was established by comparison of NMR and ECD spectra of its reduced product with those for compound 7. In total, 19 alkaloids were characterized, including the indole alkaloid mitragynine (1) and its diastereoisomers speciociliatine (2), speciogynine (3), and mitraciliatine (4); the indole alkaloid paynantheine (5) and its diastereoisomers isopaynantheine (6) and epiallo-isopaynantheine (7); the N(4)-oxides mitragynine-N(4)-oxide (8), speciociliatine-N(4)-oxide (9), isopaynantheine-N(4)-oxide (10), and epiallo-isopaynantheine-N(4)-oxide (11); the 9-hydroxylated oxindole alkaloids speciofoline (12), isorotundifoleine (13), and isospeciofoleine (14); and the 9-unsubstituted oxindoles corynoxine A (15), corynoxine B (16), 3-epirhynchophylline (17), 3-epicorynoxine B (18), and corynoxeine (19). With the ability to analyze the spectroscopic data of all of these compounds concomitantly, a decision tree was developed to differentiate these kratom alkaloids based on a few key chemical shifts in the 1H and/or 13C NMR spectra.

Drug Leads from Endophytic Fungi: Lessons Learned via Scaled Production.

Recently, the isolation and elucidation of a series of polyhydroxyanthraquinones were reported from an organic extract of a solid phase culture of an endophytic fungus, Penicillium restrictum (strain G85). One of these compounds, ω-hydroxyemodin (1: ), showed promising quorum-sensing inhibition against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) in both in vitro and in vivo models. The initial supply of 1: was 19 mg, and this amount needed to be scaled by a factor of 30 to 50 times, in order to generate material for further in vivo studies. To do so, improvements were implemented to enhance both the fermentation of the fungal culture and the isolation of this compound, with the target of generating > 800 mg of study materials in a period of 13 wk. Valuable insights, both regarding chemistry and mycology, were gained during the targeted production of 1: on the laboratory-scale. In addition, methods were modified to make the process more environmentally friendly by judicious choice of solvents, implementing procedures for solvent recycling, and minimizing the use of halogenated solvents.

Delitpyrones: α-Pyrone Derivatives from a Freshwater Delitschia sp.

In research focused on the discovery of new chemical diversity from freshwater fungi, a peak library was built and evaluated against a prostate cancer cell line, E006AA-hT, which was derived from an African American, as this population is disproportionately affected by prostate cancer. The chemical study of the bioactive sample accessioned as G858 (Delitschia sp.) led to the isolation of eight new α-pyrone derivatives (1:  - 7: , and 11: ), as well as the new 3S*,4S*-7-ethyl-4,8-dihydroxy-3,6-dimethoxy-3,4-dihydronaphthalen-1(2H)-one (15: ). In addition, the known compounds 5-(3-S-hydroxybutyl)-4-methoxy-6-methyl-2H-pyran-2-one (8: ), 5-(3-oxobutyl)-4-methoxy-6-methyl-2H-pyran-2-one (9: ), pyrenocine I (10: ), 5-butyl-6-(hydroxymethyl)-4-methoxy-2H-pyran-2-one (12: ), sporidesmin A (13: ), 6-ethyl-2,7-dimethoxyjuglone (14: ), artrichitin (16: ), and lipopeptide 15G256ε (17: ) were also obtained. The structures of the new compounds were elucidated using a set of spectroscopic (NMR) and spectrometric (HRMS) methods. The absolute configuration of the most abundant member of each subclass of compounds was assigned through a modified Mosher's ester method. For 15: , the relative configuration was assigned based on analysis of 3 J values. Compounds 1, 2, 5:  - 14, 16: , and 17: were evaluated against the cancer cell line E006AA-hT under hypoxic conditions, where compound 13: inhibited cell proliferation at a concentration of 2.5 µM.

Silymarin Prevents Memory Impairments, Anxiety, and Depressive-Like Symptoms in a Rat Model of Post-Traumatic Stress Disorder.

Post-traumatic stress disorder (PTSD) is a debilitating psychopathological disease that is triggered by exposure to traumatic events. It is usually associated with substantial comorbidities, such as cognitive impairment, anxiety, and depression. Silymarin has been recently reported to exert neuroprotective activities against neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Herein, the beneficial effects of silymarin in ameliorating PTSD-like symptoms such as memory impairments, anxiety, and depression were evaluated using a single-prolonged stress (SPS) rat model of PTSD. Male Wistar rats were randomly assigned into four groups: control, silymarin, SPS, or SPS + silymarin. Rats were administrated silymarin, 100 mg/kg i. p. for 4 wk. Rats in all groups were tested for short- and long-term memory in the radial arm water maze (RAWM), for anxiety-like behaviors using the open field test (OFT) and elevated plus maze (EPM) test, and for depression-like symptoms using the tail suspension test (TST). Conventional analyses of the RAWM, EPM, OFT, and TST were conducted using analysis of variance. Additionally, the anxiety-related behavior parameters of the EPM and OFT were entered to principal component analysis. Regression scores based on the first two extracted components, which accounted for 61% of the variance, were indicative of the anxiolytic activity of silymarin. Collectively, the results suggest that silymarin treatment prevents SPS-induced long-term memory impairments, anxiety, and depressive-like symptoms in rat models.

Pheophorbide Derivatives Isolated from Açaí Berries (Euterpea oleracea) Activate an Antioxidant Response Element In Vitro.

Activity-guided fractionation was used to isolate and identify two components of the Brazilian açaí berry (Euterpe oleracea Mart.) with the ability to induce antioxidant response element (ARE)-dependent gene transcription in human hepatoma (HepG2) cells. Using an ARE-Luciferase reporter construct in cultured HepG2 cells, a suite of fractions from dried and powdered açaí berries were evaluated for transcriptional up-regulation of the luciferase gene. Active fractions were further refined until several pure compounds were isolated and identified. These compounds belong to the pheophorbide class of molecules, and are composed of the methyl and ethyl esters of the parent pheophorbide A, all of which are classified as photosensitizers. Using standard pheophorbides, dose response studies were carried out, and ARE-activation could be observed at concentrations as low as 8.2 µM and 16.9 µM for pheophorbide A methyl ester and pheophorbide A, respectively. These studies not only suggest a possible source of antioxidant properties for the açaí berry, but may also explain the recently identified photosensitizing abilities of açaí products as well.

Conventional and accelerated-solvent extractions of green tea (camellia sinensis) for metabolomics-based chemometrics.

Metabolomics has emerged as an important analytical technique for multiple applications. The value of information obtained from metabolomics analysis depends on the degree to which the entire metabolome is present and the reliability of sample treatment to ensure reproducibility across the study. The purpose of this study was to compare methods of preparing complex botanical extract samples prior to metabolomics profiling. Two extraction methodologies, accelerated solvent extraction and a conventional solvent maceration, were compared using commercial green tea [Camellia sinensis (L.) Kuntze (Theaceae)] products as a test case. The accelerated solvent protocol was first evaluated to ascertain critical factors influencing extraction using a D-optimal experimental design study. The accelerated solvent and conventional extraction methods yielded similar metabolite profiles for the green tea samples studied. The accelerated solvent extraction yielded higher total amounts of extracted catechins, was more reproducible, and required less active bench time to prepare the samples. This study demonstrates the effectiveness of accelerated solvent as an efficient methodology for metabolomics studies.

Biosynthesis of Fluorinated Peptaibols Using a Site-Directed Building Block Incorporation Approach.

Synthetic biological approaches, such as site-directed biosynthesis, have contributed to the expansion of the chemical space of natural products, making possible the biosynthesis of unnatural metabolites that otherwise would be difficult to access. Such methods may allow the incorporation of fluorine, an atom rarely found in nature, into complex secondary metabolites. Organofluorine compounds and secondary metabolites have both played pivotal roles in the development of drugs; however, their discovery and development are often via nonintersecting tracks. In this context, we used the biosynthetic machinery of Trichoderma arundinaceum (strain MSX70741) to incorporate a fluorine atom into peptaibol-type molecules in a site-selective manner. Thus, fermentation of strain MSX70741 in media containing ortho- and meta-F-phenylalanine resulted in the biosynthesis of two new fluorine-containing alamethicin F50 derivatives. The fluorinated products were characterized using spectroscopic (1D and 2D NMR, including 19F) and spectrometric (HRESIMS/MSn) methods, and their absolute configurations were established by Marfey's analysis. Fluorine-containing alamethicin F50 derivatives exhibited potency analogous to the nonfluorinated parent when evaluated against a panel of human cancer cell lines. Importantly, the biosynthesis of fluorinated alamethicin F50 derivatives by strain MSX70741 was monitored in situ using a droplet-liquid microjunction-surface sampling probe coupled to a hyphenated system.

Comparison of Metabolomics Approaches for Evaluating the Variability of Complex Botanical Preparations: Green Tea (Camellia sinensis) as a Case Study.

A challenge that must be addressed when conducting studies with complex natural products is how to evaluate their complexity and variability. Traditional methods of quantifying a single or a small range of metabolites may not capture the full chemical complexity of multiple samples. Different metabolomics approaches were evaluated to discern how they facilitated comparison of the chemical composition of commercial green tea [Camellia sinensis (L.) Kuntze] products, with the goal of capturing the variability of commercially used products and selecting representative products for in vitro or clinical evaluation. Three metabolomic-related methods-untargeted ultraperformance liquid chromatography-mass spectrometry (UPLC-MS), targeted UPLC-MS, and untargeted, quantitative 1HNMR-were employed to characterize 34 commercially available green tea samples. Of these methods, untargeted UPLC-MS was most effective at discriminating between green tea, green tea supplement, and non-green-tea products. A method using reproduced correlation coefficients calculated from principal component analysis models was developed to quantitatively compare differences among samples. The obtained results demonstrated the utility of metabolomics employing UPLC-MS data for evaluating similarities and differences between complex botanical products.

Prealamethicin F50 and related peptaibols from Trichoderma arundinaceum: Validation of their authenticity via in situ chemical analysis.

In the field of natural products chemistry, a common question pertains to the authenticity of an isolated compound, i.e. are the interesting side chains biosynthesized naturally or an artefact of the isolation/purification processes? The droplet-liquid microjunction-surface sampling probe (droplet-LMJ-SSP) coupled to a hyphenated system (UPLC-UV-HRESIMS) empowers the analysis of natural product sources in situ, providing data on the biosynthetic timing and spatial distribution of secondary metabolites. In this study the droplet-LMJ-SSP was utilized to validate the authenticity of two new peptaibols (2 and 3) as biosynthesized secondary metabolites, even though both them had structural features that could be perceived as artefacts. Compounds 2 and 3 were isolated from the scaled up fermentation of Trichoderma arundinaceum (strain MSX70741), along with a new member of the trichobrevin BIII complex (1), and four known compounds (4-7). The structures of the isolates were established using a set of spectroscopic and spectrometric methods, and their absolute configurations were determined by Marfey's analysis. The cytotoxic activity of compounds 1, 3, 4 and 6 was evaluated against a panel of cancer cell lines, where cytotoxic activity in the single digit µM range was observed.

Synthesis of poly(1,2-glycerol carbonate)-paclitaxel conjugates and their utility as a single high-dose replacement for multi-dose treatment regimens in peritoneal cancer.

Current chemotherapeutic dosing strategies are limited by the toxicity of anticancer agents and therefore rely on multiple low-dose administrations. As an alternative, we describe a novel sustained-release, biodegradable polymeric nanocarrier as a single administration replacement of multi-dose paclitaxel (PTX) treatment regimens. The first synthesis of poly(1,2-glycerol carbonate)-graft-succinic acid-paclitaxel (PGC-PTX) is described, and its use enables high, controlled PTX loadings of up to 74 wt%. Moreover, the polymer backbone is composed of biocompatible building blocks-glycerol and carbon dioxide. When formulated as nanoparticles (NPs), PGC-PTX NPs exhibit PTX concentrations >15 mg mL-1, sub-100 nm diameters, narrow dispersity, storage stability for up to 6 months, and sustained and controlled PTX release kinetics over an extended period of 70 days. A safely administered single dose of PGC-PTX NPs contains more PTX than the median lethal dose of standard PTX. In murine models of peritoneal carcinomatosis, in which the clinical implementation of multi-dose intraperitoneal (IP) treatment regimens is limited by catheter-related complications, PGC-PTX NPs exhibit improved safety at high doses, tumor localization, and efficacy even after a single IP injection, with comparable curative effect to PTX administered as a multi-dose IP treatment regimen.