Muscarinic Receptor Binding Activity Research Articles

Development of a pharmacological evidence-based anticholinergic burden scale for medications commonly used in older adults.

The present study aimed to develop a pharmacological evidence-based anticholinergic burden scale (ABS) through a direct assessment of muscarinic receptor-binding activities of 260 medications commonly used in older adults. The muscarinic receptor-binding activities of 260 drugs were assessed by the displacement of specific [N-methyl-3 H]scopolamine methyl chloride binding in the rat brain. The maximum blood concentrations (Cmax ) of drugs after their administration to subjects were cited from their interview forms. In total, 96 of 260 drugs displayed concentration-dependent muscarinic receptor binding in rat brain. Based on muscarinic receptor-binding activity (IC50 ) and Cmax after the administration at clinical doses in humans, we rated ABS 3 (strong) for 33 drugs and ABS 2 (moderate) for 37 drugs. There was an approximate similarity between muscarinic receptor-binding activities (IC50 ) and Cmax of 33 drugs (ABS 3) after their administration at clinical doses in humans. Furthermore, 26 drugs were defined as ABS 1 (weak) by muscarinic receptor-binding activity. The remaining 164 drugs exhibited slight or no significant muscarinic receptor-binding activities at high concentration of 100 μM, and they were defined as ABS 0. There was a marked similarity for 28 drugs (ABS 3) between the present ABS data and their previous scoring data in the literature. To our knowledge, the present study developed the first comprehensive pharmacological evidence-based ABS of drugs based on muscarinic receptor-binding activity, which provides guidance as to which drugs may be discontinued to reduce anticholinergic burden. Geriatr Gerontol Int 2023; 23: 558-564.

Muscarinic receptor binding activity in rat tissues by vibegron and prediction of its receptor occupancy levels in the human bladder.

To examine the effects of vibegron, a selective β3 -adrenoceptor agonist, used to treat overactive bladder, on muscarinic receptors in the rat bladder, and to predict the occupancy levels of muscarinic receptors by vibegron in the bladders of humans orally administered a clinical dose. Muscarinic receptors in the rat bladder and other tissues were examined by a radioligand binding assay using [N-methyl-3 H]scopolamine chloride. The occupancy levels of muscarinic receptors by vibegron in bladders of humans after its oral administration were predicted from the estimation of unbound concentrations in human plasma and urine in the literature. Vibegron (0.1-100μmol/L) inhibited specific [N-methyl-3 H]scopolamine chloride binding in the bladder and other tissues of rats in a concentration-dependent manner. The 50% inhibitory concentration value of vibegron in the bladder was approximately twofold higher than that in the heart, and approximately 315- and 3.5-fold lower than those in the submaxillary gland and brain, respectively. Therefore, the binding affinity of vibegron for muscarinic receptors was higher in the heart and bladder than in the submaxillary gland and brain. By using the rat bladder receptor binding affinity, occupancy levels of muscarinic receptors in the human bladder were predicted to be 51-91% until 24h after its oral administration at 50mg of vibegron. This is the first study to suggest that vibegron binds to muscarinic receptors in the rat bladder and other tissues, with a potentially higher affinity for the M2 subtype than the M1 and M3 subtypes. These results might be clinically relevant for pharmacotherapy with vibegron for overactive bladder.

TheN-Oxide Metabolite Contributes to Bladder Selectivity Resulting from Oral Propiverine: Muscarinic Receptor Binding and Pharmacokinetics

We characterized contribution of N-oxide metabolites [1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide (M-1) and 1-methyl-4-piperidyl benzilate N-oxide (M-2)] to the binding of muscarinic receptors in relation to the pharmacokinetics of propiverine in rats. The in vitro muscarinic receptor binding activity of M-2 was equipotent to that of propiverine, whereas M-1 was much less active. After the oral administration of propiverine (24.8-248 micromol/kg), there was relatively selective and longer-lasting binding of muscarinic receptors in the rat bladder compared with the submaxillary gland as shown by a significant increase in the apparent dissociation constant (K(d)) for specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS). In addition, the intravesical instillation of M-2 produced a significant increase in K(d) for specific [(3)H]NMS binding in the rat bladder. Extremely high concentrations of M-1 and M-2 were detected in plasma after the oral administration of propiverine. The concentration of unbound M-2 was much higher than that of M-1 and propiverine in the rat plasma. The sum of maximal plasma unbound propiverine equivalents (C(max)) after the oral administration of propiverine at doses of 24.8, 74.3, and 248 micromol/kg was 66.0, 303, and 509 nM, respectively. The sum of corresponding area under the time-concentration curve from 0 to 12 h was 194, 2123, and 4645 nM . h, respectively. In fact, the unbound concentration of M-2 comprised more than 90% of sum of unbound propiverine equivalents in the plasma. After oral treatment with propiverine, the bladder showed the highest concentration of M-2, indicating specific distribution of this metabolite into the target organ. Thus, M-2 may contribute greatly to the relatively selective and long-lasting occupation of bladder muscarinic receptors after oral administration of propiverine.

The Forefront for Novel Therapeutic Agents Based on the Pathophysiology of Lower Urinary Tract Dysfunction: Bladder Selectivity Based on In Vivo Drug–Receptor Binding Characteristics of Antimuscarinic Agents for Treatment of Overactive Bladder

We have reviewed the binding of antimuscarinic agents, used to treat urinary dysfunction in patients with overactive bladder, to muscarinic receptors in target and non-target tissues in vivo. Transdermal administration of oxybutynin in rats led to significant binding in the bladder without long-term binding in the submaxillary gland and the abolishment of salivation evoked by oral oxybutynin. Oral solifenacin showed significant and long-lasting binding to muscarinic receptors in mouse tissues expressing the M3 subtype. Oral tolterodine bound more selectively to muscarinic receptors in the bladder than in the submaxillary gland in mice. The muscarinic receptor binding activity of oral darifenacin in mice was shown to be pronounced and long-lasting in the bladder, submaxillary gland, and lung. In vivo quantitative autoradiography using (+)N-[11C] methyl-3-piperidyl benzilate in rats showed significant occupancy of brain muscarinic receptors on intravenous injection of oxybutynin, propiverine, solifenacin, and tolterodine. The estimated in vivo bladder selectivity compared to brain was significantly greater for solifenacin and tolterodine than oxybutynin. Darifenacin occupied few brain muscarinic receptors. Similar findings were also observed with positron emission tomography in conscious rhesus monkeys. The newer generation of antimuscarinic agents may be advantageous in the bladder selectivity after systemic administration.

Muscarinic receptor binding activity of polyoxygenated flavones from Melicope subunifoliolata

The bark extract of Melicope subunifoliolata (Stapf) T.G. Hartley showed competitive muscarinic receptor binding activity. Six polymethoxyflavones [melibentin (1); melisimplexin (3); 3,3′,4′,5,7-pentamethoxyflavone (4); meliternatin (5); 3,5,8-trimethoxy-3′,4′,6,7-bismethylenedioxyflavone (6); and isokanugin (7)] and one furanocoumarin [5-methoxy-8-geranyloxypsoralen (2)] were isolated from the bark extract. Compounds 2 and 6 were isolated for the first time from M. subunifoliolata. The methoxyflavones (compounds 1, 3, 4, 5, 6, and 7) show moderate inhibition in a muscarinic receptor binding assay, while the furanocoumarin (compound 2) is inactive. The potency of the methoxyflavones to inhibit [ 3H]NMS-muscarinic receptor binding is influenced by the position and number of methoxy substitution. The results suggest these compounds are probably muscarinic modulators, agonists or partial agonists/antagonists.

Comparative Evaluation of Central Muscarinic Receptor Binding Activity by Oxybutynin, Tolterodine and Darifenacin Used to Treat Overactive Bladder

We characterized muscarinic receptor binding in the mouse cerebral cortex after oral administration of anticholinergic agents used to treat overactive bladder. Muscarinic receptors in the mouse cerebral cortex and bladder after oral administration of anticholinergic agents were measured using [(3)H]N-methylscopolamine. In vitro binding affinities of tolterodine and its metabolite 5-hydroxymethyl metabolite in the mouse cerebral cortex and bladder were considerably greater than those of oxybutynin and darifenacin. Also, muscarinic receptor binding affinity of oxybutynin and its metabolite N-desethyl-oxybutynin in the cerebral cortex compared with that in the bladder was 2 to 3 times higher, whereas that of tolterodine and 5-hydroxymethyl metabolite was approximately 2 times lower. Oral administration of oxybutynin (76.1 micromol/kg), tolterodine (6.31 micromol/kg) and darifenacin (59.1 micromol/kg) showed binding activity that was approximately equal to that of bladder muscarinic receptors. Oral administration of oxybutynin (76.1 micromol/kg) showed significant binding of cerebral cortical muscarinic receptors in mice, as indicated by about a 2-fold increase in K(d) values for specific [(3)H]N-methylscopolamine binding 0.5 and 2 hours later. On the other hand, tolterodine and darifenacin given at oral doses that would exert a similar extent of bladder receptor binding activity as oxybutynin showed only a low level of binding activity of central muscarinic receptors in mice. Significant binding of brain muscarinic receptors in mice was observed by the oral administration of oxybutynin but not tolterodine and darifenacin.

Human Muscarinic Receptor Binding Characteristics of Antimuscarinic Agents to Treat Overactive Bladder

We characterized the binding affinities of several antimuscarinic agents in human muscarinic receptors. Competitive inhibitory effects of antimuscarinic agents on specific NMS [H] (PerkinElmer Life Sciences, Boston, Massachusetts) binding were examined in human tissue homogenates and in CHO-K1 cell membranes expressing human muscarinic receptor subtypes. Oxybutynin, propiverine, tolterodine, the respective metabolites DEOB, DPr-P-4(N-->O) and 5-HM, and darifenacin inhibited in concentration dependent fashion specific [(3)H]NMS binding in homogenates of the human bladder and parotid gland as well as in membranes of CHO-K1 cell lines expressing human muscarinic M(1) to M(5) receptor subtypes. Based on inhibition constant values the inhibitory effects of tolterodine, 5-HM and DPr-P-4(N-->O) were 1.4 to 1.7 times greater in the bladder than in the parotid gland, whereas the inhibitory effects of oxybutynin, DEOB, propiverine and darifenacin were 2 to 10 times greater in the parotid gland. Consequently tolterodine, 5-HM and DPr-P-4(N-->O) compared with oxybutynin, DEOB, propiverine and darifenacin were found to show 3 to 4 times greater affinity to muscarinic receptors in the human bladder than in the parotid gland. Tolterodine and 5-HM were 2-fold more potent for inhibiting specific [(3)H]NMS binding at cell membranes expressing the M(2) vs the M(3) subtype. Conversely oxybutynin, DEOB, propiverine, DPr-P-4(N-->O) and darifenacin showed 2 to 22 times higher affinity to the M(3) than to the M(2) subtype. Compared with oxybutynin, tolterodine, 5-HM and DPr-P-4(N-->O) may bind more selectively to muscarinic receptors in the human bladder than in the parotid gland.

In vivo demonstration of muscarinic receptor binding activity of N-desethyl-oxybutynin, active metabolite of oxybutynin

The present study was undertaken to characterize in vivo muscarinic receptor binding of N-desethyl-oxybutynin (DEOB), active metabolite of oxybutynin (anticholinergic agent), in the bladder, submaxillary gland, heart and colon of rats, in relation to the plasma concentrations and inhibition of salivation. In the in vitro experiment, DEOB, as well as oxybutynin, inhibited the concentration-dependently specific [ 3H]N-methylscopolamine (NMS) binding in rat tissues and the affinity of DEOB in the rat bladder, submaxillary gland and colon was significantly (about 2 times) greater than that of oxybutynin. Following i.v. injection of DEOB (2.73–27.3 μmol/kg), there were dose- and time-dependent increases in the apparent dissociation constant (Kd) for specific [ 3H]NMS binding in the bladder, submaxillary gland, heart and colon of rats, compared with control values, and the effect was similar to that by i.v. injection of oxybutynin (2.54–25.4 μmol/kg). Plasma concentrations of DEOB and oxybutynin in these rats showed dose- and time-dependent increases. The pilocarpine-induced salivary secretion in rats was equipotently reduced by the i.v. injection of DEOB and oxybutynin. In conclusion, it has been shown that intravenously injected DEOB, as well as oxybutynin, binds significantly to muscarinic receptors in rat tissues including the bladder and salivary gland and the receptor binding activity of DEOB is roughly similar to that of oxybutynin.

Muscarinic Receptor Activity of Some Malaysian Plant Species

Muscarinic receptor binding activity was tested on 224 plant extracts obtained from more than 50 plant families found in Malaysia. The plant extracts were evaluated by a 96-well microplate filtration–based radioligand competitive assay, centered on the ability of the plant extracts to competitively displace the radioligand, [3H]N.-methylscopolamine, from binding to the muscarinic membrane receptors. The screening assay was initially carried out at 50 µg/assay point, and those showing inhibition at and above 61% were retested at 10 µg/assay point. The extracts of Ficus septica. Burm. f. (Moraceae) [65.85±3.75% inhibition; mean (n = 3)±SD], Polyalthia microtus. Miq. (Annonaceae) (32.63±1.38% inhibition), and Popowia odoardoi. Diels (Annonaceae) (35.79±7.11% inhibition) at 10 µg/assay point exhibited muscarinic properties, which are worthy of further investigation.

DEMONSTRATION OF BLADDER SELECTIVE MUSCARINIC RECEPTOR BINDING BY INTRAVESICAL OXYBUTYNIN TO TREAT OVERACTIVE BLADDER

The current study was done to elucidate the in vivo mechanism of action of intravesical instillation of oxybutynin to treat overactive bladder. In rats receiving oral and intravesical oxybutynin we measured muscarinic receptors in the bladder and other tissues by radioligand binding assay using [3H]NMS ([N-methyl-3H] scopolamine methyl chloride) with the simultaneous measurement of plasma concentrations of oxybutynin and its active metabolite N-desethyl-oxybutynin. Pilocarpine induced salivary secretion was also measured. Following oral administration of oxybutynin there was a significant increase in the apparent dissociation constant (Kd) for specific [3H]NMS binding in the bladder, submaxillary gland, heart and colon of rats at 1 and 3 hours with a consistent decrease in the maximal number of binding sites (Bmax) in the submaxillary gland. Furthermore, a marked and prolonged decrease in pilocarpine induced salivary secretion in rats was observed by oral oxybutynin. In contrast, intravesical instillation of oxybutynin produced a significant increase in Kd for specific [3H]NMS binding in the bladder of rats at 0.5 to 4 hours later and also in the submaxillary gland only at 0.5 hours later. The enhancement in Kd was much larger and longer lasting in the bladder than in the submaxillary gland. Moreover, intravesical oxybutynin had little muscarinic receptor binding activity in the heart and colon, and little significant suppression of pilocarpine induced salivation in rats. The plasma concentrations of oxybutynin and N-desethyl-oxybutynin were much higher in rats receiving oxybutynin orally than intravesically. Intravesical oxybutynin in rats may cause selective binding of bladder muscarinic receptors via a direct local effect, while oral oxybutynin may exert predominant binding of salivary gland receptors.

Ontogenetic and imprinting-induced changes in chick brain protein metabolism and muscarinic receptor binding activity.

Over the 20-min period following exposure of young chicks to a flashing light as an imprinting stimulus there is an increased incorporation of [14C]leucine into an acidic (tubulin-enriched) protein fraction of the anterior dorsal forebrain in birds which have learnt the characteristics of the stimulus as compared with, either birds which have been exposed to an imprinting stimulus but learn poorly, or chicks kept in the dark. This brain region has been implicated in several studies as the locus for a number of biochemical modulations that accompany learning. The amount of [14C]leucine incorporated does not seem to be determined by precursor pool availability; it does, however, correlate with a well-validated measure of the extent to which birds have learnt to recognise the characteristics of the stimulus, as shown by a two-choice discrimination test. There is no change in the total content of tubulin dimer as assayed by colchicine binding under these conditions. Additionally, in birds which show evidence of learning, the binding of quinuclidinyl benzilate, an irreversible muscarinic ligand, is altered in both the posterior dorsal forebrain and midbrain regions. None of these effects could be simply the result of visual stimulation. The meaning of these changes is discussed.

Stability of muscarinic-cholinergic receptor activity in the deafferented retinotectal pathway

The high-affinity antagonist, 3-quinuclidinyl bensilidate (QNB), was used to analyze muscarinic-cholinergic receptor activity in the optic tectum of goldfish and optic lobe of developing chicks and adult pigeons after deafferentation. After deafferentation no significant loss of total or specific muscarinic receptor binding activity was observed inc ontrast to prior experiments where there was a marked and rapid loss of nicotinic-cholinergic receptor binding activity, as measured by alpha-bungarotoxin binding. The relative stability of the muscarinic site as opposed to the instability of the nicotinic site in these experiments is discussed.