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Nuciferine (NF), one of the main and effective components in Nelumbo nucifera Gaertn. leaf extracts, is a promising drug candidate for the treatment of obesity-related diseases, while metformin is a first line therapeutic drug for type 2 diabetes mellitus. Since nuciferine and metformin are likely to be co-administered, the aim of the present study was to evaluate whether co-administration of nuciferine would influence the liver (target tissue) distribution and the anti-diabetic effect of metformin by inhibiting hepatic organic cation transporter 1 (OCT1) and multidrug and toxin extrusion 1 (MATE1). The data demonstrated that nuciferine significantly reduced metformin accumulation in MDCK cells stably expressing human OCT1 (MDCK-hOCT1) or hMATE1 (MDCK-hMATE1), and primary cultured mouse hepatocytes. Furthermore, the presence of nuciferine in the basal compartment caused a concentration-dependent reduction of intracellular metformin accumulation in MDCK-hOCT1/hMATE1 cell monolayers. Compared with the metformin treatment-alone group, co-administration of nuciferine (40 mg/kg) markedly reduced the metformin concentration in mouse livers at 30 and 60 min after a single oral dose of metformin (200 mg/kg), and subsequently impaired the glucose-lowering effect of metformin (200 mg/kg), but the glucose-lowering effect became no different at 90 and 120 min. Therefore, nuciferine influenced the liver concentration and glucose-lowering effect of metformin only for a period of time after dose, administration of nuciferine and metformin with an interval might prevent the drug-drug interaction mediated by OCT1 and MATE1.

Background and purpose The concentrative nucleoside transporter 2 (CNT2) mediates the uptake of both natural nucleosides and nucleoside-derived drugs. Therefore, it is important both physiologically and pharmacologically. However, CNT2 expression is significantly repressed in colorectal cancer (CRC). Here, we have elucidated the mechanism(s) underlying CNT2 repression in CRC. Experimental approach Repression of CNT2 in tumour samples from patients with CRC was identified using Western blot and RT-qPCR. The histone acetylation state at the CNT2 promoter region was then evaluated with chromatin immunoprecipitation and trichostatin A (TSA) treatment. To find the key enzyme responsible for hypoacetylation at the CNT2 promoter region, siRNA knockdown and RT-qPCR were used. Effects of combining HDAC inhibitors and cladribine were studied in HCT15 and HT29 cells. Key results Histone deacetylase 7 was significantly up-regulated in CRC, leading to histone hypoacetylation at the CNT2 promoter region, especially at sites H3K9Ac, H3K18Ac and H4Ac. This hypoacetylation condensed the chromatin structure and reduced CNT2 expression. All these effects were reversed by treatment with TSA, a histone deacetylase inhibitor. In HCT15 and HT29 cells, inhibition of histone deacetylase increased cell uptake and decreased IC50 for cladribine. Conclusions and implications Histone hypoacetylation due to increased levels of histone deacetylase 7 results in CNT2 repression in CRC tumour tissue and could lead to decreased uptake of and consequent resistance to nucleoside anti-cancer agents. Such resistance could be overcome by combining inhibitors of histone deacetylase with the nucleoside anti-cancer agent.

Chiral molecules are of great importance in the life science since individual enantiomers may differ in biological activity, mechanism, and toxicity, making it necessary to explore efficient chiral analysis methods. Chromatography approaches are often used to differentiate enantiomers while mass spectrometry (MS) was thought to be blind in chiral analysis. With the development of MS technique, it began to play a more and more crucial part in chiral observation. In this review, we will give a detailed introduction of the analysis methods related to MS for chiral drugs, including its mechanism, applications, and future development.

In this study, a mass spectrometry (MS)-based kinetic method (KM) is shown to be successful at analyzing a multichiral center drug stereoisomer, entecavir (ETV), both qualitatively and quantitatively. On the basis of the KM, the bivalent complex ion [MII (A)(ref*)2 ]2+ (MII = divalent metal ion, A = analyte, and ref* = chiral reference) was set as precursor ion in MS/MS. The experiment results suggest strong chiral selectivity between ETV and its isomers when using ZnII coordinated with the chiral reference R-besivance (R-B). The logarithm of the fragment ion abundance ratio and the enantiomeric percentage (%) exhibits a strong linear relation because of the competitive loss of the reference and analyte. The product ion pair [ZnII (R-B)A-H]+ (m/z 733) and [ZnII (R-B)2 -H]+ (m/z 849), together with [R-B + H]+ (m/z 394) and [A + H]+ (m/z 278), can realize the identification of ETV and all of its chiral isomers. Theoretical calculation were also performed using the B3LYP functional with the 6-31G* and LanL2DZ basis set to clarify the mechanism of structural difference of these bivalent complex ions. The results reveal that MS-KM can be used to detect optical impurities without a chiral chromatographic column and fussy sample pretreatment. The established method has been used to determine stereoisomeric impurities of less than 0.1% in ETV crude drug, a demonstration of its simple and effective nature for rapid detection of stereoisomeric impurities.

A facile method based on electrospray mass spectrometry was established and validated for the differentiation of enantiomeric tadalafil isomers without using chiral chromatographic separation. The enantiomers were coupled with a chiral selector to form diastereomeric complex ions. Nickel-tadalafil complexes, [NiII (tadalafil)(l-Trp)-H]+ , produced a characteristic fragment ion at m/z 524 by loss of 1-methyl-1,6-dihydropyrazine-2,5-dione via collision-induced dissociation. The relative abundance of this fragment ion to the precursor contributed to differentiate tadalafil enantiomers, and energy-resolved product-ion spectra were applied to determine the molar composition of tadalafil in the mixture (R,R and S,S) as well. In addition, the other two forms of stereomeric isomers of tadalafil (R,S and S,R) could be also distinguished and analyzed by this method. The method was validated in different types of mass spectrometers (AB quadrupole time-of-flight and Bruker ion trap) and also verified by a chiral high-performance liquid chromatography coupled with quadrupole time-of-flight. The chiral determination of tadalafil using MS method proved to be rapid (1-min run time for each sample) and to have the same accuracy and precision comparable to chiral liquid chromatography mass spectrometry methods. This method provides an alternative to commonly used chromatographic technique for chiral determination and is particularly useful in rapid screening in enantioselective synthesis and enantiomeric impurity detection in pharmaceutical industry. Copyright © 2017 John Wiley & Sons, Ltd.

Extramural venous invasion (EMVI) is associated with a poor prognosis and a poor overall survival rate in rectal cancer. It can independently predict local and distant tumor recurrences. Preoperative EMVI detection in rectal cancer is useful for determining the treatment strategy. EMVI status is beneficial for the post-treatment evaluation and analysis of rectal cancer. Magnetic resonance imaging (MRI) is a non-invasive diagnostic modality with no radiation effects. High-resolution MRI can detect EMVI with high accuracy. In addition, MRI results are equal to or even better than pathological results in the detection of medium to large EMVI in rectal cancer. MRI-detected EMVI (mrEMVI) can be used as a potential biomarker that facilitates treatment methods. This review highlights the importance of MRI before and after rectal cancer treatment. In addition, we analyze the prognostic correlation between mrEMVI and circulating tumor cells (CTC) in rectal cancer. This article may help shed light on the significance of mrEMVI.

Protein phosphorylation is a vital post-translational modification. This study investigated the effect of phosphorylation on human uridine diphosphate (UDP)-glucuronosyltransferase 1A3 (UGT1A3) activity. Curcumin and calphostin C suppressed the activity and phosphorylation of recombinant UGT1A3 expressed in Sf9 cells. These results indicate that UGT1A3 undergoes phosphorylation, which is required for its catalytic activity. Calphostin C is a highly specific protein kinase C (PKC) inhibitor, so three predicted PKC phosphorylation sites in UGT1A3 were examined. Site-directed mutation analysis at residues 28, 43 and 436 (from serine to glycine) was conducted. Compared with the wild-type, the S43G-mutant showed significantly decreased UGT1A3 catalytic activity. Furthermore, the UGT1A3 activity of wild-type and S43G-mutant was down-regulated by calphostin C, whereas the calphostin C inhibitory effect was much weaker on the S43G-mutant than the wild-type. In conclusion, phosphorylation plays an important role in UGT1A3 activity, and the serine at site 43 in UGT1A3 is most likely a phosphorylation site. Copyright © 2015 John Wiley & Sons, Ltd.

Rationale Electrospray ionization tandem mass spectrometry (ESI-MSn) is an invaluable tool for the study of gas-phase reactions. When N-phenylbenzamide is analyzed in negative ion mode, the nucleophilic deprotonated site of nitrogen or oxygen, together with the adjacent electrophilic phenyl carbon in the same molecule, provides a useful opportunity to study the intramolecular nucleophilic reaction in the gas phase. Methods All MSn experiments of deprotonated N-phenylbenzamides were conducted on an ion trap mass spectrometer using ESI in negative ion mode. The accurate masses of fragments were measured on an ESI quadrupole time-of-flight mass spectrometer in negative ion mode. Theoretical calculations were conducted at the B3LYP/6-31++G(d,p) level of density functional theory using the Gaussian 03 program. Results When the polarity of the substituent on the aniline ring was changed, gas-phase Smiles rearrangement reactions could be initiated by different atoms in the anionic center. Upon collisional activation, loss of CO from deprotonated N-phenylbenzamides could be observed, which can be interpreted as a nitrogen anion triggering the Smiles rearrangement reaction through a three-membered ring transition state. As the aniline ring was substituted by a strong electron-withdrawing group (e.g., NO2, COCH3, or CF3) at the para position, a characteristic phenolate anion was obtained, which was derived from the Smiles rearrangement reaction initiated by the oxygen anion through a four-membered ring transition state. Conclusions In the fragmentation of deprotonated N-phenylbenzamides, the gas-phase Smiles rearrangement reaction initiated by either the nitrogen or the oxygen atom can proceed. The findings in this study have not only enriched knowledge on the gas-phase Smiles rearrangement reactions, but also provided valuable information for understanding the rearrangements of deprotonated aromatic amides in gas phase. Copyright © 2015 John Wiley & Sons, Ltd.

The noncovalent complex interactions between cyclofructans, a new class of cyclic oligosaccharide hosts, and various amino acids have been characterized by means of electrospray ionization mass spectrometry and nuclear magnetic resonance. The 1 : 1 stoichiometry of cyclofructans and amino acid complexes was confirmed by their mass-to-charge ratio in positive mode. Cyclofructans (CFs)–amino acid complexes and cyclodextrin–amino acid complexes exhibited distinctive different fragment behaviors in collision-induced dissociation experiments. Coupled with the results of 1H NMR and nuclear overhauser effect spectroscopy, cyclofructan–amino acid complexes were deduced to be rim complexes via formation hydrogen bonding and ion–dipole forces. The interaction pattern could be controlled by changing the pH condition. In neutral solution, amino acids are located on the positive side of CFs, although moved to the negative side pocket constructed by 3-OH oxygen of furanose ring and the crown ether oxygen in acid condition. In addition, theory calculation for geometry optimization of Trp and CFs was performed, which was in good agreement with the experimental results. Copyright © 2014 John Wiley & Sons, Ltd.

RATIONALE Exploring the fragmentation mechanism of amide ions in mass spectrometry has attracted great interest because of the desire to analyze the amino acid sequences of peptides and proteins. However, the collision-induced dissociation (CID) mechanism of deprotonated small amides has been rarely studied in electrospray ionization mass spectrometry (ESI-MS). The fragmentation of deprotonated N,2-diphenylacetamides exhibited some characteristic fragment ions, which are not derived from the conventional cleavage route. Therefore, clarification of their fragmentation mechanism is very important and useful for structural analysis of related amides and peptides. METHODS All CID experiments were carried out using an electrospray ionization ion trap mass spectrometer in negative ion mode. In addition, the accurate masses of fragments were measured on an ESI quadrupole time-of-flight (Q-TOF) mass spectrometer in negative ion mode. Deuterium-labeled 2-phenyl-N-(4-trifluoromethylphenyl)acetamide was synthesized and its ESI fragmentation spectrum had been obtained. Theoretical calculations were carried out by the density functional theory (DFT) method at the B3LYP level of theory with the 6-31G++(d,p) basis set. RESULTS Deprotonated N,2-diphenylacetamides mainly generate four kinds of ions in CID: benzyl anion, aniline anion, phenyl-ethenone anion and isocyanato-benzene anion bearing respective substituent groups. The benzyl anion and the aniline anion can be generated by direct decomposition. The phenyl-ethenone anion and the isocyanato-benzene anion were proposed to be yielded from proton transfer within an ion-neutral complex, and the intensities of two competitive product ions are well correlated with the substituent constants. The mechanism was also supported by theoretical calculations. CONCLUSIONS The characteristic fragment ions of deprotonated N,2-diphenylacetamides were proposed to be produced via an ion-neutral complex mechanism, which was proved by deuterium-labeling experiments, theoretical calculations and substituent effects. Copyright © 2014 John Wiley & Sons, Ltd.

Fluoxetine (FLX) is one of the most widely prescribed selective serotonin reuptake inhibitors. Although FLX is used as racemate in the clinic, the clinical pharmacokinetics of FLX and its N-demethylation metabolite norfluoxetine (NFLX) show obvious cytochrome P450 (CYP) polymorphism dependency and exhibit marked stereoselectivity. However, the kinetic profiles of CYP variants to FLX remain unclear. In the present study, some variants of human CYP2C8, CYP2C9, and CYP2D6 were first expressed in insect cells, and their catalytic roles with respect to FLX enantiomers were then investigated. CYP2C8.4 and CYP2C9.10 showed significantly lower activity and CYP2C8.3 showed significantly higher activity toward both R- and S-FLX compared with the wildtype, while CYP2C9.3, CYP2C9.13, and CYP2C9.16 showed significantly lower activity only toward R-FLX. Five CYP2C9 variants and CYP2D6.1 exhibited significantly stereoselective kinetic profiles prior to R-FLX, and CYP2C8.3 showed a slight stereoselectivity. Interestingly, obvious substrate inhibition was observed in the CYP2C9 wildtype and its three variants only in the case of R-FLX. Together, these findings suggest that CYP2C9 and CYP2D6 polymorphism may play an important role in the clearance of FLX and also in the stereoselective kinetic profiles of FLX enantiomers.

CYP2C8 plays an important role in the metabolism of various drugs, such as paclitaxel, repaglinide and ibuprofen. Polymorphisms in the CYP2C8 gene were shown to influence interindividual differences in the pharmacokinetics of paclitaxel, repaglinide and ibuprofen enantiomers. In this study, three CYP2C8 allelic variants (CYP2C8.2, CYP2C8.3 and CYP2C8.4) and wild-type CYP2C8 (CYP2C8.1) were co-expressed for the first time with human cytochrome P450 oxidoreductase (POR) and cytochrome b5 by using a baculovirus-assisted insect cell expression system. Further, the effects of genotype-phenotype correlations of CYP2C8 alleles on the metabolism of paclitaxel, repaglinide and ibuprofen enantiomers were evaluated. The CLint values of CYP2C8.2, CYP2C8.3 and CYP2C8.4 for paclitaxel were 47.7%, 64.3% and 30.2% of that of CYP2C8.1 (p

The stereoselective uptake of propranolol enantiomers was investigated by using the K562 and K562 adriamycin-resistant cell line (K562/ADR) as a model. An enantioselective RP-HPLC method was applied to determine the accumulation of propranolol (PPL) stereoisomers in K562 and K562/ADR cells. The concentration, time and temperature dependent studies showed that the accumulation of S-(-)-PPL was higher than R-(+)-PPL in K562 cells and uptake of R-(+)-PPL was significantly higher than that of S-(-)-PPL in K562/ADR cells. The results indicate the enantioselective accumulation of propranolol enantiomers in K562 and K562 / ADR cells.

Tetrahydropalmatine (THP), with one chiral center, is an alkaloid that possesses analgesic and many other pharmacological actives. The aim of the present study is to investigate stereoselective metabolism of THP enantiomers in human liver microsomes (HLM) and elucidate which cytochrome P450 (CYP) isoforms contribute to the stereoselective metabolism in HLM. Additionally, the inhibitions of THP enantiomers on activity of CYP enzymes are also investigated. The results demonstrated that (+)-THP was preferentially metabolized by HLM. Ketoconazole (inhibitor of CYP3A4/5) inhibited metabolism of (−)-THP or (+)-THP at same degree, whereas the inhibition of fluvoxamine (inhibitor of CYP1A2) on metabolism of (+)-THP was greater than that of (−)-THP; moreover, the metabolic rate of (+)-THP was 5.3-fold of (−)-THP in recombinant human CYP1A2. Meanwhile, THP enantiomers did not show obvious inhibitory effect on the activity of various CYP isoforms (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2E1, and 3A4/5), whereas (−)-THP, but not (+)-THP, significantly inhibited the activity of CYP2D6 with the Ki value of 6.42 ± 0.38 μM. The results suggested that THP enantiomers were predominantly metabolized by CYP3A4/5 and CYP1A2 in HLM, and (+)-THP was preferentially metabolized by CYP1A2, whereas CYP3A4/5 contributed equally to metabolism of (−)-THP or (+)-THP. Besides, the inhibition of CYP2D6 by (−)-THP may cause drug–drug interaction, which should be considered. Chirality 25:43–47, 2013. © 2012 Wiley Periodicals, Inc.

BACKGROUND Proton-coupled oligopeptide transporters (POTs) serve as integral membrane protein for the cellular uptake of di/tripeptide. Prostate has a large requirement of nutriment for its function to produce and secrete prostatic fluid. Besides, prostate suffered from limited therapy effect of drug treatment. Thus present study was performed to evaluate the expression of POTs in prostate of mice and human with the aim to provide information for potential role of POTs in absorption of nutriment and peptidomimetic drugs in prostate. METHODS Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot methods were applied to study the mRNA, protein expression of POTs in prostate, human prostate cancer cells (PC-3), and human prostate epithelial cells (RWPE-1). RESULTS qRT-PCR study showed different characteristic of POTs mRNA expression in mouse prostate. Among these transporters, protein expression of PepT2 was detected and increasing during the development of mouse prostate, while PepT1, PHT1, and PHT2 protein was not detected. Furthermore, different characteristic of regulation by inflammation on POTs mRNA expression was found in RWPE-1 and PC-3. In addition, mRNA expression of PepT2 and PHT1 in prostate of patients with PCa was demonstrated be lower compared with BPH. CONCLUSIONS These findings provide the first evidence for the expression of POTs in prostate of mice and patients with BPH or PCa and suggest that POTs are likely to play a role in the transport of di/tripeptides and peptidomimetics in prostate. Prostate 73: 287–295, 2013. © 2012 Wiley Periodicals, Inc.

Besifloxacin hydrochloride is a novel chiral broad-spectrum fluoroquinolone developed for the treatment of bacterial conjunctivitis. R-besifloxacin hydrochloride is used in clinics as a consequence of its higher antibacterial activity. To establish an enantiomeric impurity determination method, some chiral stationary phases (CSPs) were screened. Besifloxacin enantiomers can be separated to a certain extent on Chiral CD-Ph (Shiseido Co., Ltd., Japan), Chiral AGP, and Crownpak CR (+) (Daicel Chemical IND., Ltd., Japan). However, the selectivity and sensitivity were both unsatisfactory on these three CSPs. Therefore, Chiral AGP, Chiral CD-Ph, and Crownpak CR (+) were not used in the enantiomeric impurity determination of besifloxacin hydrochloride. The separation of enantiomers of besifloxacin was further performed using a precolumn derivatization chiral high-performance liquid chromatography method. 2,3,4,6-Tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate was used as the derivatization reagent. Besifloxacin enantiomer derivates were well separated on a C(18) column (250 × 4.6 mm, 5 µm) with a mobile phase that consisted of methanol-KH(2)PO(4) buffer solution (20 mM; pH 3.0) (50:50, v/v). Selectivity, sensitivity, linearity, accuracy, precision, stability, and robustness of this method were all satisfied with the method validation requirement. The method was suitable for the quality control of enantiomeric impurity in besifloxacin hydrochloride.

Tetrahydropalmatine (THP), with one chiral center, is an active alkaloid ingredient in Rhizoma Corydalis. The aim of the present paper is to study whether THP enantiomers are metabolized stereoselectively in rat, mouse, dog, and monkey liver microsomes, and then, to elucidate which Cytochrome P450 (CYP) isoforms are predominately responsible for the stereoselective metabolism of THP enantiomers in rat liver microsomes (RLM). The results demonstrated that (+)-THP was preferentially metabolized by liver microsomes from rats, mice, dogs, and monkeys, and the intrinsic clearance (Cl(int)) ratios of (+)-THP to (-)-THP were 2.66, 2.85, 4.24, and 1.67, respectively. Compared with the metabolism in untreated RLM, the metabolism of (-)-THP and (+)-THP was significantly increased in dexamethasone (Dex)-induced and β-naphthoflavone (β-NF)-induced RLM; meanwhile, the Cl(int) ratios of (+)-THP to (-)-THP in Dex-induced and β-NF-induced RLM were 5.74 and 0.81, respectively. Ketoconazole had stronger inhibitory effect on (+)-THP than (-)-THP, whereas fluvoxamine had stronger effect on (-)-THP in untreated and Dex-induced or β-NF-induced RLM. The results suggested that THP enantiomers were predominately metabolized by CYP3A1/2 and CYP1A2 in RLM, and CYP3A1/2 preferred to metabolize (+)-THP, whereas CYP1A2 preferred (-)-THP.

Introduction Metabonomic analysis is an important molecular phenotyping method for characterising plant ecotypic variations; hence, it may become a powerful tool for quality control and discrimination of traditional Chinese medicine (TCM). Objective To discriminate and assess the quality of Curcuma phaeocaulis, C. kwangsiensis and C. wenyujin from different ecotypes. The identification of the compositions of essential oils from the three Curcuma species was included in this study. Methodology Metabolomics analysis was carried out on all samples by gas chromatography–mass spectrometry (GC-MS) coupled with multivariate statistical analysis. Characterisation of phytochemicals in essential oils was performed by automated matching to the MS library and comparison of their mass spectra (NIST05 database). Results Principal component analysis (PCA) effectively distinguished the samples from different species and ecotypes. Partial least squares discrimination analysis (PLS-DA) was successfully employed in classifying the GC-MS data of authentic, commercial and introduction cultivation samples. Furthermore, the components contributing significantly to the discrimination, namely curzerenone, germacrone, curdione and epicurzerenone, were screened by PCA and PLS-DA loading plots and further can be used as chemical markers for discrimination and quality control among different groups of samples. Copyright © 2011 John Wiley & Sons, Ltd.

Ginkgolic acids have been shown to possess allergenic as well as genotoxic and cytotoxic properties. The question arises whether the metabolism of ginkgolic acids in the liver could decrease or increase their toxicity. In this study, the invitro metabolism of ginkgolic acid (15:1, GA), one component of ginkgo acids, was investigated as a model compound in Sprague-Dawley rat liver microsomes. The metabolites were analyzed by ultra-performance liquid chromatography coupled with photodiode array detector/negative-ion electrospray ionization tandem mass spectrometry (UPLC-PDA/ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. The result showed that the benzene ring remained unchanged and the oxidations occurred at the side alkyl chain in rat liver microsomes. At least eight metabolites were found. Among them, six phase I metabolites were tentatively identified. This study might be useful for the investigation of toxicological mechanism of ginkgolic acids. Copyright © 2009 John Wiley & Sons, Ltd.

Cytochrome P-450 3A (CYP3A) together with its nuclear receptors plays a critical role in drug metabolism. The present study investigated the effects of undernutrition in utero on hepatic mRNA and protein expression of the enzyme CYP3A23/3A1 and nuclear receptors including pregnane X receptor (PXR; NR1I2), constitutive androstane receptor (CAR; NR1I3) and nuclear factor-4alpha (HNF4alpha; HNF4A) in neonatal rats. At gestational day 2, pregnant rats were randomly divided into two groups: nourished (fed ad libitum) and undernourished (50% of nourished group). The pups delivered by nourished rats were designated as the normal-birth-weight group (NBW, n=15) and those delivered by undernourished rats were designated as the low-birth-weight group (LBW, n=15). Hepatic mRNA expression was detected by quantitative real-time PCR and the corresponding protein expression was examined by immunohistochemistry (IHC). Compared with NBW pups, LBW pups tended to have lower mRNA expression levels of CYP3A23/3A1, PXR and CAR but higher levels of HNF4alpha. Only the CAR mRNA expression differences were significant (p

The application of mass spectrometry in drug discovery, especially in drug metabolites, is very important. This present paper is at first focused on the elucidation of fragmentation patterns of the phenolic bisbenzyltetrahydroisoquinoline alkaloid, neferine, together with its analogues isoliensinine and liensinine with anti-HIV activities using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. All title compounds displayed major diagnostic fragments that formed by the cleavage of the C1'--C9' bond resulting in positive group CD, and the loss of 4-ethyl-1-phenol or 4-ethyl-1-methoxybenzene following rearrangements. Their ESI-MS/MS spectra also showed the relatively stable fragment ions formed by the elimination of H2O, CH3NH2, CH3OH, and CH3-N==CH2. Secondly, the metabolites of neferine from dog hepatic microsomal incubations were analyzed and characterized by high-performance liquid chromatography (HPLC) and data-dependent ESI-MS/MS. Based on fragmentation patterns and compared with their retention times in LC, molecular weights and ultraviolet (UV) absorbances with standard compounds, six metabolites were identified as isoliensinine, liensinine and four novel bisbenzyltetrahydroisoquinoline alkaloids named as 6-O-desmethylneferine, 2'-N-desmethylneferine, 2'-N-6-O-didesmethylneferine, and 6,13-O-didesmethylneferine. All metabolites were desmethyl or didesmethyl products of neferine. The possible metabolic pathways for neferine have been proposed. The results suggest that N-demethylation and O-demethylation are two important metabolic pathways of neferine in dog hepatic microsomal incubations. This is critical for screening and development of phenolic bisbenzyltetrahydroisoquinoline alkaloids with anti-HIV activities such as neferine and its analogues isoliensinine and liensinine.

MDR1-encoded P-glycoprotein (P-gp) is a drug efflux transporter mainly expressed in liver, kidney, intestine, brain (at the level of the blood-brain barrier), and placenta. It thus plays important roles in drug absorption, distribution, and excretion. Cetirizine is a second-generation nonsedating antihistamine used to treat allergic disease of respiratory system, skin and eyes. To evaluate P-gp expression and function in Caco-2 cells pretreated with cetirizine enantiomers, we assessed the sensitivity of Caco-2 cells to paclitaxel using the MTT assay and the polarized transport of rhodamine-123 and doxorubicin across Caco-2 monolayers. RT-PCR and flow cytometry were used to assay MDR1 mRNA and P-gp protein respectively. The sensitivity of Caco-2 cells to paclitaxel decreased significantly after cells were pretreated with 100 microM R-cetirizine but increased upon treatment with S-cetirizine. The efflux of rhodamine-123 and doxorubicin was enhanced significantly after Caco-2 monolayers were pretreated with 100 microM R-cetirizine but was reduced by S-cetirizine. The MDR1 mRNA and P-gp levels in Caco-2 cells were increased by 100 microM R-cetirizine and decreased by 100 microM S-cetirizine. These results suggest that R-cetirizine up-regulates MDR1 expression while S-cetirizine down-regulates MDR1 expression.

This study aimed to determine the transport characteristics of chiral drug enantiomers across Caco-2 cell monolayers as a model of human intestinal epithelial membrane. Esmolol was chosen as a model drug, and the study focused on the transepithelial transport of esmolol enantiomers in this in vitro model system. Separation and quantitation of (S)- and (R)-esmolol were performed by RP-HPLC with the use of GITC as a precolumn derivatizing agent. Bidirectional transport studies of 5.0-400.0 micromol/l esmolol demonstrated that the two enantiomers were transported mainly by a passive, transcellular mechanism. At concentrations of 5.0-100.0 micromol/l, enantioselective permeability of esmolol was observed. In the absorptive transport, Papp of (S)-esmolol was smaller than (R)-esmolol and vice versa for secretory transport. The enantioselectivity disappeared when the drug concentration was increased to 200.0 micromol/l. In conclusion, the transport characteristics of (S)- and (R)-esmolol were distinctly different. An enantioselective carrier-mediated mechanism in addition to passive diffusion was involved in the transport process of esmolol across Caco-2 cell monolayers.

Antibody-drug conjugate (ADC) targeting antigens expressed on the surface of tumor cells are an effective approach for delivering drugs into the cells via antigen-mediated endocytosis. One of the well-known tumor antigens, the CD20 of B-lymphocyte, has long been suggested to be noninternalizing epitope, and is thus not considered a desirable target for ADCs. Here, sortase A (srtA)-mediated transpeptidation is used to specifically conjugate triple glycine-modified monomethyl auristatin E (MMAE), a highly toxic antimitotic agent, to anti-CD20 ofatumumab (OFA) equipped with a short C-terminal LPETG (5 amino acids) tag at heavy chain (HL), which generates ADCs that show extremely strong potency in killing CD20 positive cancer cells. One of the srtA-generated ADCs with a cleavable dipeptide linker (valine-citrulline, vc), OFA-HL-vcMMAE, shows IC50 values ranging from 5 pg mL-1 to 4.1 ng mL-1 against CD20+ lymphoma cells. Confocal laser scanning microscopy confirms that OFA-HL-vcMMAE internalization by Ramos cells is significantly improved compared to OFA alone, consistent with the high antitumor activity of the new ADC. OFA-HL-vcMMAE, at 5 mg kg-1 dose, is able to eliminate tumors with mean volume ≈400 mm3 while no obvious drug-related toxicity is observed. The results show that srtA-generated OFA-MMAE conjugate system provides a viable strategy for targeting CD20+ B lineage lymphomas.

Stereoselective metabolism of propranolol side-chain glucuronidation was studied for two recombinant human uridine diphosphate glucuronosyltransferases (UGTs), UGT1A9 and UGT2B7. The S- and R-propranolol side-chain glucuronides produced in the incubation mixtures were assayed simultaneously by RP-HPLC with fluorescent detector. The excitation and emission wavelengths were set at 310 nm and 339 nm, respectively. UGT1A9 prefers catalyzing S-enantiomer to R-enantiomer and the intrinsic clearance (CLint) ratios of S-enantiomer to R-enantiomer are 3.8 times and 6.5times for racemic propranolol and individual enantiomers, respectively. UGT2B7, however, catalyzes slightly less S-enantiomer than R-enantiomer and the CLint ratio of S-enantiomer to R-enantiomer is 0.8 times. The high concentration of racemic propranolol (>0.57 mmol/l) and individual enantiomers (>0.69 mmol/l) exhibited substrate inhibition of glucuronidation for UGT2B7, but only the S-enantiomer (>0.44 mmol/l) in racemic propranolol exhibited substrate inhibition for UGT1A9. The substrate inhibition constants (Ksi) were all similar (P > 0.05). Drug–drug interactions were also found between S- and R-enantiomer glucuronidation metabolisms by UGT1A9 and UGT2B7. Chirality 2010. © 2009 Wiley-Liss, Inc.

The transport and uptake of individual propranolol (PPL) enantiomers were studied in human intestinal Caco-2 cell monolayers, and a reversed-phase HPLC-UV assay was used for quantitative analysis. S-PPL and R-PPL across Caco-2 cell monolayers was determined in the concentrations range of 10-500 microM in both apical (AP) to basolateral (BL) and BL to AP directions. S-PPL exhibited greater permeability than R-PPL in the AP to BL direction, whereas in the BL to AP direction S-enantiomer transported less than R-enantiomer. Uptake of R-PPL was significantly higher than that of S-PPL either from AP side or from BL side. The statistically significant differences in uptake were observed at the concentrations range from 10 to 50 microM. Furthermore, the apparent Michaelis constant (K(m)) and maximal velocity (V(max)) also showed significant difference between the two enantiomers. Moreover, the AP to BL transport of PPL enantiomer was markedly decreased by lowering the pH of the apical side but it did not affect the stereoselectivity of PPL across Caco-2 cell monolayers. The transport and uptake of PPL in the BL to AP direction was not influenced by several protein inhibitors. The results suggest that PPL enantiomers showed stereoselective transport and uptake across the Caco-2 cell monolayers. A special transport mechanism capable of directing the PPL enantiomers might be present in the Caco-2 monolayers.