Prediction of Safety Margin and Optimization of Dosing Protocol for a Novel Antibiotic using Quantitative Systems Pharmacology Modeling.

Elevations of liver enzymes have been observed in clinical trials with BAL30072, a novel antibiotic. In vitro assays have identified potential mechanisms for the observed hepatotoxicity, including electron transport chain (ETC) inhibition and reactive oxygen species (ROS) generation. DILIsym, a quantitative systems pharmacology (QSP) model of drug-induced liver injury, has been used to predict the likelihood that each mechanism explains the observed toxicity. DILIsym was also used to predict the safety margin for a novel BAL30072 dosing scheme; it was predicted to be low. DILIsym was then used to recommend potential modifications to this dosing scheme; weight-adjusted dosing and a requirement to assay plasma alanine aminotransferase (ALT) daily and stop dosing as soon as ALT increases were observed improved the predicted safety margin of BAL30072 and decreased the predicted likelihood of severe injury. This research demonstrates a potential application for QSP modeling in improving the safety profile of candidate drugs.

Mechanisms of hepatotoxicity associated with the monocyclic ß-lactam antibiotic BAL30072.

BAL30072 is a new monocyclic ß-lactam antibiotic under development which provides a therapeutic option for the treatment of severe infections caused by multi-drug-resistant Gram-negative bacteria. Despite the absence of liver toxicity in preclinical studies in rats and marmosets and in single dose clinical studies in humans, increased transaminase activities were observed in healthy subjects in multiple-dose clinical studies. We, therefore, initiated a comprehensive program to find out the mechanisms leading to hepatocellular injury using HepG2 cells (human hepatocellular carcinoma cell line), HepaRG cells (inducible hepatocytes derived from a human hepatic progenitor cell line), and human liver microtissue preparations. Our investigations demonstrated a concentration- and time-dependent reduction of the ATP content of BAL30072-treated HepG2 cells and liver microtissues. BAL30072 impaired oxygen consumption by HepG2 cells at clinically relevant concentrations, inhibited complexes II and III of the mitochondrial electron transport chain, increased the production of reactive oxygen species (ROS), and reduced the mitochondrial membrane potential. Furthermore, BAL 30072 impaired mitochondrial fatty acid metabolism, inhibited glycolysis, and was associated with hepatocyte apoptosis. Co-administration of N-acetyl-L-cysteine partially protected hepatocytes from BAL30072-mediated toxicity, underscoring the role of oxidative damage in the observed hepatocellular toxicity. In conclusion, BAL30072 is toxic for liver mitochondria and inhibits glycolysis at clinically relevant concentrations. Impaired hepatic mitochondrial function and inhibition of glycolysis can explain liver injury observed in human subjects receiving long-term treatment with this compound.

Experiments for a systematic comparison between stable-isotope-(deuterium) labeling and radio-((14)C) labeling for the elucidation of the in vitro metabolic pattern of pharmaceutical drugs.

A systematic comparison between two labeling approaches for the investigation of the in vitro metabolic pattern of pharmaceutical drugs was performed by examining the use of (i) radiolabeled drugs analyzed with LC-MS-offline radiodetection and (ii) stable-isotope labeled drugs, used in a defined mixture with the unlabeled drug and analyzed by LC-MS with recognition of the specific isotopic pattern. (14)C was used for the radioisotope-approach and deuterium for the stable-isotope approach. Olanzapine, diclofenac and ketoconazole were chosen as model drugs, as they are commercially available in their non-, radio- and stable-isotope labeled forms. For all three model drugs, liver microsome- and hepatocyte-incubations (both from rat) were performed with various concentrations and incubation times for both, the radio- and the stable-isotope approaches. The metabolic pattern, including structure elucidation of all detected metabolites, was performed independently for all individual compounds and incubations. Subsequently, the metabolic patterns of the radio-, and the stable-isotope approaches were compared. In conclusion, all metabolites found with the radioisotope approach could also be found with the stable-isotope approach. Although the stable-isotope approach does not provide a quantitative result, it can be considered to be a highly suited analytical alternative for early in vitro metabolism investigations, especially when radiolabeled drug analogues are not yet available and quantitative results are not yet necessary.

Investigation of the species-dependent in vitro metabolism of BAL30630 by stable isotope labeling and isotope exchange experiments analyzed by capillary liquid chromatography coupled to mass spectrometry.

The in vitro metabolic profile of BAL30630, an antifungal piperazine propanol derivative, which inhibits the 1,3-beta-D-glucansynthase, was investigated by incubation with microsomes of several species and with rat hepatocytes. For the spotting of the metabolites, mixtures of BAL30630 with a stable isotope (deuterium) labeled analogue were incubated. The metabolic pattern comprises several oxidized metabolites. Based on isotope exchange experiments, their structures could be assigned to epoxide- and hydroxylated metabolites. In hepatocyte incubations, several glucuronides formed from these oxidized metabolites could be observed. From the analysis of the metabolic pattern in microsomes, products of carbamate hydrolysis were characterized. This hydrolysis was highly species dependent. In activated incubations and in rat hepatocytes, those metabolites were further oxidized. In incubations without NADPH activation, the resulting hydrolytic metabolites could be enriched without the subsequent oxidation. Final structural elucidation of the metabolites was performed using accurate mass determination and isotope exchange experiments, in which incubations were analyzed by deuterium exchange and capillary HPLC-QTof-MS and MS/MS. The use of non-radioactive, stabile isotope labeled drug analogues in combination with isotope exchange studies was essential in particular for a defined assignment of the functional groups in the structures of the investigated metabolites.

Elucidation of the in vitro metabolic profile of stable isotope labeled BAL19403 by accurate mass capillary liquid chromatography/quadrupole time-of-flight mass spectrometry and isotope exchange.

The in vitro metabolic pattern of BAL19403, a novel macrolide antibiotic, was investigated by capillary liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) in incubations with human microsomes. For the elucidation of the metabolic pathway, BAL19403 labeled with four deuterium atoms (D4) was used, and detection of metabolites performed using mixtures of the unlabeled (H4) BAL19403 and its D4 analogue (1:1) as substrate. All metabolites appeared with similar chromatographic behavior. MS/MS spectra of BAL19403 and its metabolites are dominated by non-informative fragment ions. Therefore, the structure of the metabolites was elucidated mainly by accurate mass measurements with subsequent proposals of elemental compositions. Main biotransformations were N-demethylation, lactone ring hydrolysis, and oxidation. Additionally, N-dealkylation of the aromatic moiety was identified. This dealkylation results not only in formation of an aldehyde, according to the classical pathway, but also in formation of the corresponding alcohol and carboxylic acid. Final elucidation of their structures was possible, since this dealkylation takes place vicinal to the deuterium-labeled part of BAL19403 and interferes with D/H exchange. The degree of D/H exchange, determined by analysis of the metabolite isotopic pattern, was used to elucidate the adjacent functional group.

Proteolytic processing by matrix metalloproteinases and phosphorylation by protein kinase CK2 of fetuin-A, the major globulin of fetal calf serum.

Bovine fetuin-A is a member of a glycoprotein family with a wide spectrum of functions. Until now the bovine protein has been thought to be a single-chain protein. Recently we have shown that native bovine plasma fetuin-A partially exists as a disulfide-bridged two-chain protein with a heavy N-terminal and a lighter C-terminal chain similar to the structure of human fetuin-A homologue (alpha2HS glycoprotein), and also is partially phosphorylated at residues Ser120, Ser302, Ser305 and Ser306 (Wind et al., Anal. Biochem. 317 (2003) 26-33). Both fetuin-A modifications, the phosphorylation at the four sites as well as the proteolysis which causes longer or shorter light chains (termed lc-1 and lc-2, respectively), are probably brought about by targeted enzymatic activities which still need to be defined. In this study we show that authentic bovine fetuin-A disulfide-bridged two-chain forms, which include the original C-terminus, were liberated from the single-chain precursor by metalloproteinases MMP-3 (stromelysin-1) and MMP-7 (matrilysin), but not by elastase, cathepsin E and cathepsin G. Peptide sequencing suggested cleavage sites chiefly at the Pro277-Ser278 or Arg294-His295 peptide bonds. Fetuin-A radioactive phosphorylation in vitro by protein kinase CK2 caused (32)P incorporation into the fetuin-A light chain lc-1 but not lc-2 or the fetuin-A heavy chain, as revealed by MMP assisted proteolysis. Analysis by nanoESI-MS pinpointed phosphorylation at the native phospho-residues Ser302, Ser305 and Ser306 by increased relative abundance following in vitro phosphorylation. Moreover, CK2 phosphorylation of synthetic C-terminal fetuin-A peptides, used as effective controls to the native protein, strongly implies that CK2 is involved in the in vivo phosphorylation of fetuin-A. The phosphorylation of N-terminally truncated peptide homologs seemed highly dependent on the sequence context N-terminal of the phosphorylation sites, thus providing a likely explanation for the non-phosphorylation of the light chain lc-2 in native fetuin-A.

Investigation of low-abundant in vitro metabolites of stable isotope-labelled BAL4815 by accurate mass capillary-LC-ESI-qTof-MS and MS/MS.

The metabolic profile of BAL4815, an antifungal azole drug, was determined using in vitro rat hepatocyte incubations and subsequent analysis by capillary LC-qTof-MS and MS/MS including accurate mass determination. For the detection of the metabolites, a mixture of the drug and its deuterium-labelled analogue was used for incubations. Metabolic stability of BAL4815 was high in cultured rat hepatocytes. However, several low-abundant metabolites were detected by the use of capillary LC-qTof-MS and manual investigation of the data. The peak intensity of the most abundant metabolite was close to the limit of detection. Except for an apparent oxidation product, the masses of the other detected metabolites could not be assigned to a single and frequently occurring biotransformation. Accurate mass determination and possible elemental compositions suggested that metabolism occurred through a combination of glutathionylation and defluorination. This was verified using accurate mass MS/MS. The use of accurate mass measurements and the derived suggestions for the elemental compositions were essential to elucidate this atypical metabolic pathway. A mass accuracy better than 8 ppm could be achieved for most assigned MS and MS/MS signals with intensities less than 6 cps in the spectra.

Analysis of CheA histidine phosphorylation and its influence on protein stability by high-resolution element and electrospray mass spectrometry.

A combination of electrospray mass spectrometry (ESI-MS) and element mass spectrometry (ICPMS) with phosphorus detection was used to characterize histidine phosphorylation (His-48) of the chemotaxis protein CheA quantitatively. The phosphorylation at His-48 was found to be responsible for a stabilization of the protein. For this investigation, the acceptor domain and the kinase domain of the bacterial chemotaxis protein CheA were recombinantly expressed as single proteins. Using in vitro kinase assay conditions, the acceptor domain CheA-H was phosphorylated by the kinase domain CheA-C. The degree of histidine phosphorylation was determined by nanoelectrospray mass spectrometry of intact CheA-H, and was found to be limited to a maximum value of approximately 50%. The site specificity of CheA-H phosphorylation was controlled by nanoESI-MS/MS of the [M + 16H](16+) ion of intact (pHis)-CheA-H and allowed localization of the pHis residue to the region between residues 32 and 86, containing candidates His-48 and His-67, for which His-48 phosphorylation has been described. Analysis of the tryptic digest of in vitro histidine-phosphorylated CheA-H by capillary chromatography coupled to ESI-MS and to ICPMS with phosphorus detection revealed a truncated (pHis)-CheA-H protein as the only phosphorus-containing analyte. Since the truncated (pHis)-CheA-H in the digest was found to exhibit a higher degree of phosphorylation than could be generated by in vitro phosphorylation without trypsin treatment, it is concluded that histidine phosphorylation at His-48 strongly interferes with structural properties of the CheA-H domain in particular with respect to proteolytic degradation by trypsin.

Plk1 docking to GRASP65 phosphorylated by Cdk1 suggests a mechanism for Golgi checkpoint signalling.

GRASP65, a structural protein of the Golgi apparatus, has been linked to the sensing of Golgi structure and the integration of this information with the control of mitotic entry in the form of a Golgi checkpoint. We show that Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and that phosphorylated GRASP65 interacts with the polo box domain of the polo-like kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression of the wild-type GRASP65 C-terminus but not the phosphorylation defective mutant in normal rat kidney cells causes a delay but not the block in mitotic entry expected if this were a true cell cycle checkpoint. These findings identify a Plk1-dependent signalling mechanism potentially linking Golgi structure and cell cycle control, but suggest that this may not be a cell cycle checkpoint in the classical sense.

Stable isotope phospho-profiling of fibrinogen and fetuin subunits by element mass spectrometry coupled to capillary liquid chromatography.

We have used one-dimensional polyacrylamide gel electrophoresis, tryptic digestion, and capillary liquid chromatography-mass spectrometry with inductively coupled plasma ionization and phosphorus-31 detection or electrospray ionization for the analysis of protein phosphorylation. We have analyzed human fibrinogen with two well-characterized phosphorylation sites and bovine fetuin with unknown phosphorylation status. Both serine-3 and serine-345 (both in Aalpha) of fibrinogen were clearly recognized. In bovine fetuin, four phosphorylated sites were newly characterized (serine-138, serine-320, serine-323, and serine-324). The novel strategy provides a fast and quantitative overview of the presence of protein phosphorylation sites.

Spotting and quantification of phosphoproteins purified by gel electrophoresis and laser ablation-element mass spectrometry with phosphorus-31 detection.

Laser ablation inductively coupled plasma-mass spectrometry (ICP-MS) with (31)P detection has been used for spotting of phosphoproteins after one-dimensional polyacrylamide gel electrophoresis (1-D PAGE) and membrane transfer. By analyzing a mixture of myoglobin, alpha-casein and reduced fibrinogen it is demonstrated that phosphoproteins are specifically recognized by this method. A special washing step was found to be necessary to remove phosphate noncovalently bound to proteins. The (31)P signal was found to contain quantitative information both with respect to relative and absolute amounts of phosphorus present in phosphoproteins. Normalizing the (31)P signal from a single laser ablation trace by the total amount of phosphoprotein applied to the gel, a detection limit of 5 pmol of phosphorus is estimated.

Identification of phosphorylation sites in the polo-like kinases Plx1 and Plk1 by a novel strategy based on element and electrospray high resolution mass spectrometry.

A novel strategy for the determination of protein phosphorylation sites is described and applied to the polo-like kinases Plx1 (Xenopus laevis) and Plk1 (Homo sapiens). The strategy comprises the sequential application of the following techniques: proteolytic digestion, capillary liquid chromatography (LC)-inductively coupled plasma mass spectrometry with phosphorus detection, capillary LC-electrospray mass spectrometry and electrospray tandem mass spectrometry. In this approach, phosphopeptides are generated, their elution time in capillary LC is determined, candidate phosphopeptides at the corresponding elution times are identified, and positive identification and sequencing of phosphopeptides is performed in the last step of the analysis. Using this technique, Ser25/26, Ser326, and Ser340 were identified as phosphorylation sites in recombinant Plx1, and Ser340 was identified as the major phosphorylation site in a kinase-dead mutant of Plx1 expressed in okadaic acid-treated Sf9 insect cells. A site corresponding to Ser326 in Plx1 was also shown to be phosphorylated in the human polo-like kinase Plk1 (Ser335). Element mass spectrometry with phosphorus detection provides a quantitative phosphorylation profile of all phosphorylation sites accessible by LC.

Cell cycle-regulated phosphorylation of the Xenopus polo-like kinase Plx1.

Polo-like kinases (Plks) control multiple important events during M phase progression, but little is known about their activation during the cell cycle. The activities of both mammalian Plk1 and Xenopus Plx1 peak during M phase, and this activation has been attributed to phosphorylation. However, no phosphorylation sites have previously been identified in any member of the Plk family. Here we have combined tryptic phosphopeptide mapping with mass spectrometry to identify four major phosphorylation sites in Xenopus Plx1. All four sites appear to be phosphorylated in a cell cycle-dependent manner. Phosphorylations at two sites (Ser-260 and Ser-326) most likely represent autophosphorylation events, whereas two other sites (Thr-201 and Ser-340) are targeted by upstream kinases. Several recombinant kinases were tested for their ability to phosphorylate Plx1 in vitro. Whereas xPlkk1 phosphorylated primarily Thr-10, Thr-201 was readily phosphorylated by protein kinase A, and Cdk1/cyclin B was identified as a likely kinase acting on Ser-340. Phosphorylation of Ser-340 was shown to be responsible for the retarded electrophoretic mobility of Plx1 during M phase, and phosphorylation of Thr-201 was identified as a major activating event.