Evaluation of the Pharmacokinetics and Safety of AMG 986 Tablet and Capsule Formulations in Healthy Adult Subjects: A Phase I, Open-Label, Randomized Study.

BACKGROUND AND OBJECTIVE: AMG 986 is a first-in-class, novel apelin receptor small molecule agonist initially developed for the treatment of heart failure. The current phase I study was conducted to evaluate the pharmacokinetics and safety of a single-dose 200-mg capsule formulation of AMG 986 relative to the tablet formulation in 12 healthy subjects. METHODS: In a two-period, two-way crossover design, eligible subjects were randomized 1:1 to tablet/capsule or capsule/tablet treatment sequences; each treatment sequence lasted for approximately 6 days and comprised six subjects. RESULTS: Following a single oral dose of AMG 986, the geometric mean maximum observed concentration (Cmax) values were 9670 ng/mL and 6920 ng/mL and the geometric mean area under the curve from time zero to 120 h (AUC0-120h) values were 68,000 ng*h/mL and 59,900 ng*h/mL for the tablet and capsule, respectively. The geometric least squares means (90% confidence interval [90% CI]) for the ratios of capsule/tablet were 0.88 (90% CI 0.81-0.96) and 0.72 (90% CI 0.57-0.91) for AUC0-120h and Cmax, respectively. AMG 986 had an acceptable safety profile; all adverse events were grade 1 or 2 in severity. CONCLUSION: There was a modest 12% decrease in AUC0-120h and a 28% decrease in Cmax with the AMG 986 capsule versus the tablet. These differences are not considered to be clinically relevant, suggesting the capsule formulation can be used in subsequent clinical studies of AMG 986.

Application of 1D 15 N and band-selective 2D 1 H-15 N CLIP-HSQMBC to detect 35/37 Cl isotope effect on nitrogen for unequivocal structure elucidation of the N-Cl moiety in molecules.

An impurity, designated MS204, was isolated from a scale-up production of an intermediate toward the synthesis of an active pharmaceutical ingredient. Structural elucidation of this chloro-containing impurity was performed based on the analysis of the MS and NMR data. Band-selective 2D 1 H-15 N CLIP-HSQMBC experiment was developed to unequivocally identify the ionic N-Cl moiety in the molecule by discovering the two isotope-shifted nitrogen peaks as 3 to 1 ratio separated by about 1 Δ15 N(37/35 Cl) = 19.6 ppb (1.19 Hz) due to the Cl isotope effect. 1D 15 N and 2D 1 H-15 N CLIP-HSQMBC experiments were applied to commercially available compounds to further confirm the techniques by detecting the isotope shift of nitrogen peaks for the N-Cl moiety in molecules.

Liquid chromatography-fluorescence and liquid chromatography-mass spectrometry detection of tryptophan degradation products of a recombinant monoclonal antibody.

Light exposure is one of several conditions used to study the degradation pathways of recombinant monoclonal antibodies. Tryptophan is of particular interest among the 20 amino acids because it is the most photosensitive. Tryptophan degradation forms several products, including an even stronger photosensitizer and several reactive oxygen species. The current study reports a specific peptide mapping procedure to monitor tryptophan degradation. Instead of monitoring peptides using UV 214 nm, fluorescence detection with an excitation wavelength of 295 nm and an emission wavelength of 350 nm was used to enable specific detection of tryptophan-containing peptides. Peaks that decreased in area over time are likely to contain susceptible tryptophan residues. This observation can allow further liquid chromatography-mass spectrometry (LC-MS) analysis to focus only on those peaks to confirm tryptophan degradation products. After confirmation of tryptophan degradation, susceptibility of tryptophan residues can be compared based on the peak area decrease.

Conformational changes of recombinant monoclonal antibodies by limited proteolytic digestion, stable isotope labeling, and liquid chromatography-mass spectrometry.

Limited proteolytic digestion is a method with a long history that has been used to study protein domain structures and conformational changes. A method of combining limited proteolytic digestion, stable isotope labeling, and mass spectrometry was established in the current study to investigate protein conformational changes. Recombinant monoclonal antibodies with or without the conserved oligosaccharides, and with or without oxidation of the conserved methionine residues, were used to test the newly proposed method. All of the samples were digested in ammonium bicarbonate buffer prepared in normal water. The oxidized deglycosylated sample was also digested in ammonium bicarbonate buffer prepared in (18)O-labeled water. The sample from the digestion in (18)O-water was spiked into each sample digested in normal water. Each mixed sample was subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). The molecular weight differences between the peptides digested in normal water versus (18)O-water were used to differentiate peaks from the samples. The relative peak intensities of peptides with or without the C-terminal incorporation of (18)O atoms were used to determine susceptibility of different samples to trypsin and chymotrypsin. The results demonstrated that the method was capable of detecting local conformational changes of the recombinant monoclonal antibodies caused by deglycosylation and oxidation.

In vitro and in vivo modifications of recombinant and human IgG antibodies.

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.

Plasma-spray ionization (PLASI): a multimodal atmospheric pressure ion source for liquid stream analysis.

A new ion generation method, named plasma-spray ionization (PLASI) for direct analysis of liquid streams, such as in continuous infusion experiments or liquid chromatography (LC), is reported. PLASI addresses many of the analytical limitations of electrospray ionization (ESI) and has potential for real time process stream analysis and reaction monitoring under atmospheric conditions in non-ESI friendly scenarios. In PLASI-mass spectrometry (MS), the liquid stream is pneumatically nebulized and partially charged at low voltages; the resultant aerosol is thus entrained with a gaseous plasma plume from a distal glow discharge prior to MS detection. PLASI-MS not only overcomes ESI-MS limitations but also generates simpler mass spectra with minimal adduct and cluster formation. PLASI utilizes the atomization capabilities of an ESI sprayer operated below the ESI threshold to generate gas-phase aerosols that are then ionized by the plasma stream. When operated at or above the ESI threshold, ionization by traditional ESI mechanisms is achieved. The multimodal nature of the technique enables readily switching between plasma and ESI operation. It is expected that PLASI will enable analyzing a wide range of analytes in complex matrices and less-restricted solvent systems, providing more flexibility than that achievable by ESI alone.

Desorption electrospray ionization (DESI) with atmospheric pressure ion mobility spectrometry for drug detection.

Desorption electrospray ionization (DESI) was coupled to an ambient pressure drift tube ion mobility time-of-flight mass spectrometer (IM-TOFMS) for the direct analysis of active ingredients in pharmaceutical samples. The DESI source was also coupled with a standalone IMS demonstrating potential of portable and inexpensive drug-quality testing platforms. The DESI-IMS required no sample pretreatment as ions were generated directly from tablets and cream formulations. The analysis of a range of over-the-counter and prescription tablet formations was demonstrated for amphetamine (methylphenidate), antidepressant (venlafaxine), barbiturate (Barbituric acid), depressant (alprazolam), narcotic (3-methylmorphine) and sympatholytic (propranolol) drugs. Active ingredients from soft and liquid formulations, such as Icy Hot cream (methyl salicylate) and Nyquil cold medicine (acetaminophen, dextromethorphan, doxylamine) were also detected. Increased sensitivity for selective drug responses was demonstrated through the formation of sodiated adduct ions by introducing small quantities of NaCl into the DESI solvent. Of the drugs and pharmaceuticals tested in this study, 68% (22 total samples) provided a clear ion mobility response at characteristic mobilities either as (M + H)(+), (M - H)(-), or (M + Na)(+) ions.

An unusual dimeric small heat shock protein provides insight into the mechanism of this class of chaperones.

Small heat shock proteins (sHSPs) are virtually ubiquitous stress proteins that are also found in many normal tissues and accumulate in diseases of protein folding. They generally act as ATP-independent chaperones to bind and stabilize denaturing proteins that can be later reactivated by ATP-dependent Hsp70/DnaK, but the mechanism of substrate capture by sHSPs remains poorly understood. A majority of sHSPs form large oligomers, a property that has been linked to their effective chaperone action. We describe AtHsp18.5 from Arabidopsis thaliana, demonstrating that it is dimeric and exhibits robust chaperone activity, which adds support to the model that suboligomeric sHSP forms are a substrate binding species. Notably, like oligomeric sHSPs, when bound to substrate, AtHsp18.5 assembles into large complexes, indicating that reformation of sHSP oligomeric contacts is not required for assembly of sHSP-substrate complexes. Monomers of AtHsp18.5 freely exchange between dimers but fail to coassemble in vitro with dodecameric plant cytosolic sHSPs, suggesting that AtHsp18.5 does not interact by coassembly with these other sHSPs in vivo. Data from controlled proteolysis and hydrogen-deuterium exchange coupled with mass spectrometry show that the N- and C-termini of AtHsp18.5 are highly accessible and lack stable secondary structure, most likely a requirement for substrate interaction. Chaperone activity of a series of AtHsp18.5 truncation mutants confirms that the N-terminal arm is required for substrate protection and that different substrates interact differently with the N-terminal arm. In total, these data imply that the core α-crystallin domain of the sHSPs is a platform for flexible arms that capture substrates to maintain their solubility.

Rapid identification and absence of drug tests for AG-013736 in 1mg Axitinib tablets by ion mobility spectrometry and DART™ mass spectrometry.

Axitinib (AG-013736) is a potent investigational drug that has antitumor activity in patients with metastatic renal cell carcinoma and other types of cancers. In this study, ion mobility spectrometry and "direct analysis in real time" (DART™) mass spectrometry were used to rapidly identify AG-013736 in drug substance samples and 1mg Axitinib tablets. The plasmagrams of the sample solutions exhibited a major peak with a reduced ion mobility that was within ±0.0002cm(2)V(-1)s(-1) of that for AG-013736 in an external reference standard solution. The DART ionization source was coupled with both a time-of-flight mass spectrometer and a lower-resolution ion trap mass spectrometer. Samples were analyzed by this technique in as little as 5s with minimal to no sample preparation required. The isotopic masses of the protonated dimer ions of AG-013736 were used to identify AG-013736 in the active tablet. Both techniques were also used to develop low-level limit tests for rapidly verifying the presence or absence of AG-013736 in blinded clinical supplies of active and matching placebo tablets of Axitinib.

Insights into small heat shock protein and substrate structure during chaperone action derived from hydrogen/deuterium exchange and mass spectrometry.

Small heat shock proteins (sHSPs) and the related alpha-crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95-156 and 228-252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP.

Symmetrical gas-phase dissociation of noncovalent protein complexes via surface collisions.

Previous gas-phase dissociation experiments of protein-protein complexes have resulted in product ion distributions that are asymmetric by charge and mass, providing limited insight into the chemical nature of subunit organization and interaction. In these experiments, a symmetric charge distribution results from an "energy sudden" collision of protein-protein complexes with a surface, indicating that it may be possible to probe the suboligomeric structure of noncovalent complexes in the gas phase. It is proposed that energy sudden surface activation of cytochrome C homodimers results in dissociation without significant unfolding of one of the monomeric subunits. Previously proposed mechanisms for the dissociation of protein-protein complexes are discussed in the context of these results. These experiments demonstrate the potential to preserve the structural details of subunit interaction within a protein-protein complex and help elucidate the asymmetric nature of macromolecular dissociation in the gas phase.

Properties of the dark and signaling states of photoactive yellow protein probed by solution phase hydrogen/deuterium exchange and mass spectrometry.

The perturbations on conversion from the dark state to the signaling state in photoactive yellow protein have been determined by solution-phase hydrogen/deuterium exchange and mass spectrometry. Both the wild type and M100A mutant are used in this study, with the mutant providing over 90% conversion to the bleached state under steady-state illumination. We found perturbations in both the wild type and the mutant on illumination, consistent with a more flexible structure in the long-lived signaling (I2') state. In the case of the wild type, the conformational changes detected are mainly around the chromophore region. With the M100A mutant, differences in H/D exchange between the light and dark are more extensive as compared to wild type; not only are the chromophore surroundings affected, but significant increases in deuterium uptake in the N-terminus and central beta-sheet are observed as well. On the basis of the data obtained from this study and previous findings, a sequence of events that leads to the perturbation of PYP following chromophore photoisomerization is proposed.

Local stability of Rhodobacter capsulatus cytochrome c2 probed by solution phase hydrogen/deuterium exchange and mass spectrometry.

The hydrogen/deuterium exchange kinetics of Rhodobacter capsulatus cytochrome c2 have been determined using mass spectrometry. As expected, the relative domain stability was generally similar to that of the cytochrome c2 structural homolog, horse heart cytochrome c, but we were able to find evidence to support the presence of a second, small beta-sheet not found in the horse cytochrome, which stabilizes a structural region dominated by Omega loops. Importantly, we find that the so-called hinge region, comprised of 15 amino acids, which include the methionine sixth heme ligand (M96), is destabilized on oxidation, and this destabilization is propagated to a portion of the second Omega loop, most likely through perturbation of two hydrogen bonds that couple these two domains in the three dimensional structure. The mutation of a lysine at position 93 to proline amplifies the destabilization observed on oxidation of the wild-type cytochrome c2 and results in further destabilization observed in regions 52-60, 75-82, and 83-97. This suggests that hydrogen bond interactions involving two bound waters, the T94 hydroxyl, the front heme propionate and the Y75 hydroxyl, are significantly compromised upon mutation. In summary, these observations are consistent with the approximately 20-fold increase in the movement of the hinge away from the heme face in the oxidized cytochrome c2 as determined by ligand binding kinetics. Thus, H/D exchange kinetics can be used to identify relatively subtle structural features and at least in some cases facilitate the understanding of the structural basis of the dynamic properties of proteins.

Mass spectrometry of peptides and proteins.

This tutorial article introduces mass spectrometry (MS) for peptide fragmentation and protein identification. The current approaches being used for protein identification include top-down and bottom-up sequencing. Top-down sequencing, a relatively new approach that involves fragmenting intact proteins directly, is briefly introduced. Bottom-up sequencing, a traditional approach that fragments peptides in the gas phase after protein digestion, is discussed in more detail. The most widely used ion activation and dissociation process, gas-phase collision-activated dissociation (CAD), is discussed from a practical point of view. Infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) are introduced as two alternative dissociation methods. For spectral interpretation, the common fragment ion types in peptide fragmentation and their structures are introduced; the influence of instrumental methods on the fragmentation pathways and final spectra are discussed. A discussion is also provided on the complications in sample preparation for MS analysis. The final section of this article provides a brief review of recent research efforts on different algorithmic approaches being developed to improve protein identification searches.

A new and efficient method for the selective olefination of aldehydes with ethyl diazoacetate catalyzed by an iron(II) porphyrin complex.

Olefination of aromatic and aliphatic aldehydes with ethyl diazoacetate was achieved in excellent yields with triphenylphosphine and catalytic amounts of iron(II) meso-tetra(p-tolyl)porphyrin. The reaction conditions are mild and the process is efficient and highly selective (>90%) for the synthesis of the trans-olefin isomer. Results of mechanistic studies are discussed.