Factors Influencing ADME Properties of Therapeutic Antisense Oligonucleotides: Physicochemical Characteristics and Beyond.

Therapeutic antisense oligonucleotides (ASOs) represent a diverse array of chemically modified singlestranded deoxyribonucleotides that work complementarily to affect their mRNA targets. They vastly differ from conventional small molecules. These newly developed therapeutic ASOs possess unique absorption, distribution, metabolism, and excretion (ADME) processes that ultimately determine their pharmacokinetic, efficacy and safety profiles. The ADME properties of ASOs and associated key factors have not been fully investigated. Therefore, thorough characterization and in-depth study of their ADME properties are critical to support drug discovery and development processes for safe and effective therapeutic ASOs. In this review, we discussed the main factors affecting the ADME characteristics of these novels and evolving therapies. The major changes to ASO backbone and sugar chemistry, conjugation approaches, sites and routes of administration, etc., are the principal determinants of ADME and PK profiles that consequentially impact their efficacy and safety profiles. In addition, species difference and DDI considerations are important in understanding ADME profile and PK translatability but are less studied for ASOs. We, therefore, have summarized these aspects based on current knowledge and provided discussions in this review. We also give an overview of the current tools, technologies, and approaches available to investigate key factors that influence the ADME of ASO drugs and provide future perspectives and knowledge gap analysis.

Development and application of a High-Resolution mass spectrometry method for the detection of fentanyl analogs in urine and serum.

Introduction: The use of illicitly manufactured synthetic opioids, specifically fentanyl and its analogs, has escalated exponentially in the United States over the last decade. Due to the targeted nature of drug detection methods in clinical laboratories and the ever-evolving list of synthetic opioids of concern, alternative analytical approaches are needed. Methods: Using the fentanyl analog screening (FAS) kit produced by the Centers for Disease Control and Prevention (CDC), we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) synthetic opioid spectral library and data acquisition method using information dependent acquisition of product ion spectra. Chromatographic retention times, limits of detection and matrix effects, in urine and serum, for the synthetic opioids in the FAS kit (n = 150) were established. All urine and serum specimens sent to a clinical toxicology laboratory for comprehensive drug testing in 2019 (n = 856) and 2021 (n = 878) were analyzed with the FAS LC-HRMS library to determine the prevalence of fentanyl analogs and other synthetic opioids, retrospectively (2019) and prospectively (2021). Results: The limit of detection (LOD) of each opioid ranged from 1 to 10 ng/mL (median, 2.5 ng/mL) in urine and 0.25-2.5 ng/mL (median, 0.5 ng/mL) in serum. Matrix effects ranged from -79 % to 86 % (median, -37 %) for urine, following dilution and direct analysis, and -80 % to 400 % (median, 0 %) for serum, following protein precipitation. The prevalence of fentanyl/fentanyl analogs in serum samples increased slightly from 2019 to 2021 while it remained the same in urine. There were only 2 samples identified that contained a fentanyl analog without the co-occurrence of fentanyl or fentanyl metabolites. Analysis of the established MS/MS spectral library revealed characteristic fragmentation patterns in most fentanyl analogs, which can be used for structure elucidation and drug identification of future analogs. Conclusions: The LC-HRMS method was capable of detecting fentanyl analogs in routine samples sent for comprehensive drug testing. The method can be adapted to accommodate testing needs for the evolving opioid epidemic.

Development of a high-throughput differential mobility separation-tandem mass spectrometry (DMS-MS/MS) method for clinical urine drug testing.

INTRODUCTION: Differential mobility separation (DMS) is an analytical technique used for rapid separation of ions and isomers based on gas phase mobility prior to entering a mass spectrometer for analysis. The entire DMS process is accomplished in fewer than 20 ms and can be used as a rapid alternative to chromatographic separation. OBJECTIVE: The primary objective was to evaluate the utility of DMS-tandem mass spectrometry (DMS-MS/MS) as a replacement for immunoassay-based clinical toxicology testing. METHODS: A sensitive DMS-MS/MS method was developed and validated for simultaneous identification of 33 drugs and metabolites in human urine samples. After DMS optimization, the method was validated and used to screen 56 clinical urine samples. These results were compared to results obtained by immunoassay. RESULTS: The DMS-MS/MS method achieved limits of detection ranging from 5 to 100 ng/mL. Moreover, the total analysis time was 2 min per sample. For the method performance evaluation, DMS-MS/MS results were compared with previously obtained urine toxicology immunoassay results. DMS-MS/MS showed higher sensitivity and identified 20% more drugs in urine, which were confirmed by LC-MS/MS. CONCLUSION: The DMS-MS/MS as applied in our lab demonstrated the capability for rapid drug screening and provided better analytical performance than immunoassay.

Health risk assessment of aflatoxin M1 in infant formula milk in IR Iran.

In this study, two accurate, precise, selective and sensitive methods were developed for determining aflatoxin M1 (AFM1) in infant formula milk using immunoaffinity column clean-up followed by high performance liquid chromatography (HPLC) with fluorescence detection. The validated methods were used for determination of AFM1 in 29 samples of 6 different infant formula milk brands and the risk of AFM1 in infants aged zero to 6 months old was assessed using cancer risk, Margin of Exposure (MOE) and Hazard Index (HI). Only one sample (3.4%) was contaminated with AFM1. Although the results showed that MOE values for the mean and median exposure to AFM1 was <10,000 in infants, the additional cancer risk due to mean and median exposure to AFM1 in infant <6 months were 0.00010 and 0.00012 additional cases per year per 105 individuals, respectively, which indicates no health concern. In addition, HI values for the mean and median exposure to AFM1 for infants were quite below one which indicates no health concern. To the best of our knowledge, this is the first report on risk assessment of AFM1 in infant formula milk consumed by Iranian infants <6 months old, presenting a low risk for the evaluated groups.

Quantitative biochemical screening for marijuana use and concordance with tobacco use in urban adolescents.

BACKGROUND: Assessing the prevalence and level of exposure (dose) of tobacco and marijuana use is important in studies of harm from use of these substances. We used biochemical analysis of urine to quantitatively assess exposure to nicotine and delta 9-tetrahydrocannabinol (THC) in adolescents receiving medical care in a public hospital METHODS: Participants were 686 adolescents between 12 and 21 years old seen at Zuckerberg San Francisco General Hospital between 2012 and 2014. Urine samples were assayed using high sensitivity liquid chromatographic assays for cotinine, a major metabolite of nicotine, and 11-nor-9-carboxy-delta 9-THC (THC-COOH), a major metabolite of THC. A commonly used immunoassay screen for THC-COOH was also performed. RESULTS: The THC-COOH immunoassay substantially underestimated THC exposure, as measured with the high sensitivity assay. THC use was detected in 25% of participants, with higher prevalence with increasing age and in non-Hispanic blacks. Active tobacco smokers had an 80% prevalence of THC use (odds ratio for cigarette smoking predicting THC use 13.2). Urine cotinine and THC-COOH were significantly correlated (r = 0.60). CONCLUSIONS: The use of a high sensitivity chromatographic urine assay provides a much more complete picture of adolescent tobacco use compared to a commonly used immunoassay. The immunoassay provides high specificity but moderate sensitivity. We confirm high concordance of tobacco and marijuana use and the high predictive value of cigarette smoking in predicting marijuana use, and provide novel data on the quantitative correlation between level of exposure to nicotine and THC. Quantitative screening of nicotine and THC exposure may enhance our understanding of addiction and harm from single and dual product use.

Correlation of Breath and Blood Δ9-Tetrahydrocannabinol Concentrations and Release Kinetics Following Controlled Administration of Smoked Cannabis.

BACKGROUND: Cannabis use results in impaired driving and an increased risk of motor vehicle crashes. Cannabinoid concentrations in blood and other matrices can remain high long after use, prohibiting the differentiation between acute and chronic exposure. Exhaled breath has been proposed as an alternative matrix in which concentrations may more closely correspond to the window of impairment; however, efficient capture and analytically sensitive detection methods are required for measurement. METHODS: Timed blood and breath samples were collected from 20 volunteers before and after controlled administration of smoked cannabis. Cannabinoid concentrations were measured using LC-MS/MS to determine release kinetics and correlation between the 2 matrices. RESULTS: Δ9-Tetrahydrocannabinol (THC) was detected in exhaled breath for all individuals at baseline through 3 h after cannabis use. THC concentrations in breath were highest at the 15-min timepoint (median = 17.8 pg/L) and declined to <5% of this concentration in all participants 3 h after smoking. The decay curve kinetics observed for blood and breath were highly correlated within individuals and across the population. CONCLUSIONS: THC can be reliably detected throughout the presumed 3-h impairment window following controlled administration of smoked cannabis. The findings support breath THC concentrations as representing a physiological process and are correlated to blood concentrations, albeit with a shorter window of detection.

A Liquid-Chromatography High-Resolution Mass Spectrometry Method for Non-FDA Approved Benzodiazepines.

Benzodiazepines (BZDs) are widely used for treatment of anxiety and insomnia, however, this class of drugs is also commonly abused. Many different BZDs and analogs have been produced that are not FDA-approved. We tested 15 of these with the ThermoFisher CEDIA® BZD-immunoassay. With the exception of ketazolam, all compounds showed significant reactivity, highlighting the need for mass spectrometry confirmation assays. We developed a liquid-chromatography high-resolution mass spectrometry method for the detection of these 15 non-FDA approved BZDs. The limit of detection for most compounds ranged from 1 to 50 ng/mL, with mostly positive matrix effects observed in urine and negative matrix effects in serum. In a clinical research case, clonazolam and etizolam were detected in serum at 10.2 and 281 ng/mL, with an apparent elimination half-life of 3.6 and 4.8 hours, respectively. Although we did not detect non-FDA approved BZDs in 211 urine samples that were previously determined to be BZD-positive by immunoassay, abuse of these drugs is on the rise and clinical and forensic toxicology laboratories should consider developing methods to detect them.

Direct identification of microribonucleic acid miR-451 from plasma using liquid chromatography mass spectrometry.

In recent years, small endogenous RNAs have come to the forefront of both basic and translational research. For example, many studies have pointed to the potential role of microRNAs (miRNAs) as disease biomarkers. However, precise quantitative methods for the analysis of miRNAs are still lacking. In this study, we report the first mass spectrometry-based quantitation of miR-451, a circulatory microRNA. Using a highly selective sample preparation method with an average recovery of 83.6% and a novel mobile phase chemistry, we were able to reach an LOQ of 0.5 ng/mL. Because of such high sensitivity, we could detect and quantify the endogenous miR-451 from both human and rat plasma. Considering the increased precision of LC-MS compared to other methods, these results usher in a new era of miRNA biomarker discovery and validation.

Sensitive liquid chromatography/tandem mass spectrometry method for the determination of two novel highly lipophilic anticancer drug candidates in rat plasma and tissues.

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for determination of two highly lipophilic anticancer drug candidates, LG1980 and GH501, in rat plasma and tissues (liver, kidney and femur bones). LG1980 and GH501 were extracted from rat plasma and tissue homogenates using liquid-liquid extraction. The method provided a linear range of 1.0-200.0 ng/mL for GH501 in plasma and LG1980 in plasma and liver. For both analytes in other tissue homogenates the linear range was 2.0-400.0 ng/mL. The method was validated with precision within 15% relative standard deviation, accuracy within 15% relative error and a consistent recovery. This method has been successfully applied in two preclinical studies for LG1980 and GH501 to determine their concentrations in rat plasma, liver, kidney and bone over 24 h after intravenous injection of compounds.

RP-HPLC Method Development and Validation for Determination of Eptifibatide Acetate in Bulk Drug Substance and Pharmaceutical Dosage Forms.

A new, rapid, economical and isocratic reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of eptifibatide acetate, a small synthetic antiplatelet peptide, in bulk drug substance and pharmaceutical dosage forms. The developed method was validated as per of ICH guidelines. The chromatographic separation was achieved isocratically on C18 column (150 x 4.60 mm i.d., 5 µM particle size) at ambient temperature using acetonitrile (ACN), water and trifluoroacetic acid (TFA) as mobile phase at flow rate of 1 mL/min and UV detection at 275 nm. Eptifibatide acetate exhibited linearity over the concentration range of 0.15-2 mg/mL (r2=0.997) with limit of detection of 0.15 mg/mL The accuracy of the method was 96.4-103.8%. The intra-day and inter-day precision were between 0.052% and 0.598%, respectively. The present successfully validated method with excellent selectivity, linearity, sensitivity, precision and accuracy was applicable for the assay of eptifibatide acetate in bulk drug substance and pharmaceutical dosage forms.

Gas chromatography/negative chemical ionization mass spectrometry of transfluthrin in rat plasma and brain.

RATIONALE: Transfluthrin is a relatively non-toxic rapid-acting synthetic pyrethroid insecticide. It is widely used in household and hygiene products. A sensitive and accurate bioanalytical method is required for quantification of its concentration in plasma and its potential target organ, the brain for studies to assess its health effects and toxicokinetics in mammals. METHODS: The samples were prepared by liquid-liquid extraction. Gas chromatography mass spectrometry (GC/MS) analysis was performed for the determination of transfluthrin in biological samples with an overall method run time of 15 min. Transfluthrin was quantified using selected-ion monitoring (SIM) in the negative chemical ionization (NCI) mode. Chromatographic separation was achieved using a Zebron® ZB5-MS GC column operating with 1 mL/min constant flow helium. Cis-Permethrin was used as the internal standard. RESULTS: The method was validated to be precise and accurate within the linear range of 1.0-400.0 ng/mL in plasma and 4.0-400.0 ng/mL in brain homogenate, based on a 100 µL sample volume for both matrices. This method was applied to samples following administration of a 10 mg/kg oral dose to male adult rats. The plasma concentrations were observed to be 11.70 ± 5.69 ng/mL and brain concentrations 12.09 ± 3.15 ng/g when measured 2 h post-dose. CONCLUSIONS: A rapid GC/NCI-MS method was demonstrated to be sensitive, specific, precise and accurate for the quantification of transfluthrin in rat plasma and brain. The optimized method was successfully used to quantify the rat plasma and brain concentrations of transfluthrin 2 h after the oral dosing of Sprague-Dawley rats.

Simultaneous determination of cis-permethrin and trans-permethrin in rat plasma and brain tissue using gas chromatography-negative chemical ionization mass spectrometry.

A sensitive method for the simultaneous determination of cis-permethrin (cis-PERM) and trans-permethrin (trans-PERM) in small volumes (100µL) of rat plasma and brain homogenate was developed, using a liquid-liquid extraction for sample preparation and gas chromatography-negative chemical ionization mass spectrometry (GCNCI-MS) for detection. Quantitation of trace levels of the insecticide in small volumes of biological samples is essential to support toxicokinetic studies in small animals. There are currently no validated methods in the literature for determining cis-PERM and trans- PERM in volumes as low as 100µL of rat plasma or brain homogenate. The method provided a linear range of 0.2-150.0ng/mL for analytes in both matrices. The intra- and inter-batch precision (as% relative standard deviation, RSD) and accuracy (as relative error, RE) of the method were better than 20% at the limit of quantitation and better than 15% across the remaining linear range. The validated method was applied in a toxicokinetic study in adult rats with oral dosing of 10mg/kg (cis-PERM) and 100mg/kg (trans-PERM) in corn oil. cis-PERM and trans- PERM were monitored in rat plasma and brain tissue samples for 6h following dosing, and both analytes were detected in all plasma and brain samples.

Influence of Maturation on In Vivo Tissue to Plasma Partition Coefficients for Cis- and Trans-Permethrin.

Permethrin, the most widely used household insecticide in the United States, is marketed as a mixture of its cis (CIS) and trans (TRANS) isomers. The major objective of this investigation is to develop and utilize a reliable approach to determine in vivo partition coefficients (PCs) for CIS and TRANS in immature and adult Sprague-Dawley rats. Adult, postnatal day (PND) 21, and PND 15 rats were infused with environmentally relevant concentrations of CIS or TRANS via a subcutaneous osmotic pump for 48 or 72 h. The adult and PND 21 rats also received an oral loading dose. Systemic steady-state or equilibrium was attained in each age group within 72 h of the protocol. CIS and TRANS were both distributed to tissues according to their neutral lipid content, with adipose tissue exhibiting much higher tissue:plasma PCs than skeletal muscle, liver, or brain. Liver:plasma and brain:plasma PCs were consistently at or lower than unity. Tissue:plasma PCs were generally higher for CIS than for TRANS, although the isomers are of comparable lipophilicity. Significantly higher blood levels of CIS apparently saturate plasma binding, resulting in greater tissue deposition of the isomer. CIS and TRANS tissue:plasma PCs were found to be inversely related to the rats' age, although TRANS brain:plasma PCs were comparable in immature and mature animals. These data support the conclusion that age-dependent partitioning is an important determinant of the pharmacokinetics of permethrin. Such partitioning could influence the risk assessment of these insecticides in infants and children when incorporated into physiologically based pharmacokinetic models.

Hazards in chromatographic bioanalysis method development and applications.

Bioanalytical methods are employed for the quantitative determination of drugs and their metabolites in biological matrices, in all stages of the drug development process. However, because of the highly complex nature of these matrices there is a wide range of potential biological, chemical and physical hazards that can influence the quality of the data produced by these methods. The present review focuses on the evaluation of the most important and frequent errors that may be encountered during bioanalytical method development/validation and analysis of clinical or preclinical samples mainly using chromatography. Additionally, the most practical ways for avoiding and managing these hazards during routine bioanalysis are presented.

Development of a surrogate matrix for cerebral spinal fluid for liquid chromatography/mass spectrometry based analytical methods.

RATIONALE: In recent years, several liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods have been reported for the quantitative determination of drugs and metabolites in cerebral spinal fluid (CSF). Artificial CSF (aCSF) is often used as a surrogate for preparing calibration curves and quality control samples in these methods. However, aCSF does not accurately represent the composition of real CSF because it is missing all of the proteins and lipids, which may alter the electrospray ionization (ESI) response when performing LC/MS/MS analyses. In the current study we compared the mass spectral response of several compounds with a range of physiochemical properties in aCSF (essentially a mixture of salts and buffers), diluted plasma (ranging from 1:5 to 1:200) and real CSF to find the best surrogate for CSF in LC/MS/MS methods. METHODS: A number of analytes from polar to non-polar, high protein binding to low protein binding, employing different sample preparation methods, were prepared in diluted plasma, actual CSF or aCSF and tested using LC/MS/MS. The analytes included cotinine and its metabolites, quetiapine, norquetiapine, chlorpromazine, efavirenz and lamivudine. The similarity of MS responses from these compounds in aCSF and diluted plasma to CSF was assessed by comparing the slopes of the calibration curves generated from using linear regression modeling. RESULTS: For all compounds, the lowest percent difference in response ratio (0 to 17%) was observed from 1:200 diluted plasma. Our results indicated that, irrespective of the inherent physiochemical properties of the analytes or the method of sample preparation, 1:200 diluted plasma performed as the best surrogate for CSF in LC/MS/MS methods. CONCLUSIONS: The percent difference in response ratio has been established to demonstrate how different compounds behave between CSF, aCSF and dilute plasma. Although among the compounds tested some of them showed a very similar MS response in actual and aCSF, there were analytes that demonstrated significant differences in ESI-MS signal when sprayed from these two matrices. However, even in such cases, 1:200 diluted plasma generated results with no significant difference from CSF. Therefore, we recommend that in order to develop robust and dependable bioanalytical LC/MS methods from CSF samples, it is more appropriate to prepare calibration curves and quality control samples in diluted plasma instead of aCSF. Copyright © 2016 John Wiley & Sons, Ltd.

Formulation and Taste Masking of Ranitidine Orally Disintegrating Tablet.

Orally Disintegrating Tablets (ODT) have the advantages of both solid dosage form specially the stability and ease of handling and liquid dosage forms including ease of swallowing and pre-gastric absorption. We focused on taste masking and formulation of ranitidine ODT which disintegrates rapidly in the mouth within 60 sec using super-disintegrants, special polymers, water soluble and even insoluble excipients, sweeteners and essence. Various formulations were designed and made in four series. The amount of ranitidine in each formulation was 150 mg, and the final weight of tablets was around 500 mg. Prepared formulations were evaluated in terms of several physicochemical tests including powder/granule flowability, appearance, thickness, uniformity of weight, hardness, friability and disintegration time. Several taste masking techniques were investigated in each series of formulation, in order to cover the bitter taste of wranitidine. These included the addition of sweetener, granulation, solid dispersion with soluble and insoluble agents and complex formation with cellulose derivatives. The best formulation(s) in each group was/were chosen for taste evaluations with the help of 10 volunteers. Finally, formulation F14 was selected as the ultimate formulation, based on its better taste and shorter disintegration time (around 5 seconds). Formulation F14 contained Na CMC, avicel, Na starch glycolate, xylitol, saccharin, Na benzoate and menthol. The chosen formulation successfully passed the complementary evaluations such as assay of active ingredient and dissolution time. Na CMC was found to be acceptable in terms of decreasing disintegration time and enhanced taste masking potential and can be used in further ODT formulations.

Development and Validation of a Reversed-phase HPLC Method for Assay of the Decapeptide Cetrorelix Acetate in Bulk and Pharmaceutical Dosage Forms.

A gradient reversed-phase high performance liquid chromatography (HPLC) method was developed for the assay of cetrorelix acetate, a synthetic decapeptide with gonadotropin-releasing hormone (GnRH) antagonistic activity used in infertility treatment. The HPLC method, which is used to determine cetrorelix in bulk and pharmaceutical dosage forms, was validated per ICH guidelines. The chromatographic separation was achieved on a C18 reversed-phase column using acetonitrile, water and trifluoroacetic acid (TFA) as mobile phase and wavelength was set at 275 nm. The calibration curve was linear (r2 = 0.999) over cetrorelix concentrations ranging from 62.50 to 12.50 µg/mL (n = 6). The limits of detection (LOD) and quantification (LOQ) were calculated from the peak-to-noise ratio as 15.6 and 62.5 µg/mL, respectively. The method had an accuracy of > 97% and intra- and inter-day RSD of < 0.3% and < 1.6%, respectively and was validated with excellent specificity, sensitivity, and stability. The validated method was successfully applied for determination of cetrorelix in bulk and pharmaceutical dosage forms.