Pharmacodynamic comparison of LY3023703, a novel microsomal prostaglandin e synthase 1 inhibitor, with celecoxib.

To assess the safety, tolerability, and pharmacology of LY3023703, a microsomal prostaglandin E synthase 1 (mPGES1) inhibitor, a multiple ascending dose study was conducted. Forty-eight subjects received LY3023703, celecoxib (400 mg), or placebo once daily for 28 days. Compared with placebo, LY3023703 inhibited ex vivo lipopolysaccharide-stimulated prostaglandin E2 (PGE2 ) synthesis 91% and 97% on days 1 and 28, respectively, after 30-mg dosing, comparable to celecoxib's effect (82% inhibition compared to placebo). Unlike celecoxib, which also inhibited prostacyclin synthesis by 44%, LY3023703 demonstrated a maximal increase in prostacyclin synthesis of 115%. Transient elevations of serum aminotransferase were observed in one subject after 30-mg LY3023703 dosing (10× upper limit of normal (ULN)), and one subject after 15-mg dosing (about 1.5× ULN). Results from this study suggest that mPGES1 inhibits inducible PGE synthesis without suppressing prostacyclin generation and presents a novel target for inflammatory pain.

Data-driven analysis approach for biomarker discovery using molecular-profiling technologies.

High-throughput molecular-profiling technologies provide rapid, efficient and systematic approaches to search for biomarkers. Supervised learning algorithms are naturally suited to analyse a large amount of data generated using these technologies in biomarker discovery efforts. The study demonstrates with two examples a data-driven analysis approach to analysis of large complicated datasets collected in high-throughput technologies in the context of biomarker discovery. The approach consists of two analytic steps: an initial unsupervised analysis to obtain accurate knowledge about sample clustering, followed by a second supervised analysis to identify a small set of putative biomarkers for further experimental characterization. By comparing the most widely applied clustering algorithms using a leukaemia DNA microarray dataset, it was established that principal component analysis-assisted projections of samples from a high-dimensional molecular feature space into a few low dimensional subspaces provides a more effective and accurate way to explore visually and identify data structures that confirm intended experimental effects based on expected group membership. A supervised analysis method, shrunken centroid algorithm, was chosen to take knowledge of sample clustering gained or confirmed by the first step of the analysis to identify a small set of molecules as candidate biomarkers for further experimentation. The approach was applied to two molecular-profiling studies. In the first study, PCA-assisted analysis of DNA microarray data revealed that discrete data structures exist in rat liver gene expression and correlated with blood clinical chemistry and liver pathological damage in response to a chemical toxicant diethylhexylphthalate, a peroxisome-proliferator-activator receptor agonist. Sixteen genes were then identified by shrunken centroid algorithm as the best candidate biomarkers for liver damage. Functional annotations of these genes revealed roles in acute phase response, lipid and fatty acid metabolism and they are functionally relevant to the observed toxicities. In the second study, 26 urine ions identified from a GC/MS spectrum, two of which were glucose fragment ions included as positive controls, showed robust changes with the development of diabetes in Zucker diabetic fatty rats. Further experiments are needed to define their chemical identities and establish functional relevancy to disease development.

In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin.

The UDP-glucuronosyltransferases (UGTs) are a superfamily of membrane-bound enzymes whose active site is localized inside the endoplasmic reticulum. Glucuronidation using human liver microsomes has traditionally involved disruption of the membrane barrier, usually by detergent treatment, to attain maximal enzyme activity. The goals of the current work were to develop a universal method to glucuronidate xenobiotic substrates using microsomes, and to apply this method to sequential oxidation-glucuronidation reactions. Three assays of UGT catalytic activity estradiol-3-glucuronidation, acetaminophen-O-glucuronidation, and morphine-3-glucuronidation, which are relatively selective probes for human UGT1A1, 1A6, and 2B7 isoforms, respectively, were developed. Treatment of microsomes with the pore-forming peptide alamethicin (50 microg/mg protein) resulted in conjugation rates 2 to 3 times the rates observed with untreated microsomes. Addition of physiological concentrations of Mg(2+) to the alamethicin-treated microsomes yielded rates that were 4 to 7 times the rates with untreated microsomes. Optimized assay conditions were found not to detrimentally affect cytochrome P450 activity as determined by effects on testosterone 6beta-hydroxylation and 7-ethoxycoumarin deethylation. Formation of estradiol-3-glucuronide displayed atypical kinetics, and data best fit the Hill equation, yielding apparent kinetic parameters of K(m)(app) = 0.017 mM, V(max)(app) = 0.4 nmol/mg/min, and n = 1.8. Formation of acetaminophen-O-glucuronide also best fit the Hill equation, with K(m)(app) = 4 mM, V(max)(app) = 1.5 nmol/mg/min, and n = 1.4. Alternatively, morphine-3-glucuronide formation displayed Michaelis-Menten kinetics, with K(m)(app) = 2 mM and V(max)(app) = 2. 5 nmol/mg/min. Finally, alamethicin treatment of microsomes was found to be effective in facilitating the sequential oxidation-glucuronidation of 7-ethoxycoumarin.

Site localization of sialyl Lewis(x) antigen on alpha1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry.

A simple, fast and sensitive method was developed to verify the presence of the sialyl Lewis(x) antigen on an N-linked glycoprotein. High performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was used to identify which of the five N-linked glycosylation sites of human plasma alpha1-acid-glycoprotein (orosomucoid, OMD) contain the sialyl Lewis(x) antigen. OMD was digested with proteolytic enzymes and analyzed by reversed phase chromatography coupled with on-line ESI/MS. A tandem mass spectrometry experiment was designed to detect the presence of the sialyl Lewis(x) antigen based on the observation of an 803 mass to charge ratio ( m/z ) ion produced in the intermediate pressure region of the ESI interface. The ESI/MS signal at m/z 803 is consistent with an oxonium ion for a glycan structure containing NeuAc, Gal, GlcNAc, and Fuc. The identity of the m/z 803 ion was confirmed by ESI/MS/MS analysis of the m/z 803 fragment ion and comparison with a sialyl Lewis(x) standard. The stereochemistry and linkage positions were assigned using previous NMR analysis but could be determined with permethylation analysis if necessary. The analysis of OMD gave a pattern showing signal for the sialyl Lewis(x) antigen coeluting with each of the five N-linked glycopeptides. The ability to monitor sialyl Lewis(x) expression at each of the five sites is of interest in the study of OMD's role in inflammatory diseases.

Evidence for a novel pentyl radical adduct of the cyclic nitrone spin trap MDL 101,002.

3,4-Dihydro-3,3-dimethyl-isoquinoline-2-oxide (MDL 101,002) is a conformationally constrained cyclic analog of the known spin trap alpha-phenyl N-tert-butyl nitrone (PBN). Because of PBN's ability to scavenge free radicals, MDL 101,002 is currently being evaluated in stroke models as a means to ameliorate the oxidative insult associated with reperfusion injury. To augment our understanding of the radical scavenging mechanism of this potential drug, MDL 101,002 was incubated with soybean lipoxygenase in the presence of linoleic acid to study the interaction between MDL 101,002 and free radicals formed during lipid peroxidation. Analysis of the reaction mixture was performed by high performance liquid chromatography using normal phase conditions with detection by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Similar to the work by Iwahashi et al. [Arch. Biochem. Biophys., 1991, 285, 172], who studied the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN), an adduct that suggested the trapping of pentyl radicals by MDL 101,002 was observed. However, the apparent molecular ion for this adduct (246 Da) was 1 Da lower than would be predicted if a pentyl radical had simply added to MDL 101,002. In addition, the adduct exhibited significant absorbance at 304 nm, consistent with the unsaturated nitrone structure of MDL 101,002. To account for these observations, it is postulated that, after the initial capture of a pentyl radical, subsequent abstraction of a hydrogen atom by a neighboring radical occurs to regenerate a nitrone (1-pentyl analog of MDL 101,002). We present evidence for this adduct and offer a mechanism for its formation. These findings indicate that mass spectroscopic analysis of stable nitrone radical adducts may be useful in the identification of radical-dependent damage in vivo and possibly in clinical development of MDL 101,002 as an antioxidant pharmaceutical.

AF12198, a novel low molecular weight antagonist, selectively binds the human type I interleukin (IL)-1 receptor and blocks in vivo responses to IL-1.

Interleukin-1 (IL-1) -alpha and -beta are potent regulators of inflammatory responses. The naturally occurring interleukin-1 receptor antagonist (IL-1ra) is effective in vitro and in vivo in modulating biological responses to IL-1. We have previously reported the discovery of IL-1 antagonist peptides from the search of phage display libraries. Further characterization of this group of peptides has led to a 15-mer, AF12198, Ac-FEWTPGWYQJYALPL-NH2 (J represents the unnatural amino acid, 2-azetidine-1-carboxylic acid), with both in vitro and in vivo IL-1 antagonist activity. AF12198 selectively binds the human type I IL-1 receptor but not the human type II receptor or the murine type I receptor. In vitro, AF12198 inhibits IL-1-induced IL-8 production by human dermal fibroblasts with a half-maximal inhibition concentration or IC50 of 25 nM and IL-1-induced intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells with an IC50 of 9 nM. When given as an intravenous infusion to cynomolgus monkeys, AF12198 blocks ex vivo IL-1 induction of IL-6 and down modulates in vivo induction of IL-6. This is the first small molecule to show IL-1 receptor antagonist activity in vivo.

Application of packed capillary liquid chromatography/mass spectrometry with electrospray ionization to the study of the human biotransformation of the anti-emetic drug dolasetron.

Packed capillary liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) was used to study the human biotransformation of the anti-emetic drug dolasetron. Urine from subjects given a single 100 mg intravenous dose, containing 14C-labeled dolasetron (50 microCi), was de-salted and concentrated for LC/MS with minimal loss of radioactivity (97% recovery). Aliquots of the de-salted material were injected directly onto a C8 packed capillary column (25 cm x 0.32 mm i.d.) and eluted with an acetonitrile-water gradient, buffered with 1% acetic acid, at a flow rate of 2 microliters min-1. Five metabolites were detected by LC ESI-MS which, yielded molecular mass information but no fragmentation. The identity of each metabolite was confirmed in a subsequent analysis using product ion scans in conjunction with collisionally induced dissociation. Precursor ion scanning was also employed and did not reveal any new biotransformation products. In addition to defining the major routes of biotransformation, the data obtained were compared with a 14C radioprofile prepared in a separate experiment. Qualitative agreement in the two chromatographic profiles enabled the major clusters of radioactivity to be assigned to specific metabolites of dolasetron. An important observation in this comparison was that the signal obtained by ESI did not provide an accurate assessment of the quantity of each metabolite. This was especially true for acidic conjugates (i.e. glucuronides, sulfates), which in the case of dolasetron can exist as zwitterions (no net charge). The results demonstrate the power of packed capillary LC ESI-MS for use in drug biotransformation studies and suggest that caution should be exercised when interpreting relative metabolite abundances from ESI data in the absence of actual reference standards.

Rapid analysis of antibiotic-containing mixtures from fermentation broths by using liquid chromatography-electrospray ionization-mass spectrometry and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry.

A crucial step in the isolation of antibiotic substances is establishing whether or not the isolated material represents a new chemical entity. Because of the importance of molecular weight to this process-known as dereplication-mass spectrometry has traditionally played an active role. In this communication a strategy for utilizing liquid chromatography-mass spectrometry (LC/MS) for novelty assessment is described. Crude extracts (20-50 µg) are chromatographed by conventional bore high-performance liquid chromatography (1 mL/min) after which a postcolumn split to divert roughly one-tenth of the sample to the mass spectrometer for molecular weight determination by electrospray ionization (ESI) mass spectrometry. The majority of the effluent is sent to a UV detector and ultimately collected as 1-min fractions for biological testing. As a secondary confirmation of molecular weight, an aliquot of each fraction (< 5%) is taken for analysis by matrix-assisted laser desorption ionization (MALDI). The improved efficiency of this approach over more traditional schemes utilizing off-line fraction collection and conventional ionization methods can be explained by several factors. First, the superior sensitivity of ESI and MALDI means that less material is required for successful analysis. Second, on-line LC/MS optimizes the efficiency of sample transfer and saves both time and labor. Furthermore, the concentration dependence of ESI allows a majority of the material injected for LC/MS to be recovered for biological testing without compromising the signal available for molecular weight determination. As a validation of the above method, crude extracts containing two well-characterized antibiotics-teicoplanin and phenelfamycin-were examined. Results from these analyses are presented along with data from the analysis of a potent unknown antifungal sample.

Evidence for a lysine-specific fragmentation in fast-atom bombardment mass spectra of peptides.

A fragmentation process observed for peptides that contain lysine, or other amino acids which possess a free amino group on their sidechain, is reported. The ions generated by this process are found 16 Da below the acylium-type B ions that result from fragmentation at the C-terminal side of lysine or other amine-containing residues in fast-atom bombardment (FAB) mass spectra. These ions, which are referred to as (B-16) ions, permit differentiation between the isobaric amino acids lysine and glutamine in peptide mass spectra. High resolution measurements indicate that (B-16) ions differ in composition from the corresponding B ions by the removal of one oxygen atom. Formation is believed to occur through a cyclization process initiated by nucleophilic attack by the free amino group of the lysine sidechain at the carbon of the acylium ion (B ion). A similar process initiated directly from the protonated peptide may also occur. Analogous cyclization processes are restricted for glutamine because this residue is comparatively less nucleophilic than lysine (i.e., amide vs amine). Although (B-16) ions have been detected under high energy collisionally induced dissociation, they are formed less readily than by FAB mass spectrometry. A mechanism consistent with this observation as well as other experimental evidence is presented to account for the formation of (B-16) ions.

Metabolism of the anticoagulant peptide, MDL 28,050, in rats.

Rats were each administered a 9 mg/kg iv bolus dose of a 3H-labeled decapeptide anticoagulant, MDL 28,050. Tritium was eliminated rapidly with approximately 50% of the dose recovered in urine within the first 6 hr. Renal excretion accounted for 68% of the dose, whereas fecal excretion accounted for 16% of the dose. Continuous flow fast atom bombardment mass spectrometry was used to identify the major urinary metabolites of MDL 28,050. Trace amounts of parent drug were found, and other biotransformation products indicated that hydrolysis had occurred at four peptide bonds. Two initial sites of hydrolysis were identified as 4I-5P and 6E-7E, which resulted in the peptide fragments Suc-Y-E-P-I-OH + P-E-E-A-Cha-E-OH and Suc-Y-E-P-I-P-E-OH + E-A-Cha-E-OH, respectively. Further metabolism of these fragments resulted in the N-terminal pentapeptide and the C-terminal dipeptide.

Incorporation of tandem mass spectrometric detection to the analysis of peptide mixtures by continuous flow fast atom bombardment mass spectrometry.

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.

Interfacing microbore and capillary liquid chromatography to continuous-flow fast atom bombardment mass spectrometry for the analysis of glycopeptides.

This work describes a system to interface either microbore or packed capillary gradient liquid chromatography (LC) to fast atom bombardment mass spectrometry (FAB-MS). The interface incorporates on-line ultraviolet detection and post-column matrix addition to enable independent optimization of both LC and FAB-MS. The glycopeptide antibiotic teicoplanin was chosen as a model system because this group of compounds places severe demands on the chromatographic separation and is difficult to analyze by FAB-MS. For both microbore and capillary LC, high-quality mass spectra of the major components in teicoplanin were obtained; however, the increased sensitivity of the capillary system allowed spectra to be obtained at low picomole concentrations. The sensitivity and ease of use make capillary LC the preferred system for use in LC-FAB-MS.

Curtatoxins. Neurotoxic insecticidal polypeptides isolated from the funnel-web spider Hololena curta.

Three polypeptide neurotoxins (curtatoxins) were isolated from the venom of the spider Hololena curta by reverse-phase high performance liquid chromatography, gel permeation, and ion-exchange chromatography. The purified toxins induced an immediate paralysis in the cricket Acheta domestica that resulted in desiccation and death of the insect within 24-48 h (LD50 congruent to 4-20 micrograms/g); this toxic effect is consistent with irreversible presynaptic neuromuscular blockade. Curtatoxins are a class of sequence-related, cysteine-rich, carboxyl-terminal amidated polypeptides of 36 to 38 amino acid residues. The cysteine residues are conserved at identical sequence positions among these polypeptides and form 4 intramolecular disulfide bonds. Hydropathy calculations show that the toxins have an internal hydrophobic region flanked by hydrophilic and oppositely charged amino- and carboxyl-terminal ends. By analogy to other cysteine-rich arthropod venom proteins, the folded structure of the curtatoxins is likely important for their target specificity and mode of action at the neuromuscular junction.

High-sensitivity amino acid analysis by derivatization with O-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection: applications in protein structure determination.

An amino acid analysis method using a commercially available analyzer that accurately quantitates protein-derived amino acids in the 10-100 pmol range is described. The method utilizes the robotic capability of the analyzer's autosampler to perform precolumn derivatization of both primary and secondary amino acids with o-phthalaldehyde and 9-fluorenylmethyl chloroformate, respectively. The derivatized amino acids are then separated on a C-18 reverse-phase amino acid column and quantitated in a single run by fluorescence detection. The characterization of beta-lactoglobulin and two tryptic peptides from the bacterial enzyme diaminopimelic acid epimerase is used to demonstrate the sensitivity and utility of this method.

Fast atom bombardment mass spectrometric investigation of in vitro degradation within the disulfide-linked core of atrial natriuretic factor.

The use of high-performance liquid chromatography and fast atom bombardment mass spectrometry are shown to be an efficient combination for investigating protease-mediated digestion of synthetic analogs of the peptide hormone ANF (atrial natriuretic factor). As examples of the reported methodology, rANF5-23-NH2 and rANF7-23-NH2 were digested with the endopeptidase thermolysin. These truncated analogs were selected to investigate metabolism within the disulfide-linked core of ANF, particularly at the Cys7-Phe8 bond. While this position was the site of initial hydrolysis for rANF5-23-NH2 (t1/2 = 0.5 min), the Cys7-Phe8 bond remained intact for all observed degradation products of rANF7-23-NH2 (t1/2 = 16 min). These findings suggest that improved stability towards endopeptidase-mediated core hydrolysis may be conferred to analogs of ANF by removal of the first six residues from the N-terminus.

Comparison of thermally-assisted fast atom bombardment (TA-FAB) with conventional FAB and EI mass spectrometry for the analysis of the Helminthosporium carbonum mycotoxins.

A new technique known as thermally-assisted fast atom bombardment (TA-FAB) has been applied to the analysis of a series of cyclic tetrapeptide mycotoxins in order to demonstrate the usefulness of the method for structural elucidation. TA-FAB uses saturated aqueous solutions of highly hydroxylated compounds, such as fructose, as alternatives to the usual viscous liquid matrices employed in conventional FAB. During the TA-FAB analysis, the probe tip is resistively heated causing differences to occur in the desorption profiles for the analyte and the matrix ions enabling an optimization for analyte desorption as a function of temperature. In this study, direct comparisons are made between TA-FAB, conventional FAB, and electron impact ionization for the analysis of the Helminthosporium carbonum mycotoxins at the 1.5 micrograms level. The results demonstrate the superior capacity of TA-FAB to provide both molecular weight confirmation and significant fragmentation to aid in the structural elucidation of these important biomolecules.

Application of fast atom bombardment mass spectrometry to biological samples: analysis of urinary metabolites of acetaminophen.

Fast atom bombardment (FAB) is useful for the characterization of all major metabolites of the analgesic acetaminophen (APAP). It is particularly useful for providing mass spectra of the polar glucuronide and sulfate conjugates which eluded identification by field desorption and other more conventional methods of ionization. A protocol is described for the use of FAB in the identification of urinary APAP metabolites isolated by reversed phase high-performance liquid chromatography (HPLC) following therapeutic dosages of the drug. A tentative set of recommendations for the off-line use of HPLC and FAB is directed towards solving problems encountered when using these two analytical techniques in concert. In addition, a method for calculating the signal to background ratio (S/B) for analyte peaks in FAB spectra from selected relative ion intensities is proposed. Examples are presented that show the potential of S/B as an empirical parameter for judging the quality of FAB spectra.