Ion Trap LC/MS reveals the generation of reactive intermediates in acalabrutinib metabolism: phase I metabolic profiling and bioactivation pathways elucidation.

Acalabrutinib (CALQUENCE; ACB) is a Bruton tyrosine kinase inhibitor (BTKI) used to treat mantle cell lymphoma, small lymphocytic lymphoma (SLL), and chronic lymphocytic leukemia (CLL). On 21 November 2019, ACB was approved by the U.S. FDA for the use as a single therapy for the treatment of CLL/SLL. In silico studies were first done to propose vulnerable sites of metabolism and reactivity pathways by StarDrop software and Xenosite online software; respectively. ACB metabolites and stable adducts were characterized in vitro from rat liver microsomes (RLMs) using Ion Trap LC/MS. Generation of reactive intermediates (RIs) in the in vitro metabolism of ACB was investigated using glutathione, potassium cyanide, and methoxylamine as trapping nucleophiles for the RIs including iminopyridinone, iminium, and aldehyde, respectively, to form stable adducts that can be identified and characterized by Ion Trap LC/MS. Five phase I metabolites, seven 6-iminopyridin-3(6H)-one and five aldehyde RIs of ACB were identified. Based on literature reviews, the generation of RIs of ACB, and the subsequent drug-induced organ toxicity (DIOT) reactions may provide an explanation of ACB ADRs. Additional drug discovery investigations can be performed to facilitate the creation of novel medications with improved safety characteristics.

Duvelisib: A comprehensive profile.

Duvelisib (DUV) is chemically named as (S)-3-(1-((9H-Purin-6-yl)amino)ethyl)-8-chloro-2-phenylisoquinolin-1(2H)-one. It is a novel drug with a small molecular weight and characterized by dual phosphoinositide-3-kinase (PI3K)- and PI3K-inhibitory activity. The Food and Drug Administration (FDA) recently approved DUV for the management of small lymphocytic lymphoma (SLL) and relapsed or refractory chronic lymphocytic leukemia (CLL) in adult patients. DUV is marketed under the brand name of Copiktra® (Verastem, Inc., Needham, MA, USA). This chapter provides a critical extensive review of the literature, the description of DUV in terms of its names, formulae, elemental composition, appearance, and use in the treatment of CLL, SLL, and follicular lymphoma. The chapter also describes the methods for preparation of DUV, its physical-chemical properties, analytical methods for its determination, pharmacological properties, and dosing information.

An Assessment of aluminum contamination in neonatal parenteral nutrition solutions based on measured versus labeled content.

Aluminum can potentially cause toxicity in pediatrics and neonates receiving parenteral nutrition. Some PN solutions and ingredients in Saudi Arabia do not comply with US FDA regulations regarding aluminum exposure. This study aims to determine the aluminum concentration in samples of PN solutions and ingredients used to feed infants in Saudi Arabia. The aluminum in the samples was determined using inductively coupled plasma mass spectrometry. The concentration of metal contaminants in each sample was determined in triplicate. The aluminum content of 38 samples was investigated, 15 of which originated from components included in the prepared PN solutions. Among the 15 samples, the least measurable aluminum content was detected in potassium chloride solutions (0.81 mcg/L). In contrast, the greatest amount of aluminum was detected in potassium phosphate and calcium gluconate (141,64 mcg/L and 462.7 mcg/L), respectively. The results showed that the final PN solution (PNS) product contained more aluminum levels than the content ingredients; in addition, the study found a statistically significant relationship among 18 pediatric patients at KFMC who had intestinal failure and needed long-term parenteral nutrition. Specifically, their high aluminum levels, exceeding the normal range of 0.6 ng/ml, indicate that the current use of PN solutions will likely cause toxicity due to aluminum contamination in additives. Hence, reducing aluminum in PN solutions is imperative to ensure patient safety.

Reactive intermediates formation and bioactivation pathways of spebrutinib revealed by LC-MS/MS: In vitro and in silico metabolic study.

Spebrutinib is a new Bruton tyrosine kinase inhibitor developed by Avila Therapeutics and Celgene. Spebrutinib (SPB) is currently in phase Ib clinical trials for the treatment of lymphoma in the United States. Preliminary in-silico studies were first performed to predict susceptible sites of metabolism, reactivity pathways and structural alerts for toxicities by StarDrop WhichP450™ module, Xenosite web predictor tool and DEREK software; respectively. SPB metabolites and adducts were characterized in vitro from rat liver microsomes (RLM) using LC-MS/MS. Formation of reactive intermediates was investigated using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles for the unstable and reactive iminium, iminoquinone and aldehyde intermediates, respectively, with the aim to produce stable adducts that can be detected and characterized using mass spectrometry. Fourteen phase I metabolites, four cyanide adducts, six GSH adducts and three methoxylamine adducts of SPB were identified and characterized. The proposed metabolic pathways involved in generation of phase I metabolites of SPB are oxidation, hydroxylation, o-dealkylation, epoxidation, defluorination and reduction. Several in vitro reactive intermediates were identified and characterized, the formation of which can aid in explaining the adverse drug reactions of SPB. Several iminium, 2-iminopyrimidin-5(2H)-one and aldehyde intermediates of SPB were revealed. Acrylamide is identified as a structural alert for toxicity by DEREK report and was found to be involved in the formation of several glycidamide and aldehyde reactive intermediates.

Investigation of Fenebrutinib Metabolism and Bioactivation Using MS3 Methodology in Ion Trap LC/MS.

Fenebrutinib is an orally available Bruton tyrosine kinase inhibitor. It is currently in multiple phase III clinical trials for the management of B-cell tumors and autoimmune disorders. Elementary in-silico studies were first performed to predict susceptible sites of metabolism and structural alerts for toxicities by StarDrop WhichP450™ module and DEREK software; respectively. Fenebrutinib metabolites and adducts were characterized in-vitro in rat liver microsomes (RLM) using MS3 method in Ion Trap LC-MS/MS. Formation of reactive and unstable intermediates was explored using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles to capture the transient and unstable iminium, 6-iminopyridin-3(6H)-one and aldehyde intermediates, respectively, to generate a stable adducts that can be investigated and analyzed using mass spectrometry. Ten phase I metabolites, four cyanide adducts, five GSH adducts and six methoxylamine adducts of fenebrutinib were identified. The proposed metabolic reactions involved in formation of these metabolites are hydroxylation, oxidation of primary alcohol to aldehyde, n-oxidation, and n-dealkylation. The mechanism of reactive intermediate formation of fenebrutinib can provide a justification of the cause of its adverse effects. Formation of iminium, iminoquinone and aldehyde intermediates of fenebrutinib was characterized. N-dealkylation followed by hydroxylation of the piperazine ring is proposed to cause the bioactivation to iminium intermediates captured by cyanide. Oxidation of the hydroxymethyl group on the pyridine moiety is proposed to cause the generation of reactive aldehyde intermediates captures by methoxylamine. N-dealkylation and hydroxylation of the pyridine ring is proposed to cause formation of iminoquinone reactive intermediates captured by glutathione. FBB and several phase I metabolites are bioactivated to fifteen reactive intermediates which might be the cause of adverse effects. In the future, drug discovery experiments utilizing this information could be performed, permitting the synthesis of new drugs with better safety profile. Overall, in silico software and in vitro metabolic incubation experiments were able to characterize the FBB metabolites and reactive intermediates using the multistep fragmentation capability of ion trap mass spectrometry.

Aspartames Alter Pharmacokinetics Parameters of Erlotinib and Gefitinib and Elevate Liver Enzymes in Wistar Rats.

Background: Erlotinib (ERL) and gefitinib (GEF) are extensively metabolized by CYP450 enzymes. Aspartame (ASP), an artificial sweetener, induces CYP2E1 and CYP3A2 enzymes in the brain and could increase liver enzymes. In this work, the influence of ASP on the pharmacokinetics (PK) of ERL and GEF in Wistar rats was evaluated. Methods: The PKs of ERL and GEF were evaluated after receiving 175 mg/kg or 1000 mg/kg of ASP for four weeks using UPLC-MS/MS. Levels of liver enzymes after four weeks of ASP consumption were also evaluated. Results: ASP 175 mg/kg was able to significantly alter levels of Cmax (36% increase for ERL, 38% decrease for GEF), AUC0-72 (205% increase for ERL, 41% increase for GEF), and AUC0-∞ (112% increase for ERL, 14% increase for GEF). Moreover, ASP 175 mg/kg decreased the apparent oral clearance ERL and GEF by 58% and 13%, respectively. ASP 1000 mg/kg increased Cmax of ERL by 159% and decreased GEF's Cmax by and 73%. Both AUC0-72 and AUC0-∞ were increased by ASP 1000 for ERL and decreased for GEF. CL/F decreased by 64% for ERL and increased by 38.8% for GEF. Moreover, data indicated that ASP significantly increased levels of liver enzymes within two weeks of administration. Conclusions: Although ASP 175 and 1000 mg/kg alter ERL and GEF PKs parameters, ASP 1000 mg/kg has the highest impact on most parameters. ASP 1000 mg/kg also can significantly increase activities of liver enzymes indicating the possibility of inducing liver injury. Therefore, it might be of clinical importance to avoid the administration of aspartame containing products while on ERL or GEF therapy.

Gut Microbiota Dynamics in Relation to Long-COVID-19 Syndrome: Role of Probiotics to Combat Psychiatric Complications.

Increasing numbers of patients who recover from COVID-19 report lasting symptoms, such as fatigue, muscle weakness, dementia, and insomnia, known collectively as post-acute COVID syndrome or long COVID. These lasting symptoms have been examined in different studies and found to influence multiple organs, sometimes resulting in life-threating conditions. In this review, these symptoms are discussed in connection to the COVID-19 and long-COVID-19 immune changes, highlighting oral and psychiatric health, as this work focuses on the gut microbiota's link to long-COVID-19 manifestations in the liver, heart, kidney, brain, and spleen. A model of this is presented to show the biological and clinical implications of gut microbiota in SARS-CoV-2 infection and how they could possibly affect the therapeutic aspects of the disease. Probiotics can support the body's systems in fighting viral infections. This review focuses on current knowledge about the use of probiotics as adjuvant therapies for COVID-19 patients that might help to prevent long-COVID-19 complications.

Profiling of in vivo, in vitro and reactive zorifertinib metabolites using liquid chromatography ion trap mass spectrometry.

Zorifertinib (AZD-3759; ZFB) is a potent, novel, oral, small molecule used for the treatment of non-small cell lung cancer (NSCLC). ZFB is Epidermal Growth Factor Receptor (EGFR) inhibitor that is characterized by good permeability of the blood-brain barrier for (NSCLC) patients with EGFR mutations. The present research reports the profiling of in vitro, in vivo and reactive metabolites of ZFB. Prediction of vulnerable metabolic sites and reactivity pathways (cyanide and GSH) of ZFB were performed by WhichP450™ module (StarDrop software package) and XenoSite reactivity model (XenoSite Web Predictor-Home), respectively. ZFB in vitro metabolites were done by incubation with isolated perfused rat liver hepatocytes and rat liver microsomes (RLMs). Extraction of ZFB and its related metabolites from the incubation matrix was done by protein precipitation. In vivo metabolism was performed by giving ZFB (10 mg kg-1) through oral gavage to Sprague Dawley rats that were housed in metabolic cages. Urine was collected at specific time intervals (0, 6, 12, 18, 24, 48, 72, 96 and 120 h) from ZFB dosing. The collected urine samples were filtered then stored at -70 °C. N-Methyl piperazine ring of ZFB undergoes phase I metabolism forming iminium intermediates that were stabilized using potassium cyanide as a trapping agent. Incubation of ZFB with RLMs were performed in the presence of 1.0 mM KCN and 1.0 mM glutathione to check reactive intermediates as it is may be responsible for toxicities associated with ZFB usage. For in vitro metabolites there were six in vitro phase I metabolites, three in vitro phase II metabolites, seven reactive intermediates (four GSH conjugates and three cyano adducts) of ZFB were detected by LC-IT-MS. For in vivo metabolites there were six in vivo phase I and three in vivo phase II metabolites of ZFB were detected by LC-IT-MS. In vitro and in vivo phase I metabolic pathways were N-demethylation, O-demethylation, hydroxylation, reduction, defluorination and dechlorination. In vivo phase II metabolic reaction was direct sulphate and glucuronic acid conjugation with ZFB.

A New Validated HPLC-MS/MS Method for Quantification and Pharmacokinetic Evaluation of Dovitinib, a Multi-Kinase Inhibitor, in Mouse Plasma.

BACKGROUND: Dovitinib (TKI 258) is a small-molecule multi-kinase inhibitor for the treatment of different types of cancer. There is currently no validated method for its quantitative determination; therefore, we aimed to develop a reliable method to assay dovitinib. METHOD AND RESULTS: An electrospray ionization tandem mass spectrometry (ESI-MS/MS) method was used to separate dovitinib using an analytical C18 column (50 × 2.1 mm, 1.8 µm) at 25°C. Bosutinib was used as the internal standard (IS). Dovitinib was extracted from mouse plasma using a precipitation procedure. The mobile phase consisted of 10 mM ammonium formate: acetonitrile (68:32, v/v, pH 4.3) run at a rate of 0.3 mL min-1. MS detection was performed in the positive ion mode. Multiple reaction monitoring transitions were 393→337 and 393→309 for dovitinib, and 530→141 and 530→113 for bosutinib. The investigated method was validated as a bio-analytical method based on FDA guidelines. The linearity of the developed method was over the range of 5-500 ng mL,-1 coefficient of determination (r2= 0.9998). The average intra-day recovery and relative standard deviation (RSD) of the quality control (QC) sample were 97.24% and 1.32%, whereas the overall inter-day accuracy and precision were 97.99% and 0.54%, respectively. Dovitinib was stable during sample storage and handling conditions. Furthermore, the dilution integrity of the method was demonstrated by good recovery (97-99%) and RSD values (0.5-0.7%). CONCLUSION: This method was selectively sensitive and exhibited no matrix effect, with an acceptable accuracy and precision according to the FDA guidelines. The developed method could be efficiently used for pharmacokinetic studies of dovitinib.

New potentiometric sensors for methylphenidate detection based on host-guest interaction.

The study aims to develop simple, sensitive, and selective methods for detecting methylphenidate in its bulk, dosage form and human urine. Sensing materials include ß-cyclodextrin (ß-CD), γ-cyclodextrin (γ-CD), and 4-tertbutylcalix[8]arene as ionophores or electroactive materials have been used for construction of sensors 1, 2, and 3, respectively; Potassium tetrakis (4-chlorophenyl)borate (KTpClPB) as an ion additive was used and dioctyl phthalate as a plasticizer. The sensors displayed a fast, stable response over a wide concentration range of methylphenidate (8 × 10-6 M to 1 × 10-3 M) with 10-6 M detection limit over the pH range of 4-8. The developed sensors displayed a Near-Nernstian cationic response for methylphenidate at 59.5, 51.37, and 56.5 mV/decade for sensors ß-CD, γ-CD, or 4-tertbutylcalix[8]arene respectively. Validation of the proposed sensors is supported by high accuracy, precision, stability, fast response, and long lifetimes, as well as selectivity for methylphenidate in the presence of different species. Sensitive and practical sensors for the determination of methylphenidate in bulk, in pharmaceutical forms and urine were developed and validated for routine laboratory use. The results were comparable to those obtained by HPLC method.

Comparative study of ß-cyclodextrin, γ-cyclodextrin and 4-tert-butylcalix[8]arene ionophores as electroactive materials for the construction of new sensors for trazodone based on host-guest recognition.

BACKGROUND: Trazodone (TRZ) is a second-generation non-tricyclic antidepressant derived from a triazolopyridine derivative, which is mainly used to treat emotional disorders and conditions related to depressive disorders. PURPOSE: This study investigated the design, development and characteristics of polyvinyl chloride (PVC) membrane sensors for trazodone HCl (TRZ). METHODS: The developed sensing membranes were constructed using ß-cyclodextrin (ß-CD; sensor 1), γ-cyclodextrin (γ-CD; sensor 2) or 4-tert-butylcalix[8]arene (t-BC8; sensor 3) ionophores as sensing materials in addition to ionic sites and dioctyl phthalate in the PVC matrix. RESULTS: Sensors 1, 2 and 3 displayed fast, stable and near-Nernstian response over a relatively wide trazodone concentration range (7.0×10-6-1×10-3, 5.0×10-5-1×10-3and 8.0×10-6-1.0×10-3 M, respectively), with detection limits of 2.2×10-6, 1.5×10-5 and 2.42×10-6 M, respectively in the pH range of 3.0-6.0. The sensors demonstrated good selectivity for TRZ in the presence of different ionic compounds. The accuracy and precision of the proposed sensors were assessed by the determination of 40.7 µg/ml of TRZ, which showed average recoveries of 99.6%, 99.1% and 98.5% with mean relative standard deviations of 2.4%, 2.5% and 2.6% for sensor 1, 2 and 3 respectively. Molecular modeling was used to calculate the host-guest binding energy. The lowest free binding energy was -6.243, -5.752 and -5.7105 kcal/mol for 1:1 stoichiometry host-guest complexes of trazodone and ß-CD, γ-CD and t-BC8, respectively, which was in-line with a Nernstian response. CONCLUSION: The investigated methods can be applied for the determination of TRZ in pharmaceutical preparations. The results of investigated dosage-form of TRZ show good agreement with those using the US Pharmacopeia method.

Physical and chemical screening of honey samples available in the Saudi market: An important aspect in the authentication process and quality assessment.

Honey is becoming accepted as a reputable and effective therapeutic agent by practitioners of conventional medicine and by the general public. It has many biological activities and has been effectively used in the treatment of many diseases, e.g. gastrointestinal diseases, skin diseases, cancer, heart diseases, and neurological degeneration. Honey is an excellent source of energy containing mainly carbohydrates and water, as well as, small amounts of organic acids, vitamins, minerals, flavonoids, and enzymes. As a natural product with a relatively high price, honey has been for a long time a target for adulteration. The authenticity of honey is of great importance from commercial and health aspects. The study of the physical and chemical properties of honey has been increasingly applied as a certification process for the purpose of qualification of honey samples. The current work focusses on studying the authenticity of various types of honey sold in Riyadh market (24 samples). For this purpose, physical properties (pH, hydroxylmethylfurfural HMF, and pollen test) were measured. Besides, sugar composition was evaluated using Fehling test and an HPLC method. Elemental analysis was carried out using inductively coupled plasma (ICP). In addition, the presence of drug additives was assessed by means of GC-MS. The obtained results were compared with the Saudi Arabian standards, Codex Alimentarius Commission (2001), and harmonized methods of the international honey commission.