ABSTRACT
To support clinical development, a solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0449 concentrations in human plasma has been developed and validated. Samples (200 microl) were extracted using an Oasis MCX 10 mg 96-well SPE plate and the resulting extracts were analyzed using reverse-phase chromatography coupled with a turbo-ionspray interface. The method was validated over calibration curve range 5-5000 ng/mL. Quadratic regression and 1/x(2) weighing were used. Within-run relative standard deviation (%RSD) was within 10.1% and accuracy ranged from 88.6% to 109.0% of nominal. Between-run %RSD was within 8.6% and accuracy ranged from 92.4% to 105.3% of nominal. Extraction recovery of GDC-0449 was between 88.3% and 91.2% as assessed using quality control sample concentrations. GDC-0449 was stable in plasma for 315 days when stored at -70 degrees C and stable in reconstituted sample extracts for 117 h when stored at room temperature. Quantitative matrix effect/ion suppression experiment was performed and no significant matrix ion suppression was observed. This assay allows for the determination of GDC-0449 plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.
Subject(s)
Anilides/blood , Chromatography, Liquid/methods , Hedgehog Proteins/antagonists & inhibitors , Hedgehog Proteins/metabolism , Pyridines/blood , Signal Transduction , Solid Phase Extraction/methods , Tandem Mass Spectrometry/methods , Anilides/pharmacokinetics , Humans , Pyridines/pharmacokineticsABSTRACT
GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide) is a potent, selective Hedgehog (Hh) signalling pathway inhibitor being developed for the treatment of various cancers. The in vivo clearance of GDC-0449 was estimated to be 23.0, 4.65, 0.338, and 19.3 ml min(-1) kg(-1) in mouse, rat, dog and monkeys, respectively. The volume of distribution ranged from 0.490 in rats to 1.68 l kg(-1) in mice. Oral bioavailability ranged from 13% in monkeys to 53% in dogs. Predicted human clearance using allometry was 0.096-0.649 ml min(-1) kg(-1) and the predicted volume of distribution was 0.766 l kg(-1). Protein binding was extensive with an unbound fraction less than or equal to 6%, and the blood-to-plasma partition ratio ranged from 0.6 to 0.8 in all species tested. GDC-0449 was metabolically stable in mouse, rat, dog and human hepatocytes and had a more rapid turnover in monkey hepatocytes. Proposed metabolites from exploratory metabolite identification in vitro (rat, dog and human liver microsomes) and in vivo (dog and rat urine) include three primary oxidative metabolites (M1-M3) and three sequential glucuronides (M4-M6). Oxidative metabolites identified in microsomes M1 and M3 were formed primarily by P4503A4/5 (M1) and P4502C9 (M3). GDC-0449 was not a potent inhibitor of P4501A2, P4502B6, P4502D6, and P4503A4/5 with IC50 estimates greater than 20 microM. K(i)'s estimated for P4502C8, P4502C9 and P4502C19 and were 6.0, 5.4 and 24 microM, respectively. An evaluation with Simcyp suggests that GDC-0449 has a low potential of inhibiting P4502C8 and P4502C9. Furthermore, GDC-0449 (15 microM) was not a potent P-glycoprotein/ABCB1 inhibitor in MDR1-MDCK cells. Overall, GDC-0449 has an attractive preclinical profile and is currently in Phase II clinical trials.
Subject(s)
Anilides/pharmacokinetics , Antineoplastic Agents/pharmacokinetics , Hedgehog Proteins/antagonists & inhibitors , Microsomes, Liver/metabolism , Pyridines/pharmacokinetics , Administration, Oral , Animals , Biological Availability , Cytochrome P-450 Enzyme System/metabolism , Dogs , Drug Evaluation, Preclinical , Hepatocytes/drug effects , Hepatocytes/enzymology , Humans , Injections, Intravenous , Macaca fascicularis , Metabolic Clearance Rate , Mice , Microsomes, Liver/drug effects , Rabbits , Rats , Rats, Sprague-DawleyABSTRACT
We have evaluated (i) a multiplexed electrospray interface, (ii) serial sample introduction, and (iii) a quadrupole time-of-flight mass spectrometer for quantitative bioanalysis in compliance with good laboratory practice. These evaluations were done using a 96-well plate liquid chromatography-tandem mass spectrometry method for the quantitation of loratadine and its metabolite, descarboethoxyloratadine. The assay has a dynamic range of 1-1000 ng/ml with 5.56 pg of each analyte being injected on-column at the limit of quantitation. For the four-channel multiplexed electrospray experiments, one-run validations were performed simultaneously in rat, rabbit, mouse and dog plasma. In the four-stream serial experiments, the total run time of the assay was reduced from 3.5 to 0.35 min, resulting in a net acquisition time of 11 s. Four simulated validation runs with standard and quality control solutions were analyzed. Precision and accuracy for standards and quality control samples met US Food and Drug Administration recommended criteria for both the drug and the metabolite using those two approaches. In addition, a quadrupole time-of-flight mass spectrometer was used as a detector in the tandem mass spectrometry mode for the loratadine assay. Our results demonstrated that a dynamic range of three orders of magnitude could be achieved using the quadrupole time-of-flight mass spectrometer, making it useful for quantitation in preclinical toxicology studies.
Subject(s)
Chromatography, High Pressure Liquid/methods , Drug Design , Mass Spectrometry/methods , Pharmaceutical Preparations/blood , Animals , Dogs , Mice , Quality Control , Rabbits , Rats , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A four-channel multiplexed electrospray interface on a triple quadrupole mass spectrometer was evaluated for the simultaneous validation of LC/MS/MS methods for the quantitation of loratadine and its metabolite, descarboethoxyloratadine, in four different biological matrixes. The assays were performed in rat, rabbit, mouse, and dog plasma from 1 to 1000 ng/mL using 96-well solid-phase extraction for sample preparation. The limit of quantitation of 1 ng/mL corresponded to 5.56 pg of each analyte injected on-column. For the drug, quality control samples (n = 6 at four concentrations) had precision ranging from 0.967 to 16.0% and accuracy ranging from -8.44 to 10.5% across all four species. For the metabolite, the precision ranged from 0.684 to 11.0% and the accuracy was between 6.36 and -9.06%. Intersprayer cross talk for the multiplexed electrospray ion source was evaluated as a function of analyte concentration and was less than 0.08% at concentrations as high as 1000 ng/mL. These results demonstrate the feasibility of using parallel analysis to reduce the time required for method validation and to increase sample throughput in drug development studies.
Subject(s)
Histamine H1 Antagonists/blood , Loratadine/blood , Animals , Chromatography, High Pressure Liquid , Dogs , Histamine H1 Antagonists/pharmacokinetics , Humans , Indicators and Reagents , Loratadine/pharmacokinetics , Mice , Rabbits , Rats , Spectrometry, Mass, Electrospray IonizationABSTRACT
New peptaibols, trichokindins I-VII, have been isolated from the fungus Trichoderma harzianum. Their structures were characterized by spectrometric methods. Trichokindins, which are 18-residue peptides containing one to three isovaline residues, were found to induce Ca(2+)-dependent catecholamine secretion from bovine adrenal medullary chromaffin cells.
Subject(s)
Adrenal Medulla/cytology , Peptides/chemistry , Trichoderma/metabolism , Adrenal Medulla/metabolism , Amino Acid Sequence , Animals , Calcium/pharmacology , Catecholamines/metabolism , Cattle , Molecular Sequence Data , Peptides/isolation & purification , Peptides/pharmacology , Spectrometry, Mass, Fast Atom BombardmentABSTRACT
WIN 64821 (1) is a substance P (SP) antagonist isolated from a fungal culture (Aspergillus sp., SC319). It is a symmetrical dimer biosynthesized from four aromatic amino acid molecules: each equivalent half of the dimer is constructed from one molecule of phenylalanine (Phe) and one molecule of tryptophan (Trp). Feeding analogs of Phe, Trp, and other amino acids to intact cells of SC319 has yielded 36 biosynthetic analogs of WIN 64821. The analogs fall into three categories: substitutions on the indoline ring, substitutions on the Phe-derived phenyl ring, and replacement of the phenyl ring by an aliphatic group. In addition, these directed biosynthesis experiments generated asymmetrical dimers (derived from three amino acids) and, often, symmetrical dimers (derived from two amino acids). The relative SP binding affinities of several analogs suggest involvement of both the indoline and phenyl moieties in SP receptor binding.
Subject(s)
Aspergillus/metabolism , Indoles/metabolism , Piperazines/metabolism , Receptors, Neurokinin-1/metabolism , Substance P/antagonists & inhibitors , Chromatography, High Pressure Liquid , Circular Dichroism , Culture Media , Humans , Indoles/chemistry , Indoles/pharmacology , Magnetic Resonance Spectroscopy , Piperazines/chemistry , Piperazines/pharmacology , Spectrometry, Mass, Fast Atom Bombardment , Spectrophotometry, UltravioletABSTRACT
Using fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS), we examined 12 synthetic N-carbamoylamino acids (CAA) as tert-butyldimethylsilyl (TBDMS) derivatives. In FAB mass spectrometry and FAB MS/MS, spectra of protonated molecules for CAA provide specific cleavages involving the TBDMS carbamoyl moiety. The daughter scan spectrum of the parent ion indicated that it was useful for structural elucidation and differentiation of structural isomers of CAA. We have also identified each CAA separately in a mixture using a neutral loss scan for characteristic ions. In addition, we demonstrated that CAA in urine samples from patients with ornithine carbamoyl transferase deficiency gave collision-induced dissociation (CID) spectra which correspond well with CID spectra obtained using synthetically prepared CAA.