Effects of fadolmidine, an α2 -adrenoceptor agonist, as an adjuvant to spinal bupivacaine on antinociception and motor function in rats and dogs.

α2 -Adrenoceptor agonists such as clonidine and dexmedetomidine are used as adjuvants to local anesthetics in regional anesthesia. Fadolmidine is an α2 -adrenoceptor agonist developed especially as a spinal analgesic. The current studies investigate the effects of intrathecally administered fadolmidine with a local anesthetic, bupivacaine, on antinociception and motor block in conscious rats and dogs. The antinociceptive effects of intrathecal fadolmidine and bupivacaine alone or in combination were tested in the rat tail-flick and the dog's skin twitch models. The durations of motor block in rats and in dogs were also assessed. In addition, the effects on sedation, mean arterial blood pressure, heart rate, respiratory rate and body temperature were evaluated in telemetrized dogs. Concentrations of fadolmidine in plasma and spinal cord were determined after intrathecal and intravenous administration in rats. Co-administration of intrathecal fadolmidine with bupivacaine increased the magnitude and duration of the antinociceptive effects and prolonged motor block without hypotension. The interaction of the antinociceptive effect was synergistic in its nature in rats. Concentration of fadolmidine in plasma was very low after intrathecal dosing. Taken together, these studies show that fadolmidine as an adjuvant to intrathecal bupivacaine provides enhanced sensory-motor block and enables a reduction of the doses of both drugs. The results indicate that co-administration of fadolmidine with intrathecal bupivacaine was able to achieve an enhanced antinociceptive effect without hypotension and could thus represent a suitable combination for spinal anesthesia.

Investigation into MAO B-Mediated Formation of CC112273, a Major Circulating Metabolite of Ozanimod, in Humans and Preclinical Species: Stereospecific Oxidative Deamination of (S)-Enantiomer of Indaneamine (RP101075) by MAO B.

Ozanimod, recently approved for treating relapsing multiple sclerosis, produced a disproportionate, active, MAO B-catalyzed metabolite (CC112273) that showed remarkable interspecies differences and led to challenges in safety testing. This study explored the kinetics of CC112273 formation from its precursor RP101075. Incubations with human liver mitochondrial fractions revealed K Mapp, V max, and intrinsic clearance (Clint) for CC112273 formation to be 4.8 µM, 50.3 pmol/min/mg protein, and 12 µl/min/mg, respectively, whereas Michaelis-Menten constant (K M) with human recombinant MAO B was 1.1 µM. Studies with liver mitochondrial fractions from preclinical species led to K Mapp, V max, and Clint estimates of 3.0, 35, and 33 µM, 80.6, 114, 37.3 pmol/min/mg, and 27.2, 3.25, and 1.14 µl/min/mg in monkey, rat, and mouse, respectively, and revealed marked differences between rodents and primates, primarily attributable to differences in the K M Comparison of Clint estimates revealed monkey to be ∼2-fold more efficient and the mouse and rat to be 11- and 4-fold less efficient than humans in CC112273 formation. The influence of stereochemistry on MAO B-mediated oxidation was also investigated using the R-isomer of RP101075 (RP101074). This showed marked selectivity toward catalysis of the S-isomer (RP101075) only. Docking into MAO B crystal structure suggested that although both the isomers occupied its active site, only the orientation of RP101075 presented the C-H on the α-carbon that was ideal for the C-H bond cleavage, which is a requisite for oxidative deamination. These studies explain the basis for the observed interspecies differences in the metabolism of ozanimod as well as the substrate stereospecificity for formation of CC112273. SIGNIFICANCE STATEMENT: This study evaluates the enzymology and the species differences of the major circulating metabolite of ozanimod, CC112273. Additionally, the study also explores the influence of stereochemistry on MAO B-catalyzed reactions. The study is of significance to the DMD readers given that this oxidation is catalyzed by a non-cytochrome P450 enzyme, and that marked species difference and notable stereospecificity was observed in MAO B-catalyzed biotransformation when the indaneamine enantiomers were used as substrates.

Simultaneous determination of donepezil, 6-O-desmethyl donepezil and spinosin in beagle dog plasma using liquid chromatography‒tandem mass spectrometry and its application to a drug-drug interaction study.

Spinosin, which is traditionally used for sedation and sleep disorders, has recently shown potential effects in alleviating memory loss. As spinosin is the main bioactive component in a standardized dried 50% ethanol extract of the seeds of Zizyphus jujuba var. spinosa, a Phase IIb clinical trial is ongoing, in Korea for the combination of the above extract formulated in a tablet (DHP1401 tablet) with donepezil hydrochloride (Aricept® tablet) in patients with mild to moderate Alzheimer's disease. Therefore, to promote safety and efficacy evaluations, a reliable method for the simultaneous detection and analysis of the two drugs is needed. Toward this end, in this study, we established and validated a rapid and sensitive LC-MS/MS method for the simultaneous determination of donepezil, its pharmacologically active metabolite 6-O-desmethyl donepezil, and spinosin in beagle dog plasma (50 µL). After optimization of the system, we used methanol for simple protein precipitation. Chromatographic separation was performed using a Phenomenex Luna C18 column (100 × 2.0 mm, 3 µm) with a mobile phase consisting of 0.1% formic acid in acetonitrile-0.1% formic acid in distilled water (2:8, v/v) at a flow rate of 0.65 mL/min. All analytes were detected and quantified in selected reaction monitoring mode. All calibration curves showed good linearity (r ≥ 0.9965) over the concentration range of 0.02-20, 0.02-10, and 0.5-250 ng/mL for donepezil, for 6-O-desmethyl donepezil, and spinosin, respectively. This validated method was then successfully applied to a pharmacokinetic study in beagle dogs with no evidence for potential drug-drug interactions between DHP1401 and donepezil hydrochloride. This information and optimized assay can be useful for the anticipated co-administration of these two drugs in clinical settings.

Bioactive indanes: Development and validation of an LC-MS/MS bioanalytical method for the determination of PH46A, a new potential anti-inflammatory agent, in dog and rat plasma and its application to a pharmacokinetic study in dog.

A new chemical entity, which is a chiral indane dimer, PH46A, has been developed by our research group. As a clinical candidate. PH46A has recently completed Phase I clinical studies in man. Previously, during its pre-clinical development, in in vivo pre-clinical studies PH46A showed potent anti-inflammatory properties, which can be targeted at a range of diseases, including inflammatory bowel disease (IBD). To support the pre-clinical development of this drug candidate, we developed a LCMS/MS method for determining PH46 (the acid form of PH46A salt) in both dog and rat plasma using Compound 1 as internal standard (IS). Those species were selected for safety pharmacology and toxicology, as well as pharmacokinetics studies. The method was validated over the range 10-10000 ng/mL for both matrices and the linearity, accuracy, precision and specificity over this range were demonstrated to be acceptable. No significant matrix effects or carryover were observed for both PH46 and IS and recovery was consistent. PH46 was found to be stable in both dog and rat plasma under the test conditions, such as at room temperature for >24 h, through 3 freeze/thaw cycles, and at -20 °C for >1 month. PH46 and IS in dog and rat plasma extracts were also found to be stable in the autosampler against fresh standard extracts on re-injection after 143.5 h and 243.5 h, respectively at 4 °C. 10- and 100-fold dilutions with control matrix were found not to affect the performance of the assay. This method was successfully applied to a pharmacokinetic study in the dog. With the exception of one dog, 003 M, oral administration of PH46A in gelatine capsules was well tolerated at a dose level of 100 mg/kg. The highest Cmax was observed in animal 003 M. The rapid absorption and high plasma concentration observed for animal 003 M compared to the data for animals 001 M and 002 M may account for the sickness observed in this animal; however, the reasons for this have not been investigated.

Fate of ptaquiloside-A bracken fern toxin-In cattle.

Ptaquiloside is a natural toxin present in bracken ferns (Pteridium sp.). Cattle ingesting bracken may develop bladder tumours and excrete genotoxins in meat and milk. However, the fate of ptaquiloside in cattle and the link between ptaquiloside and cattle carcinogenesis is unresolved. Here, we present the toxicokinetic profile of ptaquiloside in plasma and urine after intravenous administration of ptaquiloside and after oral administration of bracken. Administered intravenously ptaquiloside, revealed a volume of distribution of 1.3 L kg-1 with a mean residence-time of 4 hours. A large fraction of ptaquiloside was converted to non-toxic pterosin B in the blood stream. Both ptaquiloside and pterosin B were excreted in urine (up to 41% of the dose). Oral administration of ptaquiloside via bracken extract or dried ferns did not result in observations of ptaquiloside in body fluids, indicating deglycosolidation in the rumen. Pterosin B was detected in both plasma and urine after oral administration. Hence, transport of carcinogenic ptaquiloside metabolites over the rumen membrane is indicated. Pterosin B recovered from urine counted for 7% of the dose given intravenously. Heifers exposed to bracken for 7 days (2 mg ptaquiloside kg-1) developed preneoplastic lesions in the urinary bladder most likely caused by genotoxic ptaquiloside metabolites.

Pharmacokinetics and safety of single and multiple doses of rasagiline in healthy Japanese and caucasian subjects.

As of March 2018, rasagiline is approved for the treatment of Parkinson disease in 55 countries including Japan. The present study evaluated the pharmacokinetics (PK) and safety of rasagiline in healthy Japanese and Caucasian subjects following single and multiple administrations of three rasagiline doses. In this double-blind, placebo-controlled study, 64 healthy subjects (32 Japanese and 32 Caucasian) received either rasagiline (0.5, 1.0, or 2.0 mg) or placebo for 10 days with PK sampling for single-dose administration on day 1 and for multiple administration on day 10. Regardless of administration schedule, rasagiline plasma concentrations and dose-related increases in exposure parameters were similar between Japanese and Caucasians. Rasagiline accumulation (2-fold for 0.5 mg and 3-fold for 1.0 mg and 2.0 mg doses) following multiple administration was similar across the ethnic groups. Geometric mean ratios (GMR) comparing Japanese to Caucasians for AUC0-24 , Cmax and AUCinf following single administration were 1.38, 1.17 and 1.38 for 0.5 mg; 1.22, 1.20 and 1.22 at 1.0 mg; and 1.02, 1.00 and 1.02 at for 2.0 mg. GMR for AUCtau and Cmax,ss following multiple administration were 1.43 and 1.06 at 0.5 mg, 1.06 and 1.00 at 1.0 mg, and 1.09 and 1.07 at 2.0 mg. Safety measures were unremarkable and similar between Caucasian and Japanese subjects. Comparable systemic exposure and safety parameters were demonstrated for rasagiline administered to healthy Japanese and Caucasian subjects.

Simple determination of some antidementia drugs in wastewater and human plasma samples by tandem dispersive liquid-liquid microextraction followed by high-performance liquid chromatography.

In this work, a simple method, namely, tandem dispersive liquid-liquid microextraction, with a high sample clean-up is applied for the rapid determination of the antidementia drugs rivastigmine and donepezil in wastewater and human plasma samples. This method, which is based on two consecutive dispersive microextractions, is performed in 7 min. In the method, using a fast back-extraction step, the applicability of the dispersive microextraction methods in complicated matrixes is conveniently improved. This step can be performed in less than 2 min, and very simple tools are required for this purpose. To achieve the best extraction efficiency, optimization of the variables affecting the method was carried out. Under the optimized experimental conditions, the relative standard deviations for the method were in the range of 6.9-8.7%. The calibration curves were obtained in the range of 2-1100 ng/mL with good correlation coefficients, higher than 0.995, and the limits of detection ranged between 0.5 and 1.0 ng/mL.

Phase I Dose-Escalation Trial of PT2385, a First-in-Class Hypoxia-Inducible Factor-2α Antagonist in Patients With Previously Treated Advanced Clear Cell Renal Cell Carcinoma.

Purpose The von Hippel-Lindau tumor suppressor is inactivated in the majority of clear cell renal cell carcinomas (ccRCCs), leading to inappropriate stabilization of hypoxia-inducible factor-2α (HIF-2α). PT2385 is a first-in-class HIF-2α antagonist. Objectives of this first-in-human study were to characterize the safety, pharmacokinetics, pharmacodynamics, and efficacy, and to identify the recommended phase II dose (RP2D) of PT2385. Patients and Methods Eligible patients had locally advanced or metastatic ccRCC that had progressed during one or more prior regimens that included a vascular endothelial growth factor inhibitor. PT2385 was administered orally at twice-per-day doses of 100 to 1,800 mg, according to a 3 + 3 dose-escalation design, followed by an expansion phase at the RP2D. Results The dose-escalation and expansion phases enrolled 26 and 25 patients, respectively. Patients were heavily pretreated, with a median of four (range, one to seven) prior therapies. No dose-limiting toxicity was observed at any dose. On the basis of safety, pharmacokinetic, and pharmacodynamic profiling, the RP2D was defined as 800 mg twice per day. PT2385 was well tolerated, with anemia (grade 1 to 2, 35%; grade 3, 10%), peripheral edema (grade 1 to 2, 37%; grade 3, 2%), and fatigue (grade 1 to 2, 37%; no grade 3 or 4) being the most common treatment-emergent adverse events. No patients discontinued treatment because of adverse events. Complete response, partial response, and stable disease as best response were achieved by 2%, 12%, and 52% of patients, respectively. At data cutoff, eight patients remained in the study, with 13 patients in the study for ≥ 1 year. Conclusion PT2385 has a favorable safety profile and is active in patients with heavily pretreated ccRCC, validating direct HIF-2α antagonism for the treatment of patients with ccRCC.

Effects of High- and Low-Fat Meals on the Pharmacokinetics of Ozanimod, a Novel Sphingosine-1-Phosphate Receptor Modulator.

Ozanimod (RPC1063) is an oral selective modulator of the sphingosine-1-phosphate 1 and 5 receptors under development for the treatment of relapsing multiple sclerosis and inflammatory bowel disease. The effects of high-fat and low-fat meals on the pharmacokinetics (PK) of a single oral dose of ozanimod were evaluated in 24 healthy volunteers in a randomized, open-label crossover trial. Each subject received a 1-mg dose of ozanimod hydrochloride under 3 meal conditions (fasted, high-fat, and low-fat), each separated by 7 days. Mean plasma concentration-time profiles for ozanimod and its active metabolites (RP101988 [major], RP101075 [minor]) were similar under all 3 conditions. Moreover, all PK parameters for ozanimod, RP101988, and RP101075 were similar under the 3 meal conditions. The 90% confidence intervals (CIs) for the ratios of geometric least-squares mean (fed/fasted) were within the equivalence limits of 0.80 to 1.25 for area under the concentration-time curve from time 0 to infinity (AUC0-∞ ) and maximum plasma concentration (Cmax ) for ozanimod, RP101988, and RP101075, except for the high-fat effect on RP101075 Cmax (90%CI, 0.76-0.88). Given this lack of a food effect on the exposure of ozanimod and its active metabolites, ozanimod can be taken without regard to meals.

Long Acting Ionically Paired Embonate Based Nanocrystals of Donepezil for the Treatment of Alzheimer's Disease: a Proof of Concept Study.

PURPOSE: The aim of the present study was to prepare a patient friendly long acting donepezil (D) nanocrystals (NCs) formulation, with a high payload for i.m administration. As the native D hydrochloride salt has high aqueous solubility it is necessary to increase its hydrophobicity prior to the NCs formation. METHODS: D was ionically paired with embonic acid (E) in aqueous media and was successfully characterized using techniques like DSC, PXRD, FT-IR, NMR etc. Later, we converted the bulk ion pair into NCs using high pressure homogenization technique to study further in-vitro and in-vivo. RESULTS: The bulk ion pair has a drug content of 66% w/w and an 11,000 reduced solubility in comparison to native D hydrochloride. Also, its crystalline nature was confirmed by DSC and PXRD. The possible interaction sites responsible for the ion pair formation were identified though NMR. The prepared NCs has mean particle size 677.5 ± 72.5 nm and PDI 0.152 ± 0.061. In-vitro release showed a slow dissolution of NCs. Further, excellent bio compatibility of NCs were demonstrated in 3T3 cells. Following i.m administration of single dose of NCs, the D plasma level was found to be detectable up to 18 days. In vivo pharmacodynamic studies revealed that the single dose NCs i.m injection improved spatial memory learning and retention in ICV STZ model. CONCLUSION: Our results suggest that the developed formulation has a potential to replace the current daily dosing regimen to a less frequent dosing schedule. Graphical Abstract Improved pharmacokinetic and pharmacodynamic profile after administration of single dose donpezil embonate nanocrystals in Rats.

Effect of chongmyungtang, a traditional Korean polyherbal formula, on the Pharmacokinetic profiles of donepezil in rats.

Chongmyungtang (CMT) is a famous Korean herbal medicine for improving learning and memory, which has been reported to have anti-cholinergic and neuroprotective effects. Therefore, drug-drug interactions were examined between CMT and donepezil as a first screening of combination therapy for cognitive deficits. Rats received oral co-administration of donepezil with distilled water as a control or donepezil with CMT as a combination. The distilled water or CMT was co-administered at intervals within 5min after donepezil or 1.5h intervals. The plasma samples were analyzed for donepezil concentration and its pharmacokinetic parameters of Tmax, Cmax, AUC, t1/2 and MRTinf. In the single co-administration at intervals within 5min, donepezil was detected lower in the combination than control at 0.5h and 2h post-treatment (P<0.05). In addition, the combination showed significant increases in MRTinf compared to the control (P<0.05). This suggests drug-drug interactions between donepezil and CMT in the co-administration within 5 min. However, no meaningful differences were found in the pharmacokinetic profiles of donepezil by single dosing with CMT at 1.5h intervals and even by the repeated dosing for a week at 1.5h intervals potential combination therapy of donepezil with CMT.

A literature review of blood concentrations of new psychoactive substances classified as phenethylamines, aminoindanes, arylalkylamines, arylcyclohexylamines, and indolalkylamines.

Interpretation of blood concentrations of new psychoactive substances (NPS) requires comparison of the results to previously published case reports; as only a few experimental studies for these substances exist. A large number of articles representing single or multiple cases have been published for a great number of substances, making a unified overview difficult. In this review we have collected all published blood concentrations from the NPS groups classified as phenethylamines, aminoindanes, arylalkylamines, arylcyclohexylamines, and indolalkylamines, and also included unpublished results for MPA, MXE, 4-FMA, 4-FA and 4-MA analyzed in our laboratory. In total, 71 publications on 35 different drugs were summarized. For most of the drugs, the total number of reported cases was very low (≤5). For some of the synthetic drugs, however, a higher number of blood concentrations are now available; especially for 5-IT (32 reported cases in total), MPA (31 reported cases in total) and MXE (36 reported cases in total), thus the published results are more substantial. The present compilation could be a helpful tool for forensic toxicologists when blood concentrations of NPS are assessed.

Results From the First-in-Human Study With Ozanimod, a Novel, Selective Sphingosine-1-Phosphate Receptor Modulator.

The sphingosine-1-phosphate 1 receptor (S1P1R ) is expressed by lymphocytes, dendritic cells, and vascular endothelial cells and plays a role in the regulation of chronic inflammation and lymphocyte egress from peripheral lymphoid organs. Ozanimod is an oral selective modulator of S1P1R and S1P5R receptors in clinical development for the treatment of chronic immune-mediated, inflammatory diseases. This first-in-human study characterized the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of ozanimod in 88 healthy volunteers using a range of single and multiple doses (7 and 28 days) and a dose-escalation regimen. Ozanimod was generally well tolerated up to a maximum single dose of 3 mg and multiple doses of 2 mg/d, with no severe adverse events (AEs) and no dose-limiting toxicities. The most common ozanimod-related AEs included headache, somnolence, dizziness, nausea, and fatigue. Ozanimod exhibited linear PK, high steady-state volume of distribution (73-101 L/kg), moderate oral clearance (204-227 L/h), and an elimination half-life of approximately 17 to 21 hours. Ozanimod produced a robust dose-dependent reduction in total peripheral lymphocytes, with a median decrease of 65% to 68% observed after 28 days of dosing at 1 and 1.5 mg/d, respectively. Ozanimod selectivity affected lymphocyte subtypes, causing marked decreases in cells expressing CCR7 and variable decreases in subsets lacking CCR7. A dose-dependent negative chronotropic effect was observed following the first dose, with the dose-escalation regimen attenuating the first-dose negative chronotropic effect. Ozanimod safety, PK, and PD properties support the once-daily regimens under clinical investigation.

Clinical Response to Donepezil in Mild and Moderate Dementia: Relationship to Drug Plasma Concentration and CYP2D6 and APOE Genetic Polymorphisms.

The clinical response to donepezil in patients with mild and moderate dementia was investigated in relation to the drug plasma concentration and APOE and CYP2D6 polymorphisms. In a prospective naturalistic observational study, 42 patients with Alzheimer's disease (AD) and AD with cerebrovascular disease who took donepezil (10 mg) for 12 months were evaluated. Their DNA was genotyped, and the donepezil plasma concentrations were measured after 3, 6, and 12 months. Good responders scored ≥-1 on the Mini-Mental State Examination at 12 months in comparison to the baseline score. The study results indicated the good response pattern was influenced by the concentration of donepezil, but not by APOE and CYP2D6 polymorphisms.

Concentration of Donepezil in the Cerebrospinal Fluid of AD Patients: Evaluation of Dosage Sufficiency in Standard Treatment Strategy.

Although some studies have described the pharmacokinetics and pharmacodynamics of donepezil in the peripheral compartment, studies focused on drug transport across the blood-brain barrier are still very rare. To our knowledge, the fluctuation in the cerebrospinal fluid concentration of donepezil after administration of the drug has not been described in the literature so far. We recruited 16 patients regularly taking a standard therapeutic dose of donepezil (10 mg per day). All patients (Caucasian race) were treated for at least three months with a stable dose of 10 mg per day prior to sample collection. Patients were divided into two groups depending on the time of plasma and cerebrospinal fluid sampling: 12 h (n = 9; 4 M/5F aged 78.68 ± 7.35 years) and 24 h (n = 7; 3 M/4F aged 77.14 ± 5.87 years) after donepezil administration. The cerebrospinal fluid sample was collected by standard lumbar puncture technique using a single-use traumatic needle. The samples were analysed on an Agilent 1260 Series liquid chromatograph comprising a degasser, a quaternary pump, a light-tight autosampler unit set, a thermostated column compartment, and a UV/VIS detector. Agilent ChemStation software, the statistical software Prism4, version 5.0 (GraphPad Software, USA), and IBM® SPSS® Statistics were used for the analysis of the results. The difference in plasma concentration of donepezil after 12 h (mean ± SEM; 39.99 ± 5.90 ng/ml) and after 24 h (29.38 ± 1.71 ng/ml) was nonsignificant. In contrast, the donepezil concentration in the cerebrospinal fluid was significantly higher in the 24-h interval (7.54 ± 0.55 ng/ml) compared with the 12-h interval (5.19 ± 0.83 ng/ml, which is ~70 % based on mean cerebrospinal fluid values). Based on these data, it is plausible to predict that donepezil might produce a stronger AChE inhibition in the brain at 24 h compared with 12 h following the administration. This information may help physicians individually adjust the time of drug administration in the patients according to time course of the disease symptoms.

Increased plasma donepezil concentration improves cognitive function in patients with dementia with Lewy bodies: An exploratory pharmacokinetic/pharmacodynamic analysis in a phase 3 randomized controlled trial.

OBJECTIVE: To investigate whether increasing plasma donepezil concentration further improves cognitive function and neuropsychiatric symptoms without compromising safety in patients with dementia with Lewy bodies (DLB). METHODS: We analyzed data from a 12-week phase 3 trial of donepezil (5 and 10mg/day) in patients with DLB. The contribution of factors affecting plasma donepezil concentration was evaluated using multivariate regression analysis. The relationships between plasma donepezil concentration and efficacy (cognitive function as measured by the Mini-Mental State Examination [MMSE], hallucinations and cognitive fluctuation), or safety (blood pressure, pulse rate, body weight, and parkinsonism as measured by the Unified Parkinson's Disease Rating Scale part III) were assessed by scatterplots and Pearson correlation. RESULTS: The data of 87 patients were used in the analyses. Plasma donepezil concentration increased proportionally with increasing dose from 5 to 10mg/day. The dose (contribution rate: 0.39, p<0.0001) and age (contribution rate: 0.12, p=0.0003) were statistically significant contributing factors affecting plasma donepezil concentration. Plasma donepezil concentration correlated significantly with improvement of MMSE score (p=0.040), but no significant correlations were found with the change in other tested parameters. CONCLUSIONS: Plasma donepezil concentration correlated positively with change in cognitive function without affecting safety, and was affected mainly by dose and to a lesser extent by age. Therefore, for patients in whom safety concerns are not found at donepezil 5mg/day, increasing the dose to 10mg/day to increase plasma concentration is worthwhile to further improve cognitive function.

Postmortem redistribution mechanism of donepezil in the rat.

Population aging is rapidly advancing in numerous parts of the world and, accordingly, the prevalence of Alzheimer's disease (AD) is rising. The safety of donepezil (DPZ), which is used for AD treatment, has been established in clinical trials. However, some studies have indicated that DPZ may be associated with severe cardiac side effects, and excessive doses may induce toxicity-related symptoms or death. Therefore, the measurement of blood DPZ levels is important for the postmortem investigation of related causes of death. However, postmortem drug concentrations in the blood may not always reflect those obtained antemortem because of the postmortem redistribution (PMR) of drugs. Therefore, the aim of this study was to investigate the potential PMR of DPZ using a rat model. The DPZ concentration was measured using a validated HPLC/Q-TOF-MS system in cardiac and peripheral blood, and in the brain, lungs, myocardium, liver, and thigh muscle at different postmortem intervals (0, 1, 3, 6, 12, and 24h). Overall, the DPZ tissue to peripheral blood ratio decreased throughout the postmortem period. Furthermore, the DPZ concentration increased in the peripheral and cardiac blood but decreased in both of the lungs, postmortem. Furthermore, the blood pH was significantly lowered. We used a perfusion approach to examine the rat lung and heart to further investigate the relationship between the pH and DPZ release from the lungs. The outflow concentrations when the inflow pH changed from 7.4 to 5.5 were approximately 2-fold higher than the inflow pH fixed 7.4. These findings suggest that the antemortem accumulated DPZ in the lungs is released into the pulmonary blood owing to postmortem acidification of blood, and subsequently flows into the cardiac blood, leading to the observed increase in concentration. Although we could not determine the underlying mechanism, we confirmed that PMR occurs similarly in the cardiac and peripheral blood.

Simultaneous Determination of Donepezil and Its Three Metabolites in Human Plasma Using LC-MS-MS.

A simple and selective assay for the determination of donepezil (Aricept(®)) and its three metabolites including 6-O-desmethyl (M1), 5-O-desmethyl (M2) and N-oxide (M6) metabolites in human plasma was developed and validated using liquid chromatography with tandem mass spectrometry. An analog of donepezil was used as an internal standard (IS) for all the analytes. The analytes and the IS were extracted from plasma by solid-phase extraction. The analytes were chromatographically separated on Cadenza CD-C18 column with gradient elution, then detected with electrospray positive ionization in multiple reaction monitoring mode. The established method showed linearity ranging 0.5-100 ng/mL for donepezil and 0.2-40 ng/mL for all three metabolites and was fully validated in accordance with bioanalytical guidelines. Selectivity, clear peak separation and no carryover were ensured for all the analytes. The intra- and inter-batch reproducibility assessments demonstrated that accuracy and precision were within the acceptance criteria. Minimal matrix effects and consistent extraction recovery were noted. Stability assessment demonstrated that all the analytes were stable for at least 184 days at -20°C. Assay of post-dose samples also showed clear peak separation of the analytes, indicating successful clinical application.

An HPLC-MS method for the quantification of new acetylcholinesterase inhibitor PC 48 (7-MEOTA-donepezil like compound) in rat plasma: Application to a pharmacokinetic study.

A simple, rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative determination in rat plasma of a new candidate for AD treatment, namely PC 48 (a 7-MEOTA-donepezil like compound) in rat plasma. Sample preparation involved pH adjustment with sodium hydroxide followed by solvent extraction with ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on an Ascentis Express RP-Amide column (75 mm × 2.1mm, 2.7 µm) with a gradient mobile phase consisting of 0.05 M aqueous formic acid and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry on an LTQ XL system using the MS/MS CID (collision-induced dissociation) mode. The method was linear in the range 0.1-1000 ng/ml (r(2)=0.999) with a lower limit of quantitation of 0.1 ng/mL. Extraction recovery was in the range 63.5-72.1% for PC 48 and 70.5% for reserpine (internal standard, IS). Intra- and inter-day precisions measured as relative standard deviation were below 10.8% and accuracy was from -7.2% to 7.4%. The method was successfully applied to a pharmacokinetic study involving intramuscular application of 3.86 mg/kg PC 48 to rats for the first time. Pharmacokinetic parameters for PC 48 include Cmax 39.09 ± 4.45 ng/mL,Tmax 5.00 ± 3.08 min, AUC0-t 23374 ± 4045 min ng/mL and t1/2 1065 ± 246 min.