Effects of conventional and a novel colonic-release bile acid sequestrant, A3384, on fibroblast growth factor 19 and bile acid metabolism in healthy volunteers and patients with bile acid diarrhoea.

BACKGROUND: Primary bile acid diarrhoea (BAD) is associated with increased bile acid synthesis and low fibroblast growth factor 19 (FGF19). Bile acid sequestrants are used as therapy, but are poorly tolerated and may exacerbate FGF19 deficiency. AIM: The purpose of this study was to evaluate the pharmacological effects of conventional sequestrants and a colonic-release formulation preparation of colestyramine (A3384) on bile acid metabolism and bowel function in patients with BAD. METHODS: Patients with seven-day 75selenium-homocholic acid taurine (SeHCAT) scan retention <10% were randomised in a double-blind protocol to two weeks treatment with twice-daily A3384 250 mg (n = 6), 1 g (n = 7) or placebo (n = 6). Thirteen patients were taking conventional sequestrants at the start of the study. Symptoms were recorded and serum FGF19 and 7α-hydroxy-4-cholesten-3-one (C4) measured. RESULTS: Median serum FGF19 on conventional sequestrant treatment was 28% lower than baseline values in BAD (p < 0.05). C4 on conventional sequestrant treatment was 58% higher in BAD (p < 0.001). No changes were seen on starting or withdrawing A3384. A3384 improved diarrhoeal symptoms, with a median reduction of 2.2 points on a 0-10 Likert scale compared to placebo, p < 0.05. CONCLUSIONS: Serum FGF19 was suppressed and bile acid production up-regulated on conventional bile acid sequestrants, but not with A3384. This colonic-release formulation of colestyramine produced symptomatic benefit in patients with BAD.

The ileal bile acid transporter inhibitor A4250 decreases serum bile acids by interrupting the enterohepatic circulation.

BACKGROUND: Reabsorption of bile acids from the intestine by ileal bile acid transporter is pivotal for the enterohepatic circulation of BAs and sterol homoeostasis. AIM: To assess tolerability and study, bile acid metabolism in a phase 1 trial with the selective ileal bile acid transporter inhibitor A4250. METHODS: A randomised double-blind, single-ascending dose (SAD) and multiple-ascending-dose study consisting of five cohorts comprising 40 individuals with a single administration of A4250 (0.1, 0.3, 1, 3, or 10 mg) or placebo and three cohorts comprising 24 individuals with a 1-week administration of A4250 (1 or 3 mg once daily or 1.5 mg twice daily) or placebo. For the multiple-ascending-dose study, bile acids were measured by HPLC-MS in plasma and faeces, and fibroblast growth factor 19 (FGF19) and 7α-hydroxy-4-cholesten-3-one (C4) were measured in plasma. RESULTS: No serious adverse events occurred and all participants finished the trial per protocol. At the end of the multiple-ascending-dose study, plasma total bile acids and FGF19 decreased by 47% and 76%, respectively, at 3 mg/day (P < 0.01), and by 15% and 16%, respectively, at 1.5 mg twice daily (P < 0.05). Plasma C4 and faecal bile acids increased at all dose regimens, by 555%, 664%, 292% and 338%, 421%, 420%, respectively (P < 0.01-0.05). The primary bile acids cholic and chenodeoxycholic acids constituted the majority of faecal bile acids in the A4250-treated groups. CONCLUSIONS: A4250 is well tolerated. By blocking ileal bile acid transporter in the terminal ileum, it highly efficiently interrupts the enterohepatic circulation of BAs, and should be of benefit to patients with cholestatic liver diseases. Clinical Trial registration EudraCT 2013-001175-21.

Randomised clinical trial: The ileal bile acid transporter inhibitor A3309 vs. placebo in patients with chronic idiopathic constipation--a double-blind study.

BACKGROUND: One half of patients with constipation are not satisfied with available therapies, hence there is a need for more effective and well-tolerated drugs. AIM: To evaluate the effects of a specific inhibitor of the Ileal Bile Acid Transporter (IBAT; syn apical sodium-dependent bile acid transporter; ASBT) in patients with chronic idiopathic constipation (CIC) with focus on safety, colonic transit and efficacy signals. METHODS: This was a single-centre, prospective, randomised, double-blind, placebo-controlled study with a dose-escalating design in patients with CIC. In addition to evaluation of conventional safety and tolerability parameters, (i) colonic transit time (CTT) was measured using radio-opaque markers, (ii) metabolic parameters [lipid profile, C4 (7α-hydroxy-4-cholesten-3-one) and FGF19 (Fibroblast Growth Factor 19)] were evaluated, and (iii) constipation parameters, such as changes in stool frequency and consistency, were analysed. RESULTS: Thirty patients were randomised into five dose-levels (range: 0.1-10 mg/day) or to placebo. All patients completed a 14-day treatment period, and the safety/tolerability analysis was favourable. A3309, present in picomolar concentrations in plasma, induced up to a three-fold increase in bile acid synthesis (C4) and a reduction of plasma FGF19, as well as reduction in total and LDL cholesterol. CTT was reduced in the highest dose groups; the main acceleration was identified in the left colon. Efficacy parameters showed trends for increased number of spontaneous bowel movements and improved stool consistency. CONCLUSIONS: Ileal Bile Acid Transporter inhibition is a novel mechanism for treatment of patients with chronic idiopathic constipation and has additional benefits of improving metabolic parameters (EudraCT 2008-003255-72).

Circulating ghrelin levels after food intake during different phases of the migrating motor complex in man.

BACKGROUND: The timing of the migrating motor complexes (MMC) at food intake may influence gastric emptying and release of regulatory hormones. This report studies the relationships between phases I (motor quiescence) and II (intermediate frequency contractions) of MMC and prandial gut hormone response. MATERIALS AND METHODS: Seven fasting volunteers ingested a meal during phase I or II of MMC verified by manometry, using paracetamol as a marker for gastric emptying. Blood was sampled before, during and 210 min after food intake for analysis of ghrelin, motilin, insulin and paracetamol. RESULTS: The basal level of ghrelin during phase I was 127.5 +/- 25.4 pmol L(-1) and during phase II was 132.4 +/- 24.8 pmol L(-1). After food intake during phase I, ghrelin fell to 77.2 +/- 10 pmol L(-1); in phase II it fell to 82.7 +/- 17.8 pmol L(-1) within 60 min and returned to baseline levels after 120 min. Baseline levels of motilin were 16 +/- 2 pmol L(-1) and 18 +/- 3 pmol L(-1) during phases I and II, respectively. After food, motilin decreased to 8.5 +/- 0.7 pmol L(-1) and 8.7 +/- 1.0 pmol L(-1) within 60 min and returned to baseline after 90 min. Insulin levels in phases I and II were 8.1 +/- 1.2 mU L(-1) and 8.6 +/- 0.7 mU L(-1), respectively, reaching 138.9 +/- 35.6 mU L(-1) and 167.4 +/- 30.0 mU L(-1) at 45 min postprandially. CONCLUSIONS: The nutritional status of the gastrointestinal tract at food intake had only a limited impact on plasma ghrelin. After food intake, plasma ghrelin drops, similar to motilin, and resumes preprandial levels within 120 min.

High 3H-vesamicol binding in ALS motor neurons--autoradiographic visulalization of hyperactivities?

OBJECTIVES: To evaluate if increased metabolic demand in remaining motor neurons in ALS spinal cord sections can be visualized by 3H-vesamicol binding. MATERIAL AND METHODS: As a presumed marker of the vesicular acetylcholine transporter, 3H-vesamicol was applied in quantitative autoradiography in cervical spinal cord sections from 11 ALS patients and 4 control cases. The regional binding was compared to that of the muscarinic ligand 3H-QNB. RESULTS: Our results demonstrate the same magnitude of H-vesamicol binding in the ventral horn of ALS spinal cord as compared to controls, despite the profound loss of motor neurons in that specific area in ALS. The specificity of 3H-vesamicol binding for the cholinergic transporter is high in the motor neuron area, and sigma-sites constitute a minor proportion. CONCLUSION: The lack of decrease in 3H-vesamicol binding in postmortem ALS spinal cord sections probably reflects an upregulated synthesis of vesicular membranes in remaining and hyperactive motor neurons in vivo.

In vitro alpha-adrenoceptor autoradiography of the urethra and urinary bladder of the female pig, cat, guinea-pig and rat.

OBJECTIVE: The aim of this study was to investigate the distribution of alpha1- and alpha2-adrenoceptors in the urethra and urinary bladder of the female pig, cat, guinea-pig and rat. MATERIALS AND METHODS: The binding distributions of an alpha1-adrenoceptor ligand (3H-prazosin) and an alpha2-adrenoceptor ligand (3H-rauwolscine) were determined using in vitro autoradiography. Autoradiograms were analysed by combining computer-based image analysis and light microscopy. RESULTS: In the pig, guinea-pig and rat urethra 3H-prazosin binding was highest in the muscle layer. In the cat urethra 3H-prazosin binding could not be analysed due to a negative chemography artefact. In the pig, cat and guinea-pig urethra 3H-rauwolscine binding was highest in the urothelium, followed by the sub-mucosa, with low levels in muscle. Little 3H-rauwolscine binding was observed in the rat urethra. In the urinary bladder of all species 3H-prazosin binding was low. In the rat bladder, binding was higher in the trigone than in the dome. In the pig, cat and guinea-pig bladder 3H-rauwolscine binding was highest in the mucosa. with little binding in muscle or lamina propria. In the rat bladder, there was little binding and no regional differences. CONCLUSIONS: Alpha1-adrenoceptors were predominantly located in urethral smooth muscle, indicating their contractile importance in maintaining continence. Alpha2-Adrenoceptors were present in the urethral submucosa and bladder mucosa, but not in muscle, suggesting a role in regulation of blood flow, urethral lubrication and tumescence, but not in contraction.

Selectivity of oxymetazoline for urethral pressure vs blood pressure in the anaesthetized female rabbit.

OBJECTIVE: The aim of this study was to test alpha-adrenergic reference agonists for tissue selectivity in the urethra and to pharmacologically characterize the functional alpha-adrenoceptor type of the female rabbit urethra in vivo. MATERIAL AND METHODS: The effect of alpha-adrenergic agonists and antagonists on the urethral pressure was compared with that on blood pressure and heart rate measured simultaneously in the anaesthetized female rabbit. RESULTS: Oxymetazoline, NS-49, phenylephrine and phenylpropanolamine enhanced the urethral pressure in a dose-dependent manner. Phenylephrine and phenylpropanolamine also enhanced the blood pressure with significantly lower ED50 (dose that gives half of the maximal enhancing effect) values than for the urethral pressure. This was in contrast to oxymetazoline and NS-49. The ED50 values for oxymetazoline on urethral pressure, and systolic and diastolic blood pressure were 0.00067, 0.0030 and 0.0020 mg/kg, respectively. The ED50 values for NS-49 on urethral pressure, and systolic and diastolic blood pressure were 0.019, 0.21 and 0.18 mg/kg, respectively. Clonidine and UK 14,304 had no effect on urethral or blood pressure. The oxymetazoline-evoked increase in urethral pressure was inhibited by WB-4101 with an ID50 (dose that gives half of the inhibitory effect) significantly lower than that for rauwolscine. CONCLUSIONS: The results suggest that in the female rabbit in vivo activation of alpha1-adrenoceptors increased the urethral pressure. Phenylephrine and phenylpropanolamine, in contrast to oxymetazoline and NS-49, selectively enhanced blood pressure as compared with urethral pressure. Provided that the present results also have validity in humans, it would seem possible to develop urethra-selective drugs for treatment of stress incontinence with few or no cardiovascular side-effects.

Pharmacological characterisation of the enantiomers of BM-5, a muscarinic partial agonist with opposed enantioselectivity between affinity and efficacy.

The interaction of (R) and (S) enantiomers of the chiral oxotremorine analogue BM-5 with muscarinic acetylcholine receptors was studied in vitro using radioligand binding and isolated tissue preparations. The in vivo effects of (R)-BM-5 were also studied in anaesthetised cat. No receptor or tissue selectivity was found for either enantiomer in radioligand binding studies in cells expressing human muscarinic receptors (M1-M5) or in guinea pig tissues. The affinity of (R)-BM-5 was about 40 times, or 15-60 times higher than that of (S)-BM-5 in recombinant cells or in guinea pig tissues, respectively. Both enantiomers induced contraction of the guinea pig isolated urinary bladder and ileum. (R)-BM-5 was more potent than (S)-BM-5 in bladder (EC50 590 and 3500 nM, respectively) and in ileum (EC50 39 and 2600 nM, respectively). The maximal agonist effect was lower for (R)-BM-5 than for (S)-BM-5 in bladder (2.7% and 6.6%, respectively) and in ileum (32% and 48%, respectively). Contractions were completely inhibited by atropine (1 microM). In vivo, (R)-BM-5 induced bladder contraction and salivation after intravenous administration in the anaesthetised cat (ED50 4.1 and 6.2 microg kg(-1), respectively). In conclusion, (R)- and (S)-BM-5 act as partial muscarinic agonists in the isolated bladder and ileum. (R)-BM-5 was the more potent enantiomer but had a lower maximal agonist effect giving an opposed enantioselectivity for affinity and efficacy. (R)-BM-5 showed agonist activity in vivo, confirming in vitro findings. From affinity and efficacy data it can be concluded that the effects of racemic BM-5 are mediated by the (R)-enantiomer.

Autoradiographic comparison of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine binding in relation to vesicular acetylcholine transport sites in the temporal cortex in Alzheimer's disease.

The laminar binding distribution of three nicotinic receptor agonists, [3H](-)nicotine, [3H]cytisine, and [3H]epibatidine, and their relation to the [3H]vesamicol binding, which is known to represent the vesicular acetylcholine transport sites, was performed employing in vitro autoradiography on the medial temporal cortex (Brodmann area 21). Autopsied brain tissue from nine Alzheimer patients and seven age-matched controls were used. The binding pattern of the three nicotinic ligands in the normal cortex was in general similar, showing binding maxima in the cortical layers I, III and V. The binding of [3H](-)nicotine, [3H]cytisine, and [3H]epibatidine was lower in the older controls and more uniform throughout the layers as compared with younger controls. There was a significant age-related decrease in the binding of the three nicotinic ligands within the controls (age range: 58 to 89 years; P[3H](-)nicotine = 0.002, P[3H]epibatidine = 0.010, P[3H]cytisine = 0.037). In the older controls, the [3H]epibatidine binding was much decreased as compared with that of [3H](-)nicotine and [3H]cytisine. This may indicate a higher selectivity of [3H]epibatidine for a nicotinic receptor subtype that is particularly affected by aging. The laminar binding pattern of [3H]vesamicol showed one maximum in the outer cortical layers II/III. The [3H]vesamicol binding did not change with aging. The binding of all ligands was significantly decreased in all layers of the temporal cortex in Alzheimer's disease, but the [3H]vesamicol binding decreased only half as much as the nicotinic receptors. Also, choline acetyltransferase activity was percentually more reduced than [3H]vesamicol binding in Alzheimer's disease. The cortical laminar binding pattern of all 3H-ligands was largely absent in the Alzheimer's disease cases. The less severe loss of vesicular acetylcholine transport sites as compared with the loss of the nicotinic receptors and choline acetyltransferase activity may suggest that vesamicol binding sites might be more preserved in presynaptic terminals still existing and thereby expressing compensatory capacity to maintain cholinergic activity.

Alteration of spinal cord IGF-I receptors and skeletal muscle IGF-I after hind-limb immobilization in the rat.

The effects of 4 weeks' hind-limb immobilization on the spinal cord insulin-like growth factor-I (IGF-I) receptors and skeletal muscle IGF-I level was investigated in rats. Quantitative receptor autoradiography using [125I]IGF-I as a ligand was performed to measure IGF-I receptors in cryosections from the lumbar region of the spinal cord. IGF-I receptor levels were significantly higher in all spinal cord laminae on the side ipsilateral to the immobilized limb than in the same spinal level of the controls. Using radioimmunoassay (RIA), IGF-I levels were significantly low in the soleus (SOL), but not the tibialis anterior (TIB) muscles, compared to the controls. The enhancement of the spinal cord IGF-I receptors after hind-limb immobilization may constitute part of the nervous system response to disuse.

Chronic mild unpredictable stress after noradrenergic denervation: attenuation of behavioural and biochemical effects of DSP-4 treatment.

Chronic mild unpredictable stress, which reduces rewarded behaviour in rats, is becoming increasingly popular as an animal model of depression. The effect of chronic mild stress (applied to animals housed five per cage for 15 days) on forced swimming and open field behaviour, and on beta-adrenoceptor binding was studied in naive rats and after the denervation of the locus coeruleus projections by DSP-4 (50 mg kg(-1)) treatment. In the forced swimming test, chronic mild stress reduced the immobility time on the second day of testing in both vehicle- and DSP-4-treated rats, indicating rather an antidepressant-like effect. This antidepressant-like effect of chronic mild stress in the forced swimming test was not present in individually housed rats which suggests that this paradigm is sensitive to housing conditions. Stress had no clear effect on the open field locomotion in naive animals (but caused a reduction in defecations), but completely blocked the DSP-4-induced decrease in the exploratory activity. As measured by 3H-dihydroalprenolol binding, DSP-4 treatment increased the beta-adrenoceptor affinity in the frontal cortex and the number of binding sites in the hippocampus and in the cerebral cortex (total-frontal cortex). Stress had no effect on the beta-adrenoceptor binding in the frontal cortex and cerebral cortex, but prevented the increase in affinity caused by DSP-4 treatment in the frontal cortex. In the hippocampus, chronic mild stress and DSP-4 treatment increased the number of beta-adrenoceptor binding sites. Neither chronic mild stress nor DSP-4 treatment had any effect on CCK(B) receptor binding in the cerebral cortex and striatum. These results show that chronic mild stress applied to group-housed rats can prevent the development of certain behavioural and biochemical changes caused by the denervation of the locus coeruleus projection areas.

The effect of phenylpropanolamine on the urethral pressure and heart rate is retained after repeated short-term administration in the unanaesthetized, conscious dog.

The present study was performed to test whether high dose agonist (phenylpropanolamine) administration twice a day causes desensitization of urethral alpha-adrenoceptors in vivo. Urethral pressure was measured on five consecutive days of phenylpropanolamine treatment of the unanaesthetized, conscious dog, and the method is described in detail. Phenylpropanolamine hydrochloride proper (75 mg, 5.1-6.1 mg kg-1), and a sustained-release formulation, both significantly increased urethral pressure and decreased heart rate. Interruption of administration for two to three days did not alter the effect. The present results suggest that the effect of phenylpropanolamine was retained, and that the alpha-adrenoceptors of dog urethra did not desensitize after repeated administration of the alpha-adrenoceptor agonist phenylpropanolamine twice a day.

Laminar distribution of nicotinic receptor subtypes in human cerebral cortex as determined by [3H](-)nicotine, [3H]cytisine and [3H]epibatidine in vitro autoradiography.

The subregional localization of different nicotinic acetylcholine receptor subtypes in human cerebral cortex was estimated by quantitative in vitro autoradiography using the nicotinic ligands [3H](-)nicotine, [3H]cytisine and [3H]epibatidine in large whole human forebrain hemispheres. Saturation experiments in frontal cortex revealed for [3H](-)nicotine two binding sites with affinity constants (Kd) of 0.45 and 6.3 nM and binding site densities (Bmax) of 3.0 and 14.2 pmol/g, for [3H]cytisine one binding site with Kd of 0.19 nM and Bmax of 21.8 pmol/g, and for [3H]epibatidine one binding site with Kd of 0.011 nM and Bmax of 20.0 pmol/g. The laminar binding distributions of the three ligands were compared in different cortical areas by creating binding profiles perpendicular to the entire cortical depth. The regional autoradiographic binding patterns of the three ligands were essentially similar, with higher receptor binding in cortical layers I, III and V. In the primary sensory cortex and inferior frontal sulcus, marked binding of all ligands was observed in layer III. [3H]Cytisine showed the lowest difference between maximal and minimal binding within the gray tissue in all other areas. In the primary motor cortex, [3H]epibatidine and [3H](-)nicotine showed high binding in layers III and V. The [3H](-)nicotine binding was higher than that of the other ligands in layers I and VI of the primary motor cortex, the deeper layer V of the primary sensory cortex, layer III of the superior temporal sulcus and layer VI of the parietal cortex. A distinct band of binding of [3H](-)nicotine and [3H]epibatidine but not of [3H]cytisine was found in layer IIlb of the occipital cortex and layer V of the superior temporal sulcus. [3H]Epibatidine showed higher binding than the other ligands in all layers of the medial frontal, superior frontal and superior temporal sulcus. The findings with the three nicotinic ligands suggest three binding sites in the cortex with different laminar distributions. All three ligands bound to an identical receptor site, most likely the alpha4 nicotinic receptor subunit. The morphological distribution of [3H]epibatidine and [3H](-)nicotine binding indicate that they bind to an additional site, especially in the primary motor cortex, in layer IIIb of the occipital cortex and layer V of the superior temporal sulcus. High binding of [3H](-)nicotine in layers I and VI of the primary motor cortex, the deeper layer V of the primary sensory cortex, layer III of the superior temporal sulcus and layer VI of the parietal cortex may indicate a third binding site.

Comparison of the in vitro and in vivo profiles of tolterodine with those of subtype-selective muscarinic receptor antagonists.

Tolterodine [(R)-N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamine ] is a new potent and competitive muscarinic receptor antagonist developed for the treatment of urinary urge incontinence and other symptoms of overactive bladder. In vivo, tolterodine exhibits functional selectivity for the urinary bladder over salivary glands, a profile that cannot be explained in terms of selectivity for a single muscarinic receptor subtype. The aim of this study was to compare the in vitro and in vivo antimuscarinic profiles of tolterodine with those of muscarinic receptor antagonists with distinct receptor subtype-selectivity profiles: darifenacin [(S)-2-[1-[2-(2,3-dihydrobenzofuran-5-yl)ethyl]-3-pyrrolidinyl]-2,2-d iphenylacetamide; selective for muscarinic M3 receptors]; UH-AH 37 (6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo-[b ,e][1,4]diazepine-11-one; low affinity for muscarinic M2 receptors); and AQ-RA 741 (11-([4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl)-5,11-dihydro-6H-py rido[2,3-b][1,4]benzodiazepine-6-one; high affinity for muscarinic M2 receptors). The in vitro profiles of these compounds were in agreement with previous reports; darifenacin and UH-AH 37 demonstrated selectivity for muscarinic M3/m3 over M2/m2 receptors, while the converse was observed for AQ-RA 741. In vivo, AQ-RA 741 was more potent (1.4-2.7-fold) in inhibiting urinary bladder contraction than salivation in the anaesthetised cat (i.e., a profile similar to that of tolterodine [2.5-3.3-fold]), while darifenacin and UH-AH 37 showed the reverse selectivity profile (0.6-0.8 and 0.4-0.5-fold, respectively). The results confirm that it is possible to separate the antimuscarinic effects on urinary bladder and salivary glands in vivo. The data on UH-AH 37 and darifenacin support the view that a selectivity for muscarinic M3/m3 over M2/m2 receptors may result in a more pronounced effect on salivation than on bladder contraction. The data on AQ-RA 741 may indicate that muscarinic M2/m2 receptors may have a role in bladder contraction.

Antimuscarinic potency and bladder selectivity of PNU-200577, a major metabolite of tolterodine.

PNU-200577 (labcode DD 01 [(R)-N, N-diisopropyl-3-(2-hydroxy-5-hydroxymethylphenyl)-3-phenylpropanamine ) is a major pharmacologically active metabolite of tolterodine, a new muscarinic receptor antagonist intended for the treatment of an overactive bladder. In vitro, PNU-200577 produced a competitive and concentration-dependent inhibition of carbachol-induced contraction of guinea-pig isolated urinary bladder strips (KB = 0.84 nM; pA2 = 9.14). In vivo, PNU-200577 was significantly more potent at inhibiting acetylcholine-induced urinary bladder contraction than electrically induced salivation in the anaesthetised cat (ID50 15 and 40 nmol.kg-1, respectively; P < 0.01). In radioligand binding studies carried out in homogenates of guinea-pig tissues and Chinese hamster ovary cell lines expressing human muscarinic m1-m5 receptors, PNU-200577 was not selective for any muscarinic receptor subtype. Thus, PNU-200577 is similar to tolterodine in terms of antimuscarinic potency, functional selectivity for the urinary bladder in vivo and absence of selectivity for muscarinic receptor subtypes in vitro. The results of this study clearly indicate that PNU-200577 contributes to the therapeutic action of tolterodine, in view of its high antimuscarinic potency, similar serum concentration and lower degree of protein binding.

Effect of immobilization on skeletal muscle nicotinic cholinergic receptors in the rat.

The effect of 4 weeks of hind limb immobilization on nicotinic acetylcholinergic receptors (nAChRs) in the neuromuscular junction of the soleus (SOL) and tibialis anterior (TIB) muscles was studied in rats. Quantitative measurements of the receptors was performed using [3H]alpha-bungarotoxin ([3H]alpha-BTx) receptor autoradiography. Junctional and extrajunctional nAChRs were significantly increased in the SOL and TIB after 4 weeks immobilization. However, a significant decrease in fiber cross-sectional area was observed only in the SOL muscle. Remobilization for 4 weeks reversed the changes in cholinergic receptors and muscle fibers but not in bone. Our findings suggested that lack of nerve impulses are of importance for the events that take place after immobilization leading to muscle atrophy and osteoporosis.

Tolterodine--a new bladder-selective antimuscarinic agent.

Tolterodine is a new muscarinic receptor antagonist intended for the treatment of urinary urge incontinence and other symptoms related to an overactive bladder. The aim of the present study was to compare the antimuscarinic properties of tolterodine with those of oxybutynin, in vitro and in vivo. Tolterodine effectively inhibited carbachol-induced contractions of isolated strips of urinary bladder from guinea pigs (K(B) 3.0 nM; pA2 8.6; Schild slope 0.97) and humans (K(B) 4.0 nM; pA2 8.4; Schild slope 1.04) in a concentration-dependent, competitive manner. The affinity of tolterodine was similar to that derived for oxybutynin (K(B) 4.4 nM; pA2 8.5; Schild slope 0.89) in the guinea-pig bladder. Tolterodine (21-2103 nmol/kg (0.01-1 mg/kg); intravenous infusion) was significantly more potent in inhibiting acetylcholine-induced urinary bladder contraction than electrically-induced salivation in the anaesthetised cat. In contrast, oxybutynin displayed the opposite tissue selectivity. Radioligand binding data showed that tolterodine bound with high affinity to muscarinic receptors in urinary bladder (K(i) 2.7 nM), heart (K(i) 1.6 nM), cerebral cortex (K(i) 0.75 nM) and parotid gland (K(i) 4.8 nM) from guinea pigs and in urinary bladder from humans (K(i) 3.3 nM). Tolterodine and oxybutynin were equipotent, except in the parotid gland, where oxybutynin bound with 8-times higher affinity (K(i) 0.62 nM). Binding data on human muscarinic m1-m5 receptors expressed in Chinese hamster ovary cells showed that oxybutynin, in contrast to tolterodine, exhibits selectivity (10-fold) for muscarinic m3 over m2 receptors. The K(B) value determined for oxybutynin (4.4 nM) in functional studies on guinea-pig bladder correlated better with the binding affinity at muscarinic M2/m2 receptors (K(i) 2.8 and 6.7 nM) than at muscarinic M3/m3 receptors (K(i) 0.62 and 0.67 nM). The tissue selectivity demonstrated for tolterodine in vivo cannot be attributed to selectivity for a single muscarinic receptor subtype. However, the combined in vitro and in vivo data on tolterodine and oxybutynin may indicate either that muscarinic M3/m3 receptors in glands are more sensitive to blockade than those in bladder smooth muscle, or that muscarinic M2/m2 receptors contribute to bladder contraction.

Growth factor receptors in amyotrophic lateral sclerosis.

The regional distribution of nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1) receptors in human spinal cords from controls and amyotrophic lateral sclerosis (ALS) patients was studied by quantitative autoradiography. High-affinity nerve growth factor receptors were found to be distributed to a similar extent within the various segments of the human spinal cord and predominantly within the substantia gelatinosa of the dorsal horn, whereas no significant binding could be detected in the motor-neuron areas. A similar pattern of binding was obtained in the ALS spinal cords. Moreover, no reexpression of NGF receptors could be demonstrated in the motor-neuron areas of ALS spinal cords. When comparing 125I-IGF-1 binding in the different spinal levels of normal spinal cord, the same distribution pattern was found in which the binding was highest in the central canal > dorsal horn > ventral horn > white matter. In the ALS cases, although a general upregulation of IGF-1 receptors was observed throughout the spinal cord, significant increases were observed in the cervical and sacral segments compared to controls. The cartography of IGF-1 receptors in the normal spinal cord as well as the change of these receptors in diseased spinal cord may be of importance in future treatment strategies of ALS.

Insulin-like growth factor-1 receptors in human spinal cord: changes in amyotrophic lateral sclerosis.

Neurotrophic factors are important for neuronal survival and maintenance in the adult nervous system. The regional distribution of insulin-like growth factor-1 (IGF-1) receptors in human spinal cords from controls and amyotrophic lateral sclerosis (ALS) patients was studied by immunohistochemistry and quantitative autoradiography. When comparing 125I-IGF-1 binding in the different spinal levels of normal spinal cord the same distribution pattern was found in which the binding was highest in the central canal > dorsal horn > ventral horn > white matter. In the ALS cases although a general upregulation of IGF-1 receptors was observed throughout the spinal cord, significant increases were observed in the cervical and sacral segments compared to controls. IGF-1 receptor immunoreactivity showed a similar pattern to that for 125I-IGF-1 binding, with immunoreactivity being found in the gray matter of the spinal cord and enhanced immunoreactivity occuring in ALS patients compared to controls. In agreement with the distribution of IGF-1 receptors, IGF-1 immunoreactivity was found within the gray matter of the spinal cord. The cartography of IGF-1 receptors in the normal spinal cord as well as the change of these receptors in diseased spinal cord may be of importance in future treatment strategies of ALS.