Athletes are frequently prescribed combination therapies to manage bacterial infections and musculoskeletal injuries, often involving amoxicillin (AMX), ibuprofen (IBU), and chlorzoxazone (CHL). However, their co-administration presents analytical challenges due to the structural diversity of these drugs within complex matrices. Simultaneous quantification of these agents is particularly demanding in the context of pharmacokinetic studies, therapeutic drug monitoring, and pharmaceutical quality control, where sensitivity, selectivity, and matrix compatibility are essential. To address this challenge, the present study introduces a rapid, robust micellar liquid chromatographic (MLC) method demonstrating an environmentally favorable profile relative to conventional acetonitrile-based RP-HPLC comparators, for the simultaneous determination of AMX, CHL, and IBU in their combined pharmaceutical dosage forms, and human plasma samples. The method utilizes a short RP-C18 column i.e., 75 mm, with UV detection at 220 nm, achieving complete separation of all three analytes in under 6 min using a low impact micellar mobile phase composed of sodium lauryl sulfate (SDS), Brij-35, and potassium dihydrogen phosphate at pH 3.0. Method validation was performed according to ICH guidelines, demonstrating linearity (R[2] > 0.999), accuracy (98.6-101.1%), adequate sensitivity (LOD: 0.44-0.74 µg/mL; LOQ: 1.32-2.25 µg/mL), and excellent baseline resolution between adjacent peaks (Rs > 4.5) across the concentration range of 2.5-25 µg/mL for all three analytes. Environmental sustainability was quantitatively assessed; the proposed method achieved an Eco-scale score of 87, compared to 68-78 for previously reported RP-HPLC methods, providing a quantitative basis for the green chemistry comparison. In alignment with green chemistry principles, the method's environmental sustainability was further evaluated using multiple metrics including the Analytical Eco-Scale, GAPI, AGREE, BAGI, and RGB12 tools. The collective results indicate that the proposed method's environmental profile compares favorably with previously reported RP-HPLC procedures, though it is acknowledged that the applied greenness metrics do not fully capture potential environmental persistence of non-ionic surfactants such as Brij-35 at high concentrations. Within the validated scope, the method demonstrates preliminary suitability for pharmaceutical quality control and therapeutic concentration monitoring; full bioanalytical validation per FDA/EMA guidance is recommended prior to pharmacokinetic applications. It is acknowledged that acetonitrile use in the plasma preparation step is included in the environmental assessments, and that long-term sustainability of the surfactant system at high concentrations warrants further evaluation.

Cytochrome P450 2E1 (CYP2E1) plays a pivotal role in the metabolism and detoxification of low molecular-weight xenobiotics, as well as in their bioactivation into toxic and DNA-reactive intermediates. Prominent examples include drugs and chemicals like acetaminophen and N-nitrosamines. Chlorzoxazone and 4-nitrophenol are widely used as probe substrates to assess CYP2E1 activity, however, their specificity remains controversial. This study aimed to reevaluate the selectivity of chlorzoxazone and 4-nitrophenol toward CYP2E1 using recombinant Supersomes™ expressing individual human CYP isoforms. Incubations were performed with chlorzoxazone and 4-nitrophenol at 100 and 500 µM, followed by quantification of 6-hydroxychlorzoxazone and 4-nitrocatechol via UPLC-MS/MS. Chlorzoxazone turnover was detected across nearly all major liver CYP isoforms, with CYP1A1 showing the highest contribution (39%), followed by CYP2D6 (14%), CYP1A2 (10%), and CYP2E1 (10%). Similarly, 4-nitrophenol hydroxylation was not limited to CYP2E1, with significant contributions from CYP1A2 (20%), CYP2A6 (19%), and CYP2D6 (16%), while CYP2E1 accounted for 26% of total activity. In conclusion, our results underscore the limited selectivity of chlorzoxazone and 4-nitrophenol and highlight the need for more specific substrates for accurate CYP2E1 enzyme characterization.

In addition to other conditions involving increased muscle spasms, centrally acting muscle relaxants (guaifenesin, tolperisone, methocarbamol, chlorzoxazone, diazepam, baclofen and tizanidin) provide relief for a significant proportion of patients suffering from low back pain. Of these, tolperisone has recently been restricted by official decision due to the high incidence of anaphylactic reactions associated with its use. The aim of our work is to analyze the frequency of side effects of this group of drugs in order to help clinicians choose the appropriate medication. According to data from the World Health Organization, tolperisone is considered a safe drug, except for the aforementioned serious allergic side effect. Mortality (including suicide) associated with the administration of these medicines is the highest among the reported side effects for diazepam, followed by tizanidine and methocarbamol. Ineffectiveness was most commonly reported for guaifenesin, followed by baclofen and tizanidine. Tolperisone was the most favorable drug in terms of both mortality and ineffectiveness. We present in detail the gastrointestinal, major neurological, and psychiatric side effects that make it difficult, and in some cases impossible, to take the drugs, and we also mention the disturbing skin and eye symptoms. Orv Hetil. 2026; 167(31): 1224-1230.

Chlorzoxazone is a centrally acting muscle relaxant commonly used for low back pain. Chlorzoxazone has been linked to rare instances of acute liver injury, a few of which have been fatal.

Few liver microsomal cytochromes P450 (P450s), especially human P450s, are recombinantly expressed in bacteria as full-length proteins. Lack of the N-terminal membrane anchor hinders studies of the interactions of microsomal P450s with redox partners. Herein, we describe the successful expression of full-length human CYP2E1, referred to as hCYP2E1FLAL, in bacteria to give a yield of ∼704 nmol/L culture. This high-level expression was achieved due to the codon optimization strategy employed to reduce free energy for translation and growth temperature. The expressed hCYP2E1FLAL was purified with a single affinity column and contained no P420, the inactive form of P450. Compared with N-terminal truncated human CYP2E1 (hCYP2E1dH), hCYP2E1FLAL exhibits elevated enzyme activity toward prototypic substrates, chlorzoxazone and p-nitrophenol, with ∼2-fold increase in kcat in the absence of cytochrome-b5 (cyt b5). The elevated enzyme activity is more pronounced in the presence of cyt b5, with a 4.3-fold increase for chlorzoxazone and a 2.5-fold increase for p-nitrophenol. Moreover, hCYP2E1FLAL binds NADPH-cytochrome P450 oxidoreductase (POR) more tightly with an apparent KM of 0.36 ± 0.026 μM, compared to 1.2 ± 0.13 μM for truncated hCYP2E1dH. Interestingly, the presence of cyt b5 further decreased the KM of POR to 0.13 ± 0.014 μM for hCYP2E1FLAL as opposed to 0.40 ± 0.041 μM for hCYP2E1dH, suggesting that cyt b5 promotes the interactions of CYP2E1 with POR. Taken together, these results suggest that the N-terminal transmembrane anchor enhances hCYP2E1-catalyzed reactions, likely by favorable interactions with POR and cyt b5 to form active complexes with redox partners, and that the full-length enzyme is the appropriate form for further redox partner interaction studies in membrane systems. SIGNIFICANCE STATEMENT: In vitro drug metabolism by CYP2E1 is often inferred from rodent CYP2E1 or truncated human CYP2E1 due to the lack of recombinant full-length human CYP2E1. This study developed a high-level bacterial expression of full-length human CYP2E1. Comparative studies of full-length and truncated human CYP2E1 in hydroxylation of chlorzoxazone and p-nitrophenol reveal that the N-terminal anchor of human CYP2E1 is critical for promoting interactions with NADPH-cytochrome P450 oxidoreductase and cytochrome-b5 and, hence, enzyme activity.

The vast majority of small-molecule active pharmaceutical ingredients (APIs) are formulated in the crystalline state, for reasons including thermodynamic stability, ease of purification and characterisation, and better control over polymorphism. However, the selective crystallisation of polymorphic APIs provides a significant hurdle to overcome, especially in the case of API co-crystals. Herein we report a series of low-molecular-weight organogels (LMWGs) which can be used to selectively crystallise APIs. In solution, these LMWGs (2-10 mg mL-1) self-assemble through hydrogen bonding to form stable gels which feature nano-structured morphologies. When utilised as crystallisation media, these LMWGs can influence crystal growth, as evidenced by the discovery of two novel 1 : 1 co-crystals of chlorzoxazone with nicotinamide and chlorzoxazone with isonicotinamide. This work highlights the potential of LMWGs as another means of controlling API crystallisation.

The influence of the expired drug chlorzoxazone (N-acetyl-para-aminophenol) on the corrosion behavior of 304 stainless steel (304SS) in 1.0 M hydrochloric acid was examined. Several electrochemical measurements were investigated, including open circuit potential (OCP), cyclic voltammetry (CV), potentiodynamic polarization (PD), electrochemical potentiokinetic reactivation (EPR), chronopotentiometry (CP), chronoamperometry (CA), linear sweep voltammetry (LSV), and electrochemical impedance spectroscopy (EIS) using Nquist and Bode plots. These methods were analyzed to evaluate the corrosion rate, passivation characteristics, and stability of protective films with different drug concentrations (50-400 ppm). Open-circuit measurements indicated that the incorporation of the drug altered the potential to more noble levels, suggesting surface protection. CV and LSV measurements revealed a significant reduction in current density with increasing drug concentration, validating the robust adsorption of the inhibitor molecules and establishing a stable passive film. PD and EPR findings indicated substantial decreases in corrosion current density (I corr) and increases in polarization resistance (R p), especially at 400 ppm, where the corrosion rate was the lowest among different drug loadings. Chronopotentiometry demonstrated consistent potential responses and improved passivation with the inhibitor, whilst chronoamperometry showed a significant reduction in dissolving current relative to uncoated steel. The data collectively indicate that the drug compound functions as an effective, concentration-dependent corrosion inhibitor, establishing a protective barrier that mitigates both anodic and cathodic processes, thus improving the electrochemical stability of stainless steel in acidic environments. This study opens a new avenue for reusing expired drugs as corrosion inhibitors.

Huntington's disease (HD) is a hereditary neurodegenerative condition, passed down in an autosomal dominant manner, characterized by the gradual decline of motor functions, such as chorea, along with psychiatric symptoms and cognitive deterioration. In HD, the striatum is the main area impacted, although cerebellar atrophy has also been observed independently of striatal degeneration. Consequently, HD patients may exhibit symptoms consistent with cerebellar cognitive affective syndrome (CCAS) due to cerebellar involvement. CCAS manifestations have been noted in various rodent models of motor dysfunctions linked to cerebellar atrophy. Previous research has identified anxiety, memory deficits, and depressive-like behavior in the YAC128 transgenic murine model of HD. This investigation examines the effects of prolonged administration of the small-conductance calcium-activated potassium channel (SK channel) positive modulator chlorzoxazone (CHZ), both alone and in combination with folic acid (FA), on cerebellar Purkinje cell (PC) electrophysiology and morphology, along with locomotor and memory decline and emotional state changes in YAC128 mice. The findings indicate that both CHZ alone and the mixture of CHZ and FA similarly ameliorated cerebellar-specific deficits, encompassing the precision of PC electrophysiological activity and beam walk performance in YAC128 mice. However, solely the mixture of CHZ and FA significantly alleviated depressive-like symptoms and improved recognition memory. These findings imply that a therapeutic approach addressing both motor and cognitive-affective deficits is essential for the holistic management of movement disorders associated with cerebellar atrophy, including HD.

Pharmacokinetics (PKs) changes in the aging state are relatively common in clinical practice, but their underlying mechanisms remain unclear. This study aims to explore the potential effects of gut microbiota on PK of P450 probe drugs in old and young mice. A cocktail of probe drugs including phenacetin (PHE), midazolam (MID), dextromethorphan tartrate (DEX), and chlorzoxazone was gavage administered to control and pseudo-sterile old and young mice, and the PK parameters were compared. Subsequently, fecal microbiota transplantation (FMT) from young to old mice was performed to assess the impact of FMT on PK of the probe drugs. We observed that gut microbiota significantly affected the systemic exposure of PHE and MID, whereas age-related increase in DEX exposure in the old mice could be reversed by clearance of microbiota. No changes in PK parameters of the probe drugs were observed in old mice with FMT from young mice, suggesting that the alterations in PHE, MID, and DEX metabolism in the old mice could not be explained by unique microbiota from young mice. Our findings provide valuable guidance on how to improve the individualized medication for the elderly population. SIGNIFICANCE STATEMENT: This article offers new insights into the role of gut microbiota in the pharmacokinetic changes with aging, which is conducive to individualized medication for elderly patients and provides new insight for the research and development of drugs for elderly population.

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy (TMA) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury (AKI). Adult-onset cases that initially manifest with predominantly neuropsychiatric symptoms are uncommon and may delay diagnosis. We describe the case of a 57-year-old female who developed acute cognitive decline, psychomotor retardation, and tremor following the recent use of a compound chlorzoxazone preparation. Laboratory evaluation revealed thrombocytopenia, hemolysis, and renal impairment, while imaging demonstrated reversible white matter abnormalities. Infectious causes and thrombotic thrombocytopenic purpura (TTP) were excluded, and persistently low complement C3 levels supported complement activation. The clinical course and Naranjo probability score suggested a probable association between drug exposure and HUS. The patient improved with comprehensive supportive management, without complement inhibitor therapy. This report highlights the diagnostic challenge of HUS with predominant neuropsychiatric manifestations and underscores the importance of maintaining a high index of suspicion for TMA in patients with unexplained neurological symptoms and concurrent renal or hematologic abnormalities.