Development and validation of an UHPLC-MS/MS method for simultaneous quantification of ibrutinib and its dihydrodiol-metabolite in human cerebrospinal fluid.

Ibrutinib is an orally administered first-in-class irreversible Bruton's tyrosine kinase (BTK) covalent inhibitor for the treatment of patients with B-cell malignancies. Several isolated clinical observations reported its efficacy in central nervous system dissemination. Herein, we described the development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) procedure for the quantification of ibrutinib and its active metabolite PCI-45227 in cerebrospinal fluid (CSF). This is the first complete validated method for quantification of ibrutinib and PCI-45227 in CSF. The compounds were eluted on a Waters BEH C18 column (50.0 × 2.1 mm; 1.7 µm) using a gradient elution with a mobile phase composed of ammonium formate buffer 5 mM pH 3.2 and acetonitrile +0.1% formic acid with a flow rate of 400 µL·min-1. Two deuterated internal standards were used to obtain the most accurate quantification. The CSF samples were prepared by a simple and rapid dilution. The method was validated by testing the selectivity, response function, intra-day and inter-day precisions, trueness, limits of detection (LOD) and lower limits of quantification (LLOQ). The validation results proved that the methods were suitable to quantify ibrutinib and PCI-45227 in real biological CSF samples from 0.50 (ibrutinib) or 1.00 (PCI-45227) to 30.00 ng·mL-1. Furthermore, the developed method was adapted to allow the quantification of both compounds in plasma and the results were compared to those reported in literature. The plasmatic samples were treated by protein precipitation and the method was validated to quantify ibrutinib and PCI-45227 in real biological plasmatic samples from 5.00 to 491 ng·mL-1. Lastly, for both matrices, accuracy profiles were plotted from the trueness and precision results using a 20% α-risk (ß = 80%) and the tolerance intervals were comprised within the acceptance limits fixed at ±25% for the LLOQ and ±15% for the other concentrations. Finally, these methods were successfully applied to quantify ibrutinib and PCI-45227 in real human CSF and plasma samples.

Roux-en-Y gastric bypass increases systemic but not portal bile acid concentrations by decreasing hepatic bile acid uptake in minipigs.

Roux-en-Y gastric bypass (RYGB) surgery is widely used in the management of morbid obesity. RYGB improves metabolism independently of weight loss by still unknown mechanisms. Bile acids (BAs) are good candidates to explain this benefit, since they regulate metabolic homeostasis and their systemic concentrations increase upon RYGB. Here we analyzed the mechanisms underlying the increase in systemic BA concentrations after RYGB and the role of the liver therein. To this aim, we used the Göttingen-like minipig, a human-size mammalian model, which allows continuous sampling and simultaneous analysis of pre-hepatic portal and systemic venous blood. BA concentrations and pool composition were measured in portal blood, containing intestinal reabsorbed BAs and compared to systemic blood during a standardized meal test before and after RYGB. Systemic total BA concentrations increased after RYGB, due to an increase in conjugated BAs. Interestingly, the ratio of portal:systemic conjugated BAs decreased after RYGB, indicating a role for the liver in systemic BA concentrations changes. In line, hepatic expression of BA transporter genes decreased after RYGB. Our results show that the increase in systemic BAs after surgery is due to decreased selective hepatic recapture. Thus, alterations in hepatic function contribute to the increase in systemic BAs after RYGB.

LR12-peptide quantitation in whole blood by RP-HPLC and intrinsic fluorescence detection: Validation and pharmacokinetic study.

A simple, sensitive, selective and robust HPLC method based on intrinsic fluorescence detection was developed for the quantitation of a dodecapeptide (designated as LR12), inhibitor of Triggering Receptor Expressed on Myeloid cells-1, in rat whole blood. Sample treatment was optimized using protein precipitation and solid-phase extraction. Chromatographic separation was carried out in a gradient mode using a core-shell C18 column (150 × 4.6 mm, 3.6 µm) with mobile phases of acetonitrile and water containing trifluoroacetic acid at 1.0 mL/min. The method was validated using methodology described by the US Food and Drug Administration guidelines for bioanalytical methods. Linearity was demonstrated within the 50-500 ng/mL range and the lower limit of quantitation was 50 ng/mL. Finally, a preliminary pharmacokinetic study after intraperitoneal injection of LR12 in rats was conducted to evaluate both LR12 monomer and its corresponding disulfide dimer, the main product of degradation. Beyond the fact that this paper describes the first fully validated method for LR12 analysis in blood samples, the approach followed here to optimize pre-analytical steps could be beneficial to develop HPLC and/or MS methods for other pharmaceutical peptides.

Trace elements in e-liquids - Development and validation of an ICP-MS method for the analysis of electronic cigarette refills.

Electronic cigarette use has rapidly increased in recent years. In assessing their safety, and in view of coming regulations, trace elements (TE) are among the potentially toxic compounds required to be evaluated in electronic cigarette refill fluids ("e-liquids"). An analytical method using inductively coupled plasma with mass spectrometric detection (ICP-MS) was developed and rigorously validated in order to determine concentrations of 15 TE in 54 e-liquids from a French brand. Despite a significant matrix effect from the main e-liquid constituents, and difficulties related to the current lack of reference materials, our method demonstrated satisfactory linearity, precision and robustness, and permitted the quantification of low concentrations of these 15 elements: lower limits of quantification (LLQ) obtained were ≤4 ppb for all elements except for Ni, Cu and Zn (16 ppb, 20 ppb and 200 ppb, respectively). All TE concentrations in all tested samples were <510 ppb, mostly near or below the LLQs. This method is transposable and is timely for laboratories seeking to meet a prospective demand in light of current or future regulations.

Influence of Roux-en-Y gastric bypass on plasma bile acid profiles: a comparative study between rats, pigs and humans.

BACKGROUND: Roux-en-Y gastric bypass (RYGBP) is the most widely used bariatric surgery procedure, which induces profound metabolic and physiological effects, such as substantial improvements in obesity, type 2 diabetes and their comorbidities. Increasing evidence identifies bile acids (BAs) as signaling molecules that contribute to the metabolic improvement after RYGBP. However, how and to what extent BAs mediate the metabolic effects of RYGBP still remains unclear and requires mechanism of action studies using preclinical models. In this study, we compared plasma BA profiles before and after RYGBP in two animal models, rats and pigs, with humans to evaluate their translational potential. METHODS: Plasma BAs were profiled in rats, pigs and humans by liquid chromatography coupled with tandem mass spectrometry before and after RYGBP. RESULTS: RYGBP increased baseline plasma total BA concentrations in humans and in the two animal models to a similar extent (∼3-fold increase), despite differences in presurgery BA levels and profiles between the models. However, qualitatively, RYGBP differently affected individual plasma BA species, with similar increases in some free species (cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)), different increases in glyco-conjugated species depending on the model and globally no increase in tauro-conjugated species whatever the model. CONCLUSIONS: The tested animal models share similar quantitative RYGBP-induced increases in peripheral blood BAs as humans, which render them useful for mechanistic studies. However, they also present qualitative differences in BA profiles, which may result in different signaling responses. Such differences need to be taken into account when translating results to humans.

Separation of diastereoisomers of Ara-C phosphotriesters using solid phase extraction and HPLC for the study of their decomposition kinetic in cell extracts.

Separations of the diastereoisomers of three nucleoside 5'-phosphotriester derivatives of Ara-C (tBuSATE, hydroxy tBuSATE and bishydroxy tBuSATE phenylphosphotriester derivatives; pronucleotides) were performed by HPLC using derivatized cellulose and amylose chiral stationary phases. An optimal baseline separation (Rs>1.5) was readily obtained with an amylose based chiral column (AD-H) used in normal phase mode. This stereospecific HPLC method has been associated to a solid phase extraction step using a C18 cartridge and an internal standard for the quantification of one nucleoside 5'-phosphotriester derivative in cell extracts. After optimization, this method was validated in terms of specificity, recovery, linearity, precision and accuracy and detection limit. It was applied to the determination of the apparent rate constants of disappearance and half-lives of each diastereoisomer. This enabled us to conclude that the enzymatic activity involved in the first step of the decomposition pathway of the hydroxyl tBuSATE phenylphosphotriester of Ara-C is stereoselective and is related to the nature of the pyrimidic base.

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis.

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.

Chiral resolution of the enantiomers of new selective CB(2) receptor agonists by liquid chromatography on amylose stationary phases.

Analytical HPLC methods using derivatized amylose chiral stationary phases, Chiralpak AD-H and Chiralpak AS, were developed for the direct enantioseparation of eight substituted 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives with one stereogenic center. Baseline separation (Rs>1.5) was always achieved on amylose based Chiralpak AD-H column to the difference with Chiralpak AS. Using UV detection, a linear response was observed within a 180-420 micromol L(-1) concentration range (r2>0.991) for three racemic compounds 1, 3 and 4 with best pharmacological potentials; repeatability, limit of detection (LD) and quantification (LQ) were also determined: LD varied, for the solutes, from 0.36 to 2.56 micromol L(-1). Finally, the enantiopurity of these compounds was determined. Additionally, the effect of temperature variations upon isomer separations was investigated.

Determination of pKa values of benzoxa-, benzothia- and benzoselena-zolinone derivatives by capillary electrophoresis. Comparison with potentiometric titration and spectrometric data.

Acidity constants of benzoxa-, benzothia- and benzoselena-zolinone derivatives were determined by capillary electrophoresis, potentiometry and spectrophotometry experiments. These three analytical techniques gave pK(a) results that were in good agreement. A convenient, accurate and precise method for the determination of pK(a) was developed to measure changes in acidity constants induced by heteroatom or 6-benzoyl substituted derivatives. pK(a) values were determined simultaneously for two compounds characterized by different electrophoretic mobility (micro(e)) and pK(a) value and in the presence of an analogous neutral marker.

Diastereoisomeric resolution of a pronucleotide using solid phase extraction and high performance liquid chromatography: application to a stereoselective decomposition kinetic in cell extracts.

A stereospecific HPLC methodology has been developed for the diastereoisomeric resolution of a mononucleotide prodrug in cell extracts. This method involves the use of solid phase extraction on a C18 cartridge. Diastereoisomers and internal standard resolutions were performed on a cellulose based chiral column (Chiralcel OD-H) used in the normal phase mode. The method was validated in terms of specificity, recovery, linearity (diasteroisomers mixture concentration: 3-60 micromol L(-1)), precision and accuracy and detection limit (1.67 and 1.33 micromol L(-1) for first and second eluted diastereoisomer). This method was applied to the determination of the apparent rate constants of disappearance and half-lives of each stereoisomers. This permits to conclude to the stereoselectivity of the enzymatic activity involved in the decomposition pathway of 2.

Solid-phase synthesis and pharmacological evaluation of a library of peptidomimetics as potential farnesyltransferase inhibitors: an approach to new lead compounds.

Oncogenic Ras proteins whose activation is farnesylation by farnesyltransferase have been seen as important targets for novel anticancer drugs. Inhibitors of this enzyme have already been developed as potential anti-cancer drugs, particularly by rational design based on the structure of the CA(1)A(2)X carboxyl terminus of Ras. Synthesis of a peptidomimetics library via solid-phase synthesis using the Multipin method is described here. The most active hits on cellular assays were resynthesized and enzymatic activity was measured. Compounds A1, A5 and A7 present significant activity on the isolated enzyme (IC(50)=117, 57.3 and 28.5 nM) and their molecular docking in the active site of the enzyme provides details on key interactions with the protein.

Column selection and method development for the separation of nucleoside phosphotriester diastereoisomers, new potential anti-viral drugs. Application to cellular extract analysis.

Analytical HPLC methods using derivatized cellulose and amylose chiral stationary phases used in normal and reversed-phase modes were developed for the diastereoisomeric separation of mononucleotide prodrugs (pronucleotides) of 3'-azido-2',3'-dideoxythymidine (AZT). The resolutions were performed with two silica-based celluloses using normal and reversed-phase methodologies: Tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H and Chiracel OD-RH) and Tris-methylbenzoate (Chiralcel OJ and OJ-R). Two amyloses phases, Tris-3,5-dimethylphenylcarbamate (Chiralpak AD) and Tris-(S)-1-phenylethylcarbamate (Chiralpak AS), were used in normal-phase mode. Additionally, we developed separation using two stationary phases with immobilized cyclodextrins in reversed-phase and polar-organic modes. The mobile phase and the chiral stationary phase were varied to achieve the best resolution. Different types and concentration of aliphatic alcohols, acetonitrile or water in the mobile phase were also tested for the different separation modes. An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. The different columns gave complementary results in term of resolution. Limits of detection and quantification were 0.12-0.20 and 0.40-0.67 microm, respectively. This analytical method was applied in a preliminary study for the pronucleotide 2 quantification in cellular extract.

Determination of ionization constants of N-imidazole derivatives, aromatase inhibitors, using capillary electrophoresis and influence of substituents on pKa shifts.

Capillary electrophoresis (CE) was used as a method to determine the acidity constants of eight aromatase inhibitors. This method was validated by comparison of results obtained with a traditional method, UV spectroscopy, and additionally with computational calculations. We confirmed here, with our series of compounds, that capillary electrophoresis is an attractive method for pKa measurements which is based on migration time or mobilities of the ionic species over a range of pH values. The precision of pKa measurements of N-imidazole derivatives is useful to observe pKa shifts induced by chemical modifications introduced on adjacent aromatic rings such as heterocycle (benzoxa- or benzothiazolinone) or substituted benzyle. The knowledge of these pKa values is a great interest to predict migration of solutes and qualitative interactions with ionized cyclodextrines as chiral selectors in further enantioseparative CE studies.

A flexible approach to the design of new potent substance P receptor ligands.

The development of small-molecule antagonists of the substance-P-preferring tachykinin NK1 receptor offers an excellent opportunity to exploit these molecules as novel therapeutic agents in diverse pathologies such as depression, emesis or asthma. GR71251 has previously been identified as a potent and selective substance-P-receptor antagonist. We have therefore undertaken the synthesis of new pseudopeptidic analogues based on the C-terminal sequence of GR71251. The evaluation of binding affinities toward NK1 and NK2 receptors has enabled us to propose new selective NK1 ligands with high affinity. Structure-activity relationships showed that the Trp-OBzl(CF3)2 moiety is essential for NK1 affinity and that the introduction of building units such as spirolactam, lactam or proline, leading to a constrained peptide, increased selectivity for NK1 receptors. These compounds constitute a useful starting point for new substance P antagonists and represent an attractive lead series for further studies on the design of specific NK1 antagonists.

Conformation of the tripeptide Cbz-Pro-Leu-Trp-OBzl(CF3)2 deduced from two-dimensional 1H-NMR and conformational energy calculations is related to its affinity for NK1-receptor.

Chemical modifications of dual NK1/NK2 ligand Cbz-Gly-Leu-Trp-OBzl(CF3)2 (1) enabled us to create a high NK1 selective ligand Cbz-Pro-Leu-Trp-OBzl(CF3)2 (2). A determination of the conformational behavior of tripeptide 2 in solution is described. The 1D and 2D 1H-NMR techniques (COSY and ROESY) were used to assign resonances. Observed interproton distance restraints were considered to characterize conformational behavior. Spectral data indicate that tripeptide 2 presents a rigidified structure in DMSO stabilized by H-bond in two gamma-turns. Agreement with experimental data was obtained by averaging the 1H-NMR parameters over several combinations of low-energy conformations.

DNA interaction of the tyrosine protein kinase inhibitor PD153035 and its N-methyl analogue.

The brominated anilinoquinazoline derivative PD153035 exhibits a very high affinity and selectivity for the epidermal growth factor receptor tyrosine kinase (EGF-R TK) and shows a remarkable cytotoxicity against several types of tumor cell lines. In contrast, its N-methyl derivative, designated EBE-A22, has no effect on EGF-R TK but maintains a high cytotoxic profile. The present study was performed to explore the possibility that PD153035 and its N-methyl analogue might interact with double-stranded DNA, which is a primary target for many conventional antitumor agents. We studied the strength and mode of binding to DNA of PD153035 and EBE-A22 by means of absorption, fluorescence, and circular and linear dichroism as well as by a relaxation assay using human DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to show that both drugs bind to DNA and behave as typical intercalating agents. In particular, EBE-A22 unwinds supercoiled plasmid, stabilizes duplex DNA against heat denaturation, and produces negative CD and ELD signals, as expected for an intercalating agent. Extensive DNase I footprinting experiments performed with a large range of DNA substrates show that EBE-A22, but not PD153035, interacts preferentially with GC-rich sequences and discriminates against homooligomeric runs of A and T which are often cut more readily by the enzyme in the presence of the drug compared to the control. Altogether, the results provide the first experimental evidence that DNA is a target of anilinoquinazoline derivatives and suggest that this N-methylated ring system is a valid candidate for the development of DNA-targeted cytotoxic compounds. The possible relevance of selective DNA binding to activity is considered. The unexpected GC-selective binding properties of EBE-A22 entreat further exploration into the use of N-methylanilinoquinazoline derivatives as tools for designing sequence-specific DNA binding ligands.

New bis-catechols 5-lipoxygenase inhibitors.

Three polyhydroxy-2-phenylnaphthalenes (1-3) and the oxy analogue of tetrahydroxypavinan (4) were prepared and evaluated for their antioxidant properties (inhibition of diphenylpycrylhydrazyl radical (DPPH), reduction of iron (III) ion) and inhibition of 5-lipoxygenase (5-LO) activity. Their three-dimensional structures were established on the basis of spectroscopic data and semiempirical calculations. Compounds 1 and 2 were found as potent 5-LO inhibitors as nordihydroguaiaretic acid (NDGA), whereas 4 is 2.5 times less potent than NDGA. The reliability of the 3-D structures with the 5-LO inhibition properties is discussed. Their antioxidant properties show that tested compounds are expected to act as redox inhibitors.

Apoptotic response of HL-60 human leukemia cells to the antitumor drug NB-506, a glycosylated indolocarbazole inhibitor of topoisomerase 1.

The antitumor drug NB-506 is a glycosylated indolocarbazole derivative targeting topoisomerase I. This DNA-intercalating agent, which is currently undergoing phase I/II clinical trials, was shown to induce apoptosis in HL-60 human leukemia cells. We compared the cellular dysfunctions induced by NB-506 and the reference topoisomerase I poison camptothecin (CPT) at the nuclear, mitochondrial, and cytoplasmic levels. The two drugs NB-506 and CPT were almost equally toxic to HL-60 cells and produced similar cell cycle changes with a considerable increase in the fraction of cells with DNA content less than G1. The sub-G1 fraction, which can be considered as the apoptotic cell population, appeared more rapidly with CPT than with NB-506 but in both cases, the cell cycle perturbation was accompanied by a marked decrease in the mitochondrial transmembrane potential and the intracellular pH. In contrast, no change in the intracellular calcium concentration was detected. Treatment of HL-60 cells with NB-506 resulted in an increase in the activity of the intracellular protease caspase-3, as determined by a DEVD-based colorimetric assay and direct monitoring of poly(ADP-ribose) polymerase (PARP) cleavage by Western blot analysis. The initiator caspase-8 was also stimulated by NB-506 but, as for caspase-3, the extent of the caspase activation was weaker with NB-506 compared to CPT. With both drugs, the protease activation resulted in DNA degradation, as independently confirmed via the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and characterization of internucleosomal DNA fragmentation. Collectively, these findings identify some of the molecular events leading to NB-506-induced apoptosis and as such, provide important mechanistic insights into the mode of action of topoisomerase I-targeted indolocarbazole antitumor drugs.

Formaldehyde-induced alkylation of a 2'-aminoglucose rebeccamycin derivative to both A.T and G.C base pairs in DNA.

Rebeccamycin derivatives represent a promising class of antitumor agents. In this series, two glycosylated indolocarbazoles, NB-506 and NSC-655649, are currently undergoing clinical trials. Their anticancer activities are associated with their capacities to interact with DNA and to inhibit DNA topoisomerases. Previous studies revealed that the planar indolocarbazole chromophore can intercalate into DNA, locating the appended carbohydrate residue in one of the two helical grooves, probably the minor groove as is the case with the anthracyclines and other DNA-binding antibiotics. The sugar residue contributes significantly to the DNA binding free energy of NB-506. However, the exact positioning of the glycosyl residue of rebeccamycin derivatives in the drug-DNA complex remains poorly understood. To better understand how glycosylated indolocarbazoles interact with DNA, we investigated the interaction of a rebeccamycin derivative (85) bearing a 2'-amino group on the sugar residue. We show that the presence of the 2'-amino function permits the formation of covalent drug-DNA complexes in the presence of formaldehyde. Complementary biochemical and spectroscopic measurements attest that 85 reacts covalently with the 2-amino group of guanines exposed in the minor groove of the double helix, as is the case with daunomycin. In contrast to daunomycin, 85 also forms cross-links with an oligonucleotide containing only A.T base pairs. The covalent binding to A.T base pairs was detected using a gel mobility shift assay and was independently confirmed by thermal denaturation studies and by fluorescence measurements using a series of synthetic polynucleotides. The HCHO-mediated alkylation reaction of the drug with A.T base pairs apparently involves the 6-amino group of adenines exposed in the major groove whereas the covalent attachment to G.C base pairs implicates the 2-amino group of guanines situated in the opposite minor groove. Therefore, the results suggest that either the drug is able to switch grooves in response to sequence or it can simultaneously bind to both the minor and major grooves of the double helix. This study will help to guide the rational design of new DNA-binding antitumor indolocarbazole drugs and also provides a general experimental approach for probing minor versus major groove interactions between small molecules and DNA.

Synthesis and pharmacological evaluation of new pyrazolidine-3, 5-diones as AT(1) angiotensin II receptor antagonists.

On the basis of the structure of the non-peptide receptor antagonist irbesartan, a new series of AT(1) ligands was designed. In these compounds the central imidazolone nucleus of irbesartan was replaced by a pyrazolidine-3,5-dione structure. The key intermediate N-alkylpyrazolidine-3,5-diones were synthesized according to a new and general method. The most active compounds possess a spirocyclopentane ring at position 4, a linear butyl chain at position 1, and the [2'-(5-tetrazolyl)biphenyl-4-yl]methyl or [2'-(benzoylaminosulfonyl)biphenyl-4-yl]methyl group at position 2. Affinity toward the AT(1) and AT(2) receptors was assessed by the ability of the compounds to competitively displace [(3)H]AII from its specific binding sites. The most active compounds, 28 and 48, displayed high affinity for the AT(1) receptor, good selectivity AT(1) versus AT(2), and potent in vitro antagonist activity.