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.

Simple validated method of LC-MS/MS determination of BZ agent in rat plasma samples.

Agent BZ (3-quinuclidinyl benzilate) is a centrally acting synthetic anticholinergic agent, considered as a potential military incapacitating chemical warfare agent. Despite its significance as a model compound in pharmacological research and its potential misuse in chemical attacks, few modern analytical methods for BZ determination in biological samples have been published. The goal of the present work is to develop and validate a sensitive and rapid LC-MS/MS method for the determination of agent BZ in rat plasma. The sample preparation was based on solid-phase extraction on C-18 cartridges. The reversed-phase HPLC coupled with the mass spectrometer with electrospray ionization in the positive ion-selective reaction monitoring mode was employed in the BZ analysis. Atropine was used as an internal standard. The presented method is selective, accurate, precise, and linear (r2 = 0.9947) in a concentration range from 0.5 ng/mL to 1 000 ng/mL and sensitive enough (limit of detection 0.2 ng/mL; limit of quantification 0.5 ng/mL) to determine the BZ plasma levels in rats exposed to 2 mg/kg and 10 mg/kg of BZ. The highest level of BZ in plasma was observed 5 minutes after intramuscular administration (154.6 ± 22.3 ng/mL in rats exposed to 2 mg/kg of BZ and 1024 ± 269 ng/mL in rats exposed to 10 mg/kg). After 48 h, no BZ was observed at detectable levels. This new method allows the detection and quantification of BZ in biological samples after exposure of an observed organism and it will be further optimized for other tissues to observe the distribution of BZ in organs.

Influence of anticholinergic activity in serum on clinical symptoms of Alzheimer's disease.

Alzheimer's disease (AD) is well known as a disease characterized by degeneration of cholinergic neuronal activity in the brain. It follows that patients with AD would be sensitive to an 'anticholinergic burden', and also that medicine with anticholinergic properties would promote various clinical symptoms of AD. Despite the relevance of this important phenomenon to the clinical therapeutics of AD patients, few reports have been seen concerning the relationship between anticholinergic burden and clinical AD symptoms. Therefore, we wished to investigate the relationship between serum anticholinergic activity (SAA) and the severity of clinical symptoms of AD patients. Twenty-six out of 76 AD patients referred by practitioners to our hospital were positive for anticholinergic activity in their serum, and the remaining 50 patients were negative. Cognitive and psychiatric symptoms in AD patients were compared between the positive SAA (SAA+) group and the negative SAA (SAA-) group. The SAA+ group showed a significantly (p < 0.05) lower total score on the Mini-Mental State Examination, and significantly (p < 0.05) higher scores on the Functional Assessment Staging and the Behavioral Pathology in Alzheimer's Disease Rating Scale (BEHAVE-AD). In particular, certain subscales of the BEHAVE-AD, i.e. the items of paranoid and delusional ideation, hallucinations and diurnal rhythm disturbances, had higher scores in the SAA+ group. Moreover, it was shown that many more psychotropic medicines were prescribed to the SAA+ group. By means of logistic regression analysis, the items of paranoid and delusional ideation and diurnal rhythm disturbances in the BEHAVE-AD were positively correlated with SAA in patients. We hypothesized that SAA in AD patients would be associated with clinical symptoms, especially delusion and diurnal rhythm disturbances.

Anticholinergicity and cognitive processing in chronic schizophrenia.

Patients with chronic schizophrenia suffer from alterations in cholinergic functioning due to several factors, including the disease diathesis and pharmacologic treatments. Acetylcholine-cognition relationships are well explored in normals but are unclear in schizophrenia. Prior work indicated serum anticholinergicity does not cause global cognitive impairment in this group (Tracy et al., 1998a), raising the possibility that anticholinergicity normalizes an abnormal hyperactive cholinergic state. Serum anticholinergic levels were determined in 38 chronic schizophrenia patients using an established radioreceptor assay method. Six cognitive functions associated with cholinergic tone in normals were tested. The potential role of autonomic arousal and cigarette smoking were also assessed as both have been linked to cholinergic functioning. Regression analyses showed measures of inhibitory executive control and effortful memory accounted for a greater proportion of the variance in the anticholinergicity measure compared to the other variables. The data demonstrate a relationship between high anticholinergicity and worse performance on two types of attention-resource demanding cognitive processes and do not support the notion that reduced cholinergic tone normalizes a hyperactive cortical acetylcholine substrate. Relevant neuroanatomic structures and implications for models of cognitive deficits in schizophrenia are discussed.

Kinetic analysis of 3-quinuclidinyl 4-[125I]iodobenzilate transport and specific binding to muscarinic acetylcholine receptor in rat brain in vivo: implications for human studies.

Radioiodinated R- and S-Quinuclidinyl derivatives of RS-benzilate (R- and S-125IQNB) have been synthesized for quantitative evaluation of muscarinic acetylcholine receptor binding in vivo. Two sets of experiments were performed in rats. The first involved determining the metabolite-corrected blood concentration and tissue distribution of tracer R-IQNB (active enantiomer) and S-IQNB (inactive enantiomer) in brain 1 min to 26 h after intravenous injection. The second involved the measurement of brain tissue washout over a 2-min period after loading the brain by an intracarotid artery injection of the ligands. Various pharmacokinetic models were tested, which included transport across the blood-brain barrier (BBB), nonspecific binding, low-affinity binding, and high-affinity binding. Our analysis demonstrated that the assumptions of rapid equilibrium across the BBB and rapid nonspecific binding are incorrect and result in erroneous estimates of the forward rate constant for binding at the high-affinity receptor sites (k3). The estimated values for influx across the BBB (K1), the steady-state accumulation rate in cerebrum (K), and the dissociation rate constant at the high-affinity site (k4) of R-IQNB were independent of the specific compartmental model used to analyze these data (K1 approximately 0.23 ml/min/g, K approximately 0.13 ml/min/g, and k4 approximately 0.0019 min-1 for caudate). In contrast, the estimated values of k3 and the efflux rate constant (k2) varied over a 10-fold range between different compartmental models (k3 approximately 2.3-22 min-1 and k2 approximately 1.6-16 min-1 in caudate), but their ratios were constant (k3/k2 approximately 1.4). Our analysis demonstrates that the estimates of k3 (and derived values such as the binding potential) are model dependent, that the rate of R-IQNB accumulation in cerebrum depends on transport across the BBB as well as the rate of binding, and that uptake in cerebrum is essentially irreversible during the first 360 min after intravenous administration. Graphical analysis was consistent with compartmental analysis of the data and indicated that steady-state uptake of R-IQNB in cerebrum is established within 1-5 min after intravenous injection. We propose a new approach to the analysis of R-IQNB time-activity data that yields reliable quantitative estimates of k3, k4, and the nonspecific binding equilibrium constant (Keq) by either compartmental or graphical analysis. The approach is based on determining the free unbound fraction of radiolabeled ligand in blood and an estimate of K1.(ABSTRACT TRUNCATED AT 400 WORDS)

Cerebrovascular transport of [125I]quinuclidinyl benzilate, [3H]cyclofoxy, and [14C]iodoantipyrine.

The transport rate constants across rat brain capillaries of a muscarinic acetylcholine receptor antagonist, [125I]quinuclidinyl benzilate (IQNB), an opiate receptor antagonist, [3H]cyclofoxy (CF), and a highly diffusible blood flow indicator, [14C]iodoantipyrine (IAP), were determined by the indicator-diffusion technique and a model that includes a distribution of capillary transit times and a homogeneous distribution of the test compound in the tissue parenchyma. The mean influx extraction ratio (E1) of IAP was greater than 0.91, and E1 for CF and IQNB was 0.56-0.79 and 0.34-0.46, respectively. The order of lipid solubility was IQNB greater than IAP greater than CF; the apparent discrepancy (high lipid solubility and low permeability) of IQNB was partly due to intravascular binding to plasma protein and red blood cells. The observed initial tissue distribution volumes (lambda 1, ml/g brain) for IQNB (0.09-0.17), CF (0.51), and IAP (0.71) were compared with those estimated for the unbound free ligand in blood (lambda a, ml/g brain; IQNB = 1.3-2.3, CF = 0.88, and IAP = 1.4). These findings suggest that the binding of lipid-soluble radioligands and drugs to plasma proteins and red blood cells can be a major determinant of transport across the blood-brain barrier and the apparent distribution volume of the ligand in brain tissue.

Parametric studies on phencyclidine enhancement of 3H quinuclidinyl benzilate accumulation in vivo.

The purpose of these experiments was to define the temporal parameters involved in the phencyclidine (PCP) enhancement of 3H quinuclidinyl benzilate (QNB) accumulation in mouse brain. PCP enhanced QNB accumulation in brain if given intraperitoneally (IP) 1 and 4, but not 16 hours before intravenous (IV) administration of QNB. This effect was found in hypothalamus, striatum, cortex and hippocampus, but not cerebellum. PCP given after QNB did not alter QNB accumulation. The PCP enhancement persisted for at least 72 hours after QNB administration. These results confirm previous studies demonstrating that PCP must be present prior to QNB administration to enhance the accumulation and show that the effect persists for an extended period of time.

Identification and characterization of human erythrocyte muscarinic receptors.

Cholinergic muscarinic but not nicotinic receptors could be detected on the outer surface of the human red blood cell membrane by direct labelled ligand binding. This 3H-QNB binding could be inhibited by atropine. The erythrocyte cholinergic receptor was similar to brain muscarinic receptor in regard to stimulation of cGMP production.