Development and validation of an improved HPLC-MS/MS method for comparative pharmacokinetics of penehyclidine hydrochloride following a single intravenous or intramuscular injection.

Penehyclidine hydrochloride (PHC) is an anticholinergic drug with both antimuscarinic and antinicotinic activity. In order to compare the pharmacokinetics of two administration routes (intravenous injection (i.v.) and intramuscular injection (i.m.)) of PHC, an improved High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC-MS/MS) bioanalytical method was developed for the quantification of PHC in plasma and urine using verapamil as the internal standard (I.S.). Chromatography was performed using a Thermo Hypersil GOLD column (30mm×2.1mm, 3µm), with a gradient elution of 1‰ formic acid-10mmol/L ammonium acetate and acetonitrile at 0.3mL/min. Detection and quantitation were performed by electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H](+) MRM transition of PHC at m/z 316.2→128.3 was used for PHC quantitation, and the transition at m/z 454.6→303.2 was used to monitor I.S. The lower limit of quantification (LLOQ) was 0.05 ng/mL. The intraday precision was <6.71% and the interday precision was <11.69%. The pharmacokinetic parameters of i.v. and i.m. administration routes were as follows (i.v. vs i.m.): t1/2 15.73 vs 17.24h, Tmax 0.06 vs 0.26h, AUC0-t 69.35 vs 67.90hng/mL, AUC0-inf 78.24 vs 79.67hng/mL, Cmax 37.5 vs 9.1ng/mL, Ae0-24h 22.7 vs 25.21µg. There were no significant differences between parameters t1/2 and AUC (P>0.05), but significant differences were observed in Cmax, Tmax and Ae0-24h between the two administration routes (P<0.05). The mean absolute bioavailability of the i.m. administration route was 98.4% (95% confidence interval, 93.4-103.6%). Safety results showed that PHC appeared to be well tolerated in both i.v. and i.m. administration routes and pharmacokinetic results showed that PHC was nearly completely absorbed via i.m. administration route.

A randomized, crossover study to investigate the pharmacokinetics and safety of inhaled fluticasone furoate and umeclidinium, administered separately and in combination via dry powder inhaler in healthy adult volunteers.

INTRODUCTION: A combination of fluticasone furoate (FF) and umeclidinium (UMEC) has been considered for development for the treatment of asthma. The primary objectives were to investigate the plasma and urine pharmacokinetics (PK) of FF/UMEC in combination compared with FF and UMEC monotherapies. METHODS: This randomized, double-blind, three-period crossover, single-center study in healthy volunteers assessed the PK of FF 400 mcg and UMEC 500 mcg administered separately and in combination (four inhalations of FF/UMEC 100/125 mcg, FF 100 mcg, or UMEC 125 mcg) via dual-strip dry powder inhaler. Subjects were randomized based on codes generated using a validated computerized system (Randall, GlaxoSmithKline). RESULTS: Eighteen subjects were enrolled; 17 received all three scheduled doses of study medication. Plasma FF and UMEC concentrations peaked at 0.5 and 0.08 h post-dose, respectively, for FF/UMEC and the monotherapies. FF and UMEC co-administration resulted in slightly lower or similar systemic exposure for both drugs versus the monotherapies. In post hoc sensitivity analyses (performed because two subjects administered inhalations incorrectly), the ratio of adjusted geometric means (maximum plasma concentration and area under the curve) was closer to unity than in the planned analyses. Cumulative urinary UMEC excretion (Ae) was similar for FF/UMEC and UMEC. Post hoc sensitivity analyses on Ae(0-24) suggested a small carryover effect but results were similar to those of the population as a whole. Urinary UMEC excretion following FF/UMEC was low (~1.5% over 24 h) and unlikely to have impacted upon PK comparisons. Three adverse events were reported; none were severe or led to withdrawal. There were no clinically significant effects on electrocardiogram, vital sign, or laboratory parameters. CONCLUSION: Fluticasone furoate and umeclidinium co-administration was well tolerated and was not associated with meaningful changes in systemic or urinary PK versus the monotherapies. FUNDING: GlaxoSmithKline.

Liquid chromatography-electrospray quadrupole linear ion trap mass spectrometry method for the quantitation of palonosetron in human plasma and urine: Application to a pharmacokinetic study.

The new analytical method for the determination of palonosetron in human plasma and urine has been developed based on liquid chromatography-mass spectrometry. The method utilized tramadol as the internal standard (IS). Separation was carried out on a Zorbax Eclipse TC-C(18) column using methanol-1mM ammonium formate in water (containing 0.1% formic acid, v/v, pH=2.8) as mobile phase for gradient elution. Detection is carried out by multiple reaction monitoring (MRM) on 3200 Qtrap mass spectrometry. The method has a chromatographic run time of 5.5 min and is linear within the concentration range 0.01-5.00 ng/mL for plasma and 0.10-30.00 ng/mL for urine both with a LOD of 0.003 ng/mL. Intra- and inter-day RSD of the concentration was 3.66-6.60%, 1.29-7.71% for plasma and 2.39-5.76%, 2.06-7.13% for urine. The relative error (RE) was -4.58% to 3.26% for plasma and -1.47% to 2.53% for urine. The recovery rates of palonosetron and IS both for plasma and urine were more than 90%. Palonosetron was stable under all the conditions tested. The method was successfully used to analyze palonosetron in human plasma and urine over a period of 168 h after intravenously pumping a single dose of 0.25mg to volunteers. No significant differences were found between the pharmacokinetic parameters and urine accumulated excretory rate for male and female volunteers (P>0.05). A two-compartment model was obtained after administrations. Palonosetron was eliminated at a slow rate in volunteers. The mean urine accumulated excretory rate was 25.97 ± 12.87%. Inter-individual differences could not be neglected due to the high coefficient of variety in several pharmacokinetic parameters and the urine accumulated excretion.

Overcoming non-specific adsorption issues for AZD9164 in human urine samples: consideration of bioanalytical and metabolite identification procedures.

A key challenge in the development of robust bioanalytical methods, for the determination of drug analyte in human urine samples, is the elimination of potential analyte losses as a result of non-specific adsorption to container surfaces in which the samples are collected, stored or processed. A common approach to address adsorption issues is to treat the urine samples with additives that serve to increase analyte solubility and/or minimise interaction with the container surfaces. A series of adsorption experiments were performed on human urine samples containing an adsorption-prone in-house development compound (AZD9164). A roller-mixing methodology was employed to maximise sample interaction with container surfaces and quantification of analyte was performed by LC-MS/MS following minimal sample preparation. In the absence of any urine additive, adsorptive losses averaged 35% but were highly variable between different lots of urine. In the presence of a range of additives, including the surfactants Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) and sodium dodecylbenzenesulphonate (SDBS), analyte adsorption was shown to be eliminated. Of particular academic interest was the finding that adsorptive losses could also be reduced upon the addition of phospholipid. The presence of additive generally had no marked impact on the analyte MS response but the use of an isotopically labelled internal standard satisfactorily compensated for instances in which ion suppression was observed, e.g. in the presence of Tween 80. Since metabolite profiling/identification investigations are often performed on urine samples originating from early clinical pharmacology studies, the elution of selected additives was also monitored by MS. CHAPS, dimethylacetamide (DMA) and HP-ß-cyclodextrin eluted as single chromatographic peaks in, or just after, the column void volume whilst polymeric Tween 80, and to a lesser extent SDBS, eluted over a wide retention time window. The potential of the latter surfactants to obscure the detection of unknown metabolites is significant and therefore their use in urine samples, upon which metabolite investigations are to be performed, is not recommended. Upon consideration of other factors such as additive cost and toxicity, CHAPS was selected for use in development of the validated assay.

Determination of palonosetron in human urine by LC-MS/MS.

BACKGROUND: Palonosetron is used for the prevention of chemotherapy-induced nausea and vomiting. However, quantification of this drug in human urine has been rare. RESULTS: A one-step dilution method for the analysis of palonosetron in human urine using LC coupled to positive MS/MS has been developed and validated according to US FDA guidelines. The method uses 200 µl of urine and covers a working range from 2.5-1000 ng/ml with a LLOQ of 2.5 ng/ml. CONCLUSION: This new LC-MS/MS assay is sensitive and specific despite using an external standard method. It is suitable for clinical studies of palonosetron.

Multiple species metabolism of PHA-568487, a selective alpha 7 nicotinic acetylcholine receptor agonist.

The quinuclidine PHA-0568487(1) is an agonist of the alpha 7 nicotinic acetylcholine receptor that was designed to mitigate the bioactivation associated with the core scaffold and subsequently remove associated liabilities with in vivo tolerability. The drug metabolites of 1 in nonclinical species were identified in plasma and urine of rats, dogs and monkeys receiving oral administrations of 1. The in vitro biotransformation of 1 was subsequently investigated in multiple species employing cryopreserved hepatocytes, hepatic subcellular fractions and recombinantly-expressed human P450 enzymes. In addition, in vitro metabolism of synthetically prepared metabolite precursors were instrumental in the elucidation of several secondary metabolites. The results indicated that the principal biotransformation of 1 was oxidation of the benzo[1,4]dioxane moiety (M8, M10) followed by subsequent oxidation to a range of secondary metabolites (M1-7, M9, M11, M13-15, and M17-18). The carboxylic acids M1 and M2 resulting from the oxidative cleavage of the dioxane ring were the principal metabolites observed in the plasma, urine and hepatocyte incubations across all species (M1 & M2). Quinuclidine oxidation was another pathway of importance, yielding an N-oxide (M12) which was also observed in all species.P450 2D6 and FMO1 catalyze the oxidation of the quinuclidine nitrogen. The N oxidation of the quinuclidine moiety is consistent with previously published accounts of this scaffold's metabolism and, interestingly, may implicate the uncommon quinuclidine moiety as an entity directing the metabolism of this scaffold (e.g., 1) via FMO1 and P450 2D6 oxidation.

Structural elucidation of in vivo metabolites of penehyclidine in rats by the method of liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and isotope ion cluster.

The structural elucidation of metabolites of penehyclidine in rats, a novel anti-cholinergic drug, by the method of liquid chromatography-electrospray ionization mass spectrometry, gas chromatography-mass spectrometry with electron impact ion source and stable isotope ion cluster was described. Identification and elucidation of the phase I and phase II metabolites were performed by comparing the daughter ion pairs of stable isotope cluster, changes of the protonated molecular masses, full scan MS(n) spectra and retention times with those of the parent drug, penehyclidine and penehyclidine deuterium-labeled. Penehyclidine was easily biotransformed by the pathways of oxidative, hydroxylated, methoxylated and phase II conjugated reactions to form several metabolites that retained the some features of the parent molecules. Twelve metabolites (penehyclidine monoxide, hydroxypenehyclidine, penehyclidine dioxide, hydroxypenehyclidine monoxide, dihydroxypenehyclidine, dihydroxypenehyclidine (position isomer), dihydroxypenehyclidine monoxide, trihydroxypenehyclidine, methoxypenehyclidine dioxide, dimethoxypenehyclidine, trihydroxymethoxypenehyclidine and glucuronide conjugated hydroxypenehyclidine) were identified. The results from electrospray ion and electron impact ion data with the stable isotope cluster showed the qualitative differences in the mass spectral patterns, suggesting that these technologies should be used in parallel to ensure comprehensive metabolites detection and characterization. The described method has wide applicability to rapidly screen and provide structural information of metabolites.

Metabolism and disposition of a selective alpha(7) nicotinic acetylcholine receptor agonist in humans.

The metabolism and disposition of N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide (1), an alpha(7) nicotinic acetylcholinergic receptor agonist, were elucidated in humans (4 female, 4 male; all white) after an oral dose of [(3)H]1. Overall, 1 was well tolerated, with >94% of administered radioactivity excreted renally by 48 h postdose; lyophilization of all urine and plasma samples confirmed (3)H stability within [(3)H]1. Across genders, 1 underwent low-to-moderate oral clearance comprising both renal (67%) and metabolic (33%) components, with the biotransformation of 1 occurring predominantly via oxidation of its furanopyridine moiety to carboxylic acid 2, and minimally by modification of its quinuclidine nitrogen to N-oxide 4 or N-glucuronide M5. Experiments using human in vitro systems were undertaken to better understand the enzyme(s) involved in the phase 1 biotransformation pathways. The formation of 2 was found to be mediated by CYP2D6, a polymorphically expressed enzyme absent in 5 to 10% of white people, whereas the generation of 4 was catalyzed by CYP2D6, FAD-containing monooxygenase 1 (FMO1), and FMO3. It is of interest that, although no overall gender-related differences in excretory routes, mass recoveries, pharmacokinetics, or metabolite profiles of 1 were evident, the observation of one of eight subjects (13%) showing disparate (relative to all other volunteers) systemic exposures to 1, and urinary and plasma quantitative profiles nearly devoid of 2 with the highest levels of 1, seem consistent with both the identification of CYP2D6 as the only major recombinant cytochrome P450 transforming 1 to 2 and the demographics of white CYP2D6 poor metabolizers. Data also reported herein suggest that 4 is generated predominantly by renal FMO1 in humans.

Pharmacokinetics and metabolism of radiolabelled SNI-2011, a novel muscarinic receptor agonist, in healthy volunteers. Comprehensive understanding of absorption, metabolism and excretion using radiolabelled SNI-2011.

The pharmacokinetics and metabolism of SNI-2011 ((+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine]monohydrochloride hemihydrate, cevimeline, CAS 153504-70-2), a novel muscarinic acetylcholine receptor agonist developed for the treatment of Sjögen's syndrome, were investigated in six healthy volunteers after a single oral administration of 14C-SNI-2011. After administration, plasma concentrations of the radioactivity and SNI-2011 reached to Cmax at approximately 2 h, and then decreased with t 1/2 of 9 and 4 h, respectively. Cmax and AUC0-infinity of the radioactivity in plasma were 2.2 and 5.0 times higher than those of SNI-2011, respectively. The main excretion route of the radioactivity was urine, and 97.3% of the dose excreted in urine within 168 h, indicating that 14C-SNI-2011 was completely absorbed. The mean recoveries of the metabolites in urine at 24 h after administration were 16.0% for SNI-2011, 35.8% for SNI-2011 trans-sulfoxide (SNI-t-SO), 8.7% for SNI-2011 cis-sulfoxide, 4.1% for SNI-2011 N-oxide, furthermore, two unknown metabolites, UK-1 and UK-2, were detected 14.6% and 7.7%, respectively. LC/MS analysis and hydrolysis studies revealed that UK-1 and UK-2 were glucuronic acid conjugates of SNI-2011 and SNI-t-SO, respectively.

[Identification of the metabolites of penehyclidine hydrochloride raceme in rats by LC-MS/MS and ion cluster].

AIM: To study the metabolites of penehyclidine hydrochloride (PH) raceme, a new anticholinerigic drug invented by the Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences. METHODS: Three healthy rat urine samples were collected within 24 h after a single i.m. dose of PH raceme and PH-d5 [(5 + 5) mg.kg-1] simultaneously. The eight metabolites of PH raceme were identified by the methods of LC-MS/MS, GC-MS, FAB-MS and the stable isotope ion cluster. Mass spectrometry was operated in the positive mode for the method of LC-MS/MS. RESULTS: M1 and M1* were identified as the oxygenated products of PH in the cyclopentyl group; M2 and M2* were as the hydroxylated products of PH in the cyclopentyl group; M3 and M3* were as the oxygented and hydroxylated products of PH at the meta-position of cyclopentyl group; M4 and M4* were identified as the dihydroxylated metabolites of PH, the hydroxylated position were at the cyclopentyl group and quiniuclidinol ring of PH. Among them, M1 and M1*, M2 and M2*, M3 and M3*, M4 and M4* were the isomers of each other. CONCLUSION: These characteristics can be used for future structure elucidation in studies of the metabolites of PH optical isomers. The structure data of PH metabolites provide important information for the clinical use and for developing better anticholinerigic drug.

Pharmacokinetics of the M1-agonist talsaclidine in mouse, rat, rabbit and monkey, and extrapolation to man.

1. Talsaclidine is an M1-agonist under development for the treatment of Alzheimer's disease. The aim of the study was to investigate the absorption, distribution, metabolism and excretion (ADME) of single intravenous and oral doses of [14C]-talsaclidine in mouse, rat, rabbit and monkey. Previous data in humans showed that the drug was mainly excreted into the urine as the unchanged parent drug. The hypothesis was tested if animal data of drugs, which are mainly excreted renally, could be extrapolated to human. 2. The apparent volume of distribution at steady-state (V(ss)) was comparable in all animal species (2-5 l x kg(-1)) indicating an extensive distribution of the drug into tissues. The plasma protein binding was low and comparable in all species including man (< or = 7%). Elimination in terms of clearance was rapid-to-moderate depending on the species. The total plasma clearance (Cl) decreased in the order: mouse (128 ml x min(-1) x kg(-1))> rat (73.9) > monkey (10.6). Urinary excretion is the dominant route of excretion (> or = 86%). 3. A good correlation was achieved with human and animal data in allometric scaling of CI and V(ss). This confirms the hypothesis that renal filtration is scalable over the species and, given a comparable protein binding, animal data is predictive for man.

[Biotransformation of 10-(3-quinuclidinylmethyl)phenothiazine (LM 209), a new anti-allergy agent and the distribution and excretion of its metabolites]. / Biotransformation de la (quinuclidinyl-3 méthyl)-10 phénothiazine (LM 209), un nouvel anti-allergique, et distribution et excrétion des métabolites

1. Metabolism of 10-(3-quinuclidinylmethyl)phenothiazine and the distribution and excretion of the metabolites, especially the sulphoxides, were studied in rat and dog after oral and intravenous administration. 2. Urine and bile contained relatively little unchanged drug. The sulphoxide, sulphone, N-oxide and N-oxide sulphoxide derivatives were identified as well as glucuronide and sulphate conjugates, suggesting the formation of hydroxylated products. Faeces contained mainly unchanged drug but also some sulphoxide and N-oxide. In the lung, brain and cerebro spinal fluid only unchanged drug and traces of sulphoxide were found, whereas in liver sulphone and N-oxide were also present. 3. After administration of the 35S-labelled sulphoxide, the distribution of radio-activity was very different from that observed with LM 209. The biological half-life (t0-5) of SM 209 was 3 to 4 times higher than that of the sulphoxide. LM 209 is better absorbed and its diffusion in the organism is superior. 4. Metabolism of LM 209 by liver microsome preparation was more rapid than metabolism of the sulphoxide. 5. These findings indicate that the activity of LM 209 is due more to the molecule itself than to its major metabolite.