A Phase I Dose Escalation Study of the Safety, Tolerability, and Pharmacokinetics of Ipilimumab in Chinese Patients with Select Advanced Solid Tumors.

LESSONS LEARNED: The overall safety profiles of ipilimumab 3 mg/kg and 10 mg/kg administered every 3 weeks, were consistent between Chinese patients with solid tumors in the current study and patients from previous U.S. ipilimumab monotherapy studies. No new safety signals were identified. The mean systemic exposures to ipilimumab (assessed by first dose area under the curve during the dosing interval and maximum serum concentration) were numerically lower in the Chinese patient population than in U.S. patients for both 3 mg/kg and 10 mg/kg doses; however, the range of serum concentrations in the Chinese and U.S. populations overlapped (3 mg/kg and 10 mg/kg), suggesting that ipilimumab pharmacokinetics was ethnically insensitive in this study. BACKGROUND: This phase I, open-label study assessed ipilimumab safety, tolerability, pharmacokinetics (PK), immunogenicity, and antitumor activity in Chinese patients with unresectable, metastatic, recurrent malignant melanoma (MM) or nasopharyngeal carcinoma (NPC). METHODS: Of 39 patients enrolled, 25 received ipilimumab (11 patients received 3 mg/kg, and 14 patients received 10 mg/kg). Reasons for not receiving treatment were withdrawal of consent (3 patients), no longer meeting the criteria (10 patients), and one recorded as "other." During the induction phase, patients received ipilimumab (3 mg/kg, i.v.), on day 1 of a 3-week cycle, to a maximum of four doses or progressive disease (PD). During the maintenance phase at week 24, patients received ipilimumab (3 mg/kg, i.v.) on day 1 of a 12-week cycle, to a maximum of 3 years or PD. Considering the co-primary safety and PK endpoints, the successive dosing required nine patients with two or fewer dose-limiting toxicities during the 42-day observation period to proceed with a new cohort of nine patients at 10 mg/kg. RESULTS: Ipilimumab safety and PK profiles were similar in Chinese and predominantly White populations. Ipilimumab was well tolerated. Most adverse events (AEs) were grades 1-2 and experienced by 11 patients treated with 3 mg/kg and 14 patients treated with 10 mg/kg. There were no new safety concerns. Incidence of anti-ipilimumab antibodies was low (1 of 10 in the 3 mg/kg patients and 2 of 13 in the 10 mg/kg patients) and without safety implications. In the 3 mg/kg group, 8 of 11 patients had PD. In the 10 mg/kg group (all NPC, 0 MM patients), 11 of 14 patients had PD. Three patients had stable disease (one at 3 mg/kg and two at 10 mg/kg). CONCLUSION: Ipilimumab was well tolerated in Chinese patients, showing similar safety and PK to previous studies in predominantly White populations.

Antiseizure, antidepressant, and antipsychotic medication prescribing in elderly nursing home residents.

OBJECTIVE: The incidence of epilepsy is highest in the elderly and the prevalence of epilepsy is higher in nursing home residents than in other cohorts. Co-medications that act in the central nervous system (CNS) are frequently prescribed in this population. The objective was to identify the most commonly prescribed antiseizure drugs (ASDs) and determine the frequency of use of antipsychotic and antidepressant medications in elderly nursing home residents receiving ASDs. METHODS: Data were obtained from a pharmacy database serving 18,752 patients in Minnesota and Wisconsin nursing homes. Prescribing information was available on ASD, antidepressant, and antipsychotic drugs on one day in October 2013. The frequency distribution by age, formulation, trademarked/generic drugs, route of administration, and multiple drug combinations were determined. RESULTS: Overall, 66.8% of 18,752 residents received at least one CNS-active drug as classified by the Generic Product Identifier classification system. For those 65years and older, ASDs were prescribed for 14.3% residents. Gabapentin comprised 7.3%; valproate 3.0%; levetiracetam 1.8%; and phenytoin 0.9%. An antidepressant was used in 64.2% of persons prescribed an ASD. Antidepressant use varied for specific ASDs and ranged from 50 to 75%. An antipsychotic medication was used in 30% of persons prescribed an ASD and ranged from 16.8 to 54.2% for specific ASDs. Both antidepressant and antipsychotic use occurred in 22.2% of persons prescribed an ASD, respectively. SIGNIFICANCE: The pattern of CNS-active drug use has changed from previous years in this geographic region. Use of phenytoin has declined markedly, but antidepressant use has increased substantially. The CNS side effect profile of these medications and the possible long-term consequences in this population can greatly complicate their therapy.

Peripherally cross-linking the shell of core-shell polymer micelles decreases premature release of physically loaded combretastatin A4 in whole blood and increases its mean residence time and subsequent potency against primary murine breast tumors after IV administration.

PURPOSE: Determine the feasibility and potential benefit of peripherally cross-linking the shell of core-shell polymer micelles on the premature release of physically loaded hydrophobic drug in whole blood and subsequent potency against solid tumors. METHODS: Individual Pluronic F127 polymer micelles (F127 PM) peripherally cross-linked with ethylenediamine at 76% of total PEO blocks (X-F127 PM) were physically loaded with combretastatin A4 (CA4) by the solid dispersion method and compared to CA4 physically loaded in uncross-linked F127 PM, CA4 in DMSO in vitro, or water-soluble CA4 phosphate (CA4P) in vivo. RESULTS: X-F127 PM had similar CA4 loading and aqueous solubility as F127 PM up to 10 mg CA4 / mL at 22.9 wt% and did not aggregate in PBS or 90% (v/v) human serum at 37°C for at least 24 h. In contrast, X-F127 PM decreased the unbound fraction of CA4 in whole blood (fu) and increased the mean plasma residence time and subsequent potency of CA4 against the vascular function and growth of primary murine 4T1 breast tumors over CA4 in F127 PM and water-soluble CA4P after IV administration. CONCLUSIONS: Given that decreasing the fu is an indication of decreased drug release, peripherally cross-linking the shell of core-shell polymer micelles may be a simple approach to decrease premature release of physically loaded hydrophobic drug in the blood and increase subsequent potency in solid tumors.

Nod2 deficiency protects mice from cholestatic liver disease by increasing renal excretion of bile acids.

BACKGROUND & AIMS: Chronic liver disease is characterized by fibrosis that may progress to cirrhosis. Nucleotide oligomerization domain 2 (Nod2), a member of the Nod-like receptor (NLR) family of intracellular immune receptors, plays an important role in the defense against bacterial infection through binding to the ligand muramyl dipeptide (MDP). Here, we investigated the role of Nod2 in the development of liver fibrosis. METHODS: We studied experimental cholestatic liver disease induced by bile duct ligation or toxic liver disease induced by carbon tetrachloride in wild type and Nod2(-/-) mice. RESULTS: Nod2 deficiency protected mice from cholestatic but not toxin-induced liver injury and fibrosis. Most notably, the hepatic bile acid concentration was lower in Nod2(-/-) mice than wild type mice following bile duct ligation for 3 weeks. In contrast to wild type mice, Nod2(-/-) mice had increased urinary excretion of bile acids, including sulfated bile acids, and an upregulation of the bile acid efflux transporters MRP2 and MRP4 in tubular epithelial cells of the kidney. MRP2 and MRP4 were downregulated by IL-1ß in a Nod2 dependent fashion. CONCLUSIONS: Our findings indicate that Nod2 deficiency protects mice from cholestatic liver injury and fibrosis through enhancing renal excretion of bile acids that in turn contributes to decreased concentration of bile acids in the hepatocyte.

Model-Informed Clinical Pharmacology Profile of a Novel Fixed-Dose Combination of Nivolumab and Relatlimab in Adult and Adolescent Patients with Solid Tumors.

PURPOSE: Progression-free survival (PFS) was significantly improved with nivolumab 480 mg plus relatlimab 160 mg fixed-dose combination (FDC) every 4 weeks (Q4W) versus nivolumab alone in patients with previously untreated advanced melanoma in RELATIVITY-047. In addition, RELATIVITY-020 (Part D) demonstrated a manageable safety profile and potential for durable response with nivolumab plus relatlimab in previously treated patients. Here, we evaluate the clinical pharmacology profile (CPP) of nivolumab plus relatlimab to support the approved regimen for adult and adolescent patients with advanced melanoma and its continued clinical development in solid tumors. EXPERIMENTAL DESIGN: The pharmacokinetics (PK) and immunogenicity of relatlimab and nivolumab were assessed using data from RELATIVITY-047 and RELATIVITY-020. Patients with advanced solid tumors received relatlimab alone or nivolumab plus relatlimab as single-agent vials (SAV) or FDC. PK was characterized using a population PK (popPK) model. RESULTS: Relatlimab demonstrated nonlinear and time-varying PK. Nonlinearity in relatlimab PK represented approximately 31% of total CL of relatlimab 160 mg Q4W. Relatlimab PK was dose proportional at doses ≥160 mg Q4W. Geometric mean exposures were similar for SAV and FDC cohorts receiving equivalent dosing regimens. No dose adjustment was required for covariates. Incidence of relatlimab antidrug antibodies was <6% for nivolumab plus relatlimab and had no clinically meaningful impact. There was no PK-related drug interaction of nivolumab plus relatlimab. CONCLUSIONS: The CPP of relatlimab alone or in combination with nivolumab supports the approved dosing in advanced melanoma and the continued evaluation of nivolumab and relatlimab across other solid tumors. See related commentary by Gopalakrishnan and Amaria, p. 2862.

Pharmacokinetics, protein binding and metabolism of a quinoxaline urea analog as an NF-κB inhibitor in mice and rats by LC-MS/MS.

13-197 is a novel NF-κB inhibitor that shows promising in vitro efficacy data against pancreatic cancer. In this study, we characterized the pharmacokinetics, tissue distribution, protein binding and metabolism of 13-197 in mice and rats. A valid, sensitive and selective LC-MS/MS method was developed. This method was validated for the quantification of 13-197, in the range of 0.1 or 0.2-500 ng/mL in mouse plasma, liver, kidney, lung, heart, spleen, brain, urine and feces. 13-197 has low bioavailability of 3 and 16% in mice and rats, respectively. It has faster absorption in mice with 12-fold shorter Tmax than in rats. Tissue concentrations were 1.3-69.2-fold higher in mice than in rats at 72 h after intravenous administration. 13-197 is well distributed to the peripheral tissues and has relatively high tissue-plasma concentration ratios, ranging from 1.8 to 3634, in both mice and rats. It also demonstrated more than 99% binding to plasma proteins in both mice and rats. Finally, <1% of 13-197 is excreted unchanged in urine and feces, and metabolite profiling studies detected more than 20 metabolites in mouse and rat plasma, urine and feces, which indicates that 13-197 is extensively metabolized and primarily eliminated by metabolism rather than by excretion.

Prediction of drug-drug interaction potential mediated by transporters between dasatinib and metformin, pravastatin, and rosuvastatin using physiologically based pharmacokinetic modeling.

PURPOSE: Recent in vitro studies demonstrated that dasatinib inhibits organic cation transporter 2 (OCT2), multidrug and toxin extrusion proteins (MATEs), and organic anion transporting polypeptide 1B1/1B3 (OATP1B1/1B3). We developed a physiologically based pharmacokinetic (PBPK) model to assess drug-drug interaction (DDI) potential between dasatinib and known substrates for these transporters in a virtual population. METHODS: The dasatinib PBPK model was constructed using Simcyp® Simulator by combining its physicochemical properties, in vitro data, in silico predictions, and pharmacokinetic (PK) results from clinical studies. Model validation against three independent clinical trials not used for model development included dasatinib DDI studies with ketoconazole, rifampin, and simvastatin. The validated model was used to simulate DDIs of dasatinib and known substrates for OCT2 and MATEs (metformin) and OATP1B1/1B3 (pravastatin and rosuvastatin). RESULTS: Simulations of metformin PK in the presence and absence of dasatinib, using inhibitor constant (Ki) values measured in vitro, produced estimated geometric mean ratios (GMRs) of the maximum observed concentration (Cmax) and area under the concentration-time curve (AUC) of 1.05 and 1.06, respectively. Sensitivity analysis showed metformin exposure increased < 30% in both AUC and Cmax when dasatinib Ki was reduced by tenfold for OCT2 and MATEs simultaneously, and < 40% with a 20-fold Ki reduction. The estimated GMRs of Cmax and AUC for pravastatin and rosuvastatin with co-administration of dasatinib were unity (1.00). CONCLUSIONS: This PBPK model accurately described the observed PK profiles of dasatinib. The validated PBPK model predicts low risk of clinically significant DDIs between dasatinib and metformin, pravastatin, or rosuvastatin.

Severity of Topiramate-Related Working Memory Impairment Is Modulated by Plasma Concentration and Working Memory Capacity.

Drug side effects that impair cognition can lead to diminished quality of life and discontinuation of therapy. Topiramate is an antiepileptic drug that elicits cognitive deficits more frequently than other antiepileptic drugs, impairing multiple cognitive domains including language, attention, and memory. Although up to 40% of individuals taking topiramate may experience cognitive deficits, we are currently unable to predict which individuals will be most severely affected before administration. The objective of this study was to show the contributions of plasma concentration and working memory capacity in determining the severity of an individual's topiramate-related cognitive impairment. Subjects were enrolled in a double-blind, placebo-controlled crossover study during which they received a single dose of either 100, 150, or 200 mg topiramate. Working memory function was assessed using a modified Sternberg working memory task with 3 memory loads administered 4 hours after dosing. After adjustment for differences in working memory capacity, each 1 µg/mL of topiramate plasma concentration was associated with a 3.6% decrease in accuracy for all memory loads. Placebo effects occurred as a function of working memory capacity, with individuals with high working memory capacity experiencing less severe placebo-related impairment compared with those with low working memory capacity. Our results demonstrate that severity of topiramate-related cognitive deficits occurs as a function of both drug exposure and baseline cognitive function. By identifying patient- and exposure-related characteristics that modulate the severity of cognitive side effects, topiramate dosing strategies may be individually tailored in the future to prevent unwanted cognitive impairment.

Iron supplements in nursing home patients associated with reduced carbamazepine absorption.

Persons in nursing homes receive a number of medications that may interfere with the pharmacokinetics of carbamazepine (CBZ). The aim of our study was to determine factors that may affect the pharmacokinetics of CBZ in elderly nursing home patients. METHODS: CBZ concentration data collected from 60 nursing homes across the US were evaluated. Inclusion criteria included residency in a nursing home for at least 2 months, age 65 years or older, a stable dosing regimen of CBZ for at least 4 weeks (considered steady state), available CBZ concentration, and complete information regarding all co-medications. Using a nonlinear mixed-effects model, the data were adequately described by a one-compartment model with first-order absorption and elimination. Goodness-of-fit plots, plausibility of parameter estimates, visual predictive check and nonparametric bootstrap were used to evaluate the models. MAIN FINDINGS: The final data set consisted of 345 CBZ concentrations from 99 subjects (38 males, 61 females). The population estimate of apparent clearance (CL/F) for a 70-kg person was 3.69 L/hr (RSE 6.9%). Residents were receiving either immediate (93.9%) or extended release (6.1%) formulation of CBZ and the Ka of each formulation was fixed to literature values. Age, sex, and co-medications had no effect on CL/F and apparent volume of distribution. Iron supplementation, which was taken by 16% of the residents, resulted in a 33% decrease in bioavailability (p < 0.001). No other medications were found to have an effect. CONCLUSIONS: Results from this pharmacokinetic study indicate that use of iron supplementation is associated with a reduction in absorption of CBZ and may need to be considered when dosing CBZ in patients taking iron supplementation.

Pharmacokinetics and Saturable Absorption of Gabapentin in Nursing Home Elderly Patients.

Pharmacokinetic data of gabapentin (GBP) in community-dwelling elderly patients show a significant effect of advanced age on GBP pharmacokinetics due to altered renal function. However, there are no data in elderly nursing home (NH) patients to evaluate gabapentin absorption and elimination. Our objective was to characterize the pharmacokinetics of GBP in elderly nursing home patients maintained on GBP therapy. This was a prospective pharmacokinetic study in elderly nursing home patients (≥60 years) receiving GBP for the management of chronic pain or epilepsy from seven nursing homes. Pharmacokinetic parameters were estimated by nonlinear mixed-effects modeling. A one-compartment model described the data and clearance (CL) was associated with estimated glomerular filtration rate (eGFR) (p < 0.0001). The GBP CL in elderly nursing home patients was 2.93 L/h. After adjusting for the effect of GFR, GBP CL was not affected by age, sex, body weight, or comorbidity scores. No significant effects of body size measures, age, and sex were detected on volume of distribution. Dose-dependent bioavailability of GBP was demonstrated, and the saturable absorption profile was described by a nonlinear hyperbolic function. Prediction-corrected visual predictive check (pc-VPC) suggests adequate fixed- and random-effects models that successfully simulated the mean trend and variability in gabapentin concentration-time profiles. In this analysis, the parameters of the hyperbolic nonlinearity appear to be similar between elderly and younger adults.

Urinary bile acids as biomarkers for liver diseases I. Stability of the baseline profile in healthy subjects.

The role of bile acids (BAs) as biomarkers for liver injury has been proposed for decades. However, the large inter- and intra-individual variability of the BA profile has prevented its clinical application. To this end, we investigated the effect of covariates such as food, gender, age, BMI, and moderate alcohol consumption on the BA profile in healthy human subjects. The BA profile was characterized by the calculation of indices that describe the composition, sulfation, and amidation of total and individual BAs. Both inter- and intra-individual variabilities of BA indices were low in serum and even lower in urine compared with those of absolute concentrations of BAs. Serum BA concentrations increased with consumption of food, whereas urinary BA concentrations were mildly affected by food. Gender differences in the urinary and serum BA profile were minimal. The serum and urinary BA profiles were also not affected by age. BMI showed minimal effect on the urine and serum BA profile. Moderate alcohol consumption did not have a significant effect on the BA profile in both urine and serum. When the effect of the type of alcohol was studied, the results indicate that moderate drinking of beer does not affect BA concentrations and has minimal effect on BA indices, whereas moderate wine consumption slightly increases BA concentrations without affecting the BA indices. In summary, urinary BA indices showed lower variability and higher stability than absolute BA concentrations in serum and showed minimal changes to covariate effects suggesting their utility as biomarkers in clinic.

Urinary bile acids as biomarkers for liver diseases II. Signature profiles in patients.

Hepatobiliary diseases result in the accumulation of bile acids (BAs) in the liver, systemic blood, and other tissues leading to an unfavorable prognosis. The BA profile was characterized by the calculation of indices that describe the composition, sulfation, and amidation of total and individual BAs. Comparison of the urinary BA profiles between healthy subjects and patients with hepatobiliary diseases demonstrated significantly higher absolute concentrations of individual and total BAs in patients. The percentage sulfation of some individual BAs were different between the two groups. The percentage amidation of overall and most individual BAs was higher in patients than controls. The percentage of primary BAs (CDCA and CA) was higher in patients, whereas the percentage of secondary BAs (DCA and LCA) was lower in patients. BA indices belonging to percentage amidation and percentage composition were better associated with the severity of the liver disease as determined by the model for end-stage liver disease (MELD) score and disease compensation status compared with the absolute concentrations of individual and total BAs. In addition, BA indices corresponding to percentage amidation and percentage composition of certain BAs demonstrated the highest area under the receiver operating characteristic (ROC) curve suggesting their utility as diagnostic biomarkers in clinic. Furthermore, significant increase in the risk of having liver diseases was associated with changes in BA indices.

A Major Human Oral Lysophosphatidic Acid Species, LPA 18:1, Regulates Novel Genes in Human Gingival Fibroblasts.

BACKGROUND: The small bioactive lipid lysophosphatidic acid (LPA) plays critical roles in both normal physiology and inflammation in many systems. However, its actions are just beginning to be defined in oral biology and pathophysiology. METHODS: Microarray analysis was used to test the hypothesis that human gingival fibroblasts (GFs) would show significant changes in wound-healing and inflammation-related gene transcripts in response to a major human salivary and gingival crevicular fluid LPA species, 18:1, and that they would express transcript for the major LPA-producing enzyme autotaxin. The microarray results were validated for three highly relevant upregulated inflammatory transcripts using quantitative reverse transcription-polymerase chain reaction (QRT-PCR). Liquid chromatography-tandem mass spectrometry was used to assay time-dependent LPA species production by GFs. RESULTS: LPA 18:1 significantly regulated 20 GF novel and 27 known genes linked to the control of inflammation (P ≤0.01). QRT-PCR validation of interleukin (IL)-8, IL-11, and suppressor of cytokine signaling 2 (SOCS2) messenger RNAs confirmed statistically significant differences from control (P ≤0.05). Autotaxin transcript was present, and GFs were found to produce multiple LPA species in a time-dependent manner. CONCLUSIONS: The upregulation of transcripts for known GF proinflammatory (IL-6, IL-8) and anti-inflammatory (IL-11) ILs, along with SOCS2, shows that LPA transiently regulates a complex set of GF genes critical to periodontal wound healing and inflammation. These results implicate LPA exerting actions on GFs that are compatible with functioning as a mediator in oral fibroblast biology and inflammatory responses. Therefore, LPA may potentially modulate/regulate periodontal inflammation.

The profile of bile acids and their sulfate metabolites in human urine and serum.

The role of sulfation in ameliorating the hepatotoxicity of bile acids (BAs) in humans remains unknown due to the lack of proper analytical methods to quantify individual BAs and their sulfate metabolites in biological tissues and fluids. To this end, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to characterize the detailed BA profile in human urine and serum. The limit of quantification was 1ng/mL and baseline separation of all analytes was achieved within in a run time of 32min. The method was validated over the dynamic range of 1-1000ng/mL. The LC-MS/MS method was more accurate, precise, and selective than the commercially available kits for the quantification of sulfated and unsulfated BAs, and the indirect quantification of individual sulfated BAs after solvolysis. The LC-MS/MS method was applied to characterize the BA profile in urine and serum of healthy subjects. Thirty three percent of serum BAs were sulfated, whereas 89% of urinary BAs existed in the sulfate form, indicating the role of sulfation in enhancing the urinary excretion of BAs. The percentage of sulfation of individual BAs increased with the decrease in the number of hydroxyl groups indicating the role of sulfation in the detoxification of the more hydrophobic and toxic BA species. Eighty percent of urinary BAs and 55% of serum BAs were present in the glycine-amidated form, whereas 8% of urinary BAs and 13% of serum BAs existed in the taurine-amidated form.

Combinatorial assessments of brain tissue metabolomics and histopathology in rodent models of human immunodeficiency virus infection.

Metabolites are biomarkers for a broad range of central nervous system disorders serving as molecular drivers and byproducts of disease pathobiology. However, despite their importance, routine measures of brain tissue metabolomics are not readily available based on the requirements of rapid tissue preservation. They require preservation by microwave irradiation, rapid freezing or other methods designed to reduce post mortem metabolism. Our research on human immunodeficiency virus type one (HIV-1) infection has highlighted immediate needs to better link histology to neural metabolites. To this end, we investigated such needs in well-studied rodent models. First, the dynamics of brain metabolism during ex vivo tissue preparation was shown by proton magnetic resonance spectroscopy in normal mice. Second, tissue preservation methodologies were assessed using liquid chromatography tandem mass spectrometry and immunohistology to measure metabolites and neural antigens. Third, these methods were applied to two animal models. In the first, immunodeficient mice reconstituted with human peripheral blood lymphocytes then acutely infected with HIV-1. In the second, NOD scid IL2 receptor gamma chain knockout mice were humanized with CD34+ human hematopoietic stem cells and chronically infected with HIV-1. Replicate infected animals were treated with nanoformulated antiretroviral therapy (nanoART). Results from chronic infection showed that microgliosis was associated with increased myoinostitol, choline, phosphocholine concentrations and with decreased creatine concentrations. These changes were partially reversed with nanoART. Metabolite responses were contingent on the animal model. Taken together, these studies integrate brain metabolomics with histopathology towards uncovering putative biomarkers for neuroAIDS.

Rapid and reliable quantitation of amino acids and myo-inositol in mouse brain by high performance liquid chromatography and tandem mass spectrometry.

Amino acids and myo-inositol have long been proposed as putative biomarkers for neurodegenerative diseases. Accurate measures and stability have precluded their selective use. To this end, a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on multiple reaction monitoring was developed to simultaneously quantify glutamine, glutamate, γ-aminobutyric acid (GABA), aspartic acid, N-acetyl aspartic acid, taurine, choline, creatine, phosphocholine and myo-inositol in mouse brain by methanol extractions. Chromatography was performed using a hydrophilic interaction chromatography silica column within in a total run time of 15 min. The validated method is selective, sensitive, accurate, and precise. The method has a limit of quantification ranging from 2.5 to 20 ng/ml for a range of analytes and a dynamic range from 2.5-20 to 500-4000 ng/ml. This LC-MS/MS method was validated for biomarker discovery in models of human neurological disorders.

Simultaneous characterization of bile acids and their sulfate metabolites in mouse liver, plasma, bile, and urine using LC-MS/MS.

Sulfation is a major metabolic pathway involved in the elimination and detoxification of bile acids (BAs). Several lines of evidence are available to support the role of sulfation as a defensive mechanism to attenuate the toxicity of accumulated BAs during hepatobiliary diseases. Individual BAs and their sulfate metabolites vary markedly in their physiological roles as well as their toxicities. Therefore, analytical techniques are required for the quantification of individual BAs and BA-sulfates in biological fluids and tissues. Here we report a simple, sensitive, and validated LC-MS/MS method for the simultaneous quantification of major BAs and BA-sulfates in mouse liver, plasma, bile, and urine. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for liver and plasma) was used to extract BAs and BA-sulfates. Base-line separation of all analytes (unsulfated- and sulfated BAs) was achieved in 25min with a limit of quantification of 1ng/ml. This LC-MS/MS method was applied to simultaneously quantify BAs and BA-sulfates in both male and female mouse tissues and fluids. Less than 3% of total BAs are present in the sulfate form in the mouse liver, plasma, and bile, which provides strong evidence that sulfation is a minor metabolic pathway of BA elimination and detoxification in mice. Furthermore, we report that the marked female-predominant expression of Sult2a1 is not reflected into a female-predominant pattern of BA-sulfation.

UPLC-MS/MS quantification of nanoformulated ritonavir, indinavir, atazanavir, and efavirenz in mouse serum and tissues.

Animal pharmacokinetic and tissue distribution assays of antiretroviral therapeutic drugs require accurate drug quantification in biological fluids and tissues. Here we report a simple, rapid, and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of commonly used antiretroviral drugs ritonavir (RTV), indinavir (IDV), atazanavir (ATV), and efavirenz (EFV) in mouse serum and tissues (liver, kidney, lung, and spleen). These antiretroviral drugs are currently the cornerstones of common therapeutic regimens for human immunodeficiency virus (HIV) infection. Chromatographic separation was achieved using a gradient mobile phase (5% acetonitrile in methanol and 7.5mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC(®)BEH Shield RP 18 column. All compounds eluted within a 7 min run time. Lopinavir was used as an internal standard. Detection was achieved by dual positive and negative ionization modes on a quadrupole linear ion trap hybrid mass spectrometer with an electrospray ionization (ESI) source. The dynamic range was 0.2-1000 ng/mL for RTV, IDV, and ATV, and 0.5-1000 for EFV. The method was validated and showed high and consistent intra-day and inter-day accuracy and precision for all analytes. This method is used to support the preclinical development studies of targeted- and sustained-release combination ART (nanoART). The current data demonstrate a 1.5-4 fold increase in serum and tissue AUC of nanoformulated ATV, RTV, and EFV administered to mice when compared to native drug. In addition, the tested formulation enhanced exposure of the same anti-HIV drugs in mouse tissues.

Metabolite profiling of praziquantel and its analogs during the analysis of in vitro metabolic stability using information-dependent acquisition on a hybrid triple quadrupole linear ion trap mass spectrometer.

Rapid determination of in vitro metabolic stability and metabolite profiling of new chemical entities using microsomes or other liver preparations is one of the most important steps in drug discovery. In this paper, we report the use of liquid chromatography-hybrid triple quadrupole/linear ion trap mass spectrometry for the simultaneous analysis of metabolic stability, metabolite profiling, and the kinetics of metabolite formation of praziquantel and three structural analogs. Multiple reaction monitoring (MRM) scans were used to quantify the disappearance of parent compounds and the formation of metabolites. MRM-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) scans were used for the identification of the metabolites formed. Metabolic stability of these anthelmintics were studied in human liver microsomes (HLM) using MRM as a survey scan, which resulted in the identification of a higher number of metabolites compared to neutral loss (NL), precursor ion (PI), and enhanced mass spectrometry (EMS) scans. MRM-IDA-EPI scans resulted in the generation of similar calibration curves to MRM-only quantitative analysis. Therefore, the quantitative capabilities of the method was not affected by the additional qualitative information obtained during the same run. The formation of major metabolites was also simultaneously monitored, which could be used to understand the kinetics and mechanism of metabolite formation. Finally, our data demonstrate that the three analogs had higher metabolic stability than the anthelmintic prototype (praziquantel).