Mapping Advantages and Challenges in Analytical Development for Fixed Dose Combination Products, a Review.

Formulations containing more than one active ingredient are increasingly gaining popularity due to advantages with regard to patient convenience as well as reduced cost of production, packaging, and transportation. Such fixed-dose combinations (FDCs) demand for enhanced analytical methodologies and tools to efficiently achieve quality control of these complex products as compared to the conventional products containing only one active constituent. Highly efficient analytical methods can measure multiple constituents at once, improving their quality control. This review article discusses the challenges in the development of such methods due to the similarities or differences in the chemical identity of the participating drug molecules in an FDC. The latest developments in multiple analyte determination using various analytical techniques (HPLC, LC-MS, NMR, IR, powder XRD and DSC) are discussed, with a focus on special considerations in each case. The article discusses challenges with sample preparation of complex FDC products, and the use of Chemometrics and Quality by Design to develop efficient analytical methods. Lastly, an equation-based approach is proposed and demonstrated to arrive at a parameter referred to as "percentage efficiency gain" that would be useful in directly accessing the relevance and commercial benefits of a simultaneous method vis-a-vis separate methods for individual components.

The molecular basis of Abelson kinase regulation by its αI-helix.

Abelson tyrosine kinase (Abl) is regulated by the arrangement of its regulatory core, consisting sequentially of the SH3, SH2, and kinase (KD) domains, where an assembled or disassembled core corresponds to low or high kinase activity, respectively. It was recently established that binding of type II ATP site inhibitors, such as imatinib, generates a force from the KD N-lobe onto the SH3 domain and in consequence disassembles the core. Here, we demonstrate that the C-terminal αI-helix exerts an additional force toward the SH2 domain, which correlates both with kinase activity and type II inhibitor-induced disassembly. The αI-helix mutation E528K, which is responsible for the ABL1 malformation syndrome, strongly activates Abl by breaking a salt bridge with the KD C-lobe and thereby increasing the force onto the SH2 domain. In contrast, the allosteric inhibitor asciminib strongly reduces Abl's activity by fixating the αI-helix and reducing the force onto the SH2 domain. These observations are explained by a simple mechanical model of Abl activation involving forces from the KD N-lobe and the αI-helix onto the KD/SH2SH3 interface.

Innovative Nanoparticulate Strategies in Colon Cancer Treatment: A Paradigm Shift.

As a major public health issue, colorectal cancer causes 9.4% of total cancer-related deaths and comprises 10% of new cancer diagnoses worldwide. In the year 2023, an estimated 153,020 people are expected to receive an identification of colorectal cancer (CRC), resulting in roughly 52,550 fatalities anticipated as a result of this illness. Among those impacted, approximately 19,550 cases and 3750 deaths are projected to occur in individuals under the age of 50. Irinotecan (IRN) is a compound derived from the chemical structure of camptothecin, a compound known for its action in inhibiting DNA topoisomerase I. It is employed in the treatment strategy for CRC therapies. Comprehensive in vivo and in vitro studies have robustly substantiated the anticancer efficacy of these compounds against colon cancer cell lines. Blending irinotecan in conjunction with other therapeutic cancer agents such as oxaliplatin, imiquimod, and 5 fluorouracil enhanced cytotoxicity and improved chemotherapeutic efficacy. Nevertheless, it is linked to certain serious complications and side effects. Utilizing nano-formulated prodrugs within "all-in-one" carrier-free self-assemblies presents an effective method to modify the pharmacokinetics and safety portfolio of cytotoxic chemotherapeutics. This review focuses on elucidating the mechanism of action, exploring synergistic effects, and innovating novel delivery approaches to enhance the therapeutic efficacy of irinotecan.

SCFAs Supplementation Rescues Anxiety- and Depression-like Phenotypes Generated by Fecal Engraftment of Treatment-Resistant Depression Rats.

Alteration of gut microbiota and microbial metabolites such as short-chain fatty acids (SCFAs) coexisted with stress-generated brain disorders, including depression. Herein, we investigated the effect of SCFAs in a treatment-resistant depression (TRD) model of rat. Rats were exposed to chronic-unpredictable mild stress (CUMS) and repeated adrenocorticotropic hormone (ACTH) injections to generate a TRD-like phenotype. The cecal contents of these animals were engrafted into healthy-recipient rats and allowed to colonize for 4 weeks (TRD-FMT group). Blood, brain, colon, fecal, and cecal samples were collected for molecular studies. Rats exposed to CUMS + ACTH showed TRD-like phenotypes in sucrose-preference (SPT), forced swim (FST), and elevated plus maze (EPM) tests. The TRD-FMT group also exhibited anxiety- and depression-like behaviors. Administration of SCFAs (acetate, propionate, and butyrate at 67.5, 25, and 40 mM, respectively) for 7 days exerted robust antidepressant and antianxiety effects by restoring the levels of SCFAs in plasma and fecal samples, and proinflammatory cytokines (TNF-α and IL-6), serotonin, GABA, norepinephrine, and dopamine in the hippocampus and/or frontal cortex of TRD and TRD-FMT animals. SCFAs treatment elevated the expression of free-fatty acid receptors 2/3, BDNF, doublecortin, and zonula-occludens, and reduced the elevated plasma levels of kynurenine and quinolinic acid and increased mucus-producing goblet cells in TRD and TRD-FMT animals. In 16S sequencing results, decreased microbial diversity in TRD rats corresponds with differences in the genus of Faecalibacterium, Anaerostipes, Allobaculum, Blautia, Peptococcus, Rombustia, Ruminococcaceae_UCG-014, Ruminococcaceae_UCG-002, Solobacterium, Subdolibacterium, and Eubacterium ventriosum. SCFAs may impart beneficial effects via modulation of tryptophan metabolism, inflammation, neurotransmitters, and microbiota-gut-brain axis in TRD rats.

Study on the hydrolytic degradation behaviour of bictegravir by LC-PDA-Q/TOF-MS/MS NMR and in silico toxicity assessment.

Forced degradation studies provide rapid access to degradation products (DPs), where structural characterization and assessment of their potential toxicity are vital for pharmaceutical safety and regulatory compliance. As per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q1A R(2) guidelines, a forced degradation study of Bictegravir (BIC), a USFDA-approved drug for HIV wild type, in hydrolytic conditions (acid, base, and neutral) revealed the formation of six DPs in RP-HPLC (Reverse Phase- High-Performance Liquid Chromatography) gradient elution program using a C18 (4.6 × 250 mm, 5 µm) column. DP-1, 2, and 3 were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS), whereas DP-4, 5, and 6 posed difficulties in characterization due to their isomeric nature. Using characteristic NOEs (Nuclear Overhauser Effect) from 2D ROESY NMR (Nuclear Magnetic Resonance) studies, we have elucidated diastereomeric DP-4/5 and isomeric DP-6/BIC configurational structures. Furthermore, in silico toxicity studies for the six degradation products were predicted for toxicity endpoints by employing DEREK, SARAH, and Pro Tox-II application tools. The DP-1 (methanamine) and DP-3 (carboxylic acid) resulting from acid-catalyzed hydrolysis, were predicted to have potential carcinogenic and mutagenic properties. These findings contribute significantly to our understanding of BIC's stability and safety profile in pharmaceutical development and underscore the rigorous characterization of stereoisomers by NMR that were further utilized for toxicity prediction.

Oxidative Folding Catalysts of Conotoxins Derived from the Venom Duct Transcriptome of C. frigidus and C. amadis.

Two novel redox conopeptides with proline residues outside and within the active site disulfide loop were derived from the venom duct transcriptome of the marine cone snails Conus frigidus and Conus amadis. Mature peptides with possible post-translational modification of 4-trans-hydroxylation of proline, namely, Fr874, Fr890[P1O], Fr890[P2O], Fr906, Am1038, and Am1054, have been chemically synthesized and characterized using mass spectrometry. The estimated reduction potential of cysteine disulfides of synthetic peptides varied from -298 to -328 mV, similar to the active site cysteine disulfide motifs of the redox family of proteins. Fr906/Am1054 exhibited pronounced catalytic activity and assisted in improving the yields of natively folded globular form α-conotoxin ImI. Three-dimensional (3D) structures of the redox conopeptides were optimized using computational methods and verified by 2D-ROESY NMR spectroscopy: C. frigidus peptides adopt an N-terminal helical fold and C. amadis peptides adopt distinct structures based on the Phe4-Pro/Hyp5 peptide bond configuration. The shift in the cis-trans configuration of the Phe4-Pro/Hyp5 peptide bond of Am1038/Am1054 was observed between reduced free thiol and oxidized disulfide forms of the optimized peptides. The report confirms the position-specific effect of hydroxyproline on the oxidative folding of conotoxins and sequence diversity of redox conopeptides in the venom duct of cone snails.

Exploration of 3-aryl pyrazole-tethered sulfamoyl carboxamides as carbonic anhydrase inhibitors.

Herein, we report the design and synthesis of two series of pyrazole-tethered sulfamoyl phenyl acetamides and pyrazole-tethered sulfamoyl phenyl benzamides. The synthesized compounds were investigated for inhibiting two human carbonic anhydrases, human carbonic anhydrases (hCA) I and II, and those of the bacterial pathogen Mycobacterium tuberculosis, mtCA 1-3. The results indicate that, among the synthesized compounds, pyrazoles with 4-aminobenzene sulfonamide were more selective toward hCA I and II over mtCAs, and compounds with 3-aminobenzene sulfonamide were selective toward mtCA 1-3 over hCA I, II. Compound 6g showed significant and selective inhibition toward hCA I and II, with Ki values of 0.0366 and 0.0310 µM, respectively. Compound 5g exhibited the best inhibition toward mtCA 2, with a Ki value of 0.0617 µM. Among the benzamides, compound 9b exhibited significant activity toward mtCA 2, with a Ki value of 0.0696 µM. Selectivity of these compounds was further supported by docking studies. When tested for antitubercular activity, many compounds showed moderate to good inhibition against the Mtb H37Rv strain, with minimum inhibitory concentration (MIC) values in the range of 4-128 µg/mL.

Multicomponent Domino Reaction for Concise Access to 2-Amino-Substituted 1,3,4 Oxadiazoles via Smiles Rearrangement.

A multicomponent domino reaction has been developed for the preparation of N-substituted 2-amino-1,3,4-oxadiazoles directly from various hydrazides (32 examples). The formation of 2-amino-1,3,4-oxadiazole involves the Smiles rearrangement of thiazolidinone, which results in the formation of carbodiimide intermediate that concomitantly undergoes amide-imidic acid tautomerism followed by cyclization. The protocol developed has wide applicability and provides the desired 2-amino-1,3,4-oxadiazole in excellent yields. The GSD studies of NMR spectra of aliphatic substrates (4di, 4dh) revealed the formation of three products, whereas, in the case of allylic and benzylic substrates, thiazolidinones were obtained as the sole products. Furthermore, to elucidate the plausible mechanism, DFT studies were performed affirming carbodiimide as the crucial intermediate for the interconversion of thiazolidinone to oxadiazole.

Nirmatrelvir and ritonavir combination: an antiviral therapy for COVID-19.

INTRODUCTION: The emergence of the Omicron SARS-CoV-2 variant of concern in late November 2021 presaged yet another stage of the COVID-19 pandemic. Paxlovid, a co-packaged dosage form of two antiviral drugs (nirmatrelvir and ritonavir) developed by Pfizer, received its first FDA Emergency Use Authorization (EUA) and conditional marketing by European Medical Agency in patients at high risk of developing severe COVID-19. AREAS COVERED: We reviewed the timeline of the drug nirmatrelvir from its discovery to authorization by FDA. After 1 year of its authorization, numerous studies and reports on paxlovid's use and post-use consequences are available. This review summarizes the complete journey of paxlovid from its development, preclinical studies, clinical trials, regulatory approvals, ongoing clinical trials, and safety measures, followed by discussions on recent updates on drug-drug interactions, adverse effects, and relapse of COVID-19. EXPERT OPINION: Paxlovid, a new oral antiviral therapy for COVID-19, has shown promising results in clinical trials and has the potential to be effective against the pandemic, particularly for individuals at high risk of severe illness. Comorbidity usage and pharmacovigilance will play a significant stake in the future of paxlovid development. Second-generation Mpro inhibitors play an important role in the upcoming problems associated with COVID-19.

Synthesis and pharmacological evaluation of 1,3-diaryl substituted pyrazole based (thio)urea derivatives as potent antimicrobial agents against multi-drug resistant Staphylococcus aureus and Mycobacterium tuberculosis.

The urgent development of newer alternatives has been deemed a panacea for tackling emerging antimicrobial resistance effectively. Herein, we report the design, synthesis, and biological evaluation of 1,3-diaryl substituted pyrazole-based urea and thiourea derivatives as antimicrobial agents. Preliminary screening results revealed that compound 7a (3,4-dichlorophenyl derivative) exhibited potent activity against S. aureus (MIC = 0.25 µg mL-1) and compound 7j (2,4-difluorophenyl derivative) against Mycobacterium tuberculosis (MIC = 1 µg mL-1). Compounds 7a and 7j were non-toxic to Vero cells with a favorable selectivity index of 40 and 200, respectively, and demonstrated good microsomal stability. Compound 7a exhibited equipotent activity (MIC = 0.25 µg mL-1) against various multidrug-resistant strains of S. aureus, which include various strains of MRSA and VRSA, and elicited bacteriostatic properties. In an enzymatic assay, 7a effectively inhibited DNA gyrase supercoiling activity at a concentration of 8 times MIC. Further, molecular modeling studies suggested that compound 7a binds at the active site of DNA gyrase with good affinity.

Effect of chronopharmacology and food on in vivo pharmacokinetic profile of mavacamten.

Aim: This study investigated the impact of food intake and circadian rhythms on the pharmacokinetics of mavacamten. Materials & methods: A sensitive bioanalytical method for quantifying mavacamten in rat plasma was developed and validated. This method was applied to assess the effect of chronopharmacology and food intake on the pharmacokinetics of mavacamten in rats. Results: A circadian variation at two doses resulted in significant changes in the volume of distribution, clearance and time of maximum plasma concentration of mavacamten (p < 0.05). In addition, food intake had an insignificant impact on the pharmacokinetic parameters except for the time of maximum plasma concentration (p < 0.05). Conclusion: These pharmacokinetic changes and human chronotype findings will help optimize dosing time.

Exo-trig selenocyclization of secondary allylic carboxamides using Woollins' reagent: en route to 2,5-disubstituted selenazolines.

We report microwave-assisted selenation and exo-trig cyclization of secondary allylic carboxamides using Woollins' reagent, a serendipitous finding observed during an attempt to synthesize N-allylbenzoselenoamide compounds. This resulted in the first reported synthesis of 2-aryl-5-methyl selenazolines. Twenty-one diversified selenazolines and three late-stage-functionalized drug molecules were synthesized in 42-93% and 25-52% yield, respectively, and these were evaluated further for their anti-proliferative activity.

LC-MS/MS: A sensitive and selective analytical technique to detect COVID-19 protein biomarkers in the early disease stage.

INTRODUCTION: The COVID-19 outbreak has put enormous pressure on the scientific community to detect infection rapidly, identify the status of disease severity, and provide an immediate vaccine/drug for the treatment. Relying on immunoassay and a real-time reverse transcription polymerase chain reaction (rRT-PCR) led to many false-negative and false-positive reports. Therefore, detecting biomarkers is an alternative and reliable approach for determining the infection, its severity, and disease progression. Recent advances in liquid chromatography and mass spectrometry (LC-MS/MS) enable the protein biomarkers even at low concentrations, thus facilitating clinicians to monitor the treatment in hospitals. AREAS COVERED: This review highlights the role of LC-MS/MS in identifying protein biomarkers and discusses the clinically significant protein biomarkers such as Serum amyloid A, Interleukin-6, C-Reactive Protein, Lactate dehydrogenase, D-dimer, cardiac troponin, ferritin, Alanine transaminase, Aspartate transaminase, gelsolin and galectin-3-binding protein in COVID-19, and their analysis by LC-MS/MS in the early stage. EXPERT OPINION: Clinical doctors monitor significant biomarkers to understand, stratify, and treat patients according to disease severity. Knowledge of clinically significant COVID-19 protein biomarkers is critical not only for COVID-19 caused by the coronavirus but also to prepare us for future pandemics of other diseases in detecting by LC-MS/MS at the early stages.

Anxiolytic- and antidepressant-like effects of Bacillus coagulans Unique IS-2 mediate via reshaping of microbiome gut-brain axis in rats.

BACKGROUND: Due to the rising cases of treatment-refractory affective disorders, the discovery of newer therapeutic approaches is needed. In recent times, probiotics have garnered notable attention in managing stress-related disorders. Herein, we examined the effect of Bacillus coagulans Unique IS-2® probiotic on anxiety- and depression-like phenotypes employing maternal separation (MS) and chronic-unpredictable mild stress (CUMS) model in rats. METHODS: Both male and female Sprague-Dawley rats were subjected to MS + CUMS. Probiotic treatment was provided for 6 weeks via drinking water. Anxiety- and depression-like phenotypes were assessed using sucrose-preference test (SPT), forced-swimming test (FST), elevated-plus maze test (EPM), and open-field test (OFT). Blood, brain, intestine, and fecal samples were obtained for biochemical and molecular studies. RESULTS: Stress-exposed rats drank less sucrose solution, showed increased passivity, and explored less in open-arms in SPT, FST, and EPM, respectively. These stress-generated neurobehavioral aberrations were alleviated by 6-week of Bacillus coagulans Unique IS-2 treatment. The overall locomotor activity in OFT remained unchanged. The decreased levels of BDNF and serotonin and increased levels of C-reactive protein, TNF-α, IL-1ß, and dopamine, in the hippocampus and/or frontal cortex of stress-exposed rats were reversed following probiotic treatment. Administration of probiotic also restored the systemic levels of L-tryptophan, L-kynurenine, kynurenic-acid, and 3-hydroxyanthranilic acid, villi/crypt ratio, goblet-cell count, Firmicutes to Bacteroides ratio, and levels of acetate, propionate, and butyrate in fecal samples. These results indicate remodeling of the microbiome gut-brain axis in Bacillus coagulans Unique IS-2 recipient rats. However, protein levels of doublecortin, GFAP, and zona occludens in the hippocampus and occludin-immunoreactivity in the intestine remained unchanged. No prominent sex-specific changes were noted. CONCLUSION: Anxiolytic- and antidepressant-like effects of Bacillus coagulans Unique IS-2 in MS + CUMS rat model may be mediated via reshaping the microbiome gut-brain axis.

Peptide Cysteine Thiols Act as Photostabilizer of Avobenzone through Stabilizing the Transition State of Keto-Enol Tautomerization.

Photostabilizers have been used to impart stability to an FDA-approved chemical UV-A filter avobenzone against the UV-A radiations and sunlight. The thiol group of glutathione plays a critical role in imparting the photostabilization activity of glutathione on avobenzone. The current report aims to evaluate the photostabilization activity of multiple thiols containing cysteine peptides on avobenzone. Cysteine-tripeptide and cysteine-pentapeptide were chemically synthesized and characterized using mass spectrometry. Synthetic peptides were assessed for their photostabilization activity on the enolic-form of the avobenzone under natural sunlight using UV spectroscopy in both protic and aprotic solvents. Unlike glutathione, which has pronounced activity in protic solvents, cysteine-pentapeptide exhibits similar photoprotection activity in both protic and aprotic solvents. Computational calculations using DFT suggest that peptide cysteine thiols may assist in the reversal of the photoketonization process of avobenzone thereby exhibiting the photoprotection activity to the enolic-form of avobenzone. Peptide cysteine thiols lower the activation energy barrier of keto-to-enol tautomerization of avobenzone by 30 kcal mol-1 by assisting the proton shuttle through a six-membered transition state. The current report emphasizes the applications of peptide thiols in cosmetics and may help in the development of peptides as aesthetic medicines.

Aldehyde oxidase mediated drug metabolism: an underpredicted obstacle in drug discovery and development.

Aldehyde oxidase (AO) has garnered curiosity as a non-CYP metabolizing enzyme in drug development due to unexpected consequences such as toxic metabolite generation and high metabolic clearance resulting in the clinical failure of new drugs. Therefore, poor AO mediated clearance prediction in preclinical nonhuman species remains a significant obstacle in developing novel drugs. Various isoforms of AO, such as AOX1, AOX3, AOX3L1, and AOX4 exist across species, and different AO activity among humans influences the AO mediated drug metabolism. Therefore, carefully considering the unique challenges is essential in developing successful AO substrate drugs. The in vitro to in vivo extrapolation underpredicts AO mediated drug clearance due to the lack of reliable representative animal models, substrate-specific activity, and the discrepancy between absolute concentration and activity. An in vitro tool to extrapolate in vivo clearance using a yard-stick approach is provided to address the underprediction of AO mediated drug clearance. This approach uses a range of well-known AO drug substrates as calibrators for qualitative scaling new drugs into low, medium, or high clearance category drugs. So far, in vivo investigations on chimeric mice with humanized livers (humanized mice) have predicted AO mediated metabolism to the best extent. This review addresses the critical aspects of the drug discovery stage for AO metabolism studies, challenges faced in drug development, approaches to tackle AO mediated drug clearance's underprediction, and strategies to decrease the AO metabolism of drugs.

Structural characterization of novel hydrolytic and oxidative degradation products of acalabrutinib by LC-Q-TOF-MS, H/D exchange and NMR.

The drug substance, acalabrutinib was subjected to hydrolytic (acid, base and neutral) and oxidative stress degradation as per ICH recommendations. Degradation products (DPs) generated from the drug substance were separated on a Shimadzu Shim-pak C-8 column utilizing a mobile phase composed of methanol: acetonitrile (90:10 v/v) and ammonium acetate buffer (10 mM, pH 3.80) in a gradient elution mode. Acalabrutinib was found to be labile under acid, basic, neutral and oxidative conditions. A total of eighteen DPs of drug substance were formed in hydrolytic (fourteen DPs) and oxidative degradation conditions (four DPs). All the DPs were characterized by comparing the LC-Q-TOF mass spectrometric fragmentation pathway of the drug substance with DPs. Further, hydrogen/deuterium (H/D) exchange studies were also carried out on the DPs to confirm the presence of labile hydrogens in their structures. Four DPs (H-12, O-2, O-3 and O-4) were isolated for chemical structural elucidation by NMR. Probable mechanisms involved in degradation of acalabrutinib were also postulated.

Green bioanalysis: an innovative and eco-friendly approach for analyzing drugs in biological matrices.

Green bioanalytical techniques aim to reduce or eliminate the hazardous waste produced by bioanalytical technologies. A well-organized and practical approach towards bioanalytical method development has an enormous contribution to the green analysis. The selection of the appropriate sample extraction process, organic mobile phase components and separation technique makes the bioanalytical method green. UHPLC-MS is the best option, whereas supercritical fluid chromatography is one of the most effective green bioanalytical procedures. Nevertheless, there remains excellent scope for further research on green bioanalytical methods. This review details the various sample preparation techniques that follow green analytical chemistry principles. Furthermore, it presents green solvents as a replacement for conventional organic solvents and highlights the strategies to convert modern analytical techniques to green methods.

Metal-organic frameworks in chiral separation of pharmaceuticals.

Stereoselective chiral molecules are responsible for specific biological functions in nature. At present, more than half of the prescribed drugs are chiral. Living organisms display divergent pharmacological responses to the enantiomers, leading to altered toxicity, pharmacokinetics, and pharmacodynamics. Thus, chiral analysis, separation, and extraction are crucial for ensuring enantiomeric purity to develop safe and effective medication. In recent times, metal-organic frameworks (MOFs) with appealing structures are gaining importance because of their fascinating properties as a sorbent and stationary phase. MOFs are crystalline porous solid materials built by interconnecting metal ions or clusters and organic linkers. This review explores the advancements in MOFs for the isolation and separation of chiral active pharmaceutical drugs.