Pharmacogenomics of oxcarbazepine in the treatment of epilepsy.

Oxcarbazepine (OXC) is one of the preferred drugs for partial seizures and generalized tonic-clonic seizures. However, clinical studies have found that there are considerable differences among different populations in OXC therapeutic efficacy or safety that result from the function changes of metabolic enzymes, transporters and other receptors involved in pharmacokinetics and pharmacodynamics in vivo. The authors collected all the information on the clinically reported associations between variants of common genes (e.g., UGT1A9, HLA-B, ABCB1) and OXC. In conclusion, these associations based on variants are beneficial for adjusting the medication regimen, which could be useful for individualized treatment with OXC.

As a new-generation aromatic antiepileptic drug, oxcarbazepine (OXC) is often used for epilepsy treatment. It is known that when OXC is absorbed, it is reduced to an active metabolite in the liver and enters the brain through the blood circulation to play an antiepileptic role. Therefore, the variations of proteins participating in the process, including drug metabolic enzymes, transporters, drug targets and other receptors, have an effect on the efficacy and safety of OXC in vivo. In this study, the associations of some variants of common genes with OXC are summarized to provide epileptic patients an appropriate dose of OXC or reduce the risk of OXC-induced toxicity, which are in favor of personalized OXC treatment for patients with epilepsy.

LC-MS3 Strategy for Quantification of Carbamazepine in Human Plasma and Its Application in Therapeutic Drug Monitoring.

This study developed a detection method based on the strategy of HPLC/MS3 and verified its suitability by quantifying carbamazepine in human plasma. The high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS3) system was performed using a Shimadzu UFLC XR liquid chromatography and a SCIEX QTRAP® 5500 linear ion trap triple quadrupole mass spectrometer. The specific operation was as follows: the sample protein was firstly precipitated using methanol, then carbamazepine and carbamazepine-D2N15 were separated on an ACQUITY UPLC HSS T3 column using the gradient elution with solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 0.25 mL/min. Each sample was run for 7 min. This method was validated for various parameters including accuracy, precision, selectivity, linearity, LLOQ, etc. Only 5 µL of sample plasma could obtain the result of LLOD 0.5 µg/mL. The intra-day and inter-day precision was <8.23%, and accuracy was between -1.74% and 2.92%. This method was successfully used for monitoring the blood concentration of epilepsy patients after carbamazepine treatment.

Influence of nanoparticle size on blood-brain barrier penetration and the accumulation of anti-seizure medicines in the brain.

Anti-seizure medicines constitute a common yet important modality to treat epilepsy. However, some of them are associated with serious side effects including hepatotoxicity and hypersensitivity. Furthermore, the blood-brain barrier (BBB) is an insurmountable obstacle for brain drug delivery. Fortunately, the introduction of the nanoparticles for drug delivery is a feasible approach to overcome these obstacles. Encapsulating drugs into nanoparticles and delivering them to specific sites shows great potential for improving the efficiency of drug delivery and reducing systemic toxicity. Several in vivo studies have investigated the effect of nanoparticle size on biodistribution in mice, but very few have investigated its effects on efficient drug delivery while crossing the BBB. Therefore, we designed a methoxy poly(lactide-co-glycolide)-b-poly(ethylene glycol) methyl ether (mPEG-PLGA) nanoparticle delivery system and explored the cell uptake efficiency of nanoparticles with different sizes and their ability to penetrate the BBB while carrying carbamazepine (CBZ). CBZ-loaded nanoparticles could significantly reduce the cytotoxicity of CBZ to L929 cells at high concentrations. Results from the endocytosis experiment involving human cerebral microvessel endothelial cell/D3 showed that the DiR-loaded mPEG5K-PLGA10K nanoparticles possessed the highest cell uptake efficiency. The endocytosis efficiency was 90% at 30 min, which far exceeded that of the other groups. Moreover, similar results were obtained from subsequent experiments where fluorescence images of the isolated organs of the mice were acquired. To summarize, our study demonstrated that drug delivery to the brain using nanocarriers is size dependent. Nanoparticles with the smallest particle size can be internalized more effectively, and easily penetrate the BBB, and accumulate in the brain.

Effects of concurrent and staggered dosing of semi-solid enteral nutrients on pharmacokinetic behavior of antiepileptic drugs after oral administration in rats.

BACKGROUND: The use of enteral nutrients plays a highly important role in accurate nutrition management, but limited information is currently available on the cautionary points of semi-solid enteral nutrients. AIM: In this study, we examined whether the pharmacokinetic profiles of sodium valproate (SVA), levetiracetam (LEV), and carbamazepine (CBZ) are affected by altering the dosing time of RACOL®-NF Semi Solid for Enteral Use (RASS), a prescribed semi-solid formula. We also investigated whether the pharmacokinetic interaction observed in this study can be avoided by staggered dosing of the chemical drug and semi-solid enteral nutrient. METHODS: The plasma concentration of SVA, LEV and CBZ after oral administration was measured by LC-MS/MS method. RESULTS: There was no difference in pharmacokinetic characteristics of SVA and LEV when the dosing time of RASS was altered. On the other hand, the plasma concentration of CBZ after oral administration at all sampling points decreased with the extension of the dosing time of RASS, which was consistent with the Cmax and AUC. However, no significant difference was observed in the pharmacokinetic profiles or parameters of CBZ between the short-term and long-term RASS dosing groups by prolonging the administered interval of CBZ and RASS for 2 hr. CONCLUSION: We concluded that the pharmacokinetic profiles of CBZ, but not SVA and LEV, after its oral administration are affected by the dosing time of RASS, but staggered administration of CBZ and RASS prevented their interaction.

Relevance of NR1I2 variants on carbamazepine therapy in Mexican Mestizos with epilepsy at a tertiary-care hospital.

Aim: We evaluated the potential influence of genetic (CYP3A5, EPHX1, NR1I2, HNF4A, ABCC2, RALBP1, SCN1A, SCN2A and GABRA1) and nongenetic factors on carbamazepine (CBZ) response, adverse drug reactions and CBZ plasma concentrations in 126 Mexican Mestizos (MM) with epilepsy. Subjects & methods: Patients were genotyped for 27 variants using TaqMan® assays. Results: CBZ response was associated with NR1I2 variants and lamotrigine cotreatment. CBZ-induced adverse drug reactions were related to antiepileptic polytherapy and SCN1A rs2298771/rs3812718 haplotype. CBZ plasma concentrations were influenced by NR1I2-rs2276707 and -rs3814058, and by phenytoin cotreatment. CBZ daily dose was also influenced by NR1I2-rs3814055 and EPHX1-rs1051740. Conclusion: Interindividual variability in CBZ treatment was partly explained by NR1I2, EPHX1 and SCN1A variants, as well as antiepileptic cotreatment in MM with epilepsy.

Pharmacokinetic Drug-Drug Interactions among Antiepileptic Drugs, Including CBD, Drugs Used to Treat COVID-19 and Nutrients.

Anti-epileptic drugs (AEDs) are an important group of drugs of several generations, ranging from the oldest phenobarbital (1912) to the most recent cenobamate (2019). Cannabidiol (CBD) is increasingly used to treat epilepsy. The outbreak of the SARS-CoV-2 pandemic in 2019 created new challenges in the effective treatment of epilepsy in COVID-19 patients. The purpose of this review is to present data from the last few years on drug-drug interactions among of AEDs, as well as AEDs with other drugs, nutrients and food. Literature data was collected mainly in PubMed, as well as google base. The most important pharmacokinetic parameters of the chosen 29 AEDs, mechanism of action and clinical application, as well as their biotransformation, are presented. We pay a special attention to the new potential interactions of the applied first-generation AEDs (carbamazepine, oxcarbazepine, phenytoin, phenobarbital and primidone), on decreased concentration of some medications (atazanavir and remdesivir), or their compositions (darunavir/cobicistat and lopinavir/ritonavir) used in the treatment of COVID-19 patients. CBD interactions with AEDs are clearly defined. In addition, nutrients, as well as diet, cause changes in pharmacokinetics of some AEDs. The understanding of the pharmacokinetic interactions of the AEDs seems to be important in effective management of epilepsy.

Fabrication of Carbamazepine Cocrystals: Characterization, In Vitro and Comparative In Vivo Evaluation.

Carbamazepine (CBZ) is an antiepileptic drug having low bioavailability due to its hydrophobic nature. In the current study, efforts are made to investigate the effect of dicarboxylic acid coformer spacer groups (aliphatic chain length) on physicochemical properties, relative humidity (RH) stability, and oral bioavailability of CBZ cocrystals. Slurry crystallization technique was employed for the preparation of CBZ cocrystals with the following coformers: adipic (AA), glutaric (GA), succinic (SA), and malonic acid (MA). Powder X-ray diffractometry and Fourier-transform infrared spectroscopy confirmed cocrystal preparation. Physicochemical properties, RH stability, and oral bioavailability of cocrystals were investigated. Among the prepared cocrystals, CBZ-GA showed maximum solubility as well as improved dissolution profile (CBZ-GA > CBZ-MA > CBZ-AA > pure CBZ > CBZ-SA) in ethanol. Maximum RH stability was shown by CBZ-AA, CBZ-SA, and CBZ-MA. In vivo studies confirmed boosted oral bioavailability of cocrystals compared to pure CBZ. Furthermore, in vivo studies depicted the oral bioavailability order of cocrystals as CBZ-GA > CBZ-MA > Tegral® > CBZ-AA > CBZ-SA > pure CBZ. Thus, pharmaceutical scientists can effectively employ cocrystallization technique for tuning physicochemical properties of hydrophobic drugs to achieve the desired oral bioavailability. Overall, results reflect no consistent effect of spacer group on physicochemical properties, RH stability, and oral bioavailability of cocrystals.

Therapeutic monitoring of carbamazepine and its active metabolite during the 1st postnatal month: Influence of drug interactions.

OBJECTIVE: To receive information about carbamazepine and its active metabolite 10,11-epoxide transport into mature milk and suckling infants. METHODS: In this cohort study, maternal serum, mature milk, and infant serum carbamazepine and epoxide levels were measured between the 6th and 29th postnatal day (carbamazepine in 1990-2017, epoxide in 1997-2017). Paired maternal serum, infant serum and milk levels were used for the assessment of ratios of this levels. The influence of combined treatment with enzyme-inducing antiepileptic drugs and valproic acid was assessed. Relationship between maternal serum, infant serum, and milk levels was also evaluated. RESULTS: Maternal carbamazepine levels were 1.4-10.4 mg/L, milk 0.5-6.7 mg/L and infant 0.5-2.6 mg/L. Maternal 10,11-epoxide levels were 0.3-5.4 mg/L, milk 0.3-3.7 mg/L and infant 0.3-0.6 mg/L. Highly significant correlations were observed exclusively between milk and maternal serum levels of both carbamazepine and 10,11-epoxide. Concomitant administration of enzyme-inducing antiepileptic drugs significantly increased the maternal apparent oral clearance of carbamazepine by approximately 130%. Carbamazepine combined with valproic acid significantly increased epoxide levels in milk and maternal serum but not in breastfed infants. CONCLUSIONS: In breastfed infants, carbamazepine levels did not reach the lower limit of the therapeutic range used for the general epileptic population, and the majority of epoxide levels were less than the lower limit of quantification. Routine monitoring of carbamazepine in these infants is not compulsory. However, observation of breastfed infants is desirable. If signs of potential adverse reactions are evident, infant serum concentrations should be monitor.

Aspectos farmacológicos diferenciales entre los bloqueadores de los canales de sodio dependientes de voltaje: implicaciones en la práctica clínica / Differential pharmacological aspects between voltage-gated sodium channel blockers: implications in clinical practice

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Evaluation of clinical and genetic factors in the population pharmacokinetics of carbamazepine.

AIMS: Carbamazepine can cause hypersensitivity reactions in ~10% of patients. An immunogenic effect can be produced by the electrophilic 10,11-epoxide metabolite but not by carbamazepine. Hypothetically, certain single nucleotide polymorphisms might increase the formation of immunogenic metabolites, leading ultimately to hypersensitivity reactions. This study explores the role of clinical and genetic factors in the pharmacokinetics (PK) of carbamazepine and 3 metabolites known to be chemically reactive or formed through reactive intermediates. METHODS: A combination of rich and sparse PK samples were collected from healthy volunteers and epilepsy patients. All subjects were genotyped for 20 single nucleotide polymorphisms in 11 genes known to be involved in the metabolism or transport of carbamazepine and carbamazepine 10,11-epoxide. Nonlinear mixed effects modelling was used to build a population-PK model. RESULTS: In total, 248 observations were collected from 80 subjects. A 1-compartment PK model with first-order absorption and elimination best described the parent carbamazepine data, with a total clearance of 1.96 L/h, central distribution volume of 164 L and absorption rate constant of 0.45 h-1 . Total daily dose and coadministration of phenytoin were significant covariates for total clearance of carbamazepine. EPHX1-416G/G genotype was a significant covariate for the clearance of carbamazepine 10,11-epoxide. CONCLUSION: Our data indicate that carbamazepine clearance was affected by total dose and phenytoin coadministration, but not by genetic factors, while carbamazepine 10,11-epoxide clearance was affected by a variant in the microsomal epoxide hydrolase gene. A much larger sample size would be required to fully evaluate the role of genetic variation in carbamazepine pharmacokinetics, and thereby predisposition to carbamazepine hypersensitivity.

Silymarin as a flavonoid-type P-glycoprotein inhibitor with impact on the pharmacokinetics of carbamazepine, oxcarbazepine and phenytoin in rats.

P-glycoprotein (P-gp) is an efflux transporter involved in drug-resistant epilepsy and some flavonoids have been targeted as effective P-gp inhibitors. Herein, we assessed the impact of silymarin on the pharmacokinetics of three antiepileptic drugs (AEDs) in rats. Animals were pretreated with silymarin, verapamil (positive control) or vehicle (negative control) 1 h before AEDs administration (carbamazepine (25 mg/kg), oxcarbazepine (OXC) (50 mg/kg), or phenytoin (100 mg/kg)). Multiple blood samples were collected after AED dosing, and a non-compartmental analysis was performed. An independent study was also conducted to investigate the effects of silymarin on the OXC plasma-to-brain distribution. Silymarin altered the pharmacokinetics of OXC, increasing its peak plasma concentration by 50% and its extent of systemic exposure by 41%, which had also impact on brain drug concentrations. These findings support that the co-administration of silymarin and OXC should continue to be explored as a strategy to reverse the pharmacoresistance in epilepsy.

Biopharmaceutical implications of excipient variability on drug dissolution from immediate release products.

Elucidating the impact of excipient variability on oral product performance in a biopharmaceutical perspective would be beneficial and allow excipient implementation on Quality by Design (QbD) approaches. The current study investigated the impact of varying viscosity of binders (hypromellose (HPMC)) and superdisintegrants (sodium starch glycolate (SSG)) and particle size distribution of lubricants (magnesium stearate (MgSt)) on the in vitro dissolution of a highly and a poorly soluble drug from immediate release formulations. Compendial (pharmacopoeia buffers) and biorelevant (media simulating the gastrointestinal fluids) media and the USP 2 and USP 4 apparatuses were used to assess the exerted excipient effects on drug dissolution. Real-time dissolution UV imaging provided mechanistic insights into disintegration and dissolution of the immediate release formulations. Varying the viscosity type of HPMC or SSG did not significantly affect drug dissolution irrespective of the compound used. Faster drug dissolution was observed when decreasing the particle size of MgSt for the highly soluble drug. The use of real-time dissolution UV Imaging revealed the influential role of excipient variability on tablet disintegration, as for the highly soluble drug, tablets containing high viscosity HPMC or low particle size MgSt disintegrated faster as compared to the control tablets while for the poorly soluble drug, slower tablet disintegration was observed when increasing the viscosity of the HPMC as compared to the control tablets. Changes in drug dissolution when varying excipients may be anticipated if the excipient change has previously affected drug solubility. The use of multivariate data analysis revealed the influential biopharmaceutical factors such as critical excipient types/properties, drug aqueous solubility, medium/hydrodynamic characteristics affecting the impact of excipient variability on in vitro drug dissolution.

The effect of cannabidiol on the pharmacokinetics of carbamazepine in rats.

Carbamazepine (CBZ) is mainly metabolized by CYP3A4 into carbamazepine-10,11-epoxide (CBZE). Cannabidiol (CBD) is a potent inhibitor of the CYP3A family. The aim of this study is to determine the effect of acute and chronic administration of CBD on the pharmacokinetics of CBZ and CBZE. Male SD rats were assigned into four acute and four chronic groups: control (CBZ only), positive control (ketoconazole), low-dose cannabidiol (l-CBD), and high-dose cannabidiol (h-CBD). Acute CBD groups were administered a single dose of CBD, while chronic CBD groups were given multiple doses of CBD for 14 days (q.d.) before CBZ administration. Plasma samples had been collected and analyzed for CBZ and CBZE, then their noncompartmental pharmacokinetic parameters before and after CBD administration were determined. The co-administration of a single l-CBD has significantly increased CBZ's [Formula: see text] by 53.1%. Furthermore, CBZE kinetics showed a significant decrease in Cmax by 31.8%. Acute h-CBD caused similar effects on CBZ's [Formula: see text] with 40.4% significant decrease in CBZE's Cmax, when compared to the control. Chronic h-CBD caused a significant decrease in CBZ's Cmax and [Formula: see text] by 75.3% and 65.7%, respectively. Besides, [Formula: see text] and Cmax of CBZE significantly decreased by 75.3% and 78.3%, respectively. These results demonstrated that the pharmacokinetics of CBZ and CBZE had been significantly affected by CBD. When CBD has been administered as a single dose, the effect is believed to be mainly caused by the inhibition of CBZ metabolism through CYP3A. The effect of chronic administration of CBD probably includes kinetic pathways other than the inhibition of CYP3A-dependent pathways. Graphical abstract.

Development and Validation of a Free Carbamazepine Assay on an Automated Chemistry Analyzer.

BACKGROUND: Carbamazepine is an effective drug for treating seizures and trigeminal neuralgia. Therapeutic drug monitoring of free carbamazepine in serum can be useful in situations that drug--protein binding is altered to guide regimen adjustment and to aid in the diagnosis of clinical toxicity. METHODS: Separation of the nonprotein bound carbamazepine was achieved via ultrafiltration through a molecular weight cut-off filter. A method for free carbamazepine measurement was developed on the automated cobas chemistry analyzers (Roche Diagnostics) by modifying the Carbamazepine Gen 4 assay (Roche Diagnostics). Assay performance characteristics were established including precision, accuracy, reportable range, analytical specificity, and stability. RESULTS: The intra- and inter-assay imprecision was 0%-1.4% and 2.4%-5.1%, respectively. The lower limit of quantitation was 0.3 µg/mL, and the assay was linear up to 10.0 µg/mL. A spike recovery study, using reference standard material, showed recovery was 93.5%--101.3% across the analytical measurement range. Method comparison with a reference laboratory method demonstrated equivalent performance with a slope of 1.01, intercept of 0.09, and correlation coefficient of 0.9948. CONCLUSION: This assay provides a simple and accurate method for monitoring free carbamazepine with a fast turnaround time.

The solubility, permeability and the dose as key factors in formulation development for oral lipophilic drugs: Maximizing the bioavailability of carbamazepine with a cosolvent-based formulation.

The purpose of this research was to investigate drug dose, solubility, permeability, and their interplay, as key factors in oral formulation development for lipophilic drugs. A PEG400-based formulation was studied for five doses of the lipophilic drug carbamazepine, accounting for biorelevant dissolution of the dose in the GIT, and in-vivo bioavailability in rats. With the three lower doses (10, 25 and 50 mg/kg), complete in-vitro dissolution was achieved and maintained throughout the experiment with this formulation, while significant precipitation was obtained with higher doses (100 and 200 mg/kg). Likewise, the studied formulation allowed complete bioavailability in-vivo with the three lower doses, while the same formulation allowed only 76% and 42% bioavailability for the 100 and 200 mg/kg doses, respectively. There was good correlation between the in-vitro and in-vivo results. In conclusion, this work demonstrates that the dose is a crucial factor in formulation development; while a given formulation may be optimal for a certain drug dose, it may no longer be optimal for higher doses of the same drug. Hence, the solubility, the permeability, and their interplay, have to be considered in light of the drug dose intended to be administered in order to achieve successful oral formulation development.

Psychotropic drug use in perinatal women with bipolar disorder.

Optimal dose management of psychotropic drugs during the perinatal period reduces the risk for recurrence of mood episodes in women with Bipolar Disorder. Physiological changes during pregnancy are associated with decreases in the plasma concentrations of the majority of mood stabilizing medications. Regular symptom and drug concentration monitoring for lithium and anticonvulsants with reflexive dose adjustment improves the probability of sustained symptom remission across pregnancy. The elimination clearance trajectory across pregnancy for psychotropics dictates the frequency of laboratory monitoring and dose adjustment. The literature on the pharmacokinetics of lithium, lamotrigine, carbamazepine and atypical antipsychotics during pregnancy and postpartum are reviewed, recommendations for symptom and laboratory monitoring are proposed and recommendations for dose adjustments are presented.

Nebivolol attenuates the anticonvulsant action of carbamazepine and phenobarbital against the maximal electroshock-induced seizures in mice.

BACKGROUND: Due to co-occurrence of seizures and cardiovascular disorders, nebivolol, a widely used selective ß1-blocker with vasodilatory properties, may be co-administered with antiepileptic drugs. Therefore, we wanted to assess interactions between nebivolol and four conventional antiepileptic drugs: carbamazepine, valproate, phenytoin and phenobarbital in the screening model of tonic-clonic convulsions. METHODS: Seizure experiments were conducted in the electroconvulsive threshold and maximal electroshock tests in mice. The chimney test served as a method of assessing motor coordination, whereas long-term memory was evaluated in the computerized step-through passive-avoidance task. To exclude or confirm pharmacokinetic interactions, we measured brain concentrations of antiepileptic drugs using the fluorescence polarization immunoassay. RESULTS: It was shown that nebivolol applied at doses 0.5-15 mg/kg did not raise the threshold for electroconvulsions. However, nebivolol at the dose of 15 mg/kg reduced the anti-electroshock properties of carbamazepine. The effect of valproate, phenytoin, and phenobarbital remained unchanged by combination with the ß-blocker. Nebivolol significantly decreased the brain concentration of valproate, but did not affect concentrations of remaining antiepileptic drugs. Therefore, contribution of pharmacokinetic interactions to the final effect of the nebivolol/carbamazepine combination seems not probable. Nebivolol alone and in combinations with antiepileptic drugs did not impair motor performance in mice. Nebivolol alone did not affect long-term memory of animals, and did not potentiate memory impairment induced by valproate and carbamazepine. CONCLUSIONS: This study indicates that nebivolol attenuated effectiveness of some antiepileptic drugs. In case the results are confirmed in clinical settings, this ß-blocker should be used with caution in epileptic patients.

Nanostructured lipid carriers-mediated brain delivery of carbamazepine for improved in vivo anticonvulsant and anxiolytic activity.

The limited brain delivery of carbamezapine (CBZ) presents a major hurdle in the successful epilepsy treatment. The potential of carbamezapine-loaded nanostructured lipid carriers (CBZ-NLCs) for improved brain delivery is investigated in the current study. CBZ-NLCs were prepared by using binary mixture of trilaurin and oleic acid as a lipid core stabilized with Poloxamer 188, Tween 80 and Span 80. CBZ-NLCs were evaluated for physicochemical properties, in vitro release, in vivo brain kinetics, anticonvulsant and anxiolytic activities. The optimized CBZ-NLCs demonstrated nanometric particle size (97.7 nm), surface charge of -22 mV and high drug incorporation (85%). CBZ-NLCs displayed biphasic release pattern with initial fast followed by sustained drug release. CBZ-NLCs significantly enhanced the AUC of CBZ (520.4 µg·h/mL) in brain compared with CBZ dispersion (244.9 µg·h/mL). In vivo anticonvulsant activity of CBZ-NLCs in PTZ-induced seizure model showed a significant increase in the onset time (143.0 sec) and reduction in duration (17.2 sec) of tonic-clonic seizures compared with CBZ dispersion (75.4 and 37.2 sec). The anxiolytic activity in light-dark box and elevated-plus maze models also demonstrated superiority of CBZ-NLCs to CBZ dispersion. From the results, CBZ-NLCs presents a promising strategy to improve brain delivery and therapeutic outcomes of CBZ in epilepsy.

Synthesis of bifunctional cabbage flower-like Ho3+/NiO nanostructures as a modifier for simultaneous determination of methotrexate and carbamazepine.

Cabbage flower-like Ho3+/NiO nanostructure (CFL-Ho3+/NiO NSs) with significant electrocatalytic oxidation has been published for the first time. First, structure and morphology of CFL-Ho3+/NiO-NSs have been described by XRD, SEM, and EDX methods. Then, CFL-Ho3+/NiO-NSs have been applied as a modifier for simultaneous electrochemical detection of methotrexate (MTX) and carbamazepine (CBZ). Functions of the modified electrode have been dealt with through electrochemical impedance spectroscopy (EIS). It has been demonstrated that the electrode response has been linear from 0.001-310.0 µM with a limit of detection of 5.2 nM and 4.5 nM (3 s/m) through DPV for MTX and CBZ. Diffusion coefficient (D) and heterogeneous rate constant (kh) have been detected for MTX and CBZ oxidation at the surface of the modified electrode. Moreover, CFL-Ho3+/NiO-NS/GCE has been employed for determining MTX and CBZ in urine and drug specimens. Outputs showed the analyte acceptable recovery. Therefore, the electrode could be applied to analyze both analytes in drug prescription and clinical laboratories. Graphical abstract Electrochemical sensor based on bifunctional cabbage flower-like Ho3+/NiO nanostructures modified glassy carbon electrode for simultaneous detecting methotrexate and carbamazepine was fabricated.