Jeffrey Roth

A Sensitive and Robust HPLC Assay with Fluorescence Detection for the Quantification of Pomalidomide in Human Plasma for Pharmacokinetic Analyses

Pomalidomide is a second generation IMiD (immunomodulatory agent) that has recently been granted approval by the Food and Drug Administration for treatment of relapsed multiple myeloma after prior treatment with two antimyeloma agents, including lenalidomide and bortezomib. A simple and robust HPLC assay with fluorescence detection for pomalidomide over the range of 1-500ng/mL has been developed for application to pharmacokinetic studies in ongoing clinical trials in various other malignancies. A liquid-liquid extraction from human plasma alone or pre-stabilized with 0.1% HCl was performed, using propyl paraben as the internal standard. From plasma either pre-stabilized with 0.1% HCl or not, the assay was shown to be selective, sensitive, accurate, precise, and have minimal matrix effects (<20%). Pomalidomide was stable in plasma through 4 freeze-thaw cycles (<12% change), in plasma at room temperature for up to 2h for samples not pre-stabilized with 0.1% HCl and up to 8h in samples pre-stabilized with 0.1% HCl, 24h post-preparation at 4°C (<2% change), and showed excellent extraction recovery (∼90%). This is the first reported description of the freeze/thaw and plasma stability of pomalidomide in plasma either pre-stabilized with 0.1% HCl or not. The information presented in this manuscript is important when performing pharmacokinetic analyses. The method was used to analyze clinical pharmacokinetics samples obtained after a 5mg oral dose of pomalidomide. This relatively simple HPLC-FL assay allows a broader range of laboratories to measure pomalidomide for application to clinical pharmacokinetics.

Thrombospondin 1 and its specific cysteine-serine-valine-threonine-cysteine-glycine receptor in fetal wounds

Thrombospondin 1 (TSP-1), an adhesive glycoprotein, plays an important role in platelet adhesion, inflammation, cell-cell interaction, and angiogenesis. TSP-1 is expressed by endothelial cells, fibroblasts, and macrophages. The unique cysteine-serinevaline-threonine-cysteine-glycine (CSVTCG) binding domain of TSP-1 also plays an important role in cell binding and modulation of cellular processes. The purpose of this study was to evaluate histologically and quantitatively TSP-1 and its CSVTCG receptor in fetal skin wounds over time. Pregnant ewes underwent laparotomy and hysterotomy. At 65 days gestation (term, 145 days), incisional and excisional wounds were created on the fetal back in a similar position on each animal. The uterus and laparotomy were closed. The wounds were harvested on days 1, 3, 7, 21, and 28. Expression of TSP-1 and its CSVTCG receptor was evaluated immunohistochemically and quantitated by computer image analysis in units of absorbance. Immunoglobulin G (negative) controls were performed and subtracted from the TSP-1 sample to eliminate background absorbance readings. Serum (negative) control was used for the CSVTCG receptor. Platelet concentrates were used as the positive control: TSP-1, 63.43; CSVTCG, 58.72. Results are expressed as absorbance+/-SEM. Results of TSP-1 are as follows: day 1, 33.02+/-0.26; day 3, 22.21+/-0.14; day 7, 20.56+/-1.07; day 21, 7.76+/-0.40; and day 28, 5.99+/-0.03. TSP-1 displays an early peak during fetal skin repair, followed by a steep decrease over the viewed time period. Results of CSVTCG receptor are as follows: day 1, 26.19+/-2.43; day 3, 30.20+/-0.64; day 7, 24.56+/-0.80; day 21, 24.70+/-0.40; and day 28, 21.65+/-1.39. Thus, CSVTCG receptor displays a slowed decrease in expression over time during fetal repair. No significant differences were noted between incisional and excisional samples. Temporal and histological differences exist in the localization and expression of TSP-1 and its CSVTCG receptor during fetal wound repair. TSP-1 is upregulated in tissues early. This corresponds with the known role of TSP-1 in cell-cell interaction, including potentiation of growth factor activity. TSP-1 also modulates matrix, allowing scar-free provisional matrix in the earlier stages of repair deposited by platelets. The potentiation of cell-associated protease activity by TSP-1 can support tissue and matrix turnover. This activity of TSP-1 may contribute to the formation of a scarless wound. TSP-1 destabilizes extracellular matrix contacts, and facilitates mitosis and migration. The action of TSP-1 as an adhesive protein allows numerous different cells to adhere to the extracellular membrane. CSVTCG receptor expression decreases during fetal repair as the cells migrate to the epithelial surface, suggesting a significant role of the CSVTCG receptor in keratinocytic maturation, differentiation, and epithelization.

Thrombospondin-1 and its CSVTCG-specific receptor in wound healing and cancer.

Growth factors play a crucial role in the regulation of cellular proliferation and matrix degradation in wound healing and cancer. We have shown that thrombospondin 1 (TSP-1) and its cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG)-specific receptor play a key role in cell invasion and matrix degradation in different carcinomas. The present study was done to determine whether TSP-1 and its receptor show a similar pattern of expression in wound healing and cancer. Expression and localization of TSP-1 and its receptor were determined in fetal wounds, adult burn wounds, and different human malignancies by immunohistochemical staining and computerized image analysis. In healing wounds, TSP-1 was expressed in the stroma early in the process, followed by a steep decline. The TSP-1 receptor localized to neovessels and highly proliferating cells (i.e., fibroblasts, basal cells), its levels remaining relatively constant. Cancer cells and tumor-associated microvessels expressed the TSP-1 receptor, whereas TSP-1 localized predominantly to the tumor-associated stroma. These data suggest a critical role for TSP-1 and its CSVTCG-specific receptor in wound healing and cancer.

THE 1998 MOYER AWARD : CHARACTERISTICS OF THROMBOSPONDIN-1 AND ITS CYSTEINE-SERINE-VALINE-THREONINE-CYSTEINE-GLYCINE RECEPTOR IN BURN WOUNDS

Thrombospondin-1 (TSP-1), an adhesive glycoprotein, plays an important role in platelet adhesion, inflammation, cell-to-cell interaction, and angiogenesis. TSP-1 is expressed by endothelial cells, fibroblasts, and macrophages. TSP-1s unique cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG) specific receptor plays an important role in the binding and modulation of cellular adhesion and invasion. This article histologically and quantitatively evaluates TSP-1 and its CSVTCG receptor in adult burn wounds over time. Tissue was obtained from burn wounds on several days and samples that were 5 microns thick were placed on slides. Expression of TSP-1 and its CSVTCG receptor were evaluated immunohistochemically and quantitated by computer image analysis in units of absorbance. Immunoglobin G (IgG) (negative) controls were performed and subtracted from the TSP-1 sample to eliminate background absorbance readings. Serum (negative) control was used for the CSVTCG receptor. Platelet concentrates were used as the positive control. A quantitative examination of the results yielded the following information, expressed as absorbance +/- standard error of the mean: TSP-1: day 1, 62.0 +/- 10.13; day 3, 76.2 +/- 6.90; day 5, 36.0 +/- 3.96; day 7, 60.4 +/- 5.67; and day 9, 29.5 +/- 2.91. TSP-1 displays an early peak, followed by a steep decrease over the time period studied. The readings for the CSVTCG receptor are as follows: day 1, 33.8 +/- 1.87; day 3, 34.5 +/- 5.39; day 7, 39.1 +/- 1.93; day 21, 39.1 +/- 1.93; day 28, 34.8 +/- 3.67. In contrast, the CVSTCG receptor continues to be present in the wound over time. Histologic findings are reported, and photographs and a histopathologic analysis are included. The information presented in this article leads to the conclusion that temporal and histologic differences exist in the localization and expression of TSP-1 and its CSVTCG receptor. TSP-1 is up-regulated in injured tissues immediately after the injury; it is rapidly down-regulated as the tissue heals. In contrast, the levels of the CSVTCG receptor remain relatively constant during the healing process. These data are consistent with TSP-1s known role in cell-to-cell interaction, including the modulation of the growth factor and protease activity.

Quantitative determination of mithramycin in human plasma by a novel, sensitive ultra-HPLC-MS/MS method for clinical pharmacokinetic application

Mithramycin is a neoplastic antibiotic synthesized by various Streptomyces bacteria. It is under investigation as a chemotherapeutic treatment for a wide variety of cancers. Ongoing and forthcoming clinical trials will require pharmacokinetic analysis of mithramycin in humans, both to see if target concentrations are achieved and to optimize dosing and correlate outcomes (response/toxicity) with pharmacokinetics. Two published methods for mithramycin quantitation exist, but both are immunoassays that lack current bioanalytical standards of selectivity and sensitivity. To provide an upgraded and more widely applicable assay, a UPLC-MS/MS method for quantitation of mithramycin in human plasma was developed. Solid-phase extraction allowed for excellent recoveries (>90%) necessary for high throughput analyses on sensitive instrumentation. However, a ∼55% reduction in analyte signal was observed as a result of plasma matrix effects. Mithramycin and the internal standard chromomycin were separated on a Waters Acquity BEH C18 column (2.1×50 mm, 1.7 μm) and detected using electrospray ionization operated in the negative mode at mass transitions m/z 1083.5→268.9 and 1181.5→269.0, respectively, on an AB Sciex QTrap 5500. The assay range was 0.5-500 ng/mL and proved to be linear (r(2)>0.996), accurate (≤10% deviation), and precise (CV<15%). Mithramycin was stable in plasma at room temperature for 24 h, as well as through three freeze-thaw cycles. This method was subsequently used to quantitate mithramycin plasma concentrations from patients enrolled on two clinical trials at the NCI.

A novel uHPLC–MS/MS method for the quantitation of AZD7451 (AZ12607092) in human plasma☆

Tropomyosin-related kinases (Trk) are tyrosine kinase receptors implicated in tumor proliferation, invasion, and survival signaling across a number of tumors, making them potentially attractive targets for the treatment of cancer. AZD7451 is a potent and selective inhibitor of Trk kinases currently undergoing a Phase I dose escalation in glioblastoma multiforme at the National Cancer Institute. A key part of early clinical testing for AZD7451 involves demonstrating that pharmacokinetic half-life and clinical exposures of AZD7451 are sufficient to inhibit Trk receptors in preclinical models. To address this need, an ultra sensitive analytical method was developed to measure the AZD7451 profile in human plasma. A liquid-liquid extraction recovered >80% of AZD7451 before quantitative analysis by ultra HPLC-MS/MS. A Varian Polaris(®) C18-A column and a mass transition of m/z 383.5→340.5 (m/z 389.6→342.0 for the internal standard [(2)H6]-AZD7451) was used, and a dynamic calibration range of 0.5-1000ng/mL was established, which provided a sensitive (<8.5% deviation), and precise (<6%) quantitative assay for AZD7451. AZD7451 demonstrated stability in human plasma at room temperature for 24h (<7% change) and after extraction at 4°C for 24h (<8% change), and was stable through 4 freeze/thaw cycles (<8% change). This method was used to measure AZD7451 plasma levels in clinical samples to confirm the sensitivity at several time points following AZD7451 treatment in subjects with glioblastoma.

Abstract 2043: Effects of 24-h carboplatin pretreatment on olaparib clearance in women's cancers using noncompartmental and population pharmacokinetic analyses

Background: Olaparib (OLA) is a PARP inhibitor approved for use in deleterious germline BRCA mutated recurrent or refractory ovarian cancer. Combining OLA with carboplatin (CARBO) could have additive effects based on platinum-DNA adducts requiring PARP for DNA repair. Preclinical data suggest greater cytotoxicity when CARBO is given prior to OLA. However, the optimal treatment sequence of these agents has not been studied previously in patients. We therefore investigated: 1) the effects of CARBO treatment on the pharmacokinetics (PK) and pharmacodynamics (PD) of OLA; 2) in vitro mechanisms of the interaction between CARBO and OLA. Methods: Clinical PK and PD data of OLA were obtained from 58 patients with confirmed recurrent or refractory women9s cancers participating in a two arm, parallel design, phase 1 trial (NCT01237067). In cycle 1 OLA tablets (200 mg BID) were given for 7 days either followed by CARBO (AUC 4) on day 8 (arm A) or after CARBO on day 1 (Arm B). In cycle 2 the arms received the reversed scheme. PK of OLA were assessed in both cycles by noncompartmental (NCA) and population pharmacokinetic (PPK) analyses. For PK/PD analyses, PAR levels were measured at baseline and 24 h after the first OLA dose. In vitro mechanistic studies were carried out by incubating whole human blood and avian DT40 PARP-1 KO cells with 10 μM CARBO for 24 h, followed by 1h-treatment of isolated PBMCs and PARP-1 KO cells with 10 μM OLA. Intracellular OLA concentrations were determined using UPLC-MS/MS. Results: Both NCA and PPK analyses showed a ∼50% increase in OLA clearance when CARBO was administered 24-h prior (P Conclusion: This is the first known PK analysis showing a significant increase in OLA clearance after pretreatment with CARBO, possibly leading to subtherapeutic plasma concentrations of OLA. Preclinical experiments are ongoing to reveal the exact pharmacological mechanisms of this interaction. Citation Format: Andrew K.L. Goey, Cody J. Peer, Tristan M. Sissung, Jeffrey Roth, Shandiz Shahbazi, Jeffers Nguyen, Christina M. Annunziata, Nicole Houston, Elise C. Kohn, Jung-Min Lee, William D. Figg. Effects of 24-h carboplatin pretreatment on olaparib clearance in women9s cancers using noncompartmental and population pharmacokinetic analyses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2043.

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