Introduction
Antagonists of acetylcholine muscarinic receptors, such as darifenacin, fesoterodine, oxybutynin, propiverine, solifenacin, tolterodine, and trospium chloride (TCl), are the cornerstone of pharmacotherapy for the symptoms of overactive bladder (OAB) [
1]. Apart from blocking muscarinic receptors in the bladder, most anticholinergic drugs can cross the blood–brain barrier (BBB) and antagonize the effects of acetylcholine in the central nervous system (CNS). In particular, blocking of muscarinic M
1 receptors in the brain was found to be associated with cognitive impairment, dizziness, and sleep disturbance, which can be especially detrimental in older patients [
2].
Most of the differences in CNS effects of antimuscarinic drugs can be explained by their different abilities to penetrate through the BBB. While most of the aforementioned antimuscarinic drugs are tertiary amines that are quite lipophilic, TCl is a highly polar quaternary amine [
3]. Therefore, TCl showed much lower penetration across the BBB than other more lipophilic antimuscarinic drugs, at least in mice and rats [
4,
5]. The low brain penetration of TCl was also confirmed in a recent study with older OAB patients who received extended-release trospium chloride 60 mg once daily over a 10-day period. In these patients, TCl showed normal peak plasma concentrations of 925 ± 478 pg/ml, but was assay undetectable in the cerebrospinal fluid (<40 pg/ml) [
6].
Apart from their physicochemical properties, the brain penetration of antimuscarinic drugs also depends on their interaction with the drug-transporting P-glycoprotein (P-gp), which limits the entry of many drugs and xenobiotics into the brain by an efflux-based transport mechanism. Recently, we showed that penetration of TCl into the brain is significantly increased in P-gp-deficient knockout mice, indicating that P-gp normally restricts the entry of this drug into the brain [
7].
However, the possibility that in elderly patients, who represent the majority of patients with OAB, the brain penetration of antimuscarinic drugs might be increased due to a histological or functional breakdown of the BBB or the down-regulation of P-gp has been discussed [
2,
8]. However, until now, no clear experimental evidence has been found to show increased penetration of antimuscarinic drugs into the brain during ageing. Therefore, in the present study, we aimed to analyse the absolute brain concentrations of TCl after its administration to mice of different ages.
Discussion
Older patients are more likely to experience adverse CNS effects under drug treatment for several reasons. In the case of anticholinergic drugs, these have been associated with cognitive deficits, behavioural changes, and sleep disturbance [
10]. An increased permeability of the BBB may contribute to such vulnerabilities. In this regard, it was claimed that TCl is a safer anticholinergic alternative due to its generally low permeability across the BBB [
11,
12]. However, the age-dependent penetration of trospium chloride into the brain has never been measured before in an experimental study.
In the CNS, different barriers exist between blood circulation and neural tissue that have to be considered for drug permeation into the brain: the blood–brain barrier (BBB), which is formed by cerebrovascular endothelial cells, and the blood–cerebrospinal fluid barrier (BCSFB), which is constituted by epithelial cells of the choroid plexus [
13]. At these interfaces, a barrier function results from the combination of (I) a histological barrier formed by tight junction complexes that tightly connect the epithelial cells and thus greatly limit the paracellular flux of polar solutes, and (II) a functional barrier comprised of different transport systems such as P-gp, which regulate the solute flux across the barrier [
14]. Since the surface area of the BBB is at least 5,000-fold greater than that of the choroid plexus [
15], the BCSFB is only of marginal importance for the overall drug delivery into the brain. In contrast, absolute drug concentrations in the brain predominantly depend on the permeability of the BBB, and therefore, this barrier is the most relevant barrier in the CNS regarding adverse CNS effects under drug treatment [
16].
Several factors can restrict the entry of drugs into the CNS including a highly polar surface area or a molecular weight of >450 Da [
17]. Additionally, interactions with drug carriers at the BBB can facilitate or restrict drug entry into the brain by carrier-mediated uptake or efflux processes, respectively [
16]. In the case of the anticholinergic drug TCl, brain penetration is highly restricted by the polar structure of the molecule and its low lipophilicity, as well as by a P-gp-mediated drug efflux in the endothelial cells of the BBB [
7]. Therefore, compared with the highly lipophilic and uncharged drug oxybutynin, TCl showed 200-fold lower absolute drug concentrations in the brain when administered at an equal dosage to laboratory mice [
4].
In recent reviews about OAB treatment with anticholinergic drugs, the fact that the BBB becomes more leaky with age and so might be more permeable to polar and P-gp-transported drugs such as TCl was discussed [
2,
8]. Indeed, several age-related changes in the cerebral microvasculature have been reported, including a lower microvascular density, a smaller capillary lumen size, or gliofibrillar proliferation [
18]. However, whether these changes actually affect drug penetration across the BBB has not been analysed. In animal studies with mice and rats, this question was addressed by studies that administered [
14C]sucrose [
19] or higher molecular weight compounds such as horseradish peroxidase [
20]. In general, these reports showed no significant alteration in BBB permeability with ageing per se. Other studies that analysed the permeability of the BBB in older human subjects (most of them by measuring the albumin liquor/plasma ratio) produced inconsistent results [
21]. Although some of these studies reported an elevation of the albumin liquor/plasma ratio with ageing, it must be emphasized that this ratio is a very artificial parameter for the assessment of absolute drug permeability across the BBB because: (I) alterations in this ratio primarily indicate a dysfunction of the BCSFB at the choroid plexus; (II) in terms of physicochemical properties and drug carrier interactions, albumin is not an appropriate surrogate for common drugs; and (III) an elevation of the albumin liquor/plasma ratio was previously shown to be associated with reduced cerebrospinal fluid production but not with increased albumin permeability at the BCSFB [
22,
23]. Therefore, an increase in the albumin liquor/plasma ratio during ageing cannot simply be interpreted as an increased permeability of the BBB [
24,
25].
However, a direct determination of the BBB drug permeability and of the absolute drug concentrations in the brain would require the analysis of brain material after drug administration, and therefore, such studies can generally not be carried out on human subjects. Therefore, we decided to analyse the age-dependent brain penetration of TCl in a mouse model, with animals ranging from 6 to 24 months of age. In these mice, the absolute drug concentrations were directly measured in the brain after a single-dose application and showed identical levels in the adult and middle-aged mice (13 ± 2 ng/g) and significantly lower levels in the aged mice (8 ± 4 ng/g). This slight decline in the brain concentration in the aged mice is probably due to the age-related changes in the cerebral microvasculature mentioned above. But due to the generally low drug concentrations in the brain compared to other organs and the relatively low degree (13 ± 2 ng/g vs. 8 ± 4 ng/g), this difference is not regarded as biologically meaningful with regard to the question of CNS side effects after treatment with antimuscarinic drugs. Furthermore, the brain/plasma concentration ratios for TCl were not different between the three age groups. Based on these data, it can be concluded that TCl permeation across the BBB is at least not increased with ageing per se.
We are aware of the potential limitations of the present study as the mouse, in terms of lifespan and metabolic activity, is a somewhat artificial model for the situation in human patients. Furthermore, this study was designed as a single-dose application study with single point detection and cannot directly be compared to a steady-state distribution after repeated drug application, which represents the situation in human patients. Nevertheless, our data are in good agreement with a recent clinical study in older OAB patients (≥65–75 years old) who received extended-release TCl treatment with 60 mg once daily over 10 days. These patients underwent memory testing using the Hopkins Verbal Learning Test-Revised and the Brief Visuospatial Memory Test-Revised, and showed no significant drug effects on learning or recall, which can be regarded as very sensitive parameters of adverse anticholinergic CNS effects. Furthermore, the TCl drug concentrations in the cerebrospinal fluid were assay undetectable (<40 pg/ml), with normal peak concentrations in plasma (~1 ng/ml) [
6]. These data clearly indicate that in older OAB patients, there is no relevant penetration of TCl into the brain under a therapeutic dosage.
Apart from the polar drug TCl, brain penetration studies have previously been carried out using the more lipophilic drug verapamil. These studies, using [
11C]verapamil positron emission tomography, found an increase in the distribution volume in certain, but not all, brain regions [
26,
27]. As verapamil is a well-established substrate of the P-gp efflux transporter, it was suggested that P-gp might be functionally down-regulated with ageing. However, studies on human brain samples showed no influence of age on the cerebrovascular expression of the P-gp protein [
28]. Even studies on P-gp expression at the BBB in rodents showed no down-regulation with age [
29], including the present study where we found identical levels of mRNA expression of P-gp and the tight junction markers occludin and claudin-5 in mice of 4/6 and 24/25 months of age. We are aware of the fact that the protein expression level in particular of P-gp might be different, although the mRNA expression level is equal in adult and aged mice. But as we directly measured the drug entry into the brain which is significantly dependent on the functional expression of P-gp in the blood–brain barrier [
7], we do not expect that the protein expression of P-gp is significantly up- or down-regulated in the aged mice.
However, it is possible that barrier functions in the CNS could be affected by several disease conditions and pathologies including multiple sclerosis, acute hypertension, cerebral ischaemia, diabetes, Alzheimer’s or Parkinson’s disease, and brain tumours or inflammation [
13,
30,
31]. Under these conditions, impairment of the neurovascular barriers could range from transient opening of the tight junctions to chronic barrier breakdown [
13,
21]. Additionally, the expression of drug efflux carriers such as P-gp at the BBB can be affected in certain diseased conditions such as Alzheimer’s and Parkinson’s disease, amyotrophic lateral sclerosis, and inflammatory processes [
32]. Therefore, in patients with multiple co-morbidities, the brain penetration of peripherally acting antimuscarinic drugs might be increased.
In conclusion, we demonstrated in a mouse model that the absolute brain concentrations of the hydrophilic anticholinergic drug TCl were not increased with normal ageing. Furthermore, we found that, irrespective of the age of the mice, the brain was the organ with by far the lowest drug concentrations in the body of the mice, indicating that the BBB is a very effective permeation barrier against this drug. Based on our in vivo data, we conclude that TCl permeation across the BBB is not increased in ageing per se, and therefore, the occurrence of adverse CNS drug effects is also not expected to increase.