Browsing by Subject "Chromatography, High Pressure Liquid"
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Item Open Access Amino acid racemization reveals differential protein turnover in osteoarthritic articular and meniscal cartilages.(Arthritis Res Ther, 2009) Stabler, Thomas V; Byers, Samuel S; Zura, Robert D; Kraus, Virginia ByersINTRODUCTION: Certain amino acids within proteins have been reported to change from the L form to the D form over time. This process is known as racemization and is most likely to occur in long-lived low-turnover tissues such as normal cartilage. We hypothesized that diseased tissue, as found in an osteoarthritic (OA) joint, would have increased turnover reflected by a decrease in the racemized amino acid content. METHODS: Using high-performance liquid chromatography methods, we quantified the L and D forms of amino acids reported to racemize in vivo on a biological timescale: alanine, aspartate (Asp), asparagine (Asn), glutamate, glutamine, isoleucine, leucine (Leu), and serine (Ser). Furthermore, using a metabolically inactive control material (tooth dentin) and a control material with normal metabolism (normal articular cartilage), we developed an age adjustment in order to make inferences about the state of protein turnover in cartilage and meniscus. RESULTS: In the metabolically inactive control material (n = 25, ages 13 to 80 years) and the normal metabolizing control material (n = 19, ages 17 to 83 years), only Asp + Asn (Asx), Ser, and Leu showed a significant change (increase) in racemization with age (P < 0.01). The age-adjusted proportions of racemized to total amino acid (D/D+L expressed as a percentage of the control material) for Asx, Ser, and Leu when compared with the normal articular cartilage control were 97%, 74%, and 73% in OA meniscal cartilage and 97%, 70%, and 78% in OA articular cartilage. We also observed lower amino acid content in OA articular and meniscal cartilages compared with normal articular cartilage as well as a loss of total amino acids with age in the OA meniscal but not the OA articular cartilage. CONCLUSIONS: These data demonstrate comparable anabolic responses for non-lesioned OA articular cartilage and OA meniscal cartilage but an excess of catabolism over anabolism for the meniscal cartilage.Item Open Access Determination of metarrestin (ML-246) in human plasma for a first-in-human clinical pharmacokinetic application by a simple and efficient uHPLC-MS/MS assay.(Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2023-05) Richardson, William J; Zimmerman, Sara M; Reno, Annieka; Corvalan Cabanas, Natalia; Arisa, Oluwatobi; Rudloff, Udo; Figg, William D; Peer, Cody JMetarrestin is a first-in-class small molecule inhibitor targeting the perinucleolar compartment, a subnuclear body associated with metastatic capacity. Promising preclinical results led to the clinical translation of the compound into a first-in-human phase I trial (NCT04222413). To characterize metarrestin's pharmacokinetic profile in humans, a uHPLC-MS/MS assay was developed and validated to determine the disposition of the drug in human plasma. Efficient sample preparation was accomplished through one-step protein precipitation paired with elution through a phospholipid filtration plate. Chromatographic separation was achieved with gradient elution through an Acuity UPLC® BEH C18 column (50 × 2.1 mm, 1.7 µm). Tandem mass spectrometry facilitated the detection of metarrestin and tolbutamide, the internal standard. The effective calibration range spanned 1-5000 ng/mL and was both accurate (range -5.9 % to 4.9 % deviation) and precise (≤9.0 %CV). Metarrestin proved stable (≤4.9 % degradation) under various assay-imposed conditions. Matrix effects, extraction efficiency, and process efficiency were assessed. Further, the assay was successfully able to determine the disposition of orally administered metarrestin in patients from the lowest dose cohort (1 mg) for 48 h post-administration. Thus, the validated analytical method detailed in this work is simple, sensitive, and clinically applicable.Item Open Access Pharmacology of riluzole in acute spinal cord injury.(Journal of neurosurgery. Spine, 2012-09) Chow, Diana SL; Teng, Yang; Toups, Elizabeth G; Aarabi, Bizhan; Harrop, James S; Shaffrey, Christopher I; Johnson, Michele M; Boakye, Maxwell; Frankowski, Ralph F; Fehlings, Michael G; Grossman, Robert GObject
The aim of this paper was to characterize individual and population pharmacokinetics of enterally administered riluzole in a Phase 1 clinical trial of riluzole as a neuroprotective agent in adults 18-70 years old with acute spinal cord injury (SCI).Methods
Thirty-five individuals with acute SCI, American Spinal Injury Association Impairment Scale Grades A-C, neurological levels from C-4 to T-12, who were enrolled in the Phase 1 clinical trial sponsored by the North American Clinical Trials Network for Treatment of Spinal Cord Injury, received 50 mg riluzole twice daily for 28 doses. The first dose was administered at a mean of 8.7 ± 2.2 hours postinjury. Trough plasma samples were collected within 1 hour predose, and peak plasma samples were collected 2 hours postdose on Days 3 and 14 of treatment. Riluzole concentrations were quantified by high-performance liquid chromatography assay. The data were analyzed for individual and population pharmacokinetics using basic structural and covariate models. The pharmacokinetic measures studied were the peak concentration (C(max)), trough concentration (C(min)), systemic exposure (AUC(0-12)), clearance (CL/F), and volume of distribution (V_F) normalized by the bioavailability (F).Results
The C(max) and AUC(0-12) achieved in SCI patients were lower than those in ALS patients on the same dose basis, due to a higher CL and larger V. The pharmacokinetics of riluzole (C(max), C(min), AUC(0-12), CL, and V) changed during the acute and subacute phases of SCI during the 14 days of therapy. It was consistently observed in patients at all clinical sites that C(max), C(min), and AUC(0-12) (128.9 ng/ml, 45.6 ng/ml, and 982.0 ng × hr/ml, respectively) were significantly higher on Day 3 than on Day 14 (76.5 ng/ml, 19.1 ng/ml, and 521.0 ng × hr/ml, respectively). These changes resulted from lower CL (49.5 vs 106.2 L/hour) and smaller V (557.1 vs 1297.9/L) on Day 3. No fluid imbalance or cytochrome P 1A2 induction due to concomitant medications was identified during the treatment course to account for such increases in V and CL, respectively. Possible mechanisms underlying these changes are discussed.Conclusions
This is the first report of clinical pharmacokinetics of riluzole in patients with SCI. The C(max) and AUC(0-12) achieved in SCI patients were lower than those in ALS patients on the same dose basis, due to a higher clearance and larger volume of distribution in SCI patients. The finding in SCI patients of an increase in the clearance and distribution of riluzole between the 3rd and 14th days after SCI, with a lower plasma concentration of riluzole on the 14th day, stresses the importance of monitoring changes in drug metabolism after SCI in interpreting the safety and efficacy of therapeutic drugs that are used in clinical trials in SCI. Clinical trial registration no.: NCT00876889.Item Open Access PLN-encoded phospholamban mutation in a large cohort of hypertrophic cardiomyopathy cases: summary of the literature and implications for genetic testing.(American heart journal, 2011-01) Landstrom, AP; Adekola, BA; Bos, JM; Ommen, SR; Ackerman, MJBACKGROUND:hypertrophic cardiomyopathy (HCM) is a major cause of sudden death in young athletes and one of the most common inherited cardiovascular diseases, affecting 1 in 500 individuals. Often viewed as a disease of the cardiac sarcomere, mutations in genes encoding myofilament proteins are associated with disease pathogenesis. Despite a clinically available genetic test, a significant portion of HCM patients remain genetically unexplained. We sought to determine the spectrum and prevalence of mutations in PLN-encoded phospholamban in a large cohort of HCM cases as a potential cause of mutation-negative HCM. METHODS:comprehensive genetic interrogation of the promoter and coding region of PLN was conducted using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing. RESULTS:one L39X nonsense mutation was identified in 1 of 1,064 HCM proband cases with a family history of HCM, previously found to be negative for the current HCM genetic test panel. This mutation cosegregated with incidence of HCM in a multigenerational family. Compared with similar studies, we identified an overall yield of PLN-HCM mutations of 0.65%, similar to 3 genes that are part of current HCM genetic test panels. We did not observe any PLN coding sequence genetic variation in 600 reference alleles. CONCLUSIONS:overall, mutations in PLN are rare in frequency, yet the small size of the genetic locus may make it amenable to inclusion on HCM gene test panels, especially because the frequency of background genetic variation among otherwise healthy subjects appears negligible. The exact role of mutations in PLN and other calcium-handling proteins in the development of HCM warrants further investigation.Item Open Access Primary vascularization of the graft determines the immunodominance of murine minor H antigens during organ transplantation.(J Immunol, 2011-10-15) Kwun, Jean; Malarkannan, Subramaniam; Burlingham, William J; Knechtle, Stuart JGrafts can be rejected even when matched for MHC because of differences in the minor histocompatibility Ags (mH-Ags). H4- and H60-derived epitopes are known as immunodominant mH-Ags in H2(b)-compatible BALB.B to C57BL/6 transplantation settings. Although multiple explanations have been provided to explain immunodominance of Ags, the role of vascularization of the graft is yet to be determined. In this study, we used heart (vascularized) and skin (nonvascularized) transplantations to determine the role of primary vascularization of the graft. A higher IFN-γ response toward H60 peptide occurs in heart recipients. In contrast, a higher IFN-γ response was generated against H4 peptide in skin transplant recipients. Peptide-loaded tetramer staining revealed a distinct antigenic hierarchy between heart and skin transplantation: H60-specific CD8(+) T cells were the most abundant after heart transplantation, whereas H4-specific CD8(+) T cells were more abundant after skin graft. Neither the tissue-specific distribution of mH-Ags nor the draining lymph node-derived dendritic cells correlated with the observed immunodominance. Interestingly, non-primarily vascularized cardiac allografts mimicked skin grafts in the observed immunodominance, and H60 immunodominance was observed in primarily vascularized skin grafts. However, T cell depletion from the BALB.B donor prior to cardiac allograft induces H4 immunodominance in vascularized cardiac allograft. Collectively, our data suggest that immediate transmigration of donor T cells via primary vascularization is responsible for the immunodominance of H60 mH-Ag in organ and tissue transplantation.Item Open Access Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.(Am J Physiol Endocrinol Metab, 2016-03-15) Shankaran, Mahalakshmi; Shearer, Todd W; Stimpson, Stephen A; Turner, Scott M; King, Chelsea; Wong, Po-Yin Anne; Shen, Ying; Turnbull, Philip S; Kramer, Fritz; Clifton, Lisa; Russell, Alan; Hellerstein, Marc K; Evans, William JBiomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM.Item Open Access Quantitation of the next-generation imipridone ONC206 in human plasma by a simple and sensitive UPLC-MS/MS assay for clinical pharmacokinetic application.(Journal of pharmaceutical and biomedical analysis, 2022-05) Goodell, Jennifer C; Zimmerman, Sara M; Peer, Cody J; Prabhu, Varun; Yin, Tyler; Richardson, William J; Azinfar, Arya; Dunn, John A; Mullin, Mark; Theeler, Brett J; Gilbert, Mark; Figg, William DONC206 is an imipridone derivative that is being developed clinically as a single agent given orally in a first-in-human trial (NCT04541082). This ongoing clinical trial requires pharmacokinetic analysis of ONC206 to fully characterize its pharmacologic profile. There is currently no published bioanalytical method for ONC206 quantitation. To understand the clinical pharmacokinetics of ONC206, a sensitive yet simple uHPLC-MS/MS method for quantitation of ONC206 in human plasma was developed. Protein-precipitation allowed rapid and sensitive bioanalytical measurement of ONC206 in human plasma. A Phenomenex Kinetex C18 (50 ×2.1 mm, 1.3 µm, 100 Å) analytical column achieved symmetrical and sharp chromatography peaks of ONC206 and the internal standard, [2H]7-ONC206, which were detected using multiple reaction monitoring. The assay calibration range was 1-500 ng/mL and was best fit by a linear regression model (r2 > 0.99732 ± 0.0010). The method proved accurate (< ± 9% deviation), precise (<11%CV), selective and specific with no interference and low inter-lot matrix variability. ONC206 demonstrated excellent short-term, long-term, and multiple freeze-thaw cycle stability in solution and human plasma. This fully validated method was used to quantitate ONC206 plasma concentrations from patients enrolled in the aforementioned clinical trial at the NCI to demonstrate its clinical applicability.Item Open Access Succinylated octopamine ascarosides and a new pathway of biogenic amine metabolism in Caenorhabditis elegans.(J Biol Chem, 2013-06-28) Artyukhin, Alexander B; Yim, Joshua J; Srinivasan, Jagan; Izrayelit, Yevgeniy; Bose, Neelanjan; von Reuss, Stephan H; Jo, Yeara; Jordan, James M; Baugh, L Ryan; Cheong, Micheong; Sternberg, Paul W; Avery, Leon; Schroeder, Frank CThe ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in Caenorhabditis elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLC-MS/MS-based targeted metabolomics, we identified novel ascarosides incorporating a side chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine-derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.