Cathepsin A contributes to left ventricular remodeling by degrading extracellular superoxide dismutase in mice.

In the heart, the serine carboxypeptidase cathepsin A (CatA) is distributed between lysosomes and the extracellular matrix (ECM). CatA-mediated degradation of extracellular peptides may contribute to ECM remodeling and left ventricular (LV) dysfunction. Here, we aimed to evaluate the effects of CatA overexpression on LV remodeling. A proteomic analysis of the secretome of adult mouse cardiac fibroblasts upon digestion by CatA identified the extracellular antioxidant enzyme superoxide dismutase (EC-SOD) as a novel substrate of CatA, which decreased EC-SOD abundance 5-fold. In vitro, both cardiomyocytes and cardiac fibroblasts expressed and secreted CatA protein, and only cardiac fibroblasts expressed and secreted EC-SOD protein. Cardiomyocyte-specific CatA overexpression and increased CatA activity in the LV of transgenic mice (CatA-TG) reduced EC-SOD protein levels by 43%. Loss of EC-SOD-mediated antioxidative activity resulted in significant accumulation of superoxide radicals (WT, 4.54 µmol/mg tissue/min; CatA-TG, 8.62 µmol/mg tissue/min), increased inflammation, myocyte hypertrophy (WT, 19.8 µm; CatA-TG, 21.9 µm), cellular apoptosis, and elevated mRNA expression of hypertrophy-related and profibrotic marker genes, without affecting intracellular detoxifying proteins. In CatA-TG mice, LV interstitial fibrosis formation was enhanced by 19%, and the type I/type III collagen ratio was shifted toward higher abundance of collagen I fibers. Cardiac remodeling in CatA-TG was accompanied by an increased LV weight/body weight ratio and LV end diastolic volume (WT, 50.8 µl; CatA-TG, 61.9 µl). In conclusion, CatA-mediated EC-SOD reduction in the heart contributes to increased oxidative stress, myocyte hypertrophy, ECM remodeling, and inflammation, implicating CatA as a potential therapeutic target to prevent ventricular remodeling.

Cathepsin A Mediates Ventricular Remote Remodeling and Atrial Cardiomyopathy in Rats With Ventricular Ischemia/Reperfusion.

After myocardial infarction, remote ventricular remodeling and atrial cardiomyopathy progress despite successful revascularization. In a rat model of ventricular ischemia/reperfusion, pharmacological inhibition of the protease activity of cathepsin A initiated at the time point of reperfusion prevented extracellular matrix remodeling in the atrium and the ventricle remote from the infarcted area. This scenario was associated with preservation of more viable ventricular myocardium and the prevention of an arrhythmogenic and functional substrate for atrial fibrillation. Remote ventricular extracellular matrix remodeling and atrial cardiomyopathy may represent a promising target for pharmacological atrial fibrillation upstream therapy following myocardial infarction.

Olfactory function in patients with multiple sclerosis: a diffusion tensor imaging study.

BACKGROUND: Previous research has shown that multiple sclerosis (MS) patients can develop olfactory disturbances. OBJECTIVE: The purpose of our study was to investigate how olfactory function in MS patients correlates with cerebral magnetic resonance imaging (MRI) including diffusion tensor imaging (DTI). METHODS: Olfactory performance was tested in 30 MS patients and 30 controls to determine odour threshold (T), odour discrimination (D), and odour identification (I) summarised in the TDI score. The lesion load (number and total volume of lesions) was measured on proton-density (PD)- and T2-weighted images of the olfactory brain and the total brain. Fractional anisotropy (FA) of the lesions and the surrounding normal-appearing brain tissue (NABT) was quantified using DTI. RESULTS: The median FA of white matter lesions was 0.29 and was on average 11.1% lower than in the surrounding NABT. The normalised TDI score and the normalised I subscore were significantly poorer in the MS group compared to controls (p<0.0001), while the T and D subscores were similar in both groups. The median FA of lesions in the olfactory brain correlated inversely with the decreased I subscore (p=0.001). There was also a strong correlation between the TDI score and the Expanded Disability Status Scale (EDSS) (p=0.001). CONCLUSION: A strong inverse relationship between decreased odour identification ability of MS patients and FA values in the olfactory brain indicates that the reduction in I is more strongly affected by lesions in areas with high FA values, i.e., with an increased amount of affected white matter tracts.

Structural correlates of taste and smell loss in encephalitis disseminata.

BACKGROUND: Olfactory dysfunction in MS patients is reported in the literature. MRI of the olfactory bulb (OB) is discussed as a promising new testing method for measuring olfactory function (OF). Aim of this study was to explore reasons for and optimize the detection of olfactory dysfunction in MS patients with MRI. MATERIALS AND METHODS: OB and olfactory brain volume was assessed within 34 MS patients by manual segmentation. Olfactory function was tested using the Threshold-Discrimination-Identification-Test (TDI), gustatory function was tested using Taste Strips (TST). RESULTS: 41% of the MS patients displayed olfactory dysfunction (8% of the control group), 16% displayed gustatory dysfunction (5% of the control group). There was a correlation between the OB volume and the number and volume of MS lesions in the olfactory brain. Olfactory brain volume correlated with the volume of lesions in the olfactory brain and the EDSS score. The TST score correlated with the number and volume of lesions in the olfactory brain. CONCLUSION: The correlation between a higher number and volume of MS lesions with a decreased OB and olfactory brain volume could help to explain olfactory dysfunction.

Peak inspiratory flow rates generated through the Novolizer and the Turbuhaler dry powder inhaler devices by children with stable asthma.

We compared the peak inspiratory flows (PIF) generated through a novel dry powder inhaler device, the Novolizer (PIF-N), and the Turbuhaler (PIF-T). Forty-six pediatric patients with stable bronchial asthma were randomized in an open-label, multicenter, crossover trial. No drug was administered during the inhalation maneuvers that were spaced by 10 min. There was neither a carryover nor a sequence effect. The patients were characterized by mean age of 8.5 years, mean FEV(1) of 1.79 L, and mean PIF without any device (baseline, PIF-B) of 185 L/min. Through the devices mean PIF-N of 94 L/min and mean PIF-T of 69 L/min were achieved, calculated from the maxima of three inhalations. This resulted in p < 0.0001 for the difference. The median PIFN/PIF-T ratio was estimated as 1.39. Each child achieved a higher PIF-N than PIF-T and was able to release the feedback mechanisms of the Novolizer indicating sufficient inhalation performance. We conclude that the PIF through the Novolizer is higher than the PIF through the Turbuhaler in stable asthmatic children. The flow rates achieved through the Novolizer allow for sufficient lung deposition even in children as young as 6 years.

Severe traumatic brain injury in Austria V: CT findings and surgical management.

OBJECTIVES: The aim of this paper is to describe CT findings and surgical management of patients with severe traumatic brain injury (TBI) in Austria. PATIENTS AND METHODS: Data sets from 415 patients treated by 5 Austrian hospitals were available. The analysis focused on incidence, surgical management, and outcome of different types of intracranial lesions, and outcome of surgical interventions with and without monitoring of intracranial pressure (ICP). For the first analysis we assigned the patients to 16 groups based on the type of lesion as evaluated by CT scan. For the second analysis we created 4 groups based on surgical treatment (yes/no) and ICP monitoring (yes/no). RESULTS: The mean age was 48.9 years with a male to female ratio of 299:116. The most frequent single lesions were contusions (CONT) and diffuse brain edema. Combined lesions were far more common than single lesions; the most frequently observed combinations included CONT and subarachnoid hemorrhage (SAH) with or without subdural hematoma (SDH). Surgery was done in 276 (66.5%) patients. Osteoplastic surgery (OPS; n = 221) was the most common method followed by osteoclastic surgery (OCS; n = 91) and decompressive craniectomy (DEC; n = 15). ICU mortality was 29.7% for all patients who had any kind of surgery, which was lower than that of patients who were treated non-operatively (33.1%). The ICU mortality of patients with SDH was lower with OCS (18.8%) than with OPS (36.0%). Patients who received ICP monitoring but did not require surgery had the lowest 90 day mortality (17.5%). CONCLUSIONS: ICP monitoring seems to be beneficial in both operatively and non-operatively treated patients with severe TBI. Patients with SDH who were operated on had significantly better outcomes. In patients with SDH, their outcome after osteoclastic surgery was significantly better than after osteoplastic procedures.

Lack of a pharmacokinetic interaction between steady-state roflumilast and single-dose midazolam in healthy subjects.

AIMS: The aim of this study was to investigate the effects of roflumilast, an investigational PDE4 inhibitor for the treatment of COPD and asthma, on the pharmacokinetics of the CYP3A probe drug midazolam and its major metabolites. METHODS: In an open, randomized (for midazolam treatment sequence) study, 18 healthy male subjects received single doses of midazolam (2 mg oral and 1 mg i.v., 1 day apart) alone, repeated doses of roflumilast (500 microg once daily for 14 days) alone, and repeated doses of roflumilast together with single doses of midazolam (2 mg oral and 1 mg i.v., 1 day apart). RESULTS: A comparison of clearance and peak and systemic exposure to midazolam following administration of roflumilast indicated no effect of roflumilast dosed to steady state on the pharmacokinetics of midazolam. Point estimates (90% CI) were 0.97 (0.84, 1.13) for the AUC of i.v. midazolam and 0.98 (0.82, 1.17) for that of oral midazolam with and without roflumilast. CONCLUSIONS: Therapeutic steady state concentrations of roflumilast and its N-oxide do not alter the disposition of the CYP3A substrate midazolam in healthy subjects. This finding suggests that roflumilast is unlikely to alter the clearance of drugs that are metabolized by CYP3A4.

Tramadol SR Formulations : Pharmacokinetic Comparison of a Multiple-Units Dose (Capsule) versus a Single-Unit Dose (Tablet).

INTRODUCTION: Different oral sustained-release (SR) formulations of tramadol have been introduced in pain treatment in order to prolong the dosage interval to improve convenience for the patient. The objective of this study was to compare tramadol pharmacokinetics and intra- and intersubject variability after replicate single-dose administrations of a multiple-units SR formulation (capsule) and a single-unit formulation (tablet). METHODS: This was a randomised, single-dose, single-centre study with an open-label, four-period, two-sequence, two-formulation, replicate crossover design in healthy subjects under fed conditions. The main outcome measures were the intra- and intersubject variance of the area under the concentration-time curve from 0 to 12 hours (AUC(12)) and maximum concentration (C(max)), as well as the mean AUC(12) and C(max) for tramadol. Study drugs were a tramadol SR multiple-units formulation (capsule) and a tramadol SR single-unit formulation (tablet), each containing tramadol hydrochloride 100mg. The time interval from 0 to 12 hours of AUC(12) of the single-dose design corresponds to the recommended twice-daily dosage interval for both study drugs during long-term treatment. RESULTS: The two formulations were equivalent in the area under the curve (AUC(infinity): 2411 vs 2527 mug . h/L). However, capsules led to a lower C(max) (148.6 vs 183.2 mug/L), to a later time to reach C(max) (5.9 vs 4.9 hours), and to a longer half-value duration (13.4 vs 10.4 hours). In addition, intrasubject variability of AUC(12) was significantly smaller for capsules than for tablets (p = 0.041). Capsules also produced smaller intra- and intersubject variability in plasma concentrations during the first 2.5 and 3.0 hours after administration, respectively (p < 0.05). CONCLUSION: Although tramadol SR capsules and tramadol SR tablets led to an equivalent systemic exposure to the drug, capsules provided a smoother and more extended plasma profile. In addition, in the case of capsules, bioavailability was subjected to lower variability in terms of both rate and extent of absorption.

Phenotyping of N-acetyltransferase type 2 by caffeine from uncontrolled dietary exposure.

BACKGROUND AND OBJECTIVE: The standard approach for phenotyping of the human arylamine N-acetyltransferase 2 (NAT2) uses urinary caffeine metabolite ratios after a caffeine test dose taken in after methylxanthine abstinence. We tested whether these standardization measures were still needed when a more sensitive quantification technique was used. METHODS: A new liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the quantification of the caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), and 1-methylurate (1U) was developed. Urine samples from 77 healthy volunteers collected before and 5-6 h after oral intake of 150-200 mg caffeine were analyzed. The lower limits of quantification were 0.1 microg/ml for caffeine, 1X, 1U, and AFMU, and 0.2 microg/ml for AAMU. RESULTS: The urinary NAT2 ratios (AFMU+AAMU) / (AFMU+AAMU+1X+1U) before and after caffeine intake correlated well in 65 volunteers (r(2)=0.827; P< 0.0001). In 12 participants (16%), metabolite concentrations in urine before caffeine intake were below the quantification limit. NAT2 genotyping, done in 41 volunteers for four SNPs, corroborated the phenotyping results. CONCLUSION: NAT2 activity can be determined from a spontaneous urine probe in most subjects by quantification of caffeine metabolites arising from non-standardized dietary caffeine exposure using LC-MS/MS. This may facilitate the phenotyping procedure.

A rapid and simple CYP2D6 genotyping assay--case study with the analgetic tramadol.

There is substantial evidence for a causal relationship between genetic variability of the CYP2D6 gene and changes in the pharmacokinetics of drugs. Therefore, knowledge of single-nucleotide polymorphisms (SNPs) prior to drug administration is highly desired for assisting in the development of individualized pharmacotherapy. We therefore developed a robust assay that detects common CYP2D6 alleles within 60 minutes of blood withdrawal and links carriers of the variant CYP2D6*3 and *4 alleles to the pharmacokinetics of tramadol. This new genotyping assay employs fluorescence resonance energy transfer (FRET) analysis, which permits parallel identification of the CYP2D6*3 and CYP2D6*4 alleles within 60 minutes of blood withdrawal. We determined the genotypes of 100 healthy unrelated individuals and studied the pharmacokinetics of tramadol in 24 CYP2D6 genotyped healthy subjects. The total allelic frequencies of homozygote carriers were 0.015 and 0.25 for the CYP2D6*3 and *4 alleles, respectively, and the plasma area under the curve (AUC) was 84% above those of extensive metabolizers (homozygous EM group): 3,941.2 ng/mL.h (95% confidence interval [CI], 2,928.9 ng/mL.h to 4,953.5 ng/mL.h) versus 2,142.6 ng/mL.h (95% CI, 1,829.6 ng/mL.h to 2,455.7 ng/mL.h). Likewise, the AUC for the O-desmethyl-tramadol metabolite (M1) was significantly reduced in poor metabolizers (PMs): 300.2 ng/mL.h (95% CI, 260.3 ng/mL.h to 340.0 ng/mL.h) versus 842,6 ng/mL.h (95% CI, 715.1 ng/mL.h to 970.0 ng/mL.h). We observed a statistically significant correlation between plasma tramadol AUC and production of the O-desmethyl metabolite in CYP2D6 genotyped healthy volunteers. Our assay can be used reliably in clinical pharmacology studies and may be used for dose adjustment.

Respiratory and miotic effects of morphine in healthy volunteers when P-glycoprotein is blocked by quinidine.

AIM: Our objective was to evaluate whether P-glycoprotein inhibition after quinidine pretreatment results in modified central nervous effects of morphine. METHODS: Twelve healthy volunteers received 7.5 mg morphine as an intravenous infusion over a 3-hour period. In a randomized, double-blind, 2-way crossover fashion, subjects received either 800 mg quinidine or placebo 1 hour before the start of morphine administration. The miotic and respiratory depressive effects of morphine were assessed by means of pupillometry and the respiratory response to carbon dioxide rebreathing, respectively. Quinidine effects were assessed by electrocardiogram recordings. Plasma concentrations of morphine and its glucuronide metabolites were measured throughout the observation period of 5 hours. RESULTS: Morphine significantly reduced both the respiratory response to carbon dioxide and the pupil diameter. Throughout the observation period, quinidine had significant effects on the corrected QT interval (QTc increase of >60 milliseconds), indicating clinically relevant quinidine action. However, quinidine pretreatment did not enhance the respiratory depressive effects of morphine, nor did it alter the miotic effects of morphine to a statistically significant or clinically relevant extent. Plasma concentrations of morphine and its glucuronides were not significantly changed by quinidine pretreatment. CONCLUSIONS: Whereas morphine clearly produced miosis and respiratory depression, pretreatment with quinidine as an inhibitor of P-glycoprotein did not result in an enhancement of central nervous opioid effects in healthy volunteers.

Drug interaction of spirapril hydrochloride monohydrate and hydrochlorothiazide. A clinical study to compare the pharmacokinetics after administration of spirapril hydrochloride monohydrate tablets, hydrochlorothiazide tablets and fixed combination bi-layer tablets.

The potential influence of concomitantly administered hydrochlorothiazide (CAS 58-93-5) on the pharmacokinetics of spirapril (CAS 94841-17-5)/spiraprilat (CAS 83602-05-5) and of concomitantly administered spirapril on the pharmacokinetics of hydrochlorothiazide was investigated in an open, randomised, 3-way crossover study in 12 healthy male subjects. The test drug was a newly developed bi-layer tablet containing a fixed combination of spirapril hydrochloride monohydrate and hydrochlorothiazide (Quadroplus). The reference formulations were tablets containing solely spirapril hydrochloride monohydrate (Quadropril) or hydrochlorothiazide (produced exclusively for study medication). For spirapril, spiraprilat and hydrochlorothiazide the 90% confidence intervals of the AUC(0-infinity) as a measure for the extent of absorption were entirely included within the equivalence range of 0.8 to 1.25 and the 90% confidence intervals of the Cmax as a measure for the rate of absorption were entirely included within the extended equivalence range of 0.7 to 1.43. Therefore, bioequivalence was concluded for the test and reference formulations. The results suggest that hydrochlorothiazide does not interact in the fixed combination with the pharmacokinetics of spirapril and vice versa.

Pharmacokinetic/pharmacodynamic modeling of luteinizing hormone (LH) suppression and LH surge delay by cetrorelix after single and multiple doses in healthy premenopausal women.

Cetrorelix (CET) is a potent luteinizing hormone-releasing hormone (LH-RH) antagonist and is used to prevent premature ovulation in IVF (in vitro fertilization) procedures. The objective of the present study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model for the LH suppression and LH surge delay after single doses (SD) and multiple doses (MD) of CET in healthy premenopausal women without ovarian stimulation. CET was given by subcutaneous route (SD, 0.25, 0.5, or 1 mg) on cycle day 3 and as similar multiple once-a-day doses from cycle day 3 to day 16 in two consecutive menstrual cycles. The concentration-time data of CET and LH were used for PK/PD modeling. A two-compartment model described the PK of CET with median terminal half-life estimates of 9.2 and 54.5 hours after SD and MD, respectively. An indirect-response Emax model was used to describe the LH suppression and the LH surge delay. LH suppression was linked to plasma concentrations of CET, while the delay in the LH surge was linked to the PK of CET through a hypothetical effect compartment. Since the SD regimen on day 3 did not cause significant delay, these values were used as controls in the analysis of surge delay in MD data. The IC50 (for suppression) estimate was 0.73 ng/ml for SD, and EC50 (surge delay) was 1.42 ng/ml for MD. The PK/PD model adequately described the LH suppression and the surge delay.

Effects of age and sex on the disposition of retigabine.

BACKGROUND: The novel antiepileptic drug retigabine is the first selective M-current potassium channel opener for KCNQ2/3 and KCNQ3/5 channels. Retigabine undergoes phase II metabolism (N-glucuronidation, acetylation) exclusively and renal excretion. OBJECTIVE: Our objective was to evaluate the effects of age and sex on the pharmacokinetics of retigabine. METHODS: Healthy young (age range, 18-40 years) and elderly (age range, 66-81 years) white subjects (12 men and 12 women in each group) received a single 200-mg oral dose of retigabine. After dosing, blood was collected over a 72-hour period to determine plasma concentrations of retigabine and its acetylated metabolite, AWD21-360. Pharmacokinetics was compared for age group and sex by ANOVA. RESULTS: In young men, retigabine was rapidly absorbed, with the maximum concentration occurring within 2 hours, and was eliminated with an apparent clearance of 0.67 L x h(-1) x kg(-1) and a mean terminal half-life of 8.5 hours. Subjects were similarly exposed to AWD21-360. Compared with young men, young women had higher retigabine maximum concentration (56%) and exposure (20%) but similar clearance (0.68 L x h(-1) x kg(-1)); these differences were related to differences in body weight. Although maximum concentration was similar in elderly subjects, retigabine elimination was slower (30% lower apparent clearance normalized for weight), resulting in higher exposure (42%) and a longer half-life (30%). Because phase II metabolism is scarcely affected by age, these differences may be related to the known decline of renal function with age. CONCLUSIONS: Although there are no substantial sex-related differences in the disposition of retigabine, a relevant decrease in clearance resulting in higher exposure occurs in elderly patients. The results suggest that decline of renal function with age may account for some of the observed changes.

Novel formulations of cetrorelix acetate in healthy men: pharmacodynamic effects and noncompartmental pharmacokinetics.

The objective of this study was to investigate the effects on the pharmacodynamics and noncompartmental pharmacokinetics after weekly subcutaneous administration of novel formulations of cetrorelix acetate in healthy men. In a randomized parallel-group study, single subcutaneous doses of cetrorelix acetate (concentration: 2.5 mg peptide base/ml) dissolved in aqueous gluconic acid (CET/glu, dose: 5 or 10 mg peptide base) or in water (CET/wat, dose: 10 mg peptide base) were given to 36 subjects once weekly in the morning for 4 weeks. Cetrorelix plasma, serum testosterone, luteinizing hormone, andfollicle-stimulating hormone concentrations were monitored after each administration for 1 week, with extensive profiling after the first and fourth administration. Cetrorelix plasma concentrations were analyzed by radioimmunoassay and serum hormone concentrations by enzyme immunoassays. At least half-maximum testosterone suppression started with all treatments within less than 1 day. Deepest and longest testosterone suppression was achieved by 10 mg CET/glu. Duration of atleasthalf-maximum suppression was after the first dose median of 82 hours and after the fourth dose median of 122 hours, respectively. Substantial suppression was also evidentfor luteinizing hormone (LH) and, to a lesser extent, forfollicle-stimulating hormone (FSH). On average, Cmax was nearly doubled after single and multiple doses, and AUC(tau) was increased by about 50% after single doses and about 30% after multiple doses of 10 mg CET/glu as compared to 10 mg CET/wat. For tmax and t1/2, no significant differences were found between formulations. It was concluded that testosterone suppression increased with weekly subcutaneous administrations of 10 mg CET/glu. Compared to CET/wat, bioavailability and duration of suppression were increased with CET/glu.