3-Quinuclidinyl benzilate (agent BZ) toxicokinetics in rats.

3-Quinuclidinyl benzilate (BZ) ranks among incapacitating military warfare agents. It acts as a competitive inhibitor on muscarinic receptors leading to non-lethal mental impairment. The present study aimed to investigate toxicokinetics of BZ in rats. Moreover, BZ can be exploited to produce a pharmacological model of Alzheimer's disease; thus, this paper focuses mainly on the BZ distribution to the brain. Wistar rats were administered i.p. with BZ (2 and 10 mg/kg). The BZ concentration was determined using LC-MS/MS in plasma, urine, bile, brain, kidney and liver. The sample preparation was based on a solid phase extraction (liquids) or protein precipitation (organ homogenates). The plasma concentration peaked at 3 min (204.5 ± 55.4 and 2185.5 ± 465.4 ng/ml). The maximal concentration in the brain was reached several minutes later. Plasma elimination half-life was 67.9 ± 3.4 in the 2 mg/kg group and 96.6 ± 27.9 in the 10 mg/kg group. BZ concentrations remained steady in the brain, with slow elimination (t1/2 506.9 ± 359.5 min). Agent BZ is excreted mainly via the urine. Steady BZ concentration in the brain could explain the previously published duration of the significant impairment in passive avoidance tasks in rats after an injection of BZ.

Urinary Excretion Contributes to Long-Lasting Blockade of Bladder Muscarinic Receptors by Imidafenacin: Effect of Bilateral Ureteral Ligation.

Imidafenacin is a potent and selective antagonist of M1 and M3 muscarinic receptors that is safe, efficacious, and well tolerated for controlling the symptoms of overactive bladder (OAB). However, the precise mechanisms responsible for the bladder-selective pharmacological effects of this agent remain unclear. The in vivo pharmacologic effects of imidafenacin result from receptor occupancy. Therefore, the present study was performed to characterize in vivo muscarinic receptor binding by tritium-labeled imidafenacin with high specific activity ([3H]imidafenacin) in the bladder and other tissues of mice, and to clarify the mechanisms underlying selective binding of imidafenacin to bladder muscarinic receptors. After intravenous injection of [3H]imidafenacin, its binding to muscarinic receptors in the bladder and other tissues of mice was assessed by a radioligand binding assay. [3H]Imidafenacin showed a significantly longer duration of binding to muscarinic receptors in the bladder than in other tissues, and muscarinic receptor binding of [3H]imidafenacin was markedly suppressed in the bladder alone after bilateral ligation of the ureters. After intravenous injection, the [3H]imidafenacin concentration was markedly higher in the urine than in the plasma, suggesting that urinary excretion may contribute significantly to the selective and long-lasting binding of imidafenacin to bladder muscarinic receptors. These findings suggest that the intravesicular concentration of an antimuscarinic agent and its active metabolites may have a substantial influence on its pharmacological effect and duration of action in patients with OAB. In addition, factors that modulate urine production may influence the efficacy and safety of antimuscarinic agents.

Molecularly imprinted photonic hydrogels for fast screening of atropine in biological samples with high sensitivity.

Based on molecularly imprinted photonic hydrogels (MIPHs) that combined the colloidal-crystal with molecular imprinting technique, a novel label-free colorimetric chemosensor for convenient and fast efficient detection of atropine with high sensitivity and specificity was developed. Due to the special inverse opal arrays with a thin polymer wall in which the imprinted nanocavities of atropine moleculars distributed, the proposed MIPHs designed as water-compatible exhibited high sensitive (as low as 1 pg/mL), rapid responsive (less than 30 s) and specific detection of atropine in complex matrix. The unique three-dimensional, highly-ordered photonic hydrogels would be obviously swelling in response to the specific atropine molecular recognition process and the response would be directly transferred into visually perceptible optical signal (change in color) that could be detected by the naked eye through Bragg diffractive shifts of ordered macroporous arrays. With a broad concentration range varying from 1 pg/mL to 1 µg/mL of atropine, the distinct color changes of MIPHs almost covered the whole visible-light wavelength range from blue to red for semi-quantitative analysis. The smart chemosensor was successfully employed to determine the trace level atropine in human urine samples, providing a fast and effective alternative for semi-quantitative detection of atropine for clinical analysis and forensic investigations.

Mass balance and metabolism of aclidinium bromide following intravenous administration of [¹4C]-aclidinium bromide in healthy subjects.

Aclidinium bromide is a novel, inhaled long-acting muscarinic antagonist with low systemic activity developed for the treatment of COPD. It is an ester compound rapidly hydrolysed in plasma into inactive alcohol and acid metabolites. In this Phase I, open-label study, the rates and routes of elimination of radioactivity following intravenous administration of [¹4C]-aclidinium bromide were determined. The metabolites of aclidinium were also characterized and identified in plasma and excreta. Twelve healthy males were randomized (1:1) to receive a single intravenous 400 µg dose of [phenyl-U-¹4C]- or [glycolyl-U-¹4C]-aclidinium bromide (via 5 min infusion) to label alcohol or acid metabolites of aclidinium, respectively. Safety and tolerability were assessed over a 9-day period. Following intravenous administration, the parent compound was rapidly hydrolysed into its acid and alcohol metabolites. Primary excretion routes for [phenyl-U-¹4C]- and [glycolyl-U-¹4C]-aclidinium were renal (urine: 65% and 54%, respectively; feces: 33% and 20%, respectively), with 1% excreted as unchanged aclidinium. A total of three treatment-emergent adverse events in two subjects were reported and were related to infusion site pain. Overall, aclidinium is rapidly hydrolysed into two main metabolites, which are predominantly excreted in urine. Aclidinium bromide 400 µg administered intravenously was safe and well tolerated in healthy subjects.

Specific pharmacokinetic aspects of the urinary tract.

This chapter reviews the evidence for "specific" pharmacokinetics playing a role in currently marketed drugs intended to treat lower urinary tract (LUT) symptoms. Principles of drug targeting include intrinsic properties of drugs or organs as well as drug formulations to modify drug release or to create confinement of drug presence. Prodrugs and specific formulations to deliver high drug concentrations at the site(s) of action as well as other ways to manipulate drug distribution to achieve enrichment in target tissues are considered. In overactive bladder (OAB), specific formulations for oxybutynin have been introduced to reduce the level of side effects of the active drug. Extended release tablet formulations and a topical gel formulation have been introduced, with efficacy similar to immediate release (IR) tablets, but with a reduction in anticholinergic adverse effects. However, these modifications have not led to outstanding performance parameters compared to other anticholinergic drugs marketed as IR formulations. Urinary excretion is discussed as potential mechanism for targeting LUT symptoms, but no strong indications appear to exist that this mechanism would contribute for currently available drugs. Intravesical administration of drugs is not a preferred option and only considered for drugs like botulinum toxin, where the inconvenient application compensates for a reasonable degree of long-term efficacy in severe refractory OAB. Alpha acid glycoprotein binding is discussed as a potential factor to influence drug tissue distribution, and it is concluded that there is reasonable evidence that for tamsulosin this mechanism is responsible for the difference in free fraction of the drug observed in plasma and prostate, which could contribute to its relative absence of blood pressure effects in patients with LUT symptoms related to benign prostate hyperplasia (LUTS-BPH). The principle of irreversible inhibition of type II 5α-reductase as a tool to develop drugs to reduce prostatic levels of dihydrotestosterone is employed by both dutasteride and finasteride for treatment of LUTS-BPH. Of the mechanisms discussed, the principles employed for the 5α-reductase blockers and tamsulosin in this respect can be considered relatively specific for its urological indication.

Pharmacokinetics and safety of aclidinium bromide, a muscarinic antagonist, in adults with normal or impaired renal function: A phase I, open-label, single-dose clinical trial.

BACKGROUND: Aclidinium bromide is an inhaled, long-acting muscarinic antagonist currently in development for the treatment of chronic obstructive pulmonary disease. Renal impairment may affect drug clearance. OBJECTIVE: This study was conducted to evaluate the pharmacokinetic (PK) parameters, safety, and tolerability of aclidinium bromide and its metabolites in patients with normal and impaired renal function to determine whether dosing adjustments are required when renal dysfunction is present. METHODS: This was a Phase I, open-label, single-center, single-dose clinical trial conducted in Munich, Germany. Adults with varying degrees of renal function were assigned to 4 groups (n = 6 for each) based on creatinine clearance, including normal renal function (>80 mL/ min), mild renal insufficiency (>50-≤80 mL/min), moderate renal insufficiency (>30-≤50 mL/min), and severe renal insufficiency (<30 mL/min). Single doses of aclidinium bromide 400 µg were administered using a multidose dry powder inhaler. Blood and urine samples were obtained before dosing and at various time points up to 48 hours after dosing to analyze the PK parameters of aclidinium bromide and its metabolites. Plasma PK Parameters were AUC0₋(t), MJC0₋(∞) C(max), T(max), t(½) CL/F and apparent volume of distribution during the terminal phase Xz; urinary parameters were the amount of aclidinium or acid or alcohol metabolite excreted in urine, the percentage of the dose excreted in urine (fe), and renal clearance (CL(R)). Tolerability was assessed using physical examination, vital signs, 12-lead ECG recordings, laboratory tests, and adverse-event (AE) reports. The Wilcoxon rank sum test was used to compare the median PK values between the normal and impaired renal function groups. Pearson correlation coefficients and linear regression models were used to analyze the relationship between creatinine clearance and AUC0₋(∞) and between creatinine clearance and CL(R) for aclidinium and its metabolites. RESULTS: A total of 16 men and 8 women were included in the study. All participants were white; mean (SD) age was 55 (10.7) years and weight was 70.8 (9.2) kg. Aclidinium Cmax was observed in plasma by 5 minutes after dosing (ie, median Tmax) and did not differ significantly among the renal function groups. Plasma concentrations of aclidinium declined after reaching Cmax, with median t(½) values ranging from 2.07 to 4.18 hours across all renal function groups. Most of the individual t(½) values were between 1.5 and 3.5 hours, regardless of the degree of renal insufficiency. No significant relationship between AUC0₋(∞)) and creatinine clearance was observed (Pearson correlation coefficient = -0.0446; P = NS). Urinary excretion of aclidinium was very low, with a mean 0.090% (median 0.078%) of the dose recovered from the urine in participants with normal renal function. Eight AEs were reported in 7 participants after drug administration; all were mild to moderate in severity and resolved spontaneously. There were no serious drug-related AEs and no deaths. CONCLUSIONS: The plasma PK parameters of aclidinium bromide were not significantly altered after a single inhaled dose of aclidinium bromide 400 µg in this small group of patients with various degrees of impaired renal function. The very low urinary excretion of aclidinium in all renal function groups indicates that renal function plays a minor role in aclidinium plasma clearance. Aclidinium appeared well tolerated in the population studied. These results suggest that aclidinium dose adjustment on the basis of renal function may not be necessary.

Intravesical instillation of human urine after oral administration of trospium, tolterodine and oxybutynin in a rat model of detrusor overactivity.

OBJECTIVE: To study the effects of antimuscarinics excreted into human urine on normal bladder in a rat model of detrusor overactivity. MATERIALS AND METHODS: Two 'normal' adult volunteers collected voided urine after taking trospium (20 mg, twice daily), tolterodine LA (4 mg, four times daily), or oxybutynin XL (10 mg, four times daily). The drugs were taken in a random order for 5 days with a 7-day washout period between the drugs. The urine collected from the two volunteers was mixed together and then blindly labelled and used for testing. Control human urine (no oral antimuscarinics) was also used. The effect of intravesical administration of human urine on carbachol-induced bladder overactivity was studied in female Sprague-Dawley rats anaesthetised with urethane. Cystometric variables during continuous infusion (0.04 mL/min) for >1 h each of saline, human urine, then a mixture of carbachol (30 microm) and human urine were compared in the four groups (control and the three different antimuscarinics tested; six rats per group). RESULTS: Human urine, with or with no intake of antimuscarinic agents, had no effect on normal bladder function. Bladder capacity and intercontraction intervals were significantly decreased after adding carbachol to urine containing vehicle, tolterodine or oxybutynin. However, urine collected from the humans who had taken trospium prevented the carbachol-induced reduction in bladder capacity and intercontraction intervals. Maximum voiding pressure and pressure threshold were not changed in any case. CONCLUSION: This is the first report that the urine excreted after oral ingestion of trospium (20 mg, twice daily) has a significant inhibitory effect in a rat model of detrusor overactivity. This suggests that antimuscarinic agents have a local bladder effect during the bladder-storage phase in addition to the smooth muscle-mediated voiding phase.

Acute intoxication with orphenadrine and clozapine.

This report describes a fatal intoxication with two different drugs: clozapine and orphenadrine. A 38-year-old man was found dead in the bedroom of his residence. Near the body were found various empty pharmaceutical boxes, employed in schizophrenic therapy, two of them containing clozapine and orphenadrine. High concentrations of clozapine and orphenadrine detected in blood, urine and gastric content were related to death. These compounds were identified and quantitated by liquid-liquid extraction followed by gas chromatographic/mass spectrometry (GC/MS) analysis.

Effects of antimuscarinic drugs on both urinary frequency and cognitive impairment in conscious, nonrestrained rats.

Recent studies indicate a risk of learning and memory impairments when patients with senile dementia are treated with antimuscarinic drugs. In this study, we compared the effectiveness of propiverine hydrochloride (propiverine) and oxybutynin chloride (oxybutynin) on the increased urinary frequency and cognitive impairment induced by nucleus basalis magnocellularis (nBM) lesioning in conscious and nonrestrained rats. For examination of bladder function, nBM-lesioned rats were given total parenteral nutrition regimens for 8 days. Propiverine administered orally at 0.3, 3 and 30 mg/kg on the postoperative day 7 significantly lessened the increase in the frequency of voiding caused by the nBM lesion, whereas oxybutynin administration did not show any improvement at 0.1 or 1 mg/kg but did so at 10 mg/kg. To examine the memory impairment, we trained nBM-lesioned rats in an 8-arm radial maze task for 20 days and then evaluated the effectiveness of oral drug administration on 19th and 20th radial maze performance. The higher rate of errors caused by nBM lesioning was significantly aggravated by oxybutynin at 30 and 100 mg/kg. Propiverine showed slight aggravation of errors, but with no statistical significance at any dose, 30, 100 or 300 mg/kg. These results suggest that propiverine has comparatively less effect on the cognitive impairment than oxybutynin.

Pharmacokinetics of tolterodine, a muscarinic receptor antagonist, in mouse, rat and dog. Interspecies relationship comparing with human pharmacokinetics.

Tolterodine ((R)-N,N-diisopropyl-3-(2-hydroxy-5-methyl-phenyl)-3-phenylpropanamine, CAS 124937-51-5) is an antimuscarinic agent developed specifically for the treatment of the overactive bladder. In this study, the pharmacokinetics of tolterodine were investigated in the mouse, rat and dog, following which allometric scaling was performed to predict oral pharmacokinetics in man. The intestinal absorption of tolterodine after oral dosing was almost complete in all three species, with peak serum concentrations observed within 1 hour post-dose. Bioavailability varied between 2-20% in rodents and 58-63% in the dog. A high volume of distribution in all three species was consistent with extravascular distribution. Tolterodine was extensively metabolised in all the animal models, but the profile of metabolism differed in the rat compared to the mouse and dog, the latter having similar metabolism routes as man. Limitation of metabolism capacity caused a non-linear increase of tolterodine concentrations with dose (repeat-dose study in the mouse), and changed the relative metabolite concentration pattern. The results suggest that the hydroxylation of tolterodine is a high affinity, low capacity pathway, while N-dealkylation follows a low affinity, high capacity pathway. The elimination of tolterodine from serum was rapid, with a half-life of less than 2 h in all species. A very high clearance in the mouse and rat (10-15 l/h.kg), and in the dog (1.4 l/h.kg), indicated additional non-metabolic clearance mechanisms for tolterodine (shown to be attributed to biliary excretion). Urinary excretion of compound-related radioactive substance was around 15%, 45% and 50%, respectively, in the rat, mouse and dog. Allometric scaling allowed a good prediction of clearance and volume of distribution to be extrapolated for comparison with tolterodine pharmacokinetics in man. In conclusion, the pharmacokinetics of tolterodine are similar in the mouse and dog, and correlate with that in man. Although the rat has a different metabolic profile, clearance fits into the allometric relationship between species, enabling prediction of total clearance of tolterodine in man from preclinical data.

Determination of tolterodine and the 5-hydroxymethyl metabolite in plasma, serum and urine using gas chromatography-mass spectrometry.

A specific and sensitive capillary gas chromatography-mass spectrometry assay for the determination of tolterodine and the 5-hydroxymethyl metabolite (Labcode DD 01) in plasma, serum and urine is described. Extraction of the analytes was performed with liquid/liquid or solid-phase extraction prior to derivatisation with a silyl reagent. The derivatives were quantified by selected ion monitoring mass spectrometry using deuterium-labelled internal standards. A single level calibration curve was utilised for quantification of plasma, serum and urine concentrations of tolterodine and DD 01. The accuracy (inter- and intra-day) for both analytes was within 87-110% in the range 0.5 and 50 ng ml-1 and precision was better than 90%. Overall, this method was shown to be reliable for pharmacokinetic assays of tolterodine and the metabolite DD 01 in samples from preclinical and clinical studies.

Phase I and II metabolites of benztropine in rat urine and bile.

Following oral administration of benztropine (IO mg/kg, body weight), the phase I metabolites, benztropine N-oxide, N-desmethylbenztropine, tropine, 4'-hydroxybenz- tropine, N-desmethyl-4'-hydroxybenztropine, 4'-hydroxvbenztropine N-oxide and methoxy-4'-hydroxybenztropine, together with unmetabolized benztropine, were isolated and identified in rat urine and bile by GC-electron impact mass spectrometry (EI GC/MS), microcolumn LC-electrospray mass spectrometry (ES LC/MS) and hplc followed by MS analysis. The mass spectra and chromatographic properties of isolated N-desmethylbenztropine, benztropine N-oxide and tropine were confirmed by comparison with authentic reference standards. Sufficient quantities of 4'-hydroxybenztropine and N-desmethyl-4'-hydroxybenztropine were isolated from the urine by tlc and examined by 1H-nmr, ES/MS and EI/MS. The structure of the methoxy-4'-hydroxybenztropine metabolite was determined by EI/MS. 4'-Hydroxybenztropine N-oxide was identified by reacting it with a reducing agent, titanous chloride, to form 4'-hydroxybenztropine, which was then confirmed by comparing its EI/MS and ES/MS behaviour with a previously isolated and 1H-nmr-authenticated sample. In addition, four intact glucuronide conjugates of benztropine were also characterized in bile and urine as phase II metabolites, including 4'-O-glucuronylbenzotropine, N-desmethyl-4'-O-glucuronylbenztropine, methoxy-4'-O-glucuronylbenztropine and 4'- O-glucuronylbenztropine N-oxide by hplc followed by ES/MS analysis. These results provide the first direct evidence of the presence of these metabolites of benztropine in rat.