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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 SPE 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.

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-6β-[(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.

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

Co-users of cannabis and tobacco frequently use cannabis, then tobacco cigarettes, in a sequential pattern within an occasion, that is, they "chase" smoked cannabis with a tobacco cigarette. The objective of this placebo-controlled, double-blind, within-subjects human laboratory study was to gather preliminary data on how smoking active versus placebo cannabis impacts tobacco cigarette smoking behavior, craving, and subjective effects. Adult daily cannabis and tobacco co-users (N = 9) were randomly assigned to two experimental visit orders (i.e., active cannabis (5.2% THC) first visit and placebo cannabis second visit, or vice versa). Participants smoked one cannabis cigarette, and approximately 30 min later were given a 5-min ad libitum period to smoke one of their own brand of tobacco cigarette. As expected, boost in plasma THC levels and cannabis-related subjective effects differed between active and placebo cannabis conditions. Tobacco cigarette puff topography measures and tobacco craving did not differ between cannabis conditions, but there appeared to be between-participants heterogeneity in cumulative total puff volume. After smoking active versus placebo cannabis, the changes in subjective effects of tobacco smoking after adjusting for pretobacco smoking levels were not significant. Results do not support the notion that immediate effects of smoked cannabis change the behavior of tobacco smoking. The strong overlap between cannabis and tobacco smoking may not be explained by primarily pharmacological factors, but may be driven by more nuanced and complex mechanisms involving pharmacological processes as well as learning factors. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

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.

Osteosarcoma (OS) metastasizes primarily and almost exclusively to the lungs. The survival rate decreases significantly for patients who develop OS lung metastasis (OSLM). Purpose: To assess the safety and efficacy of colony-stimulating factor-1 receptor inhibitor (CSF-1Ri) (Pexidartinib; PLX) in shifting the OSLM tumor microenvironment (TME) to an anti-tumorigenic state upon local delivery to lungs of a syngeneic mouse model of OSLM. Methods: The safety of PLX upon repeated pulmonary administration (9 doses, every other day at 2 mg/kg) in healthy BALB/c mice was assessed using health scoring, pulmonary mechanics, molecular and cellular composition of bronchoalveolar lavage fluid (BALF), blood count and plasma biochemistry, and H&E of lungs and liver. To establish the OSLM model, murine OS cells, expressing bioluminescent and fluorescent genes (K7M2-Luc-tdT), were used. Mice bearing OSLM were randomized into two groups (vehicle and PLX) and treated over 3 weeks. Tumor burden was evaluated using bioluminescent imaging (BLI) and lung weight. The treatment impact on the abundance and phenotype of tumor-associated macrophages (TAMs) and their CSF-1R expression as well as on the abundance of tumor-infiltrating lymphocytes and their expression to PD-1 were investigated using immunofluorescence, flow cytometry, and western blot. Results: PLX is well tolerated upon local lung administration with all safety markers indicating no alteration compared to naïve and vehicle controls. Treatment did not affect health scores. We did not detect any functional or mechanical alteration on the lungs or systemically (including liver); no marked differences in pulmonary function parameters, proinflammatory cytokines, nor infiltrating differential cell count obtained from BALF were observed. Both total blood count and liver enzymes were within normal range. As a measure of efficacy, treatment with PLX via local lung administration reduced tumor burden, indicated by the in vivo BLI and lung weight. Notably, PLX significantly decreased the abundance of total TAMs, with a more pronounced effect on pro-tumorigenic alternatively-activated (M2-like) TAMs phenotype, thus leading to a 22-fold increase in M1/M2 ratio. PLX further led to a marked decrease in tumor-promoting T regulatory cells (Treg), increase in the CD8/Treg ratio, and decrease in PD-1+ percentage in CD8 T cells in the TME, indicating a shift to a more immunogenic tumor phenotype. Conclusions: We demonstrated that repeated doses of PLX is safe upon local delivery to the lungs of murine model of OSLM, with no alternations in lungs or liver tissue. We further showed that PLX reduces tumor burden, which correlates with a change in the immune phenotype of the OSLM toward a more anti-tumorigenic profile. These results are clinically relevant as PLX has been approved by the FDA, thus opening opportunities for its repurposing to support standard of care therapy in OSLM. Citation Format: Fatemah S. Sunbul, Sulaiman S. Alhudaithi, Rashed M. Almuqbil, Hanming Zhang, Raneem R. Aldaqqa, Shane Albin, Rebecca L. Heise, Valentina Robila, Matthew S. Halquist, Sarah W. Gordon, Paula D. Bos, Sandro R. da Rocha. Remodeling the microenvironment of osteosarcoma lung metastases with inhaled CSF-1Ri immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1991.

Organophosphorus (OP) compounds are chemical threat agents and are irreversible inhibitors of the enzyme acetylcholinesterase that lead to a hypercholinergic response that could include status epilepticus (SE). SE particularly targets the heart and the brain and despite existing therapies it is still associated with significant mortality and morbidity. Here, we investigated the effect of intramuscular (i.m.) adjunct therapy consisting of atenolol (AT) and levetiracetam (LV) when administered after paraoxon (POX)-induced SE. The combination therapy was administered twice daily for 2, 7, or 14 days. POX exposure in rats produced rapid SE onset that was treated with atropine, pralidoxime chloride, and midazolam. Here, AT + LV therapy produced significant reductions in POX SE mortality assessed at 30 days post SE. AT + LV therapy exhibited muscle pathology inflammation scores that were not significantly different from saline-treated controls. Pharmacokinetic analyses revealed that the i.m. route achieved faster and stabler plasma therapeutic levels for both AT and LV under OP SE conditions compared with oral administrations. Our data provides evidence of the safety and efficacy of i.m. AT + LV therapy for reducing mortality following POX SE.

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.

As biologic drugs become an increasing segment in the overall pharmaceutical market, it is important to develop accurate and reliable methods to analyze these drugs in biological matrices. With advancements in technology, biologics’ complex molecular structures can now be selectivity distinguished and quantified using high-resolution mass spectrometry and deconvolution software. Intact (top-down) mass spectrometric techniques have been established as alternative or complementary bioanalytical techniques for instances when ligand binding assays (LBAs) alone were not well suited, as it can provide additional structural information, in its pharmacologically active form.

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 had80% 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.

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.

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 (β = -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 (β = 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.

Poor antiretroviral penetration may contribute to human immunodeficiency virus (HIV) persistence within the brain and to neurocognitive deficits in opiate abusers. To investigate this problem, HIV-1 Tat protein and morphine effects on blood-brain barrier (BBB) permeability and drug brain penetration were explored using a conditional HIV-1 Tat transgenic mouse model. METHODS. Tat and morphine effects on the leakage of fluorescently-labeled dextrans (10-, 40- and 70- kDa) into the brain were assessed. To evaluate effects on antiretroviral brain penetration, Tat+ and Tat− mice received three antiretroviral drugs (dolutegravir, abacavir, and lamivudine) with or without concurrent morphine exposure. Antiretroviral and morphine brain and plasma concentrations were determined by LC-MS/MS. FINDINGS. Morphine exposure, and to a lesser extent, Tat, significantly increased tracer leakage from the vasculature into the brain. Despite enhanced BBB breakdown evidenced by increased tracer leakiness, morphine exposure led to significantly lower abacavir concentrations within striatum and significantly less dolutegravir within hippocampus and striatum (normalized to plasma). P-glycoprotein, an efflux transporter for which these drugs are substrates, expression and function were significantly increased in the brains of morphine-exposed mice compared to mice not exposed to morphine. These findings were consistent with lower antiretroviral concentrations in brain tissues examined. Lamivudine concentrations were unaffected by Tat or morphine exposure. CONCLUSIONS. Collectively, our investigations indicate that Tat and morphine differentially alter BBB integrity. Morphine decreased brain concentrations of specific antiretroviral drugs, perhaps via increased expression of the drug efflux transporter, P-glycoprotein.

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.

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.

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.

In vitro release studies are commonly used to assess the product performance of topical dosage forms. In such studies, the mass transport of drugs through synthetic membranes into a receiving chamber filled with a release medium is measured. The release medium is also passed through filtration membranes prior to chromatographic analysis. There are no official guidelines directing membrane selection for in vitro release studies or for filtration. Considering the diversity in membrane materials and their physical properties, the aim of this study was to investigate membrane-drug binding and the effect of various membranes on the release performance of a model drug dexamethasone (DEX) using USP dissolution apparatus IV. Seven membranes of different pore sizes (0.45 and 1.2 μm) and materials (cellulose acetate, polyethersulfone, and nylon) were assessed. Two different methods, syringe filter and 24-h incubation, were used for the determination of membrane-drug binding effects at low drug concentrations and saturated concentration conditions. Cellulose acetate and nylon membranes showed significant drug binding after 24-h incubations at both drug concentrations. DEX diffusion through membranes was significantly slowed down in all the tested membranes when compared with DEX solution without membranes. The extent of the retardation varied due to the differences in membrane structures. In conclusion, materials and sources of membranes affected drug dissolution profiles and the results showed membrane-drug binding effects. Proper selection of membranes with low drug binding ability and low diffusion resistance is essential to ensure appropriate and reproducible in vitro release assessments and filtration studies. Graphical Abstract.

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. 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 obtained from the National Institute on Aging rodent tissue bank. Our sample shows significant loss of 5mC at Sult1a1, 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, 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 changes at Ugt1a6. We conclude that Sult1a1 expression is under epigenetic influence in normal aging and that 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 future, epigenetic biomarkers could prove useful to inform dosing in older adults.

INTRODUCTION: Unregulated e-cigarette devices and their nicotine content have amplified the potential of e-cigarettes as addictive agents. Several e-cigarette-related parameters have been identified altering nicotine’s absorption profile, so their potential effects on addiction should be considered. Of these factors, nicotine forms (protonated and free base) play a significant role in the addiction potential yet their impact on nicotine’s absorption has been studied with limited research. AREAS COVERED: Current review aims to emphasize on the possible mechanism behind different absorption profiles of nicotine forms considering their physical states (droplet and vapor phase) and the aerosol particle size, their analysis in e-cigarette research and the regulatory attention warranted by them to combat nicotine addiction in the population due to e-cigarettes. EXPERT OPINION: The protonated form of nicotine is being correlated with the smooth sensory effects and high nicotine absorption as compared to free base nicotine. With the introduction of nicotine salts, which yield mostly the protonated form, the youth popularity of e-cigarettes has spiked exponentially. While it is important to control nicotine levels in e-cigarette products, attention should also be given to the nicotine forms present in these products in order to address nicotine addiction in the population.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In electronic cigarette users, nicotine delivery to lungs depends on various factors. One of the important factors is e-liquid nicotine concentration. Nicotine concentration in e-liquids ranges from 0 to >50 mg/mL. Furthermore, nicotine exists in protonated and unprotonated (“free base”) forms. The two forms are believed to affect the nicotine absorption in body. Therefore, in addition to total nicotine concentration, e-liquids should be characterized for their free base nicotine yield. Two approaches are being used for the determination of free base nicotine in e-liquids. The first is applying a dilution to e-liquids followed by two methods: Henderson–Hasselbalch theory application or a Liquid-Liquid Extraction. The second is the without-dilution approach followed by 1H NMR method. Here, we carried out controlled experiments using five e-liquids of different flavors using these two approaches. In the dilution approach, the Henderson–Hasselbalch method was tested using potentiometric titration. The accuracy was found to be >98% for all five e-liquid samples (n = 3). A Liquid-Liquid Extraction was carried out using toluene or hexane as extraction solvent. The Liquid-Liquid Extraction technique was found to be limited by solvent interactions with flavors. Solvent extractions resulted in flavor dependent inaccuracies in free base nicotine determination (5 to 277% of calculated values). The without-dilution approach was carried out using 1H NMR as described by Duell et al. This approach is proposed to offer an independent and alternative scale. None of the methods have established a strong correlation between pre- and postvaporization free base nicotine yield. Here we present comparative results of two approaches using analytical techniques. Such a comparison would be helpful in establishing a standardized method for free base nicotine determination of e-liquids.

Cigarettes and electronic cigarettes (e-cigarettes) are major sources of exposure to nicotine, an addictive chemical. Although these products are being regulated by the Food and Drug Administration (FDA) under the Tobacco Control Act, specifications about the nicotine content in these products have not been established yet. In e-cigarettes, nicotine concentration ranges from 0 to > 50mg/mL, and the recent e-cigarette devices provide control to change nicotine flux for higher nicotine delivery. Due to the lack of robust regulations in manufacturing, distribution and marketing, e-cigarettes have already infiltrated the market with youth appealing flavors and devices. As a result, the country is facing a youth epidemic of e-cigarette use. The unregulated nicotine levels in both cigarettes and e-cigarettes can lead to repeated and overexposure of nicotine to youth which can lead to the addiction and detrimental effects on their cognitive functions. Over the past decade, the corrective measures being taken by the FDA for cigarette and e-cigarette regulations also should focus on nicotine exposure levels. Before it is too late to prevent youth from lifetime addiction to nicotine, it is important to address the issues of nicotine concentration, nicotine flux and the e-cigarette device regulations while offering adults with smoking disorder less harmful alternatives to cigarettes.

Precision medicine is an emerging field in healthcare that seeks to tailor preventive and therapeutic strategies to the unique physiology, biochemistry, lifestyles, and genetics of individual patients. There are several technologies that are key to the successful delivery of precision medicine including pharmacogenomics, pharmacometabolomics, improved point-of-care testing, and therapeutically-tailored medications. The inclusion of additive manufacturing (AM) technology, more commonly known as 3D printing, in the manufacture of oral dosage forms such as tablets, provides an avenue for the implementation of precision medicine in current healthcare practice via the prescription of specific dosage forms and drug combinations tailored to individual needs. Widespread commercialization of AM of pharmaceuticals has the potential to disrupt the supply chain used by the healthcare industry worldwide with the cost-saving potential of minimizing waste related to unused, expired medications. Despite the potential of this technology, many clinical and regulatory challenges will need to be addressed prior to large-scale implementation of AM fabricated therapeutics for precision medicine applications. This review investigates both the potential and the challenges of delivering AM fabricated medications for therapeutic use in precision medicine applications.

IntroductionThe present study examined how the lack of characterising flavours and low levels of humectants may affect users’ waterpipe tobacco (WT) smoking topography, subjective effects, toxicant exposure and intentions for continued use.Methods89 WT smokers completed four ad libitum smoking sessions (characterising flavor/high humectant (+F+H); characterising flavor/low humectant (+F-H); no characterising flavor/high humectant (-F+H); no characterising flavor/low humectant (-F-H)) in a randomised cross-over design. WT was commercially available; same brand but nicotine levels were not held constant. A subsample (n=50) completed a standardised, 10-puff session preceding ad libitum smoking. Participants completed questionnaires, exhaled carbon monoxide (eCO) testing and provided blood samples for plasma nicotine. Smoking topography was measured throughout the session. Post hoc analyses showed that -F+H and -F-H did not differ significantly in humectant levels. Therefore, these groups were collapsed in analyses (-F-H).ResultsWT smokers reported significantly greater satisfaction, liking, enjoyment and greater intentions for continued use when smoking +F+H compared with other WT products, with -F-H receiving the lowest ratings. Significant differences in topography were observed during standardised and ad libitum sessions, with the -F-H preparation leading to greater total inhaled volume and eCO boost, but lower nicotine boost compared with +F+H (all pDiscussionThe findings demonstrate the importance of flavours and humectants on improving WT smoking experience and increasing the likelihood that users will want to initiate and continue smoking. Moreover, it demonstrates that flavours and humectants influence smoking behaviour and toxicant exposure in some unexpected ways that are important for regulatory efforts.

NLRP3 inflammasomes have recently emerged as an attractive drug target for neurodegenerative disorders. In our continuing studies, a new chemical scaffold was designed as selective inhibitors of NLRP3 inflammasomes. Initial characterization of the lead HL16 demonstrated improved, however, nonselective inhibition on the NLRP3 inflammasome. Structure-activity relationship studies of HL16 identified a new lead, 17 (YQ128), with an IC50 of 0.30 ± 0.01 μM. Further studies from in vitro and in vivo models confirmed its selective inhibition on the NLRP3 inflammasome and its brain penetration. Furthermore, pharmacokinetic studies in rats at 20 mg/kg indicated extensive systemic clearance and tissue distribution, leading to a half-life of 6.6 h. However, the oral bioavailability is estimated to be only 10%, which may reflect limited GI permeability and possibly high first-pass effects. Collectively, these findings strongly encourage development of more potent analogues with improved pharmacokinetic properties from this new chemical scaffold.

Importance Experimental data suggest that intravenous vitamin C may attenuate inflammation and vascular injury associated with sepsis and acute respiratory distress syndrome (ARDS). Objective To determine the effect of intravenous vitamin C infusion on organ failure scores and biological markers of inflammation and vascular injury in patients with sepsis and ARDS. Design, Setting, and Participants The CITRIS-ALI trial was a randomized, double-blind, placebo-controlled, multicenter trial conducted in 7 medical intensive care units in the United States, enrolling patients (N = 167) with sepsis and ARDS present for less than 24 hours. The study was conducted from September 2014 to November 2017, and final follow-up was January 2018. Interventions Patients were randomly assigned to receive intravenous infusion of vitamin C (50 mg/kg in dextrose 5% in water, n = 84) or placebo (dextrose 5% in water only, n = 83) every 6 hours for 96 hours. Main Outcomes and Measures The primary outcomes were change in organ failure as assessed by a modified Sequential Organ Failure Assessment score (range, 0-20, with higher scores indicating more dysfunction) from baseline to 96 hours, and plasma biomarkers of inflammation (C-reactive protein levels) and vascular injury (thrombomodulin levels) measured at 0, 48, 96, and 168 hours. Results Among 167 randomized patients (mean [SD] age, 54.8 years [16.7]; 90 men [54%]), 103 (62%) completed the study to day 60. There were no significant differences between the vitamin C and placebo groups in the primary end points of change in mean modified Sequential Organ Failure Assessment score from baseline to 96 hours (from 9.8 to 6.8 in the vitamin C group [3 points] and from 10.3 to 6.8 in the placebo group [3.5 points]; difference, −0.10; 95% CI, −1.23 to 1.03;P = .86) or in C-reactive protein levels (54.1 vs 46.1 μg/mL; difference, 7.94 μg/mL; 95% CI, −8.2 to 24.11;P = .33) and thrombomodulin levels (14.5 vs 13.8 ng/mL; difference, 0.69 ng/mL; 95% CI, −2.8 to 4.2;P = .70) at 168 hours. Conclusions and Relevance In this preliminary study of patients with sepsis and ARDS, a 96-hour infusion of vitamin C compared with placebo did not significantly improve organ dysfunction scores or alter markers of inflammation and vascular injury. Further research is needed to evaluate the potential role of vitamin C for other outcomes in sepsis and ARDS. Trial Registration ClinicalTrials.gov Identifier:NCT02106975

Electronic cigarettes (e-cigarettes) are an alternate nicotine delivery system that generate a condensation aerosol to be inhaled by the user. The size of the droplets formed in the aerosol can vary and contributes to drug deposition and ultimate bioavailability in the lung. The growing popularity of e-cigarette products has caused an increase in internet sources promoting the use of drugs other than nicotine (DOTNs) in e-cigarettes. The purpose of this study was to determine the effect of various e-cigarette and e-liquid modifications, such as coil resistance, battery voltage, and glycol and drug formulation, on the aerosol particle size. E-liquids containing 12 mg/mL nicotine prepared in glycol compositions of 100% propylene glycol (PG), 100% vegetable glycerin (VG), or 50:50 PG:VG were aerosolized at three voltages and three coil resistances. Methamphetamine and methadone e-liquids were prepared at 60 mg/mL in 50:50 PG:VG and all e-liquids were aerosolized onto a 10 stage Micro-Orifice Uniform Deposit Impactor. Glycol deposition correlated with drug deposition, and the majority of particles centered between 0.172–0.5 μm in diameter, representing pulmonary deposition. The 100% PG e-liquid produced the largest aerosol particles and the 100% VG and 50:50 PG:VG e-liquids produced ultra-fine particles

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.

Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macrophage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets; the reduction in free cholesterol was mainly attributed to decreases in lysosome-compartmentalized cholesterol. Functionally, the egression of free cholesterol from lysosomes attenuated pro-inflammatory cytokine secretion. It was determined that the reduction of lysosomal free cholesterol buildup by simvastatin was due to the up-regulation of Niemann-Pick C1 (NPC1), a lysosomal residing cholesterol transporter. Moreover, the enhanced enzymatic production of 7-hydroxycholesterol by cytochrome P450 7A1 and the subsequent activation of liver X receptor α underscored the up-regulation of NPC1. These findings reveal a novel pleiotropic effect of simvastatin in affecting lysosomal cholesterol efflux in macrophages and the associated significance in the treatment of atherosclerosis.

BACKGROUND: A diet rich in saturated fat and sugars (Western diet, WD) induces myocardial expression of the NLRP3 inflammasome and dysfunction in mice. We therefore hypothesized that a diet enriched with an orally available NLRP3 inflammasome inhibitor could prevent WD-induced cardiac dysfunction in mice. METHODS: Ten-week-old CD-1 male mice were fed WD or standard diet (SD) for 8 weeks. The compound 16673-34-0, an orally active NLRP3 inhibitor, was added to the diet at a concentration of 100 mg/Kg. The plasmatic levels of the NLRP3 inflammasome inhibitor were measured. Food intake, body weight, and glucose tolerance were assessed. Cardiac systolic and diastolic functions were measured by Doppler echocardiography at baseline, 4 weeks, and 8 weeks. RESULTS: WD induced a significant increase in body weight (+14%, P = 0.02), impaired glucose tolerance (+34%, P = 0.03), and a significant increase in isovolumetric relaxation time (+129%, P = 0.03) and reduction in left ventricular ejection fraction (−10%, P = 0.03), as compared to standard chow diet (SD). The treatment with NLRP3 inhibitor in the diet prevented cardiac systolic and diastolic dysfunction (P < 0.05 for left ventricular ejection fraction, isovolumetric relaxation time, and myocardial performance index in WD with drug vs. WD without drug), without significant changes in heart rate and metabolic parameters. CONCLUSIONS: An orally available NLRP3 inhibitor prevented WD-induced cardiac dysfunction in obese mice.

BACKGROUND: Electronic cigarettes (ECIGs) are a class of tobacco products that produce different effects (e.g., nicotine delivery), depending on the device, liquid, and behavioral factors. However, the influence of the two primary ECIG liquid solvents, propylene glycol (PG) and vegetable glycerin (VG), on ECIG acute effects is unknown. METHODS: Thirty ECIG-experienced, ≥ 12-h nicotine- abstinent participants completed four conditions consisting of two ECIG-use bouts (10 puffs, 30 s interpuff-interval) differing only by liquid PG:VG ratio (2PG:98VG, 20PG:80VG, 55PG:45VG, 100PG). Device power (7.3 W) and liquid nicotine concentration (18 mg/ml) remained constant. Nicotine delivery, subjective effects, heart rate (HR), and puff topography were assessed. RESULTS: In the 100PG condition, participants took shorter and smaller puffs but obtained significantly more nicotine relative to the two VG-based conditions. Total nicotine exposure (i.e., area under the curve) was also significantly higher during use of the two PG-based liquids. However, participants reported that the 100 PG liquid was significantly less “pleasant” and “satisfying” relative to the other liquids (all ps < .05). Increases in HR and decreases in abstinence symptoms (e.g., “craving”) did not differ across conditions. CONCLUSIONS: PG:VG ratio influenced nicotine delivery, some subjective effects, and puff topography. Lower overall product satisfaction associated with the 100PG liquid suggests factors other than nicotine delivery (e.g., aerosol visibility) may play a role in maintaining ECIG use. Regulating ECIG acute effects such as nicotine delivery and subjective effects may require simultaneous attention to liquid PG:VG ratio as well as device, liquid, and behavioral factors known to influence these outcomes.

Human osteopontin (hOPN) is a secreted plasma protein which is elevated in various cancers and is indicative of poor prognosis. Here we describe investigations involving an extended peptide internal standard to track an unstable signature peptide followed by further method development and validation for quantitative measurement of hOPN from plasma using microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide 'GDSVVYGLR' was used as a signature peptide for this method. The optimized method involved immunocapture of the analyte protein using hOPN specific antibodies followed by trypsin digestion to obtain the signature peptide. Analysis was carried out on a Waters IonKey/MS system using a flow rate of 2.5μL/min. Immunocapture buffer was used as a surrogate matrix for the validation studies. The method was validated over a range of 25-600ng/mL. Intra-assay and inter-assay accuracies were within ±13%. Intra-assay and inter-assay precision were within 17%. A stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. Inherent digestion variability i.e., under controlled conditions, was within ±20% with both IS peptides. In digestion variability studies, where trypsin activity was varied (20-180%), the use of the extended SIL peptide as an internal standard limited the variability to within ±30% of the normalized response. Alternatively, when the SIL peptide was used as the internal standard, the variability ranged from -67.4% to 50.6% of the normalized response. The applicability of the validated method was demonstrated by quantification of OPN from plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. The plasma OPN concentrations in healthy individuals ranged from 38 to 85ng/mL with a mean concentration of 55.4±15.3ng/mL. A 1.5-12 fold increase in OPN concentrations, ranging from 85 to 637ng/mL, was seen in breast cancer patient samples.

As a means to evaluate the role of organic solute carriers in CNS homeostasis, a simple and rapid high-performance liquid chromatographic method utilizing ultraviolet and electrochemical detectors was developed and validated for the simultaneous determination of the neurotransmitters dopamine, norepinephrine, and serotonin and their metabolites in mouse brain homogenate. For analyte separation, the method utilized a C18 column with a mobile phase of 75 mM sodium dihydrogen phosphate (monohydrate), 1.7 mM 1-octanesulfonic acid sodium salt, 25 µM EDTA, 10% acetonitrile, and 1% triethylamine with a final pH of 3.0 adjusted using phosphoric acid. The method was linear for dopamine, norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid from 20–1000 ng/mL (r ≥ 0.993) with detection limits of 5 ng/mL for dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid, and of 20 ng/mL for serotonin and 5-hydroxyindoleacetic acid. The method wa...

Background: Oxyntomodulin (OXM1–37) is an anorectic gut-secreting peptide with a promise to treat obesity, but its needle-free delivery has yet to be successful. Results: Pulmonary delivery of OXM1–37, but not its C-terminal octapeptides, caused dose-related, transient 4–6 h food intake suppression in rats. At 0.5 mg/kg, its 30–38% food intake suppression led to 46% reduction in body weight gain by day 8. Its lung absorption was fast, elevating the systemic level rapidly, yet the bioavailability was low at 13%. In the brain, twofold neuronal c-fos activation was seen in the hypothalamus arcuate nucleus and brainstem area postrema. Conclusion: Pulmonary delivery is a promising needle-free systemic delivery option for OXM1–37 to treat obesity, as enabling effective lung absorption and brain interaction.

The blue lotus flower (Nymphea caerulea) is an Egyptian water lily containing apomorphine and nuciferine. Apomorphine has been described as a psychoactive alkaloid and is a non-selective dopamine agonist primarily used to treat Parkinson's disease as it stimulates dopamine receptors and improves motor function. Nuciferine is an alkaloid associated with dopamine receptor blockade. Today, blue lotus flower is used as a sleep aid and anxiety reliever. The rebuildable dripping atomizer (RDA) is an electronic cigarette that allows direct application of an e-liquid onto the coil in the atomizer for aerosolization, compared to a typical electronic cigarette where the e-liquid is wicked from a storage vessel to the coil. Our laboratory received a dark-brown resin material from a concerned parent. The resin had been confiscated from an adolescent who had a reported history of marijuana use. The resin was later identified as blue lotus flower (N. caerulea). This resin, together with four commercially available blue lotus products, was analyzed for content. Apomorphine was detected in two samples, and nuciferine was detected in all five samples. The confiscated resin was determined to contain no apomorphine and 4300 ng/g of nuciferine. The nuciferine resin was shown to aerosolize using aRDA electric cigarette.

The use of appropriate analgesia in laboratory mice may be suboptimal because of concerns about adverse events (AE). Target Animal Safety trials were conducted to determine the safety of an extended-release suspension of buprenorphine. Drug or control suspensions were injected subcutaneously in surgically-treated BALB/c mice anesthetized with ketamine–xylazine to mimic post-operative conditions in which the compound might commonly be administered. Single and repeat five-fold (5×) excesses of the 3.25 mg/kg intended dose were used to provoke potential AE. Trials included prospective measurements of weight changes, blood chemistry, hematology, and histopathology. Clinical and histopathology findings were similar in drug-treated and control mice in a four-day trial using a single 16.25 mg/kg, 5× overdose of the drug. In a 12-day trial, which used a total buprenorphine dose of 48.75 mg/kg, clinical and histopathology values were also similar in control and drug-treated female mice. In the male arm of the repeat-overdose trial, two of eight mice died on the morning of day 12, three days following the third 16.25 mg/kg overdose administration. Histopathology did not reveal a cause of death. In a 14-month trial using a single 3.25 mg/kg dose of the drug, no significant findings identified potential AE. These findings indicate a high tolerance to an extended-release buprenorphine suspension administered post-operatively in mice with appropriate husbandry.

A stable isotope-labeled signature peptide, whose sequence corresponds to the human osteopontin (hOPN) specific antibody epitope, was evaluated as an internal standard to compensate for immunocapture variability during quantification of hOPN by immunoaffinity-coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well from 150 to 4500 ng and analysis was carried out with internal standards added before and after the immunocapture step. The immunocapture variability ranged from -80.9 to 77.0% when the IS was added after immunocapture and from -37.5 to 20.3% when the internal standard was added before immunocapture. The lower variability demonstrates the ability of stable labeled isotope internal standard peptide to compensate for variation during immunocapture.

Objectives The aim of this study was to improve the oral bioavailability of buprenorphine by inhibiting presystemic metabolism via the oral co-administration of ‘Generally Recognized as Safe’ compounds, thus providing an orally administered drug product with less variability and comparable or higher exposure compared with the sublingual route. Methods The present studies were performed in Sprague Dawley rats following either intravenous or oral administration of buprenorphine/naloxone and oral co-administration of ‘Generally Recognized as Safe’ compounds referred to as ‘adjuvants’. Plasma samples were collected up to 22 h postdosing followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. Key findings The adjuvants increased Cmax (21 ± 16 ng/ml vs 75 ± 33 ng/ml; 3.6-fold) and AUC(0–22 h) (10.6 ± 8.11 μg min/ml vs 22.9 ± 11.7 μg min/ml; 2.2-fold) values of buprenorphine (control vs adjuvant-treated, respectively). The absolute oral bioavailability of buprenorphine doubled (from 1.24% to 2.68%) in the presence of the adjuvants. Conclusions One may suggest that the adjuvant treatment most likely inhibited the presystemic metabolic enzymes, thus decreasing the intestinal ‘first-pass effect’ on buprenorphine. Additional studies are now required to further explore the concept of inhibiting presystemic metabolism of buprenorphine by adjuvants to potentially increase the oral bioavailability of buprenorphine.

Non-oral dosage forms often comprise of complex dosage forms such as parenterals, implants, drug eluting stents, transdermal patches, liposomes, microspheres and nano-particles as injectable. With the rapid advances in genetic engineering and recombinant DNA technology, the active drug moiety can also be a bio therapeutic (i.e. peptide or protein), which adds an additional layer of complexity to the drug product. In vitro dissolution tests/models for oral dosage forms such as tablets and suspensions have long been an indicator of in vivo performance of the drug. In commercial manufacturing processes, they act as an evaluative quality control tool. A dissolution test in oral dosage forms ensures minimal inter-batch variability and hence works as a check that the dosage form meets the requisite regulatory and quality standards. These in vitro models may also work as a tool for safety (i.e., predict in vivo performance and streamline clinical studies). The predictive in vitro data from these tests could often help in minimizing steps of clinical trials. In the case of non-oral dosage forms, similar tests that ensure performance verification of the drug are often called “in vitro release tests” rather than dissolution tests [1-3]. The rationale being non-oral dosage forms are frequently placed at diverse locations in the body e.g. transdermal patches on skin, drugeluting stents in coronary arteries [4], suppositories in rectum or urethra, subcutaneous or intramuscular implants (all of which have a different physiological environment). As a result, the action of the drug largely depends on its release from the dosage form. The drug moiety may be carried in complex delivery systems e.g. in a lipid based system such as liposomes, absorbable carrier scaffolds e.g. collagen matrix, injectable biodegradable polymer based matrix which solidifies at the site of action (e.g. ATRIGEL°). In cases like transdermal patches, the release needs to occur through multiple membranes and needs to penetrate through the skin and layers of external barriers before reaching the site of action. In subcutaneous and intramuscular implants the blood flow conditions might affect the time taken by the drug to reach the targeted site. Furthermore, the release mechanism, rate of release, and the time required by the drug to reach the site of action thus varies notably in each unique case. Hence the in vivo performance of these complex (non-oral) dosage forms can be characterized more precisely by carefully designed in vitro release tests, with design space criteria that are ‘biorelevant’. Unlike oral dosage forms where a single standardized USP method or apparatus can be used for dissolution testing of a class of compounds to determine in vivo performance (i.e., systemic release). Non-oral dosage forms can release locally with minimal systemic exposure. In such cases the standardized tests may not predict local release. The testing apparatus and method parameters have to be selected or modified accordingly to design a biorelevant reproducible and predictable in vitro release test. Biorelevance in Non-Oral Dosage Forms