Laryngeal Cancer Cells Metabolize 25-Hydroxyvitamin D3 and Respond to 24R,25-dihydroxyvitamin D3 via a Mechanism Dependent on Estrogen Receptor Levels.

Studies have evaluated vitamin D3's therapeutic potential in estrogen-responsive cancers, with conflicting findings. We have shown that the proliferation of breast cancer cells is regulated by 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) depending on estrogen receptor alpha 66 (ERα66) expression, suggesting that this could also be the case for estrogen-sensitive laryngeal cancer cells. Accordingly, we examined levels of ERα isoforms in ERα66-positive UM-SCC-12 and ERα66-negative UM-SCC-11A cells and their response to 24R,25(OH)2D3. 24R,25(OH)2D3 stimulated proliferation, increased the expression of metastatic markers, and inhibited apoptosis in UM-SCC-12 cells while having the opposite effect in UM-SCC-11A cells. To evaluate if vitamin metabolites could act via autocrine/paracrine mechanisms, we assessed the expression, protein levels, and activity of vitamin D3 hydroxylases CYP24A1 and CYP27B1. Both cell types expressed both mRNAs; but the levels of the enzymes and their activities were differentially regulated by estrogen. ERα66-negative UM-SCC-11A cells produced more 24,25(OH)2D3 than UM-SCC-12 cells, but comparable levels of 1,25(OH)2D3 when treated with 25(OH)D3 These results suggest that the regulation of vitamin D3 metabolism in laryngeal cancer cells is modulated by ERα66 expression, and support a role for 24R,25(OH)2D3 as an autocrine/paracrine regulator of laryngeal cancer. The local metabolism of 25(OH)D3 should be considered when determining the potential of vitamin D3 in laryngeal cancer.

Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model.

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

NCP, a dual kappa and mu opioid receptor agonist, is a potent analgesic against inflammatory pain without reinforcing or aversive properties.

While agonists of mu (MOR) and kappa (KOR) opioid receptors have analgesic effects, they produce euphoria and dysphoria, respectively. Other side effects include respiratory depression and addiction for MOR agonists and sedation for KOR agonists. We reported that 17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-{[4'-(2'-cyanopyridyl)]carboxamido}cmorphinan (NCP) displayed potent KOR full agonist and MOR partial agonist activities (58%) with 6.5x KOR-over-MOR selectivity in vitro Herein, we characterized pharmacological effects of NCP in rodents. In mice, NCP exerted analgesic effects against inflammatory pain in both the formalin test and the acetic acid writhing test, with A50 values of 47.6 and 14.4 microg/kg (s.c.), respectively. The analgesic effects in the acetic acid writhing test were mediated by the KOR. NCP at doses much higher than those effective in reducing inflammatory pain did not produce antinociception in the hot plate and tail flick tests, inhibit compound 48/80-induced scratching, cause conditioned place aversion (CPA) or preference, impair rotarod performance, inhibit locomotor activity, cause respiratory depression, or precipitate morphine withdrawal. However, NCP (10~100 microg/kg) inhibited gastrointestinal transit with a maximum of ~40% inhibition. In MOR knockout mice, NCP caused CPA, demonstrating that its lack of CPA is due to combined actions on the MOR and KOR. Following s.c. injection, NCP penetrated into the mouse brain. In rats trained to self-administer heroin, NCP (1~320 microg/kg/infusion) did not function as a reinforcer. Thus, NCP produces potent analgesic effects via KOR without side effects except constipation. Therefore, dual full KOR/partial MOR agonists with moderate KOR-over-MOR selectivity may be promising as non-addictive analgesics for inflammatory pain. Significance Statement Developing non-addictive analgesics is crucial for reducing opioid overdose deaths, minimizing drug misuse, and promoting safer pain management practices. Herein, pharmacology of a potential non-addictive analgesic, NCP, is reported. NCP has full KOR agonist / partial MOR agonist activities with a 6.5 x selectivity for KOR over MOR. Unlike MOR agonists, analgesic doses of NCP do not lead to self-administration or respiratory depression. Furthermore, NCP does not produce aversion, hypolocomotion, or motor incoordination, side effects typically associated with KOR activation.

Advancing the use of molecularly imprinted polymers in bioanalysis: the selective extraction of cotinine in human urine.

Aim: To characterize a molecularly imprinted polymer via precipitation polymerization for the extraction of cotinine in urine. Methods: The polymer was created via precipitation polymerization. Physical characteristics of the polymer were assessed via scanning electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis. The polymer adsorption capacity was assessed and an solid-phase extraction method from urine by LC-MS/MS was developed. Results: The polymer had small, spherical morphology and little thermal decomposition. The extraction method yielded cotinine recoveries of 77-103% in urine. The molecularly imprinted polymer adsorption capacity for cotinine was 448.2 ± 2.1 µg/mg. Common interferants did not affect cotinine's extraction. Conclusion: The resulting polymer was determined to be specific for cotinine and can be used for the detection of cotinine in urine for clinical samples.

Local production of active vitamin D3 metabolites in breast cancer cells by CYP24A1 and CYP27B1.

The role of vitamin D3 and its metabolites in cancer and especially as a treatment option has been widely disputed. Clinicians noting low serum 25-hydroxyvitamin D3 [25(OH)D3] levels in their patients, recommend vitamin D3 supplementation as a method of reducing the risk of cancer; however, data supporting this are inconsistent. These studies rely on systemic 25(OH)D3 as an indicator of hormone status, but 25(OH)D3 is further metabolized in the kidney and other tissues under regulation by several factors. This study examined if breast cancer cells also possess the ability to metabolize 25(OH)D3, and if so, whether the resulting metabolites are secreted locally; if this ability reflects ERα66 status; and if they possess vitamin D receptors (VDR). To address this question, estrogen receptor alpha (ERα) positive (MCF-7) and ERα negative (HCC38 and MDA-MB-231) breast cancer cell lines were examined for expression of ERα66, ERα36, CYP24A1, CYP27B1, and VDR as well as for local production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after treatment with 25(OH)D3. The results showed that independent of ER status, breast cancer cells express the enzymes CYP24A1 and CYP27B1, which are responsible for converting 25(OH)D3 into its dihydroxylated forms. Moreover, these metabolites are produced at levels comparable to the levels observed in blood. They are positive for VDR, indicating that they can respond to 1α,25(OH)2D3, which can upregulate CYP24A1. These findings suggest that vitamin D metabolites may contribute to the tumorigenicity of breast cancer via autocrine and/or paracrine mechanisms.

Effects of Device Settings and E-Liquid Characteristics on Mouth-Throat Losses of Nicotine Delivered with Electronic Nicotine Delivery Systems (ENDS).

Currently it is not fully understood how the device settings and electronic liquid (e-liquid) composition, including their form of nicotine content, impact mouth and throat losses, and potentially lead to the variations in total nicotine delivery to the human lungs. An in situ size assessment method was developed for real-time measurements at the mouthpiece and outlet of a biorelevant mouth-throat to account for the dynamic nature of the aerosol. The aerosol size, temperature, and delivery through the mouth-throat replica and the exhaled aerosol between the puff intervals were measured at different wattages using various e-liquid compositions. The effects of body temperature and humidity on aerosol size and nicotine delivery were also explored to evaluate the importance of considering realistic in vivo conditions in in vitro measurements. Notably, in vitro tests with body temperature and humidity in mouth-throat model vs room conditions, resulted in larger aerosol size at the end of the throat (Dv50=5.83±0.33 µm vs 3.05±0.15 µm), significantly higher thoracic nicotine delivery (>90% vs 50-85%) potentially due to the lower exhaled amount (<10% vs 15-50%). Besides, higher VG/PG ratios resulted in significantly lower exhaled amount and higher mouth-throat nicotine deposition. One of the main outcomes of the study was finding significantly lower exhaled amount and higher thoracic nicotine delivery with nicotine salt form vs free-base. Considering body temperature and humidity also showed significant enhancement in nicotine delivery, so it is essential to account for biorelevant experimental conditions in benchtop testing.

A determination of the aerosolization efficiency of drugs of abuse in a eutectic mixture with nicotine in electronic cigarettes.

Eutectic mixtures can be formed by adding drugs other than nicotine (DOTNs) to nicotine-based e-liquids in electronic cigarettes (e-cigarettes). Thus, the interaction between nicotine e-liquids and DOTNs must be evaluated. Presented is the change in e-cigarette aerosolization of nicotine and methadone alone versus a 1:1 nicotine:methadone mixture to evaluate the possible formation of a eutectic mixture that can result in an increase of drug delivery. E-liquids were prepared in-house using 1:1 propylene glycol (PG):vegetable glycerin (VG) as a base plus nicotine, methadone hydrochloride, or 1:1 nicotine:methadone hydrochloride. The e-liquids were aerosolized via an automated vaping machine using parameters adopted from the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) E-cigarette Task Force method. Drug recovery was determined by capturing the aerosol from 15 puffs generated by the e-cigarette. Concentrations of nicotine and methadone aerosolized were determined by gas chromatography-mass spectrometry using nicotine (n = 3), methadone (n = 3), and combined nicotine/methadone e-liquids (n = 3), each prepared in-house at 12 mg/ml. The concentration of nicotine and methadone in 15 puffs of the single drug e-liquids were determined to be 1.60 ± 0.20 and 2.67 ± 0.12 mg, respectively. The concentration of nicotine and methadone in 15 puffs of the multidrug e-liquid were determined to be 3.66 ± 0.49 and 3.65 ± 0.10 mg, respectively. The single nicotine and methadone e-liquids had recoveries of 70 ± 0.1% and 84 ± 0.1%, respectively. In the 1:1 mixture, the recovery of both drugs increased. The development of a eutectic mixture can promote aerosolization of the drug and deliver a greater dose to the user.

Comparison of methods for quantitative analysis of ranibizumab and bevacizumab in human plasma using various bioanalytical techniques, including microfluidic immunoassay, triple quadrupole, and high-resolution liquid chromatography-tandem mass spectrometry approaches.

With the ever-growing abundance of complex therapeutic proteins reaching clinical trials and post-marketing, it is vital to develop highly accurate and robust bioanalytical methods for their quantitative analysis in matrices, to support clinical trial data as well as therapeutic drug monitoring. In bioanalysis, proteins have traditionally been evaluated using ligand binding assays (LBAs). However, in recent years, bottom-up LC-MS/MS methods have begun to gain recognition as an alternative to LBAs in situations where either there is a desire to reduce lengthy development times, or where selectivity issues prevent the immunoassay from reaching the desired outcome. In our study, a microfluidic immunoassay was compared to two bottom-up LC-MS/MS methods, including triple quadrupole and high-resolution mass spectrometry methods. The methods were designed to quantitatively analyze a monoclonal antibody, bevacizumab, and its related fab fragment, ranibizumab, in human plasma after intravitreal administration. All three methods were validated (or cross-validated) according to the 2018 Food and Drug Administration (FDA) guidance, and were then compared by quantitating eighteen patient samples on each platform. The concentrations values obtained from each method were compared using percent variability, as well as Bland-Altman and Pearson Correlation plots, to determine agreeability and linear correlation between methods. Based on the results of the validations and comparison studies, all three methods aligned well with each other. However, the LC-MS/MS methods were able to achieve significantly improve sensitivity, with a lower limit of quantitation (LLOQ) of 0.300 ng/mL, compared to 6.00 ng/mL for the LBA, due to the reduction of interferences at lower concentrations using the LC-MS/MS technique (increased selectivity). Therefore, for this specific study, we were able to establish the correlation between methods, while also demonstrating increased value in using LC-MS/MS as an alternative approach to LBAs in bioanalysis.

Intact quantitative bioanalytical method development and fit-for-purpose validation of a monoclonal antibody and its related fab fragment in human vitreous and aqueous humor using LC-HRMS.

Ranibizumab is an FDA-approved drug used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, macular edema, and myopic choroidal neovascularization. Bevacizumab is another drug often used off-label to treat wet AMD. In order to reduce unwanted angiogenesis, ranibizumab and bevacizumab target circulating VEGF-A in the eye. Concentration levels in human vitreous and aqueous humor can be used to provide valuable efficacy information. However, vitreous and aqueous humor's aqueous environment, and vitreous humor's viscosity, as well as the stickiness of the analytes can provide bioanalytical challenges. In this manuscript, we describe the development, optimization, and fit-for-purpose validation of an LC-HRMS method designed for intact quantitative bioanalysis of ranibizumab and bevacizumab in human vitreous and aqueous humor following intravitreal administration. In order to fully develop this method, evaluations were conducted to optimize the conditions, including the data processing model (extracted ion chromatograms (XICs) vs deconvolution), carryover mitigation, sample preparation scheme optimization for surrogate and primary matrices, use of internal standard/immunocapture/deglycosylation, and optimization of the extraction and dilution procedure, as well as optimization of the liquid chromatography and mass spectrometry conditions. Once the method was fully optimized, a fit-for-purpose validation was conducted, including matrix parallelism, with a linear calibration range of 10 to 200 µg/mL. The development of this intact quantitative method using LC-HRMS provides a proof-of-concept template for challenging, but valuable new and exciting bioanalytical techniques.

Characterization of molecularly imprinted polymers for the extraction of tobacco alkaloids and their metabolites in human urine.

Molecularly imprinted polymers (MIPs) are synthetic polymers designed to selectively extract target analytes from complex matrices (including biological matrices). The literature shows that MIPs have a degree of cross-selectivity from analytes within the same class of compounds. A commercially available MIP for tobacco-specific nitrosamines (TSNAs) is designed to be class selective for four TSNA compounds. This study sought to characterize the extent of cross-selectivity of the TSNA MIPs with other tobacco alkaloids. Cross-selectivity and recovery of the SupelMIP™ TSNA SPE cartridges was assessed with N-nitrosonornicotine (NNN), nicotine, cotinine and morphine. Their recoveries were compared with the recoveries of a nonimprinted polymer SPE cartridge, and two traditional SPE cartridges: a Waters mixed-mode cation exchange cartridge and a Waters hydrophilic-lipophilic balance cartridge. NNN and cotinine had the highest recoveries with the MIP cartridge, over 80%, and cotinine samples in urine had >80% recoveries. Nicotine had highly variable recoveries, possibly owing to differing chemical properties from the TSNAs. All three analytes had significantly different recoveries with the MIP cartridges compared with the traditional SPE cartridges. Morphine displayed nonspecific interactions with the MIP cartridges. Utilization of the TSNAs' cross-selectivity allows for simultaneous extraction and identification of multiple tobacco biomarkers using one extraction technique.

Design, Synthesis, and Biological Evaluation of NAP Isosteres: A Switch from Peripheral to Central Nervous System Acting Mu-Opioid Receptor Antagonists.

The µ opioid receptor (MOR) has been an intrinsic target to develop treatment of opioid use disorders (OUD). Herein, we report our efforts on developing centrally acting MOR antagonists by structural modifications of 17-cyclopropylmethyl-3,14-dihydroxy-4,5α-epoxy-6ß-[(4'-pyridyl) carboxamido] morphinan (NAP), a peripherally acting MOR-selective antagonist. An isosteric replacement concept was applied and incorporated with physiochemical property predictions in the molecular design. Three analogs, namely, 25, 26, and 31, were identified as potent MOR antagonists in vivo with significantly fewer withdrawal symptoms than naloxone observed at similar doses. Furthermore, brain and plasma drug distribution studies supported the outcomes of our design strategy on these compounds. Taken together, our isosteric replacement of pyridine with pyrrole, furan, and thiophene provided insights into the structure-activity relationships of NAP and aided the understanding of physicochemical requirements of potential CNS acting opioids. These efforts resulted in potent, centrally efficacious MOR antagonists that may be pursued as leads to treat OUD.

Direct analysis of tobacco specific nitrosamines in tobacco products using a molecularly imprinted polymer-packed column.

Tobacco specific nitrosamines (TSNAs) are highly carcinogenic by-products in tobacco samples, and their presence is regulated by the Food and Drug Administration. Molecularly imprinted polymers (MIPs) are synthetic polymers that have been "imprinted" with a template analyte in a co-polymer system, and can selectively extract analytes from complex matrices. MIPs can be incorporated into online systems, replacing traditional high performance liquid chromatography (HPLC) columns. MIP material specific for TSNAs was packed into an empty HPLC column using a slurry packing technique. The developed method with the MIP-packed HPLC column was validated on a LC-MS/MS system for the quantitation of N-nitrosonornicotine (NNN) and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in commercial tobacco products. The method was linear over .1-10 ng/ml (.4-10 µg/g) for NNN and NNK. The limit of detection (LOD) was .03 ng/ml (12 µg/g) and the limit of quantitation (LOQ), .1 ng/ml (.4 µg/g). All column uniformity parameters with the exception of theoretical plate number were within the accepted criteria (% RSD values <15%). Theoretical plate number was <250, owing to the large (50 µm) sized MIP particles. Twenty-six tobacco products contained TSNA concentrations that were consistent with reported literature values. The TSNA-MIP based HPLC column effectively replaced a traditional reverse phase HPLC column, and was used for the direct analysis of nicotine and tobacco products without extensive sample preparation prior to instrumental analysis.

Histone acetylation at the sulfotransferase 1a1 gene is associated with its hepatic expression in normal aging.

OBJECTIVES: Phase II drug metabolism is poorly studied in advanced age and older adults may exhibit significant variability in their expression of phase II enzymes. We hypothesized that age-related changes to epigenetic regulation of genes involved in phase II drug metabolism may contribute to these effects. METHODS: We examined published epigenome-wide studies of human blood and identified the SULT1A1 and UGT1A6 genes as the top loci showing epigenetic changes with age. To assess possible functional alterations with age in the liver, we assayed DNA methylation (5mC) and histone acetylation changes around the mouse homologs Sult1a1 and Ugt1a6 in liver tissue from mice aged 4-32 months. RESULTS: Our sample shows a significant loss of 5mC at Sult1a1 (ß = -1.08, 95% CI [-1.8, -0.2], SE = 0.38, P = 0.011), mirroring the loss of 5mC with age observed in human blood DNA at the same locus. We also detected increased histone 3 lysine 9 acetylation (H3K9ac) with age at Sult1a1 (ß = 0.11, 95% CI [0.002, 0.22], SE = 0.05, P = 0.04), but no change to histone 3 lysine 27 acetylation (H3K27ac). Sult1a1 gene expression is significantly positively associated with H3K9ac levels, accounting for 23% of the variation in expression. We did not detect any significant effects at Ugt1a6. CONCLUSIONS: Sult1a1 expression is under epigenetic influence in normal aging and this influence is more pronounced for H3K9ac than DNA methylation or H3K27ac in this study. More generally, our findings support the relevance of epigenetics in regulating key drug-metabolizing pathways. In the future, epigenetic biomarkers could prove useful to inform dosing in older adults.

A phase 1 clinical trial of SP16, a first-in-class anti-inflammatory LRP1 agonist, in healthy volunteers.

BACKGROUND: Endogenous serine protease inhibitors are associated with anti-inflammatory and pro-survival signaling mediated via Low-density lipoprotein receptor-related protein 1 (LRP1) signaling. SP16 is a short polypeptide that mimics the LRP1 binding portion of alpha-1 antitrypsin. METHODS: A pilot phase I, first-in-man, randomized, double blind, placebo-controlled safety study was conducted to evaluate a subcutaneous injection at three dose levels of SP16 (0.0125, 0.05, and 0.2 mg/kg [up to 12 mg]) or matching placebo in 3:1 ratio in healthy individuals. Safety monitoring included vital signs, laboratory examinations (including hematology, coagulation, platelet function, chemistry, myocardial toxicity) and electrocardiography (to measure effect on PR, QRS, and QTc). RESULTS: Treatment with SP16 was not associated with treatment related serious adverse events. SP16 was associated with mild-moderate pain at the time of injection that was significantly higher than placebo on a 0-10 pain scale (6.0+/-1.4 [0.2 mg/kg] versus 1.5+/-2.1 [placebo], P = 0.0088). No differences in vital signs, laboratory examinations and electrocardiography were found in those treated with SP16 versus placebo. CONCLUSION: A one-time treatment with SP16 for doses up to 0.2 mg/kg or 12 mg was safe in healthy volunteers.

LC-MS/MS method for simultaneous quantification of dexamethasone and tobramycin in rabbit ocular biofluids.

The complexity of Tobradex® ointment formulation (dexamethasone 0.1 wt% and tobramycin 0.3 wt%) and the high cost of pharmacokinetic (PK) studies in human aqueous humor may prevent generic drug companies from moving forward with a Tobradex®-equivalent product development. The in vitro drug release test would be an alternative approach for differentiating the generic formulations containing both dexamethasone (DEX) and tobramycin (TOB), and the results should be correlated with the in vivo ocular PK studies for further evaluation. To facilitate the in vivo ocular PK studies, a sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that can simultaneously quantify both DEX and TOB in rabbit ocular matrices including tear, aqueous humor and cornea was established and validated. The lower limit of quantification (LLOQ) was 1.5 ng/ml for DEX and 3 ng/ml for TOB with good precision and accuracy. Both intra- and inter-batch precisions were within ±15%, and the accuracy for all QCs was within the range of 85-115%. This new method was successfully applied for a pilot pharmacokinetic analysis of DEX and TOB in rabbit tears after topical administration of Tobradex® ointment.

An examination of social and environmental determinants of secondhand smoke exposure among non-smoking adolescents.

INTRODUCTION: Adolescents are at increased risk of secondhand smoke exposure (SHS) due to the limited control that they have over social and physical environments. Yet, knowledge regarding determinants of SHS among non-smoking adolescents is limited. This study identifies social and environmental factors associated with SHS among non-smoking adolescents. METHODS: To be included, parents and adolescents (aged 11-17 years) of the Adolescents, Place, and Behavior Study had to have completed surveys between March 2019 and May 2020. Adolescents had to have not reported smoking within the past 30 days and provided a saliva sample assayed for cotinine (≤3 ng/mL). A series of stepwise linear regression models were fit to the data to identify social and environmental determinants of SHS, using log-transformed salivary cotinine. RESULTS: Of the 105 adolescent and parent dyads included, 90.3% were African American, 26.9% of parents reported smoking, 33.3% resided in multi-unit housing, and 67.7% lived in homes where smoking was not permitted. Significant associations were found between parent tobacco use (ß=2.56, SE=0.98, p=0.0082) and residing in multi-unit housing (ß=1.72, SE=0.86, p=0.0460) with increased log-transformed cotinine levels among non-smoking adolescents. Adolescent age, gender, and race/ ethnicity, parental education, peer tobacco use, the number of adults and children in the home, average number of days of self-reported SHS within public spaces outside of the home, and home smoking policies were not significantly associated with cotinine. CONCLUSIONS: Results emphasize the importance of reducing secondhand smoke exposure by reducing parental smoking and altering exposures within social and home environments. Parental tobacco use and residential setting should be considered when developing interventions to reduce secondhand smoke exposure among non-smoking adolescents.

Potential factors affecting free base nicotine yield in electronic cigarette aerosols.

BACKGROUND: The free base and protonated nicotine forms in e-cigarette aerosol have shown different absorption profiles in users. Therefore, it is also important to identify the factors which can affect the ratio of these nicotine forms in the aerosol. Such factors may include nicotine concentrations, flavors, PG:VG ratios, types of nicotine chemical compounds and e-cigarette battery power outputs. The current study attempts to identify such factors using a controlled experiment. RESEARCH DESIGNS AND METHODS: The aerosol was generated using validated aerosol generating model. Various factors were tested for their effect on nicotine forms. Additionally, a degradation study of one of the nicotine compounds, nicotine salicylate, was also carried out using mass spectrometry. RESULTS: The free base nicotine in the aerosol was found to be affected by flavors, battery power output, nicotine compound type and PG:VG ratios. Based on the preliminary mass spectrometry data, degradation of nicotine salicylate was found to be one of the significant factors affecting free base nicotine in aerosol. CONCLUSIONS: Potential factors affecting free base nicotine in e-cigarette aerosol have been identified in this study. These findings would help in understanding the nicotine delivery better and assist for better regulations.

Optimization and method validation for the quantitative analysis of a monoclonal antibody and its related fab fragment in human plasma after intravitreal administration, using LC-MS/MS.

As biologic based drugs become an increasingly important sector of the pharmaceutical industry, accurate and precision techniques for bioanalysis are required to support clinical trials and beyond. Ranibizumab, a fab therapeutic, is an FDA approved drug to treat wet age-related macular degeneration (AMD), as well as other eye related diseases. Ranibizumab's mAb counterpart, bevacizumab, is often also used off-label to treat wet AMD. Ranibizumab and bevacizumab target circulating VEGF-A in the eye, reducing unwanted angiogenesis. Since these drugs are designed for local intravitreal administration, concentration levels in human plasma are expected to be significantly lower compared to vitreous fluid concentrations, presenting bioanalytical challenges. However, this is important for assessment of drug toxicity. In this manuscript, we describe the development, optimization, and validation of an LC-MS/MS method designed for quantitative bioanalysis of ranibizumab and bevacizumab in human plasma following intravitreal administration. In order to fully develop this method, evaluations were conducted to optimize the conditions, including selection of the surrogate peptide by in-silico experiments, optimizations of the immunocapture, denaturation, reduction, alkylation, and digestion extraction steps, as well as optimization of the LC-MS/MS conditions, and evaluation of a dissociation step to determine if there was interference from VEGF or ADAs. Once the method was fully optimized, it was then validated, following the 2018 FDA guidance on bioanalytical method validations. This method is now available for use during clinical trials and precision medicine, for the quantitative evaluation of systemic exposure of ranibizumab or bevacizumab in human plasma after intravitreal administration, with a linear calibration range of 0.300-100 ng/mL.

Growing Trends in the Efficient and Selective Extraction of Compounds in Complex Matrices Using Molecularly Imprinted Polymers and Their Relevance to Toxicological Analysis.

In the world of forensic and clinical toxicology, proper sample preparation is one of the key steps in identification and quantification of drugs of abuse. Traditional extraction methods such as solid-phase extraction and liquid-liquid extraction are often laborious and nonselective for the target analytes being measured. Molecularly imprinted polymers (MIPs) can be synthesized for sample extraction and their versatility allows the polymer to be employed in off-line, benchtop extractions or on/in-line instrument extractions, offering a faster and more selective sample preparation without the risk of interfering matrix effects. This review details the synthesis and applications of MIP materials for the extraction of drug compounds from biological matrices in publications from 1994 to today.