Kinetic Characteristics of Curcumin and Germacrone in Rat and Human Liver Microsomes: Involvement of CYP Enzymes

Curcumin and germacrone, natural products present in the Zingiberaceae family of plants, have several biological properties. Among these properties, the anti-NSCLC cancer action is noteworthy. In this paper, kinetics of the two compounds in rat liver microsomes (RLMs), human liver microsomes (HLMs), and cytochrome P450 (CYP) enzymes (CYP3A4, 1A2, 2E1, and 2C19) in an NADPH-generating system in vitro were evaluated by UP-HPLC–MS/MS (ultrahigh-pressure liquid chromatography–tandem mass spectrometry). The contents of four cytochrome P450 (CYP) enzymes, adjusting by the compounds were detected using Western blotting in vitro and in vivo. The t_1/2 of curcumin was 22.35 min in RLMs and 173.28 min in HLMs, while 18.02 and 16.37 min were gained for germacrone. The V_max of curcumin in RLMs was about 4-fold in HLMs, meanwhile, the V_max of germacrone in RLMs was similar to that of HLMs. The single enzyme t1/2 of curcumin was 38.51 min in CYP3A4, 301.4 min in 1A2, 69.31 min in 2E1, 63.01 min in 2C19; besides, as to the same enzymes, t1/2 of germacrone was 36.48 min, 86.64 min, 69.31 min, and 57.76 min. The dynamic curves were obtained by reasonable experimental design and the metabolism of curcumin and germacrone were selected in RLMs/HLMs. The selectivities in the two liver microsomes differed in degradation performance. These results meant that we should pay more attention to drugs in clinical medication–drug and drug–enzyme interactions.     

A Drug–Drug Multicomponent Crystal of Metformin and Dobesilate: Crystal Structure Analysis and Hygroscopicity Property

A drug–drug multicomponent crystal consisting of metformin (MET) and dobesilate (DBS) was prospectively connected by solvent cooling and evaporating co-crystallization using the multicomponent crystal strategy, not only to optimize the physicochemical properties of single drugs, but also to play a role in the cooperative effect of DBS with the potential vascular protective effects of MET against diabetic retinopathy (DR). The crystal structure analysis demonstrated that MET and DBS were coupled in a 3D supramolecular structure connected by hydrogen-bonding interactions with a molar ratio of 1:1. Almost all hydrogen bond donors and receptors of MET and DBS participated in the bonding, which hindered the combination of remaining potential hydrogen bond sites and water molecules, resulting in a lower hygroscopicity property than MET alone.     

In Vitro Investigation of the Impact of Bacterial–Fungal Interaction on Carbapenem-Resistant Klebsiella pneumoniae

Fungal–bacterial co-culturing is a potential technique for the production of secondary metabolites with antibacterial activity. Twenty-nine fungal species were screened in a co-culture with carbapenem-resistant Klebsiella pneumoniae at different temperatures. A temperature of 37 ° showed inhibition of bacterial growth. Antimicrobial susceptibility testing for K. pneumoniae was conducted to compare antibiotic resistance patterns before and after the co-culture. Genotypic comparison of the K. pneumonia was performed using next generation sequencing (NGS). It was shown that two out of five K. pneumoniae, with sequence type ST 101 isolates, lost bla-_OXA48, bla-_CTX-M-14, tir, strA and strB genes after the co-culture with Scopulariopsis brevicaulis fungus. The other three isolates (ST 383 and 147) were inhibited in the co-culture but did not show any changes in resistance. The total ethyl acetate extract of the fungal–bacterial co-culture was tested against K. pneumoniae using a disc diffusion method. The concentration of the crude extract was 0.97 mg/µL which resulted in total inhibition of the bacteria. Using chromatographic techniques, the purified compounds were identified as 11-octadecenoic acid, 2,4-Di-tert-butylphenol, 2,3-Butanediol and 9-octadecenamide. These were tested against K. pneumoniae using the well diffusion method at a concentration of 85 µg/µL which resulted in total inhibition of bacteria. The co-culture results indicated that bacteria under chemical stress showed variable responses and induced fungal secondary metabolites with antibacterial activities.     

Validation of an LC–MS/MS Method for the Quantification of the CK2 Inhibitor Silmitasertib (CX-4945) in Human Plasma

Silmitasertib (CX-4945) is currently being investigated in clinical trials against various types of cancer. The U.S. Food and Drug Administration (FDA) has already granted orphan drug designation to the compound for the treatment of advanced cholangiocarcinoma, medulloblastoma, and biliary tract cancer. Silmitasertib inhibits the serine/threonine protein kinase CK2, which exerts a proliferation-promoting and anti-apoptotic effect on cancer cells. In view of current and future applications, the measurement of silmitasertib levels in plasma is expected to play an important role in the evaluation of therapeutic and toxic concentrations in cancer patients. In the present work, we therefore present an LC–MS/MS method for the quantification of silmitasertib in human plasma. Using a simple liquid–liquid extraction with ethyl acetate and a mixture of n-hexane and ethyl acetate, this method can be performed in any laboratory with mass spectrometry. The validation was carried out according to the FDA guideline.     

Synthesis of Mono-Amino Substituted γ-CD: Host–Guest Complexation and In Vitro Cytotoxicity Investigation

Cyclodextrins (CDs) are cyclic oligosaccharides which can trap hydrophobic molecules and improve their chemical, physical, and biological properties. γ-CD showed the highest aqueous solubility with the largest cavity diameter among other CD types. The current study describes a direct and easy method for nucleophilic mono-aminos to be substituted with γ-CD and tested for their ability to host the guest curcumin (CUR) as a hydrophobic drug model. The mass spectrometry and NMR analyses showed the successful synthesis of three amino-modified γ-CDs: mono-6-amino-6-deoxy-cyclodextrine (γ-CD-NH₂), mono-6-deoxy-6-ethanolamine-γ-cyclodextrine (γ-CD-NHCH₂CH₂OH), and mono-6-deoxy-6-aminoethylamino)-γ-cyclodextrin (γ-CD-NHCH₂CH₂NH₂). These three amino-modified γ-CDs were proven to be able to host CUR as native γ-CDs with formation constants equal to 6.70 ± 1.02, 5.85 ± 0.80, and 8.98 ± 0.90 mM⁻¹, respectively. Moreover, these amino-modified γ-CDs showed no significant toxicity against human dermal fibroblast cells. In conclusion, the current work describes a mono-substitution of amino-modified γ-CDs that can still host guests and showed low toxicity in human dermal fibroblasts cells. Therefore, the amino-modified γ-CDs can be used as a carrier host and be conjugated with a wide range of molecules for different biomedical applications, especially for active loading methods.     

Heterogeneous Catalysis of Ozone Using Iron–Manganese Silicate for Degradation of Acrylic Acid

Iron–manganese silicate (IMS) was synthesized by chemical coprecipitation and used as a catalyst for ozonating acrylic acid (AA) in semicontinuous flow mode. The Fe-O-Mn bond, Fe-Si, and Mn-Si binary oxide were formed in IMS on the basis of the results of XRD, FTIR, and XPS analysis. The removal efficiency of AA was highest in the IMS catalytic ozonation processes (98.9% in 15 min) compared with ozonation alone (62.7%), iron silicate (IS) catalytic ozonation (95.6%), and manganese silicate catalytic ozonation (94.8%). Meanwhile, the removal efficiencies of total organic carbon (TOC) were also improved in the IMS catalytic ozonation processes. The IMS showed high stability and ozone utilization. Additionally, H₂O₂ was formed in the process of IMS catalytic ozonation. Electron paramagnetic resonance (EPR) analysis and radical scavenger experiments confirmed that hydroxyl radicals (•OH) were the dominant oxidants. Cl⁻, HCO₃⁻, PO₄³⁻, Ca²⁺, and Mg²⁺ in aqueous solution could adversely affect AA degradation. In the IMS catalytic ozonation of AA, the surface hydroxyl groups and Lewis acid sites played an important role.     

Development and Validation of a Bioanalytical Method for 3′- and 6′-Sialyllactose in Minipig Liver and Kidney Using Liquid Chromatography-Tandem Mass Spectrometry and Its Application to Analysis of Tissue Distribution

Breast milk contains human milk oligosaccharides (HMOs), including sialyllactose (SL). SL is composed of sialic acid and lactose, and is divided into 3′-SL and 6′-SL according to the binding position. SL has immunoprotective effects against bacteria and viruses, and acts as a probiotic in the gastrointestinal tract. In this study, we developed a bioanalytical method for simultaneous analysis of 3′-SL and 6′-SL in liver and kidney tissues of Yucatan minipigs using liquid chromatography–tandem mass spectrometry (LC-MS/MS) under conditions optimized in our previous study. LC-MS/MS was performed using a hydrophilic interaction liquid chromatography (HILIC) column (50 mm × 2.1 mm, 3 μm) with a mobile phase consisting of 10 mM ammonium acetate in water (pH 4.5) and acetonitrile with gradient elution at a flow rate of 0.3 mL/min. A surrogate matrix method using water was applied for analysis of endogenous SL. The developed method was validated with regard to selectivity, linearity, precision, accuracy, the matrix effect, recovery, parallelism, dilution integrity, carryover, and stability according to the US Food and Drug Administration guidelines. We performed a tissue distribution study of minipigs, and analyzed liver and kidney tissues using the developed method to determine the tissue distribution of 3′-SL and 6′-SL. The tissue concentrations of 3′-SL and 6′-SL were readily measurable, suggesting that the method would be useful for evaluating the tissue distributions of these compounds in minipigs.     

Improved Stability of a Stable Crystal Form C of 6S-5-Methyltetrahydrofolate Calcium Salt,Method Development and Validation of an LC–MS/MS Method for Rat Pharmacokinetic Comparison

Folate is a vitamin beneficial for humans that plays an important role in metabolism, but it cannot be well supplemented by food; it is necessary to supplement it in other ways. Based on this consideration, a novel crystal form C of 6S-5-methyltetrahydrofolate calcium salt (MTHF CAC) was obtained. To explore the difference between MTHF CAC and the crystal form Ⅰ of 6S-5-methyltetrahydrofolate calcium salt (MTHF CA) as well as an amorphous product of 6S-5-methyltetrahydrofolate glucosamine salt (MTHF GA), their stability and pharmacokinetic behaviours were compared. The results of high-performance liquid chromatography coupled with ultraviolet detection analysis indicated that MTHF CAC showed a better stability than MTHF CA and MTHF GA. After oral administration of MTHF CAC, MTHF CA, and MTHF GA to male rats, the MTHF concentrations were analysed using a validated liquid chromatography–tandem mass spectrometry, and the pharmacokinetic parameters were compared. The mean residence times (0–t) of MTHF CAC, MTHF CA, and MTHF GA were 3.7 ± 1.9 h, 1.0 ± 0.2 h (p < 0.01), and 1.5 ± 0.3 h (p < 0.05), respectively. The relative bioavailability of MTHF CAC was calculated to be 351% and 218% compared with MTHF CA and MTHF GA, respectively, which suggests that MTHF CAC can be better absorbed and utilized for a longer period of time.     

Tandem Transesterification–Esterification Reactions Using a Hydrophilic Sulfonated Silica Catalyst for the Synthesis of Wintergreen Oil from Acetylsalicylic Acid Promoted by Microwave Irradiation

SiO₂–SO₃H, with a surface area of 115 m²/g and pore volume of 0.38 cm³g⁻¹, and 1.32 mmol H⁺/g was used as a 20% w/w catalyst for the preparation of methyl salicylate (wintergreen oil or MS) from acetylsalicylic acid (ASA). A 94% conversion was achieved in a microwave reactor over 40 min at 120 °C in MeOH. The resulting crude product was purified by flash chromatography. The catalyst could be reused three times.