Topical ophthalmic atropine in horses, pharmacokinetics and effect on intestinal motility.

BACKGROUND: Topical ophthalmic atropine sulfate is an important part of the treatment protocol in equine uveitis. Frequent administration of topical atropine may cause decreased intestinal motility and colic in horses due to systemic exposure. Atropine pharmacokinetics are unknown in horses and this knowledge gap could impede the use of atropine because of the presumed risk of unwanted effects. Additional information could therefore increase safety in atropine treatment. RESULTS: Atropine sulfate (1 mg) was administered in two experiments: In part I, atropine sulfate was administered intravenously and topically (manually as eye drops and through a subpalpebral lavage system) to six horses to document atropine disposition. Blood-samples were collected regularly and plasma was analyzed for atropine using UHPLC-MS/MS. Atropine plasma concentration was below lower limit of quantification (0.05 µg/L) within five hours, after both topical and IV administration. Atropine data were analyzed by means of population compartmental modeling and pharmacokinetic parameters estimated. The typical value was 1.7 L/kg for the steady-state volume of distribution. Total plasma clearance was 1.9 L/h‧kg. The bioavailability after administration of an ophthalmic preparation as an eye drop or topical infusion were 69 and 68%, respectively. The terminal half-life was short (0.8 h). In part II, topical ophthalmic atropine sulfate and control treatment was administered to four horses in two dosing regimens to assess the effect on gastro-intestinal motility. Borborygmi-frequency monitored by auscultation was used for estimation of gut motility. A statistically significant decrease in intestinal motility was observed after administration of 1 mg topical ophthalmic atropine sulfate every three hours compared to control, but not after administration every six hours. Clinical signs of colic were not observed under any of the treatment protocols. CONCLUSIONS: Taking the plasma exposure after topical administration into consideration, data and simulations indicate that eye drops administrated at a one and three hour interval will lead to atropine accumulation in plasma over 24 h but that a six hour interval allows total washout of atropine between two topical administrations. If constant corneal and conjunctival atropine exposure is required, a topical constant rate infusion at 5 µg/kg/24 h offers a safe alternative.

Effect of Vemurafenib on the Pharmacokinetics of a Single Dose of Tizanidine (a CYP1A2 Substrate) in Patients With BRAFV600 Mutation-Positive Malignancies.

This phase 1 open-label, multicenter, 3-period, fixed-sequence study evaluated the effect of multiple doses of vemurafenib on the pharmacokinetics of 1 dose of tizanidine, a probe CYP1A2 substrate, in patients with BRAFV600 mutation-positive metastatic malignancy. Patients received 1 dose of tizanidine 2 mg on day 1 (period A), vemurafenib 960 mg twice daily on days 2-21 (period B), and 1 dose of tizanidine 2 mg and vemurafenib 960 mg twice daily on day 22 (period C). Log-transformed area under the concentration-time curve (AUC) and maximum plasma concentration (Cmax ) values for tizanidine in 16 patients were compared between periods A (tizanidine alone) and C (tizanidine plus vemurafenib) using an analysis of variance model. Multiple doses of vemurafenib increased plasma exposure of 1 dose of tizanidine, with geometric mean ratios (period C/period A) for Cmax , AUCinf , and AUClast of 2.15 (90%CI, 1.71-2.71), 4.22 (90%CI, 3.37-5.28), and 4.74 (90%CI, 3.55-6.33), respectively; 90%CIs were all outside predefined limits for lack of drug-drug interaction (0.82-1.22). This study confirmed vemurafenib as a moderate inhibitor of CYP1A2 in vivo, with a statistically significant drug-drug interaction with tizanidine. Caution should be exercised when dosing vemurafenib concurrently with CYP1A2 substrates.

In Silico, Ex Vivo and In Vivo Studies of Roflumilast as a Potential Antidiarrheal and Antispasmodic agent: Inhibition of the PDE-4 Enzyme and Voltage-gated Ca++ ion Channels.

The aim of the present study was to evaluate the possible gut inhibitory role of the phosphodiesterase (PDE) inhibitor roflumilast. Increasing doses of roflumilast were tested against castor oil-induced diarrhea in mice, whereas the pharmacodynamics of the same effect was determined in isolated rabbit jejunum tissues. For in silico analysis, the identified PDE protein was docked with roflumilast and papaverine using the Autodock vina program from the PyRx virtual screening tool. Roflumilast protected against diarrhea significantly at 0.5 and 1.5 mg/kg doses, with 40% and 80% protection. Ex vivo findings from jejunum tissues show that roflumilast possesses an antispasmodic effect by inhibiting spontaneous contractions in a concentration-dependent manner. Roflumilast reversed carbachol (CCh, 1 µM)-mediated and potassium (K+, 80 mM)-mediated contractile responses with comparable efficacies but different potencies. The observed potency against K+ was significantly higher in comparison to CCh, similar to verapamil. Experiments were extended to further confirm the inhibitory effect on Ca++ channels. Interestingly, roflumilast deflected Ca++ concentration-response curves (CRCs) to the right with suppression of the maximum peak at both tested doses (0.001-0.003 mg/mL), similar to verapamil. The PDE-inhibitory effect was authenticated when pre-incubation of jejunum tissues with roflumilast (0.03-0.1 mg/mL) produced a leftward deflection of isoprenaline-mediated inhibitory CRCs and increased the tissue level of cAMP, similar to papaverine. This idea was further strengthened by molecular docking studies, where roflumilast exhibited a better binding affinity (-9.4 kcal/mol) with the PDE protein than the standard papaverine (-8.3 kcal/mol). In conclusion, inhibition of Ca++ channels and the PDE-4 enzyme explains the pharmacodynamics of the gut inhibitory effect of roflumilast.

A Novel Ileocolonic Release Peppermint Oil Capsule for Treatment of Irritable Bowel Syndrome: A Phase I Study in Healthy Volunteers.

INTRODUCTION: Peppermint oil (PO) has been shown to reduce abdominal pain in patients with irritable bowel syndrome (IBS). PO is assumed to induce intestinal smooth muscle relaxation and desensitization of nociceptive nerve afferents. To increase colonic PO concentration, an ileocolonic release peppermint oil (IC-PO) capsule has been developed. The aim of this study was to compare pharmacokinetic parameters of the currently available small intestinal release PO (SI-PO) and the novel IC-PO. METHODS: In this randomized, double-blind, crossover study, subjects received 182 mg of either SI-PO or IC-PO in a crossover design with a washout period of more than 14 days. Blood samples were collected to determine menthol glucuronide concentrations. RESULTS: Eight healthy volunteers (50% female, median age 22) were included. The time to reach the maximum concentration (Tmax) of IC-PO was significantly longer compared to SI-PO with a median (IQR) of 360 (360-405) versus 180 (120-180) min. The lag time (Tlag) was significantly longer with a median (IQR) of 225 (204-284) for IC-PO compared to 37 (6-65) min for SI-PO. The areas under the menthol glucuronide plasma concentration-time curves were significantly smaller with a median (IQR) of 2331 µg h/L (2006-2510) for IC-PO compared to 2623 µg h/L (2471-2920) for SI-PO. No significant differences were found in peak concentrations and elimination half-lives. CONCLUSION: IC-PO has a significantly delayed peak menthol glucuronide concentration and Tlag, both pointing to the release of PO in the more distal part of the intestine. This may enhance therapeutic efficacy as it results in increased exposure of colonic mucosal afferents to the PO. A randomized controlled trial investigating the efficacy of SI and IC-PO in IBS is currently ongoing. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT02291445, EudraCT database 2014-004195-32.

Formulation and Evaluation of Fast-Disintegrating Sublingual Tablets of Atropine Sulfate: the Effect of Tablet Dimensions and Drug Load on Tablet Characteristics.

In this study, we formulated and evaluated the effects of tablet dimensions and drug load on the characteristics of atropine sulfate (AS) fast-disintegrating sublingual tablets (FDSTs). We aim to develop AS FDSTs as an alternative non-invasive and portable dosage form for the emergency treatment of organophosphate (OP) toxicity. AS autoinjector, AtroPen®, is the only self-administered dosage form available as an antidote for-out-of-hospital emergency use, but it is associated with several limitations and drawbacks. Seven FDST formulations of two tablet sizes, 150 mg (A) and 50 mg (B), and of several AS loads, 0 mg (A1, B1), 2 mg (A2, B2), 4 mg (B3), and 8 mg (B4a, B4b), were formulated and manufactured by direct compression. AS FDST characteristics were evaluated using USP and non-USP tests. Results were statistically compared at p < 0.05. All FDSTs passed the USP content uniformity and friability tests, disintegrated and released AS in ≤30 and 60 s. B1 and B2 were significantly harder than A1 and A2. Water uptake of A1 was significantly the highest. However, B1 and B2 had shorter disintegration and wetting times and higher amounts of AS dissolved than did A1 and A2 (p < 0.05). Increasing AS negatively affected FDST tensile strength (p < 0.05 for B4a) and water uptake (p < 0.05 for B3, B4a and B4b), however, without affecting AS dissolution. Formulation of AS up to 16% into smaller FDSTs was successful. Smaller FDSTs were harder and disintegrated more quickly. These AS FDSTS have the potential for further in vivo testing to evaluate their OP antidote potential.

Pharmacokinetics of Compound D, the Major Bioactive Component of Zingiber cassumunar, in Rats.

Rhizomes of Zingiber cassumunar have been used for many years in traditional Thai medicine as an anti-inflammatory agent. The major bioactive component of this plant is Compound D [E-4-(3', 4'-dimethoxyphenyl)but-3-en-1-ol], which is a strong smooth muscle relaxant, and has antihistamine and anti-inflammatory actions. There is, however, incomplete information available for the pharmacokinetics of Compound D in mammals. In this study, we examined the pharmacokinetic profiles of Compound D in male Wistar rats. A standardized extract of Z. cassumunar containing 4 % w/w Compound D was administered intravenously at 25 mg/kg or by oral gavage at 25, 75, or 250 mg/kg to Wistar rats. Blood, tissues, urine, and feces were collected from 0 to 48 h after dosing and the level of Compound D was determined by liquid chromatography-tandem mass spectrometry. The concentration of Compound D ranged from 10-100 µg/L, reached a maximum approximately 0.15 h after oral dosing. Compound D exhibited an excellent tissue to plasma ratio, ranging from 1- to 1000 in several organs at 1-4 h after oral dosing. Less than 1 % of unchanged Compound D was excreted in the urine and feces. Further studies on tissue uptake and metabolite identification are required to obtain complete pharmacokinetic information and to develop appropriate dosing strategies of Compound D and the standardized extract of Z. cassumunar.

Expression of drug transporters and drug metabolizing enzymes in the bladder urothelium in man and affinity of the bladder spasmolytic trospium chloride to transporters likely involved in its pharmacokinetics.

The cationic, water-soluble quaternary trospium chloride (TC) is incompletely absorbed from the gut and undergoes wide distribution but does not pass the blood-brain barrier. It is secreted by the kidneys, liver, and intestine. To evaluate potential transport mechanisms for TC, we measured affinity of the drug to the human uptake and efflux transporters known to be of pharmacokinetic relevance. Affinity of TC to the uptake transporters OATP1A2, -1B1, -1B3, -2B1, OCT1, -2, -3, OCTN2, NTCP, and ASBT and the efflux carriers P-gp, MRP2 and MRP3 transfected in HEK293 and MDCK2 cells was measured. To identify relevant pharmacokinetic mechanisms in the bladder urothelium, mRNA expression of multidrug transporters, drug metabolizing enzymes, and nuclear receptors, and the uptake of TC into primary human bladder urothelium (HBU) cells were measured. TC was shown to be a substrate of OATP1A2 (Km = 6.9 ± 1.3 µmol/L; Vmax = 41.6 ± 1.8 pmol/mg·min), OCT1 (Km = 106 ± 16 µmol/L; Vmax = 269 ± 18 pmol/mg·min), and P-gp (Km = 34.9 ± 7.5 µmol/L; Vmax = 105 ± 9.1 pmol/mg·min, lipovesicle assay). The genetic OATP1A2 variants *2 and *3 were loss-of-function transporters for TC. The mRNA expression analysis identified the following transporter proteins in the human urothelium: ABCB1 (P-gp), ABCC1-5 (MRP1-5), ABCG2 (BCRP), SLCO2B1 (OATP2B1), SLCO4A1 (OATP4A1), SLC22A1 (OCT1), SLC22A3 (OCT3), SLC22A4 (OCTN1), SLC22A5 (OCTN2), and SLC47A1 (MATE1). Immuno-reactive P-gp and OATP1A2 were localized to the apical cell layers. Drug metabolizing enzymes CYP3A5, -2B6, -2B7 -2E1, SULT1A1-4, UGT1A1-10, and UGT2B15, and nuclear receptors NR1H3 and NR1H4 were also expressed on mRNA level. TC was taken up into HBU cells (Km = 18.5 ± 4.8 µmol/L; Vmax = 106 ± 11.3 pmol/mg·min) by mechanisms that could be synergistically inhibited by naringin (IC50 = 10.8 (8.4; 13.8) µmol/L) and verapamil (IC50 = 4.6 (2.8; 7.5) µmol/L), inhibitors of OATP1A2 and OCT1, respectively. Affinity of TC to OCT1 and P-glycoprotein may be the reason for incomplete oral absorption, wide distribution into liver and kidneys, and substantial intestinal and renal secretions. Absence of brain distribution may result from affinity to P-gp and a low affinity to OATP1A2. The human urothelium expresses many drug transporters and drug metabolizing enzymes that may interact with TC and other drugs eliminated into the urine.

Characterization of in vitro ADME properties of diosgenin and dioscin from Dioscorea villosa.

Dioscorea villosa (wild yam) is native to North America and has been widely used as a natural alternative for estrogen replacement therapy to improve women's health as well as to treat inflammation, muscle spasm, and asthma. Diosgenin and dioscin (glycoside form of diosgenin) are reported to be the pharmacologically active compounds. Despite the reports of significant pharmacological properties of dioscin and diosgenin in conditions related to inflammation, cancer, diabetes, and gastrointestinal ailments, no reports are available on ADME properties of these compounds. This study was carried out to determine ADME properties of diosgenin and dioscin and their effects on major drug metabolizing enzymes (CYP 3A4, 2D6, 2C9, and 1A2). The stability was determined in simulated gastric and intestinal fluids (SGF, pH 1.2 and SIF, pH 6.8), and intestinal transport was evaluated in Caco-2 model. Phase I and phase II metabolic stability was determined in human liver microsomes and S9 fractions, respectively. Quantitative analysis of dioscin and diosgenin was performed by UPLC-MS system. Dioscin degraded up to 28.3 % in SGF and 12.4 % in SIF, which could be accounted for by its conversion to diosgenin (24.2 %. in SGF and 2.4 % in SIF). The depletion of diosgenin in SGF and SIF was < 10 %. Diosgenin was stable in HLM but disappeared in S9 fraction with a half-life of 11.3 min. In contrast, dioscin was stable in both HLM and S9 fractions. Dioscin showed higher permeability across Caco-2 monolayer with no significant efflux, while diosgenin was subjected to efflux mediated by P-glycoprotein. Diosgenin and dioscin inhibited CYP3A4 with IC50 values of 17 and 33 µM, respectively, while other CYP enzymes were not affected. In conclusion, dioscin showed better intestinal permeability. Conversion of dioscin to diosgenin was observed in both gastric and intestinal fluids. No phase I metabolism was detected for both compounds. The disappearance of diosgenin in S9 fraction indicated phase II metabolism.

Pharmacokinetic comparisons of two different combinations of Shaoyao-Gancao Decoction in rats: competing mechanisms between paeoniflorin and glycyrrhetinic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Shaoyao-Gancao Decoction (SGD), a well-known traditional Chinese medicine prescription, is a combination of Radix Paeoniae Alba (Paeonia lactiflora Pall, root) and Glycyrrhizae uralensis (Glycyrrhiza uralensis Fisch., root and rhizome, honeyed) for spasmolysis and emergency pain relief. Paeoniflorin (PF) and glycyrrhetinic acid (GA) are two typical active components of SGD for pain relief. AIM OF THE STUDY: To study comparative pharmacokinetics of ten bioactive compounds in SGDs with two different combinations of RP and GU, and therefore to investigate the herb-herb interaction mechanisms of Shaoyao-Gancao Decoction for better spasmolysis and emergency pain relief in rats. MATERIALS AND METHODS: Herbal IR macro-fingerprinting was implemented to provide the full chemical fingerprints of RP, GU and SGD decoctions and to investigate the variation rule of the full chemical profile of SGDs with various combinations of RP and GU. A specifically developed HPLC-MS/MS assay coupled with protein precipitation method was employed to determine the plasma concentrations of the ten analytes. Male Wistar rats were orally administered with SGD1 (RP:GU, 1:1 (w/w)) and SGD2 ((RP:GU, 4:1 (w/w)) equivalent to 9.5 g/kg body weight of GU. RESULTS: Full chemical fingerprints of RP, GU and SGDs with various combinations of RP and GU were provided in the form of IR macro-fingerprints. Except for liquiritin, there were statistically significant differences (p<0.05 or p<0.01) of these analytes between SGD1 and SGD2 in in vivo pharmacokinetic study. Compared with the results when oral administrated with SGD1, six glycosides (PF, albiflorin, oxypaeoniflorin, isoliquiritin, ononin, and glycyrrhizin) exhibited higher systematic exposure levels (AUC0-t) and slower elimination rates (CL) whereas two glycones (GA and isoliquiritigenin) were the reverse when administrated with SGD2. CONCLUSIONS: Increasing the amount of RP attenuated the inhibitory effect of GA via competing being consumed by intestinal bacteria (or ß-glucosidase) to reduce the conversion amount of glycyrrhizin to GA and subsequently to afford significantly higher bioavailability and longer efficacy of PF, glycyrrhizin, albiflorin, oxypaeoniflorin, isoliquiritin, and ononin, leading to better spasmolysis and emergency pain relief.

Metabolic and toxicological considerations for the latest drugs used to treat irritable bowel syndrome.

INTRODUCTION: The high prevalence of irritable bowel syndrome (IBS), a chronic gastrointestinal (GI) disorder, its lack of satisfactory effective drugs and its complicated pathophysiology lead to the demand of new therapeutic agents. During a new drug development process, the pharmacokinetic profiling is of a great considerable importance comparable to drug's efficacy. This involves the drug's absorption, distribution, metabolism and excretion, all of which are crucial to its usefulness. In addition, the toxicological profile and possible adverse reactions of the drug should be identified. Also its interactions should be identified at different phases of trials. Several pharmacokinetic studies are carried out to achieve drugs with the best absorption and bioavailability and the least adverse effects and lowest toxicity. AREAS COVERED: To make an update on new clinically introduced drugs for IBS and their dynamics and kinetics data, the present systematic review was accomplished. All relevant bibliographic databases were searched from the year 2003 up to May 2012 to identify all clinical trials that evaluated the potential efficacy of a novel agent in IBS. EXPERT OPINION: Some evaluated drugs, such as ramosetron (5-HT3 antagonist) and pexacerfont (CRF1 receptor antagonist), have shown some benefits in diarrhea-predominant IBS (D-IBS), while, prucalopride and mosapride (5-HT4 agonist) with prokinetic effect were found useful in constipation-predominant IBS (C-IBS). Besides, dexloxiglumide, lubiprostone and linaclotide have shown beneficial effects in C-IBS patients. Melatonin regulates GI tract motility and, asimadoline, gabapentin and pregabalin show reduction of pain threshold and visceral hypersensitivity. Glucagon-like peptide analog, calcium-channel blockers and neurokinin receptor antagonists have shown benefits in pain attacks. More time is required to indicate both efficacy and safety in long-term treatment due to multifactorial pathophysiology, variations in individual responses and insufficient assessment methods, which limit the right decision-making process about the efficacy and tolerability of these new drugs.

[Hyperthermia in spina bifida patients treated with oxybutynin]. / Temperaturerhöhung unter Oxybutynin bei Patienten mit Spina bifida.

Paraplegic spina bifida patients often suffer from disturbed sweat secretion in the paretic regions. A diminished sweat production of caudal parts of the body is compensated by an increased secretion of sweat in parts cranial to the lesion to maintain temperature homoeostais. If the sweat secretion is blocked by anticholinergic effects of urotherapeutic drugs (for instance oxybutynin) hyperthermia can result as a side effect as these casuistic examples show.An 8-year-old girl with a lumbar meningomyelocele and a neurogenic bladder reported a dry skin and hyperthermia up to 38,5°C during oral therapy with oxybutynin (0.4 mg per kg body weight) during hot summer days. Similar symptoms were shown by a 7-year-old male patient with a sacral meningomyelocele and neurogenic bladder on oral therapy of 0.35 mg oxybutynin per kg body weight. A 4-year-old female patient with lumbar spina bifida and neurogenic bladder reacted to intravesical administration of 0.4 and 0.3 mg per kg body weight during early summertime with hyperthermia up to 38°C. In this case the medication had been started in wintertime and was primarily well tolerated.Hyperthermia under treatment with anticholinergic drugs has mainly been published for geriatric patients with sometimes fatal outcome. In the pediatric literature there is only one warning regarding the use of oxybutynin in children with spina bifida living in high temperature regions. It is remarkable that hyperthermia can also happen after intravesical administration of oxybutynin in usual dosage.

Pharmacokinetic and bioequivalence studies of trospium chloride after a single-dose administration in healthy Chinese volunteers.

The study aimed to compare and evaluate the bioequivalence of a new generic preparation of trospium chloride (CAS NO:10405-02-4) capsule (20 mg, test) and the available import tablet (20 mg , reference) for the requirement of state regulatory criteria in China. A randomized- sequence, 2-period crossover study was conducted in 20 healthy Chinese male volunteers in the fasted state. Blood samples were collected before and 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60 h after administration of a single oral dose of 40 mg trospium chloride capsules or tablets, followed by a 7-day washout period. The concentration of trospium chloride was determined by a LC-MS/MS method. Drug And Statistical-Version 2.0 was used to calculate the pharmacokinetics parameters and assess bioequivalence of the two preparations. It was considered bioequivalent if the 90% CIs of the mean ratios (test: reference) for Cmax, AUC0-t and AUC0-∞ were within the range from 80% to 125%, respectively. The main pharmacokinetics parameters of test and reference were as follows: t1/2 was (15.11 ± 3.24) h and (16.00 ± 3.96) h; Tmax was (4.0 ± 1.2) h and (4.1 ± 0.9) h; Cmax was (3.76 ± 1.87) ng·mL - 1 and (3.70 ± 1.89) ng·mL - 1; AUC0-t was (33.51 ± 14.39) ng·mL - 1·h and (33.33 ± 14.88) ng·mL - 1·h, and the AUC0-∞ was (35.20 ± 14.88) ng·mL - 1·h and (35.16±15.17) ng·mL - 1·h. The ratios (test: reference) for Cmax, AUC0-t, and AUC0-∞ were 94.0%~111.7%, 96.4%~106.8%, and 96.1%~105.3%, respectively. No significant differences in pharmacokinetic parameters were found between preparations and periods (p>0.05). No obvious adverse events were monitored throughout the study based on clinical parameters and patient reports.

Shenfushu granule and atropine attenuate microvasculature loss in rat models with 5/6 nephrectomy.

OBJECTIVE: To investigate the effect of Shenfushu granule (SFSG) and atropine treatment on microvessels of the kidney and intestine after chronic renal failure (CRF) induced by 5/6 nephrectomy. METHODS: Sprague Dawley rats were randomly divided into a sham group, a model group, an SFSG group, and an SFSG + atropine group. SFSG was administered daily 1 week after inducing CRF. Rats were sacrificed at the end of the eighth week. Urinary protein and stool and serum urea nitrogen (UN) and creatinine (Cr) levels were assessed. Hematoxylin and eosin and periodic acid-Schiff staining of the kidney and examination of the vascular endothelial growth factor (VEGF) and microvessel density (MVD) levels in kidney and intestine were performed. RESULTS: The Cr and UN levels were significantly increased in blood and stool of the model group. SFSG significantly improved renal function, and the protective effects were further enhanced with the addition of atropine. Glomerular sclerosis, tubulointerstitial fibrosis, and microvessel loss were observed in CRF rats, and these pathological changes were ameliorated in the two treatment groups (p < 0.05), especially in the SFSG + atropine group. The expression of VEGF and MVD was decreased in the CRF rats compared with the sham group. SFSG treatment increased the expression of these proteins and reversed the degree of microvessel loss, glomerular sclerosis, and tubulointerstitial fibrosis (p < 0.05). Co-treatment with atropine enhanced these effects. CONCLUSIONS: SFSG alleviated renal function, upregulated the expression of VEGF and MVD in the kidney and intestine, and attenuated the degree of microvessel loss, glomerular sclerosis, and tubulointerstitial fibrosis in the early stages of CRF in rats, and addition of atropine enhanced these effects.

Muscarinic receptor/G-protein coupling is reduced in the dorsomedial striatum of cognitively impaired aged rats.

Behavioral flexibility, the ability to modify responses due to changing task demands, is detrimentally affected by aging with a shift towards increased cognitive rigidity. The neurobiological basis of this cognitive deficit is not clear although striatal cholinergic neurotransmission has been implicated. To investigate the possible association between striatal acetylcholine signaling with age-related changes in behavioral flexibility, young, middle-aged, and aged F344 X Brown Norway F1 rats were assessed using an attentional set-shifting task that includes two tests of behavioral flexibility: reversal learning and an extra-dimensional shift. Rats were also assessed in the Morris water maze to compare potential fronto-striatal-dependent deficits with hippocampal-dependent deficits. Behaviorally characterized rats were then assessed for acetylcholine muscarinic signaling within the striatum using oxotremorine-M-stimulated [(35)S]GTPγS binding and [(3)H]AFDX-384 receptor binding autoradiography. The results showed that by old age, cognitive deficits were pronounced across cognitive domains, suggesting deterioration of both hippocampal and fronto-striatal regions. A significant decline in oxotremorine-M-stimulated [(35)S]GTPγS binding was limited to the dorsomedial striatum of aged rats when compared to young and middle-aged rats. There was no effect of age on striatal [(3)H]AFDX-384 receptor binding. These results suggest that a decrease in M2/M4 muscarinic receptor coupling is involved in the age-associated decline in behavioral flexibility.

Determination of pinostrobin in rat plasma by LC-MS/MS: application to pharmacokinetics.

A rapid and sensitive method for the determination of pinostrobin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the first time. Isoliquiritigenin was used as an internal standard in rat plasma. Chromatographic separation was performed on an HiQ Sil C(18) column with isocratic elution at a flow rate of 1mL/min. The mobile phase consisted of water and methanol (9:91, v/v) containing 0.1% formic acid. The quantification limit was 10ng/mL within a linear range of 10-1000ng/mL (R=0.9984). The intra- and inter-day assay precision ranged from 3.8-5.3% to 3.2-5.2%, respectively, and the intra- and inter-day assay accuracy was between 93.2-95.1% and 95.5-104.3%, respectively. Our results indicated that the LC-MS/MS method is effective for pharmacokinetic study of pinostrobin in rat plasma.

Formulation and evaluation of effervescent floating tablets of tizanidine hydrochloride.

Tizanidine hydrochloride is an orally administered prokinetic agent that facilitates or restores motility through-out the length of the gastrointestinal tract. The objective of the present investigation was to develop effervescent floating matrix tablets of tizanidine hydrochloride for prolongation of gastric residence time in order to overcome its low bioavailability (34-40 %) and short biological half life (4.2 h). Tablets were prepared by the direct compression method, using different viscosity grades of hydroxypropyl methylcellulose (HPMC K4M, K15M and K100M). Tablets were evaluated for various physical parameters and floating properties. Further, tablets were studied for in vitro drug release characteristics in 12 hours. Drug release from effervescent floating matrix tablets was sustained over 12 h with buoyant properties. DSC study revealed that there is no drug excipient interaction. Based on the release kinetics, all formulations best fitted the Higuchi, first-order model and non-Fickian as the mechanism of drug release. Optimized formulation (F9) was selected based on the similarity factor (f2) (74.2), dissolution efficiency at 2, 6 and 8 h, and t50 (5.4 h) and was used in radiographic studies by incorporating BaSO4. In vivo X-ray studies in human volunteers showed that the mean gastric residence time was 6.2 ± 0.2 h.

Pharmacokinetics of once-daily trospium chloride 60 mg extended release and twice-daily trospium chloride 20 mg in healthy adults.

The objective of this study is to characterize the steady-state pharmacokinetics and compare the relative bioavailability of the extended-release capsule formulation of the antimuscarinic trospium chloride, developed for once-daily administration, and trospium chloride immediate-release tablets. This is a single-center, multidose, randomized, open-label, 2-period, 2-arm crossover, bioavailability study in healthy adult male and female subjects who are within 20% of their ideal body weight. Subjects receive trospium 60-mg extended-release capsules once daily and trospium 20-mg tablets twice daily for 10 days, each in a crossover manner. Twenty-four subjects are enrolled in the study. With multiple dosing of trospium 60 mg extended-release once daily versus 20 mg twice daily, lower geometric least squares mean area under the concentration-time curve from 0 to 24 hours (17 360 vs 28 590 pg.h/mL; ratio 61%; 90% confidence interval, 51-72) and maximum plasma concentration (1517 vs 2502 pg/mL; 61%; 90% confidence interval, 49-75) are observed. Furthermore, with trospium 60 mg extended-release versus 20 mg, median time to maximum plasma concentration is later (5.0 vs 4.5 hours) and half-life is longer (35.8 vs 27.2 hours). Trospium exposure is lower with multiple dosing of trospium 60 mg extended-release compared with trospium 20 mg twice daily. Thus, with the extended-release formulation, trospium concentrations are less likely to reach the threshold where adverse events may sometimes occur.

Molecular mechanisms of action and in vivo validation of an M4 muscarinic acetylcholine receptor allosteric modulator with potential antipsychotic properties.

We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M(4) muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligand-binding studies revealed that LY2033298 displayed a preference for the active state of the M(4) mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [(35)S]GTPgammaS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3beta phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M(4) mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [(3)H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M(4) mAChR knockout mice, validating the M(4) mAChR as a key target of action of this novel allosteric ligand.

Dual radiotracer analysis of cholinergic neuronal changes in prediabetic mouse pancreas.

BACKGROUND: Pancreatic neuronal changes associated with beta cell loss in type 1 diabetes mellitus are complex, involving, in part, parasympathetic mechanisms to compensate for preclinical hyperglycemia. The parasympathetic neurotransmitter acetylcholine (ACh) mediates insulin release via M3 muscarinic receptors on islet beta cells. The vesicular ACh transporter (VAChT) receptor has been shown to be a useful marker of cholinergic activity in vivo. The positron emission tomography (PET) radiotracer (+)-4-[(18)F]fluorobenzyltrozamicol ([(18)F]FBT) binds to the VAChT receptor on presynaptic cholinergic neurons and can be quantified by PET. The compound 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), available in a tritiated form, binds to M3 muscarinic receptors on beta cells and is a potential target for assessing pancreatic beta cell mass. In this study, we investigate the feasibility of dual radiotracer analysis in identifying neurofunctional changes that may signify type 1 diabetes mellitus in its early preclinical state. METHODS: Ex vivo determinations of pancreatic uptake were performed in prediabetic nonobese diabetic mice and controls after intravenous injection of [(18)F]FBT or 4-[(3)H]DAMP. Beta cell loss in prediabetic mice was confirmed using immunohistochemical methods. RESULTS: [(18)F]FBT uptake was significantly higher in prediabetic pancreata than controls: 3.22 +/- 0.81 and 2.51 +/- 1.04, respectively (P < 0.03). 4-[(3)H]DAMP uptake was significantly lower in prediabetic pancreata than controls: 0.612 +/- 0.161 and 0.968 +/- 0.364, respectively (P = 0.01). CONCLUSIONS: These data suggest that a combination of radiotracer imaging agents that bind to neuronal elements intimately involved in insulin production may be an effective method of evaluating changes associated with early beta cell loss using PET.

Hyoscine butylbromide - a review on its parenteral use in acute abdominal spasm and as an aid in abdominal diagnostic and therapeutic procedures.

BACKGROUND: Being a quaternary ammonium compound derived from scopolamine, the alkaloid hyoscine butylbromide (HBB) exerts anticholinergic effects without side effects related to the central nervous system because it does not pass the blood-brain barrier. Clinical experience with this antispasmodic dates back to the 1950s and led to its registration for treating abdominal cramps/spasm and for diagnostic imaging purposes. OBJECTIVES AND SCOPE: This review focuses on the therapeutic efficacy and safety of the parenteral administration of HBB for treating biliary and renal colic and acute spasm in the genito-urinary tract. In addition, its value for diagnostic or therapeutic procedures in the abdomen, as well as for labour and palliative care, is reviewed. With the generic and trade name of the drug combined with various search terms related to the relevant clinical applications, a thorough literature search was performed in the Medline and EMBASE databases in April 2008. FINDINGS: In most clinical studies, recommended doses of 20-40 mg HBB were injected, mainly intravenously. Fast pain reduction was achieved by HBB in renal colic; about 90% of the patients showed good to moderate analgesic responses after 30 min and the onset of action was noticeable within 10 min. Similarly, a pain reduction of 42-78% was observed in patients with biliary colic within 30 min after a single intravenous injection of 20 mg. In contrast, no analgesic efficacy of a single injection of 20 mg was found after surgical or shock-wave procedures in the urogenital area. Administration of HBB prior to, or during, radiological imaging distended the gastrointestinal (GI) tract in double-contrast barium and computed tomographic colonography studies and reduced motion artefacts in magnetic resonance imaging. This improved diagnostic image quality and organ visualisation. Pre-medication led to shorter and easier endoscopy in some, but not all, studies. Because of cervical relaxation, HBB shortened total labour duration with 17-67%. It also relieved pain and reduced GI secretions in terminal cancer patients with inoperable bowel obstruction. With regard to its safety profile, parenteral administration of HBB is associated with mild and self-limiting adverse events, typical for anticholinergic drugs. CONCLUSIONS: These clinical results of rapid action and beneficial efficacy combined with good tolerability support the use of HBB in a range of indications related to acute abdominal spasm, in labour and palliative care and for supporting diagnostic and therapeutic abdominal procedures, where spasm may be a problem.