Determination of blood concentration of higenamine by high pressure liquid chromatography

A procedure utilizing high pressure liquid chromatography coupled with UV detection is described for the determination of blood concentration of higenamine. Deproteinized serum was pretreated with C₁₈ (Sep-pak C₁₈ cartridge) and the 70% EtOH eluent was applied onto a reversed-phase column (μ Bondapak C₁₈) with a 15% acetonitrile in 0.05 N NaH₂ PO₄-trichloroacetic acid mixed buffer (pH 2.8) as a mobile phase. With the UV detection at 232 nm, the retention times of higenamine and 1,2,3,4-tetrahydropapaveroline, an internal standard, were 5.2 min and 3.9 min respectively. The blood concentration of higenamine was measured at regular intervals afteri.v. injection of higenamine to rabbit. A drastic decrease in higenamine concentration to 30% of the maximum value obtained immediately after the injection, was observed during the first 1–2 min period and thereafter the rate of decrease was slowed down. The analytical result seemed to coincide with the pharmacological effect of higenamine exerting the maximum chronotropic and hypotensive effect at the completion of the injections which were progressively recovered.     

A phase I study on pharmacokinetics and pharmacodynamics of higenamine in healthy Chinese subjects

Aim:

To investigate the pharmacokinetics, pharmacodynamics, and safety of higenamine, an active ingredient of Aconite root, in healthy Chinese volunteers.

Methods:

Ten subjects received continuous, intravenous infusion of higenamine at gradually escalating doses from 0.5 to 4.0 μg·kg⁻¹·min⁻¹, each dose was given for 3 min. Blood and urine samples were collected at designated time points to measure the concentrations of higenamine. Pharmacodynamics was assessed by measuring the subject''s heart rate. A nonlinear mixed-effect modeling approach, using the software Phoenix NLME, was used to model the plasma concentration-time profiles and heart rate.

Results:

Peak concentrations (C_max) of higenamine ranged from 15.1 to 44.0 ng/mL. The half-life of higenamine was 0.133 h (range, 0.107–0.166 h), while the area under concentration-time curve (AUC), extrapolated to infinity, was 5.39 ng·h·mL⁻¹ (range, 3.2-6.8 ng·h·mL⁻¹). The volume of distribution (V) was 48 L (range, 30.8–80.6 L). The total clearance (CL) was 249 L/h (range, 199-336 L/h). Within 8 h, 9.3% (range, 4.6%–12.4%) of higenamine was recovered in the urine. The pharmacokinetics of higenamine was successfully described using a two-compartment model with nonlinear clearance. In the pharmacodynamic model, heart rates were related to the plasma drug concentrations using a simple direct effect model with baseline. The E₀, E_max, and EC₅₀ were 68 bpm, 73 bpm and 8.1 μg/L, respectively.

Conclusion:

Higenamine has desirable pharmacokinetic and pharmacodynamic characteristics. The results provide important information for future clinical studies on higenamine.     

Excretion of mephedrone and its phase I metabolites in urine after a controlled intranasal administration to healthy human volunteers

Mephedrone is a stimulant drug structurally related to cathinone. At present, there are no data available on the excretion profile of mephedrone and its metabolites in urine after controlled intranasal administration to human volunteers. In this study, six healthy male volunteers nasally insufflated 100 mg of pure mephedrone hydrochloride (Day 1). Urine was collected at different timepoints on Day 1 and then on Days 2, 3 and 30. Samples were analysed for the presence of mephedrone and its metabolites, namely, dihydro-mephedrone, nor-mephedrone (NOR), hydroxytolyl-mephedrone, 4-carboxy-mephedrone (4-carboxy) and dihydro-nor-mephedrone (DHNM), by a validated liquid chromatography-tandem mass spectrometry method. All analytes were detected in urine, where 4-carboxy (C_max = 29.8 μg/ml) was the most abundant metabolite followed by NOR (C_max = 377 ng/ml). DHNM was found at the lowest concentrations (C_max = 93.1 ng/ml). Analytes exhibited a wide range of detection windows, but only 4-carboxy and DHNM were detectable in all samples on Day 3, extending the detection time of mephedrone use. Moreover, mephedrone had a mean renal clearance of 108 ± 140 ml/min, and 1.3 ± 1.7% of unchanged parent drug was recovered in urine in the first 6 h post administration. It is hoped that this novel information will be useful in future studies involving mephedrone and other stimulant drugs.     

Disposition of the opioid antagonist,nalmefene, in rat and dog

1. The disposition of nalmefene in rat and dog was studied using in vitro and in vivo methodology. In vitro metabolite profiles were obtained following incubation of nalmefene with liver microsomes and biological fluids were assayed to profile in vivo metabolites. Characterization of metabolites was accomplished using hplc, co-chromatography with synthetic standards, or LC/MS.

2. In rat, tissue distribution and metabolite plasma concentration-time data were obtained following intravenous bolus dosing of nalmefene.

3. The results indicate that the primary phase I metabolite of nalmefene from liver microsome incubations was the N-dealkylated metabolite, nornalmefene. Quantitative metabolite production was rat ? dog. In vivo, nornalmefene glucuronide was the major metabolite in rat urine, whereas nalmefene glucuronide(s) were predominant in dog urine.

4. More than 90% of the radioactive dose was recovered in the rat excreta and tissues 24?h after an intravenous bolus dose of ¹⁴C-nalmefene, with no apparent organ-specific retention of radioactivity.

5. Pharmacokinetic analysis of the rat plasma metabolite data indicated that terminal half-lives for nalmefene and nornalmefene were comparable (～ 1?h). However, C_max and AUC of nornalmefene were ≤ 7% that of corresponding nalmefene values.     

Disposition and bioavailability of the β1-adrenoceptor antagonist talinolol in man

In an open randomized crossover study, the pharmacokinetics and bioavailability of the selective β₁-adrenoceptor antagonist talinolol (Cordanum®—Arzneimittelwerk Dresden GmbH, Germany) were investigated in twelve healthy volunteers (five female, seven male; three poor and nine extensive metabolizers of the debrisoquine hydroxylation phenotype) after intravenous infusion (30 mg) and oral administration (50 mg), respectively. Concentrations of talinolol and its metabolites were measured in serum and urine by HPLC or GC-MS. At the end of infusion a peak serum concentration (C_max) of 631 ± 95 ng mL^?1 (mean ± SD) was observed. The area under the serum concentration-time curve from zero to infinity (AUC_0-∞) was 1433 ± 153 ng h mL^?1. The following parameters were estimated: terminal elimination half life (t_1/2), 10.6 ± 3.3 h; mean residence time, 11.6 ± 3.1 h; volume of distribution, 3.3 ± 0.5 L kg^?1; and total body clearance, 4.9 ± 0.6 mL min^?1 kg^?1. Within 36 h 52.8 ± 10.6% of the administered dose was recovered as unchanged talinolol and 0.33 ± 0.18% as hydroxylated talinolol metabolites in urine. After oral administration a C_max of 168 ± 67 ng mL^?1 was reached after 3.2 ± 0.8h. The AUC_0-∞ was 1321 ± 382 ng h mL^?1. The t_1/2 was 11.9 ± 2.4 h. 28.1 ± 6.8% of the dose or 55.0 ± 11.0% of the bioavailable talinolol was eliminated as unchanged talinolol and 0.26 ± 0.17% of the dose as hydroxylated metabolites by kidney. The absolute bioavailability of talinolol was 55 ± 15% (95% confidence interval, 36–69%). Talinolol does not undergo a relevant first-pass metabolism, and its reduced bioavailability results from incomplete absorption. Talinolol disposition is not found to be altered in poor metabolizers of debrisoquine type.     

PHARMACOKINETICS OF HIGENAMINE IN RABBITS

The pharmacokinetics of higenamine were investigated in rabbits by IV bolus, PO route, and IV infusion. Plasma higenamine concentration declined rapidly in a biexponential pattern, with a terminal half-life of 22 min. The AUC increased proportionally with increasing dose, whereas the percentage of unchanged higenamine excreted from urine remained constant when dose was increased. The means of total body clearance, mean residence time, volume of distribution at steady state, and fraction of urinary excretion were 127·7 mL min⁻¹ kg⁻¹, 9·28 min, 1·44 L kg⁻¹, and 5·48%, respectively. The mean percentage of protein binding of higenamine in plasma was 54·8% at steady state after IV infusion. The results from post-infusion also confirmed that higenamine followed a two-compartment open model in animals. After oral administration, higenamine was rapidly absorbed to reach peak concentration within 10 min. Interestingly, the plasma concentration–time profiles revealed two distinguishable groups with different C_max, extent of absorption, and urinary excretion. The average absolute bioavailabilities of higenamine calculated by AUCs and accumulated urinary excretion were 21·86 and 2·84% versus 20·19 and 5·50% for the two groups, respectively. Upon hydrolysis of urine samples with μ-glucuronidase, urinary concentrations of higenamine were greatly enhanced in both groups     

Bioavailability of progesterone from rectal dosage forms in rabbits

The systemic availability of progesterone in two rectal dosage forms was investigated in rabbits. The progesterone plasma concentration was determined as total radioactivity (progesterone and its metabolites) after a single dose of 5 mg/kg [³H]-labelled progesterone in an adeps solidus suppository (1) and in a propylene glycol enema (2) given in a randomized cross-over fashion. The equivalent dose was given intravenously (3) to the same rabbits.The maximum plasma concentration (C_max) after (1) was 0.82 ± 0.43 μg/ml and significantly lower than after (2), which was 3.81 ± 1.08 μg/ml (mean ± S.E.; P < 0.05). The time to reach the maximum plasma concentration (T_max) was for (1) 1.75 ± 0.25 h and for (2) 0.56 ± 0.32 h (mean ± S.E.; P < 0.05). The mean plasma concentration vs time curve after (3) indicates that a muhicompartment system is involved in the disposition of progesterone. The plasma half-life (t_{$\text{1}\text{2}$}) estimated from 0–6 h was 5.10 ± 1.12 (mean ± S.E.).The systemic availability (F%) of (1) from 0–6 h (AUC_0–6h) was 14 ± 8% and significantly lower than that of (2), which was 44 ± 10% (mean ± S.E.; P < 0.05).The results indicate a delayed and possibly lower absorption of progesterone from the suppositories as compared to the enema.     

Effects of chlorpromazine and atropine on acetaminophen absorption in rabbits

The effects of chlorpromazine and atropine on the gastrointestinal absorption of acetaminophen were investigated in rabbits. Two doses of chlorpromazine and atropine were injected intraperitoneally 30 min prior to the oral administration of acetaminophen. Blood samples were collected before and 0.25,0.5,0.75, 1.0,1.5,2,3,4,5 and 6 h after acetaminophen administration and were analyzed for acetaminophen contents using a HPLC method. Chlorpromazine at a 10 mg/kg dose significantly reduced the maximum plasma concentration (C_max) of acetaminophen from 64.2 ± 2.8 to 40.0 ± 6.3 μgg/ml(P < 0.05). In addition, chlorpromazine at 5 and 10 mg/kg doses significantly increased the time taken to reach the maximum plasma concentration (T_max) of acetaminophen from 0.29 ± 0.04 to 0.67 ± 0.15 and 0.96 ± 0.21h, respectively (P < 0.05). Atropine at 0.5 and 1.0 mg/kg doses also significantly reduced the C_max of acetaminophen from 69.6 ± 4.7 to 45.6 ± 3.7 and 45.9 ± 6.7 μg/ml, respectively (P < 0.05). However, atropine has little effect on T_max of acetaminophen. Both chlorpromazine and atropine did not seem to affect the area under the plasma concentration-time curve and the elimination half-life of acetaminophen. It was concluded that chlorpromazine and atropine affect the rate but not the extent of acetaminophen absorption, by delaying the gastric emptying.     

Rapid determination of isocorydine in rat plasma and tissues using liquid chromatography – tandem mass spectrometry and its applications to pharmacokinetics and tissue distribution

1. A rapid and sensitive method for the determination of isocorydine in rat plasma and tissues was developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS).

2. The biological samples were processed by extracting with diethyl ether–dichloromethane (3:2, v/v) and tetrahydropulmatine was used as the internal standard (IS). Detection of the analytes was achieved using positive ion mode electrospray ionization in the multiple reaction monitoring mode. The MS/MS ion transitions monitored were m/z 342.0→279.0 and 356.0→191.9 for isocorydine and IS, respectively.

3. The maximum plasma concentration (C_max 2496.8?±?374.4 µg/L) was achieved at 0.278?±?0.113?h (T_max) and the half-life (t_1/2) of isocorydine was 0.906?±?0.222?h after a 20?mg/kg oral administration. As for a 2?mg/kg intravenous (i.v.) administration, the C_max and clearance (CL) were 1843.3?±?338.3 µg/L and 2.381?±?0.356?L/h/kg, respectively. Based on the AUC_0–∞ obtained from oral and i.v. administration, the absolute bioavailability (F) was estimated as 33.4%. Tissue distribution results indicated that isocorydine underwent a rapid and wide distribution into tissues and it could effectively cross the blood-brain barrier.

     

Quantitation of DNA reactive pyrrolic metabolites of senecionine – A carcinogenic pyrrolizidine alkaloid by LC/MS/MS analysis

Pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals, inducing liver tumors in experimental rodents. We previously determined that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), 7-glutathione-DHP, 7-cysteine-DHP, 7-N-acetylcysteine-DHP, and 1-CHO-DHP are DNA reactive pyrrolic metabolites potentially associated with PA-induced liver tumor initiation. In this study, we developed an LC/MS/MS multiple reaction monitoring (MRM) mode method to identify and quantify these metabolites formed from the metabolism of senecionine, a carcinogenic PA, by mouse, rat, and human liver microsomes, and primary rat hepatocytes. Together with the chemically prepared standards of these metabolites, this represents an accurate and convenient LC/MS/MS analytical method for quantifying these five reactive pyrrolic metabolites in biological systems.     

Functional UDP-glucuronyltransferase 2B15 polymorphism and bisphenol A concentrations in blood: results from physiologically based kinetic modelling

Bisphenol A (BPA) is a chemical in widespread use that is under scientific discussion due to its endocrine activity. Controversies exist about how to interpret reportedly high blood concentrations measured in uncontrolled situations. Physiologically based pharmaco-/toxicokinetic modelling resulted in 10–100-fold lower blood concentrations than those reported. Moreover, in controlled situations, BPA did not exceed the level of detection (<0.3 ng/ml) in human blood or urine. Using a validated human PBK model, this study investigated the influence of functionally relevant polymorphic UGT2B15, the enzyme mediating BPA metabolism, on the BPA concentration–time profile in human blood. Maximum concentrations (C _max) and areas under the curves (AUCs) in blood from high and low metabolisers differed by a factor of 4.7 and 4.6, respectively (doses: 1 and 0.05 μg/kg/day). Low metabolisers excreted a greater proportion of BPA via the sulphate pathway compared to high metabolisers. This finding explains why C _max and AUC increased to a smaller extent, as predicted from in vitro data obtained with transfected cells possessing only the UGT2B15 variants. The highest C _max value calculated in the subject with the lowest metabolic clearance was roughly 40 pg/ml, which is far lower than reported high blood concentrations, which in turn cannot be explained by genetically impaired UGT2B15 activity. From the risk assessment perspective, our results indicate that the traditional uncertainty factor is sufficient to account for the variability in the polymorphic glucuronidation of BPA.     

Detection of lithospermate B in rat plasma at the nanogram level by LC/MS in multi reaction monitoring mode

Low bioavailability and high binding affinity to plasma proteins led to the difficulty for the quantitative detection of lithospermate B (LSB) in plasma. This study aimed to develop a protocol for detecting LSB in plasma. A method was employed to quantitatively detect LSB of 5–500 ng/mL by LC/MS spectrometry in multi reaction monitoring mode via monitoring two major fragments with m/z values of 519 and 321 in the MS2 spectrum. To set up an adequate extraction solution to release LSB captured by plasma proteins, recovery yields of LSB extracted from rat plasma acidified by formic acid or HCl in the presence or absence of EDTA and caffeic acid were detected and compared using the above quantitative method. High recovery yield (～90%) was achieved when LSB (5–500 ng/mL) mixed in rat plasma was acidified by HCl (5 M) in the presence of EDTA (0.5 M) and caffeic acid (400 μg/mL). The lower limit of detection and the lower limit of quantification for LSB in the spiked plasma were calculated to be 1.8 and 5.4 ng/mL, respectively. Good accuracy (within ±10%) and precision (less than 10%) of intra- and inter-day quality controlled samples were observed. Oral bioavailability of LSB in rat model was detected via this optimized extraction method, and the maximum plasma concentration (C_max) was found to be 1034.3 ± 510.5 μg/L at t_max around 10 min, and the area under the plasma concentration–time curve (AUC) was 1414.1 ± 851.2 μg·h/L.     

Isolation, identification and antiviral activities of metabolites of calycosin-7-O-β-D-glucopyranoside

In vivo and in vitro metabolites of calycosin-7-O-β-d-glucopyranoside in rats were identified using a specific and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MSⁿ) method. The parent compound and twelve metabolites were found in rat urine after oral administration of calycosin-7-O-β-d-glucopyranoside. The parent compound and six metabolites were detected in rat plasma. In heart, liver, spleen, lung and kidney samples, respectively, six, eight, seven, nine and nine metabolites were identified, in addition to the parent compound. Three metabolites, but no trace of parent drug, were found in the rat intestinal flora incubation mixture and feces, which demonstrated cleavage of the glycosidic bond of the parent compound in intestines. The main phase I metabolic pathways of calycosin-7-O-β-d-glucopyranoside in rats were deglycosylation, dehydroxylation and demethylation reactions; phase II metabolism included sulfation, methylation, glucuronidation and glycosylation (probably). Furthermore, two metabolites commonly found in rat urine, plasma and tissues were isolated from feces and characterized by NMR. The antiviral activities of the metabolite calycosin against coxsackie virus B₃ (CVB₃) and human immunodeficiency virus (HIV) were remarkably stronger than those of calycosin-7-O-β-d-glucopyranoside.     

(±)-Govadine and (±)-THP,Two Tetrahydroprotoberberine Alkaloids,as Selective α1-Adrenoceptor Antagonists in Vascular Smooth Muscle Cells

(±)-Govadine and (±)-THP ((±)-2,3,10,11-tetrahydroxytetrahydroprotoberberine HBr) have been shown to inhibit noradrenaline-induced contraction of rat thoracic aortae. The pharmacological activity of the compounds was determined in thoracic aortae and cardiac tissue isolated from the rat and in trachea isolated from the guinea-pig to determine the selectivity of the compounds towards different types of receptor. (±)-Govadine and (±)-THP were found to be α₁-adrenoceptor blocking agents in rat thoracic aorta as revealed by their competitive antagonism of vasoconstriction induced by noradrenaline (pA₂ = 6.57 ± 0.07 and 5.93 ± 0.06, respectively) or phenylephrine (pA₂ = 6.74 ± 0.08 and 6.06 ± 0.10, respectively). Removal of endothelium did not affect the antagonistic potencies of (±)-govadine (pA₂ = 6.83 ± 0.09) and (±)-THP (pA₂ = 6.25 ± 0.06) on phenylephrine-induced vasoconstriction. They were more potent than yohimbine (pA₂ = 6.05 ± 0.05), but less so than phentolamine (pA₂ = 7.54 ± 0.11) and prazosin (pA₂ = 9.27 ± 0.12). (±)-Govadine and (±)-THP, furthermore, inhibited [³H]inositol monophosphate formation caused by noradrenaline (3 μm ) in rat thoracic aorta. (±)-Govadine and (±)-THP were also α₂-adrenoceptor blocking agents with pA₂ values 5.50 ± 0.13 and 5.41 ± 0.11, respectively. A high concentration of (±)-govadine (30 μm ) or (±)-THP (30 μm ) did not, however, affect the contraction induced by the thromboxane receptor agonist U46619, prostaglandin F_2α (PGF_2α), 5-hydroxytryptamine (5-HT), angiotensin II, endothelin or high K⁺ in rat aorta denuded of endothelium. Neither the cyclic AMP nor cyclic GMP content of rat thoracic aorta was, furthermore, changed by (±)-govadine or (±)-THP. Contraction of guinea-pig trachea caused by carbachol, histamine, leukotriene C₄ or neurokinin A was not affected by (±)-govadine or (±)-THP. (±)-Govadine or (±)-THP also did not block β₁- or β₂-adrenoceptor-mediated responses induced by isoprenaline in rat right atria and guinea-pig trachea. It is concluded that (±)-govadine and (±)-THP are selective α₁-adrenoceptor antagonists in vascular smooth muscle.     

NMR spectroscopic studies on the metabolism and futile deacetylation of phenacetin in the rat

1. ¹H-NMR spectroscopy of urine was used to determine the % deacetylation and re-acetylation of ²H-labelled (in the acetyl) phenacetin metabolites in the rat. 2. Male Sprague- Dawley rats were each dosed with either phenacetin or phenacetin C²H₈ at 50?mg kg^?1. The total urinary recoveries for phenacetin and phenacetin-C²H₈ were 47.6 ± 16.7 and 50.1 ± 16.2% respectively (not significantly different, p > 0.05). Paracetamol sulphate and glucuronide are the major urinary metabolites of both protio and deuteriophenace tin. 3. The futile deacetylationgiven by the urinary recovery of protio-acetyl metabolitesof phenacetin-C²H₈ was 29.6 ± 0.9% for paracetamol sulphate and 36.6 ± 3.1% for paracetamol glucuronide. These observations demonstrate a high level of futile deacetylation in the paracetamol conjugates formed by metabolism of phenacetin-C²H₈ and this may indicate a high metabolic flux through the nephrotoxic intermediate 4-aminophenol. 4. The level of futile deacetylation for phenacetin was significantly higher than that found previously in studies of labelled paracetamol in rat or man, and may be important in understanding the higher nephrotoxicity of phenacetin as compared with paracetamol.     

Dermal pharmacokinetics of pyrazinamide determined by microdialysis sampling in rats

Studies have demonstrated the efficacy of pyrazinamide (PZA) against stages of the Leishmania parasite that causes cutaneous leishmaniasis. Although PZA is widely distributed in most body fluids and tissues, the amount of drug reaching the skin is unknown. This study aimed to investigate the pharmacokinetics of PZA in rat dermal tissue by dermal microdialysis. Skin pharmacokinetics was assessed by implanting a linear microdialysis probe in the dermis of ten rats. In addition, blood samples were collected to assess plasma pharmacokinetics. Unbound microdialysate (N?=?280) and plasma (N?=?120) concentrations following single intravenous doses of 25?mg/kg or 50?mg/kg PZA were quantified by a validated HPLC method. Probe calibration was performed by retrodialysis. Non-compartmental analysis and non-linear mixed-effects modelling were performed using WinNonlin and NONMEM v.7.3. PZA rapidly permeated into the dermis and reached high levels, with mean maximum concentrations (C_max) of 22.4?±?7.1?µg/mL and 48.6?±?17.3?µg/mL for the two doses studied. PZA showed significant distribution to the skin (fAUC_dermal/fAUC_plasma?=?0.82?±?0.31 and 0.84?±?0.25 for 25?mg/kg and 50?mg/kg doses, respectively). Active unbound concentrations in dermal tissue reached lower levels than free plasma concentrations, indicating that free PZA levels in plasma were in equilibrium with tissue levels. These results showed equivalent unbound drug tissue concentrations and corresponding unbound plasma levels. This study shows that PZA distributes rapidly into dermal interstitial fluid space in rats and therefore may be a potential agent in the treatment of cutaneous leishmaniasis.     

反相高效液相色谱法测定大鼠血浆中左旋黄皮酰胺及其主要代谢产物和药代动力学

目的　探讨左旋黄皮酰胺［（-）clau］及其主要代谢产物6-OH-clau在大鼠体内的药代动力学。方法　建立了RP-HPLC-UV法。固定相为Kromasil-100 C₁₈（5 μm）色谱柱,流动相为乙腈-甲醇-水（21∶16.5∶62.5), DM-9384作内标,氯仿作提取溶剂。结果　测得（-）clau的回收率为96.91%～105.74%,日内、日间RSD低于7%,最低检测浓度为24 ng.mL^-1,（-）clau和6-OH-clau分别在0.047～968 μg.mL^-1和0.049～200 μg.mL^-1,线性关系良好(γ=0.999); （-）clau和6-OH-clau血浆浓度-时间曲线符合二室开放模型,同时求得两者的药代动力学参数。结论　数据表明（-）clau在大鼠血浆中的分布、代谢转化和消除均较快。     

Identification of in‐vivo and in‐vitro metabolites of palmatine by liquid chromatography–tandem mass spectrometry

Objectives Despite its important therapeutic value, the metabolism of palmatine is not yet clear. Our objective was to investigate its in‐vivo and in‐vitro metabolism. Methods Liquid chromatography–tandem electrospray ionization mass spectrometry (LC‐ESI/MSⁿ) was employed in this work. In‐vivo samples, including faeces, urine and plasma of rats, were collected after oral administration of palmatine (20 mg/kg) to rats. In‐vitro samples were prepared by incubating palmatine with intestinal flora and liver microsome of rats, respectively. All the samples were purified via a C₁₈ solid‐phase extraction procedure, then chromatographically separated by a reverse‐phase C₁₈ column with methanol–formic acid aqueous solution (pH 3.5, 70: 30 v/v) as mobile phase, and detected by an on‐line MSⁿ detector. The structure of each metabolite was elucidated by comparing its molecular weight, retention time and full‐scan MSⁿ spectra with those of the parent drug. Key findings The results revealed that 12 metabolites were present in rat faeces, 13 metabolites in rat urine, 7 metabolites in rat plasma, 10 metabolites in rat intestinal flora and 9 metabolites in rat liver microsomes. Except for six of the metabolites in rat urine, the other in‐vivo and in‐vitro metabolites were reported for the first time. Conclusions Seven new metabolites of palmatine (tri‐hydroxyl palmatine, di‐demethoxyl palmatine, tri‐demethyl palmatine, mono‐demethoxyl dehydrogen palmatine, di‐demethoxyl dehydrogen palmatine, mono‐demethyl dehydrogen palmatine, tri‐demethyl dehydrogen palmatine) were reported in this work.     

Metabolic disposition of tacrine in primary suspensions of rat hepatocyte and in single-pass perfused liver: in vitro/in vivo comparisons

1. Incubations of tacrine (1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate, THA) with a primary suspension of rat hepatocytes for 2 min resulted in formation of the 1-hydroxy derivative as the major metabolite with smaller amounts of the 2- and 4-hydroxy metabolites.

2. Apparent V_max and K_m for THA metabolism were 12·4 ± 3·3 nmol/min/g liver and 0·98 ± 0·34 μm respectively.

3. Incubations of THA for longer time-periods (>10 min) resulted in irreversible binding of THA-derived radioactivity to hepatocellular protein. The apparent maximal rate of irreversible binding (B_max) was 76·7 ± 30·5 pmol equivalents bound/h/mg cell protein, whereas the apparent K_b for binding was 2·8 ± 1·4μm.

4. The kinetic parameters, V_max and K_m, were used to predict steady-state extraction ratios (ER_ss) for various THA input concentrations (C_in) in single-pass perfused rat liver. At low input concentrations (0·72–0·85 μm; C_in < K_m), ER_ss of THA was approximately 1. For higher C_in (14·05, 20·72, 20·88 μm; C_in), the calculated ER_ss was markedly decreased with 0·300, 0·296 and 0·261, respectively.

5. The intrinsic clearance of THA (Cl_i) estimated from in vitro hepatocyte data was 6·7 ml/min/g liver while the apparent oral THA clearance (Cl_oral) calculated from in vivo rat data was 6·6 ml/min/g liver.     

Absorption,distribution, metabolism and excretion of a new, 14C-labelled oxazolidinone MAO-A inhibitor in rat and dog

1. After oral administration of ¹⁴C-labelled (5R)-3-\[2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011) at a dose of 1?mg/kg, the blood level of radioactivity reached a maximum concentration (C_max) of 0.545 μg eq./ml after 0.25?h in the rat and of 0.900 μg eq./ml after 0.5?h in the dog. In dog plasma, C_max for radioactivity and unchanged E2011 were 0.862 μg eq./ml and 0.650 μg/ml respectively with corresponding T_max (time at C_max) of 0.75 and 0.25?h. The unchanged drug in dog plasma was below the detection limit (5 ng/ml plasma) after 24?h. 2. The tissue levels of radioactivity were measured at 0.25 (T_max), 6, 24, and 168?h after max administration to the rat and at 0.5 (T_max), 24, and 168?h in the dog. The radioactivity was max distributed in all tissues examined at T_max in the rat and dog. The radioactivity levels of the cerebral cortex in the rat and dog were 26 and 36% of the plasma level at T_max. The radioactivity in tissues decreased at almost the same rate as that in plasma. Plasma protein binding of the unchanged drug in the rat in vitro were about 70% in the range of 0.1-10 μg/ml, and those in the dog were about 45% in the same concentration range. 3. Cumulative excretion of radioactivity in the rat was 74.5% in urine and 22.5% in faeces after 7 days. In the dog, 55.5 and 36.5% of the radioactivity administered were excreted in urine and faeces respectively after 7 days. The biliary excretion of radioactivity in the cannulated rat was 23.0% within 48?h. 4. In tlc analysis of plasma and tissues of the rat and dog, the radioactivity for the unchanged drug was much higher than metabolites. In tlc analysis of urine, the same metabolites were detected in the rat and dog, and the radioactivity of a metabolite, IM1, was the highest in the both animals. Eight metabolites were detected in the plasma, tissues and excreta of the rat, and four metabolites in the dog. 5. In conclusion, the absorption, distribution, metabolism and excretion of ¹⁴C-labelled E2011 in the rat and dog have been established, and only minor differences were observed between these species.