Yashpal S. Chhonker

Quantitative analysis of endogenous compounds

Accurate quantitative analysis of endogenous analytes is essential for several clinical and non-clinical applications. LC-MS/MS is the technique of choice for quantitative analyses. Absolute quantification by LC/MS requires preparing standard curves in the same matrix as the study samples so that the matrix effect and the extraction efficiency for analytes are the same in both the standard and study samples. However, by definition, analyte-free biological matrices do not exist for endogenous compounds. To address the lack of blank matrices for the quantification of endogenous compounds by LC-MS/MS, four approaches are used including the standard addition, the background subtraction, the surrogate matrix, and the surrogate analyte methods. This review article presents an overview these approaches, cite and summarize their applications, and compare their advantages and disadvantages. In addition, we discuss in details, validation requirements and compatibility with FDA guidelines to ensure method reliability in quantifying endogenous compounds. The standard addition, background subtraction, and the surrogate analyte approaches allow the use of the same matrix for the calibration curve as the one to be analyzed in the test samples. However, in the surrogate matrix approach, various matrices such as artificial, stripped, and neat matrices are used as surrogate matrices for the actual matrix of study samples. For the surrogate analyte approach, it is required to demonstrate similarity in matrix effect and recovery between surrogate and authentic endogenous analytes. Similarly, for the surrogate matrix approach, it is required to demonstrate similar matrix effect and extraction recovery in both the surrogate and original matrices. All these methods represent indirect approaches to quantify endogenous compounds and regardless of what approach is followed, it has to be shown that none of the validation criteria have been compromised due to the indirect analyses.

LC–MS/MS method for simultaneous determination of diethylcarbamazine, albendazole and albendazole metabolites in human plasma: Application to a clinical pharmacokinetic study

HighlightsThe first LC–MS/MS method of diethylcarbamazine and albendazole along with its active metabolites.The method was successfully applied to analyze clinical samples.The highly sensitive and selective LC–MS/MS method for routine pharmacokinetic application.This method is useful for drug–drug interaction or TDM studies of diethylcarbamazine and albendazole in Lymphatic filariasis therapy. ABSTRACT Combination therapy with anti‐filarial drugs is now widely used for treatment of lymphatic filariasis. A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of diethylcarbamazine (DEC), albendazole (ABZ) and albendazole metabolites in human plasma. Separation and detection of analytes were achieved on a reversed phase column (Acquity UPLC®BEH C18 column (100 × 2.1 mm, 1.7 &mgr;m) with gradient elution using 0.05% formic acid in methanol and 0.05% formic acid as mobile phase. Solid phase extraction was utilized for elution of analytes from the matrix. Thereafter, analytes were monitored by using MS/MS with electrospray ionization source in positive multiple reaction monitoring mode. The MS/MS response was linear over the concentration range from 0.1–200 ng/mL for ABZ and ABZ‐ON, 0.5–1000 ng/mL for ABZ‐OX and 1–2000 ng/mL for DEC with a correlation coefficient (r2) of 0.998 or better. The within‐ and between‐batch precisions (relative standard deviation, % RSD) and the accuracy (% bias) were within the acceptable limits as per FDA guideline. The validated method was successfully applied to the clinical pharmacokinetic study. Due to high sensitivity and low requirement of sample volume, the method will be applicable for therapeutic drug monitoring of this regimen.

Quantitative determination of a potent geranylgeranyl diphosphate synthase inhibitor using LC–MS/MS: Derivatization and application

HighlightsA LC–MS/MS for quantitation of VSW1198 in mouse plasma and tissues.Highly sensitive and stable derivatized compounds were analyzed by LC–MS/MS.This approach could be applied to other structurally similar bisphosphonates. Abstract An isomeric mixture of homogeranyl/homoneryl triazole bisphosphonates (VSW1198) has previously been shown to be a potent inhibitor of geranylgeranyl diphosphate (GGDP) synthase (GGDPS) and of therapeutic interest for the treatment of multiple myeloma. We have developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantitation of both the E‐ and Z‐ isomers of VSW1198 in cell culture media, mouse plasma and tissues. VSW1198 and internal standard are extracted from the bio‐matrices by solid‐phase extraction, followed by derivatization using trimethylsilyldiazomethane. The chromatographic separation of analytes was achieved on a Phenomenex Gemini NX column (150 mm * 2.0 mm, 5 &mgr;) with gradient elution using 0.1% acetic acid and methanol/acetonitrile (1:1) as the mobile phase at a flow rate of 0.2 mL/min. Derivatized analytes were ionized with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode and quantitated using MS/MS. The MS/MS response was linear over the concentration range from 0.38–1500 and 0.13–500 ng/mL for the E‐ and Z‐isomers, respectively. The within‐ and between‐day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was used for quantitative determination of the compounds in preclinical studies focused on the development of VSW1198 as a novel anti‐cancer agent.

Simultaneous LC–MS/MS analysis of eicosanoids and related metabolites in human serum, sputum and BALF

The differences among individual eicosanoids in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual eicosanoids and their metabolites in serum, sputum and bronchial alveolar lavage fluid (BALF). Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 34 eicosanoids in human serum, sputum and BALF was developed and validated. This method is valid and sensitive with a limit of quantification ranging from 0.2 to 3 ng/mL for the various analytes, and has a large dynamic range (500 ng/mL) and a short run time (25 min). The intra- and inter-day accuracy and precision values met the acceptance criteria according to US Food and Drug Administration guidelines. Using this method, detailed eicosanoid profiles were quantified in serum, sputum and BALF from a pilot human study. In summary, a reliable and simple LC-MS/MS method to quantify major eicosanoids and their metabolites was developed and applied to quantify eicosanoids in human various fluids, demonstrating its suitability to assess eicosanoid biomarkers in human clinical trials.

Pharmacokinetic and Biodistribution Studies of HPMA Copolymer Conjugates in an Aseptic Implant Loosening Mouse Model

N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers were previously found to represent a versatile delivery platform for the early detection and intervention of orthopedic implant loosening. In this article, we evaluated the impact of different structural parameters of the HPMA copolymeric system (e.g., molecular weight (MW), drug content) to its pharmacokinetics and biodistribution (PK/BD) profile. Using 125I, Alexa Fluor 488, and IRDye 800 CW-labeled HPMA copolymer-dexamethasone (P-Dex) conjugates with different MW and dexamethasone (Dex) contents, we found the MW to be the predominant impact factor on the PK/BD profiles of P-Dex, with Dex content as a secondary impact factor. In gamma counter-based PK/BD studies, increased MW of P-Dex reduced elimination, leading to lower clearance, longer half-life, and higher systemic exposure (AUC and MRT). In the semiquantitative live animal optical imaging evaluation, the distribution of P-Dex to the peri-implant inflammatory lesion increased when MW was increased. This result was further confirmed by FACS analyses of cells isolated from peri-implant regions after systemic administration of Alexa Fluor 488-labeled P-Dex. Since the in vitro cell culture study suggested that the internalization of P-Dex by macrophages is generally independent of P-Dexs MW and Dex content, the impact of the MW and Dex content on its PK/BD profile was most likely exerted at physiological and pathophysiological levels rather than at the cellular level. In both gamma counter-based PK/BD analyses and semiquantitative optical imaging analyses, P-Dex with 6 wt % Dex content showed fast clearance. Dynamic light scattering analyses unexpectedly revealed significant molecular aggregation of P-Dex at this Dex content level. The underlining mechanisms of the aggregation and fast in vivo clearance of the P-Dex warrant further investigation.

Simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using LC–ESI–MS/MS: An application for pharmacokinetic studies

Hydroxychloroquine (HCQ) has been shown to disrupt autophagy and sensitize cancer cells to radiation and chemotherapeutic agents. However, the optimal delivery method, dose, and tumor concentrations required for these effects are not known. This is in part due to a lack of sensitive and reproducible analytical methods for HCQ quantitation in small animals. As such, we developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues. The chromatographic separation and detection of analytes were achieved on a reversed phase Thermo Aquasil C18 (50×4.6mm, 3μ) column, with gradient elution using 0.2% formic acid and 0.1% formic acid in methanol as mobile phase at a flow rate of 0.5mL/min. Simple protein precipitation was utilized for extraction of analytes from the desired matrix. Analytes were separated and quantitated using MS/MS with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode. The MS/MS response was linear over the concentration range from 1 to 2000ng/mL for all analytes with a correlation coefficient (R2) of 0.998 or better. The within- and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was successfully applied to a preclinical pharmacokinetic mouse study involving low volume blood and tissue samples for hydroxychloroquine and metabolites.

Abstract A16: The in vitro and in vivo activity of the pyrrolomycin marinopyrrole RL71 alone and combined with temsirolimus (TEM) in MYCN-amplified neuroblastoma (NB)

Introduction: We previously reported a series of novel marinopyrroles as Mcl-1/Bcl-2 inhibitors for potential anticancer activity. The marinopyrrole agents have not been studied in pediatric tumors including neuroblastoma (NB). Temsirolimus (TEM) is an mTOR inhibitor that has been used clinically in renal cell carcinoma and breast cancer and more recently has been used in relapse/refractory pediatric tumors, including NB. In the following study we describe the in vitro and in vivo activity of the most active of the marinopyrroles (RL71) alone or with the mTOR inhibitor TEM in a chemoresistant NB cell line. Methods: RL71, which was determined to be the most active of 39 pyrrolomycin derivatives, was tested in a MYCN amplified BE-2c NB cell line using an MTT assay and was selected for study in experiments to determine potential mechanism of action and for evaluation of in vivo activity. RL71 was tested in a series of in vitro assays to evaluate effects on apoptosis, metabolism, and cell ultrastructure. Apoptosis was evaluated using propidium iodide cell cycle analysis by flow cytometry and measurement of the sub-G1 fraction. Apoptosis was also evaluated by the measurement of cleaved PARP by Western blot. The effect of RL71 alone and combined with TEM on metabolism was determined using Seahorse Xp Metabolic Analyzer. The effect of agent(s) on OXPHOS metabolism was measured by oxygen consumption rate and effect on glycolysis by extracellular acidification rate (ECAR). Electron microscopy (EM) was used to determine changes in cell ultrastructure, particularly of mitochondria, in treated and nontreated cells. Using a mouse xenograft model, the antitumor effect for RL71 and the combination of RL71 plus TEM was tested. RL71 concentrations were measured in blood, tumor, and brain. Results: RL71 at low concentrations (500 nM) caused disruption of ATP production and increased proton leak in mitochondria after injection. This effect was associated with sharp drop in ATP cellular levels. There was also a modest decline in glycolytic capacity and a lack of compensation for loss of mitochondrial ATP production. These effects were not seen with TEM alone, nor did TEM combined with RL71 show potentiation. Measures of apoptosis, which included cleaved PARP and sub-G1 peaks on flow cytometry, were not consistently elevated in either RL71 or TEM alone or when the two were combined. EM showed mitochondrial and cell membrane disruption with RL71 treatment alone but not with TEM alone; drugs combined appeared to produce greater disruption of cell membranes. There was no clear effect on nuclear morphology on EM, further supporting that apoptosis was unlikely to be the mechanism of cell death. The cell membrane disruption was consistent with necrosis, perhaps as a result of sharp decline in ATP. Three of 5 mice dosed with the combination of RL71 plus TEM had complete responses while none of the mice in the control group, TEM-alone group, or PL71-alone group responded (p = 0.009). Concentrations of RL71 in blood, tumor, and brain generally achieved concentrations exceeding the IC50 when it was combined with TEM (IC50=25 nM). Conclusion: The combination of RL71 and TEM may be active in chemoresistant, MYCN-amplified NB. The mechanism of action of the combination continues to be evaluated; it is unlikely to be completely explained by disrupted metabolism or induction of apoptosis but likely results from nonapoptotic mechanisms of cell death such as necrosis or autophagy. Citation Format: Timothy R. McGuire, Donald W. Coulter, Yan Liu, Yashpal S. Chhonker, Daryl J. Murry, Gracey R. Alexander, Erin M. McIntyre, Jason A. Sughroue, Graham J. Sharp, Rongshi Li. The in vitro and in vivo activity of the pyrrolomycin marinopyrrole RL71 alone and combined with temsirolimus (TEM) in MYCN-amplified neuroblastoma (NB) [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A16.

Pharmacokinetics and efficacy of orally administered polymeric chloroquine as macromolecular drug in the treatment of inflammatory bowel disease

Inflammatory bowel disease is a chronic inflammation of the gastrointestinal tract with poor understanding of its pathogenesis and no effective cure. The goal of this study was to evaluate the feasibility of orally administered non-degradable polymeric chloroquine (pCQ) to locally reduce colon inflammation. The pCQ was synthesized by radical copolymerization of N-(2-hydroxypropyl)methacrylamide with methacryloylated hydroxychloroquine (HCQ). The anti-inflammatory activity of orally administered pCQ versus HCQ was tested in a mouse model of colitis induced by Citrobacter rodentium (C. rodentium). Single-dose pharmacokinetic and biodistribution studies performed in the colitis model indicated negligible systemic absorption (p ≤ 0.001) and localization of pCQ in the gastrointestinal tract. A multi-dose therapeutic study demonstrated that the localized pCQ treatment resulted in significant reduction in the colon inflammation (p ≤ 0.05). Enhanced suppression of pro-inflammatory cytokines IL-6 (p ≤ 0.01) and IL1-β and opposing upregulation of IL-2 (p ≤ 0.05) recently reported to be involved in downstream anti-inflammatory events suggested that the anti-inflammatory effects of the pCQ are mediated by altering mucosal immune homeostasis. Overall, the reported findings demonstrate a potential of pCQ as a novel polymer therapeutic option in inflammatory bowel disease with the potential of local effects and minimized systemic toxicity.