Effects of AT1- and β-adrenergic receptor antagonists on TGF-β1-induced fibrosis in transgenic mice

Background Transforming growth factor‐β1 (TGF‐β1) is involved in interstitial remodelling promoting collagen synthesis and suppressing collagen degradation by inhibition of collagenases. TGF‐β1 mediates angiotensin II‐dependent effects and modulates β1‐adrenergic signalling. To study the effect of neuroendocrine antagonism on TGF‐β‐induced hypertrophic and fibrotic phenotype, we treated TGF‐β1 (Cys223,225Ser) transgenic mice (TGF‐β1‐TG) with either the β1‐receptor blocker metoprolol (MET), the angiotensin II type I (AT1)‐receptor antagonist telmisartan (TEL) or an antibody blocking TGF‐β1 signalling (TGFβ1‐sR‐Ab). Material and Methods Transforming growth factor‐β1‐TG mice (8 weeks) overexpressing TGF‐β1 were treated with either TEL (10 mg kg^?1), MET (350 mg kg^?1) or a soluble TGF‐β1 receptor antibody (1 mg kg^?1) for 6 weeks. Morphological analyses of interstitium and cardiomyocytes were related to expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) by immunoblotting and zymography. Results In TGF‐β1‐TG mice, myocardial interstitial total collagen content was fourfold elevated compared to that of controls (P < 0·05) and was lowered under the treatment with TEL (P < 0·05). Protein expression of TIMP‐1 and ‐4 was increased in TGF‐β1‐TG but inhibited by TEL (TIMP‐1 and TIMP‐4) and MET (TIMP‐1), while collagenase activity was decreased in TGF‐β1‐TG and normalized by treatment with TEL (MMP‐1 and MMP‐13) and MET (MMP‐1) (P < 0·05). Morphometric measurements of cardiomyocyte diameter and area demonstrated similar antihypertrophic effects for all treatment groups. Conclusion The AT1‐antagonist TEL reduced myocardial hypertrophy and interstitial fibrosis in TGF‐β1‐TG mice by normalizing MMP/TIMP ratio. β1‐Adrenergic inhibition by MET as well as TGF‐β1 antagonism induced antihypertrophic rather than antifibrotic effects. Inhibition of both renin‐angiotensin system and β1‐adrenergic system may exert different but synergistic effects to reduce myocardial remodelling.     

Pharmacogenetic determinants for interindividual difference of tacrolimus pharmacokinetics 1 year after renal transplantation

What is known and objective: Tacrolimus, a widely used immunosuppressive agent in organ transplantation, has a narrow therapeutic window. It has been suggested that its interaction with lansoprazole could be dependent on polymorphisms of CYP3A5 and CYP2C19. The objective of this study was to investigate how, 1 year after renal transplantation, CYP3A5 and CYP2C19 polymorphisms, biochemical parameters and coadministration with lansoprazole, influenced tacrolimus pharmacokinetics. Methods: The pharmacokinetics of tacrolimus was studied 1 year after renal transplantation, in 75 recipients who were all receiving continuation treatment with 12‐hourly oral tacrolimus, and 30 mg lansoprazole daily (Group 1; n = 20) or, 10 mg rabeprazole daily or no proton pump inhibitor (Group 2; n = 55). Results: There were no significant differences in the dose‐adjusted area under the plasma concentration–time curve (AUC_0–12) and maximum plasma concentration (C_max) of tacrolimus between CYP2C19 genotype groups, but there were significant differences between CYP3A5 genotypes groups (*1/*1 + *1/*3 vs. *3/*3 = 45·2 ± 20·0 vs. 71·0 ± 34·1 ng·h/mL/mg, P < 0·0001 and 6·3 ± 2·6 vs. 9·3 ± 7·0 ng/mL/mg, P = 0·0017, respectively) and between co‐administration with and without lansoprazole (74·5 ± 34·0 vs. 52·4 ± 27·4 ng·h/mL/mg, P = 0·0054 and 10·9 ± 8·8 vs. 6·7 ± 3·0 ng/mL/mg, P = 0·0024, respectively). In a multiple regression analysis, the dose‐adjusted AUC_0–12 and C_max of tacrolimus were associated with CYP3A5*3/*3 and co‐administration with lansoprazole. What is new and conclusion: CYP2C19 does not seem to contribute to the interaction between tacrolimus and lansoprazole. The long‐term combination of tacrolimus and lansoprazole requires careful monitoring of patients with the CYP3A5*3/*3 genotype.     

Pelvic circumferential compression devices benefit patients with pelvic fractures who need transfers

IntroductionPatients with pelvic fracture usually require transfers to trauma centers for additional advanced treatment. Patient safety during the transfer should always be a priority. The noninvasive pelvic circumferential compression device (PCCD) can reportedly provide a tamponade effect, which reduces hemorrhage. In the present study, we evaluated the feasibility and efficiency of PCCD in patients with pelvic fracture who required transfer to trauma centers.Materials and MethodsIn the present study, we aimed to evaluate patients with pelvic fractures who were transferred from other hospitals. We investigated and compared the characteristics of these types of patients with and without pretransfer PCCD. We compared 2 groups (with and without pretransfer PCCD) of patients under different situations (unstable pelvic fracture, stable pelvic fracture, or indicated for transcatheter arterial embolization). We also analyzed the characteristics of patients with unstable pelvic fracture who were initially evaluated as having stable pelvic fracture primarily before being transferred.ResultsDuring the 53-month period, we enrolled 585 patients in the study. The patients with unstable pelvic fractures who received pretransfer PCCDs required significantly fewer blood transfusions (398.4 ± 417.6 mL vs 1954.5 ± 249.0 mL, P < .001), shorter intensive care unit length of stay (LOS; 6.6 ± 5.2 days vs 11.8 ± 7.7 days, P = .024), and shorter hospital LOS (9.4 ± 7.0 days vs 19.5 ± 13.7 days, P = .006) compared with patients who did not receive the pretransfer PCCD. The stable patients who received pretransfer PCCDs required significantly fewer blood transfusions (120.2 ± 178.5 mL vs 231.8 ± 206.2 mL, P = .018) and had shorter intensive care unit LOS (1.7 ± 3.3 days vs 3.4 ± 2.9 days, P = .029) and shorter hospital LOS (6.8 ± 5.1 days vs 10.4 ± 7.6 days, P = .018) compared with patients who did not receive the pretransfer PCCD.ConclusionPelvic circumferential compression devices benefit patients with pelvic fracture who need to be transferred to trauma centers. Pretransfer PCCDs appeared to be a feasible and safe procedure during the transfer. In discussions between the referring physicians and the receiving physicians, we recommend using pretransfer PCCDs.     

OATP1B1 388A>G polymorphism and pharmacokinetics of pitavastatin in Chinese healthy volunteers

Purpose: To investigate the contribution of the most frequent single nucleotide polymorphism (SNPs) of the organic anion transporting polypeptide 1B1 (OATP1B1) 388A>G to the pharmacokinetics of pitavastatin in Chinese healthy volunteers. Methods: Eighteen healthy volunteers participated in this study. Group 1 consisted of nine subjects who were of 388AA wild‐type OATP1B1 genotype. Group 2 consisted of seven subjects with the 388GA genotype and two 388GG homozygotes. Two milligram of pitavastatin was administered orally to the volunteers. The plasma concentration of pitavastatin was measured for up to 48 h by liquid chromatography–mass spectrometry (LC–MS). Results: The pharmacokinetic parameters of pitavastatin were significantly different between the two genotyped groups. The concentration (C_max) value was higher in the 388GA + 388GG group than that in the 388AA group (39·22 ± 8·45 vs. 22·90 ± 4·03 ng/mL, P = 0·006). The area under the curve to the last measurable concentration (AUC_0–48) and area under the curve extrapolated to infinity (AUC_0–∞) of pitavastatin were lower in the 388AA group than in the 388GA + 388GG group (100·42 ± 21·19 vs. 182·19 ± 86·46 ng h/mL, P = 0·024; 108·12 ± 24·94 vs. 199·64 ± 98·70ng h/mL, P = 0·026) respectively. The oral clearance (Cl/F) was lower in the 388GA + 388GG group than that in the 388AA group (12·46 ± 4·79 vs. 19·21 ± 3·74/h, P = 0·012). The elimination of half‐life (t_1/2) and peak concentration times (T_max) values showed no difference between these groups. Conclusions: The OATP 388A>G polymorphism causes significant alterations in the pharmacokinetics of pitavastatin in healthy Chinese volunteers and this may well be clinically significant.     

Metabolic syndrome aggravates the increased endothelial activation and low‐grade inflammation in subjects with familial low HDL

Background. Inhibition of cytokine‐induced expression of adhesion molecules is one of the atheroprotective mechanisms of high‐density lipoprotein (HDL).

Aim. We investigated whether increased endothelial activation and low‐grade inflammation are present in Finnish subjects with familial low HDL, and which factors contribute to the inflammatory parameters.

Method. High‐sensitivity C‐reactive protein (hsCRP), soluble intercellular adhesion molecule‐1 (sICAM‐1), vascular cell adhesion molecule‐1 (sVCAM‐1), and sE‐selectin were measured in 91 subjects with low HDL‐cholesterol from 41 low‐HDL families and in 112 normolipidemic controls with comparable age‐ and gender distribution. Presence of the features of the metabolic syndrome (MetS) was recorded.

Results. sVCAM‐1, sICAM‐1, sE‐selectin, and hsCRP were significantly higher in low‐HDL subjects than in the controls (sVCAM‐1: 560±147?ng/mL versus 496±95?ng/mL, P = 0.001; sICAM‐1: 247±60?ng/mL versus 215±47?ng/mL, P<0.001; sE‐selectin: 52±20?ng/mL versus 44±16?ng/mL, P = 0.022; and hsCRP: 1.73±2.05?mg/L versus 0.85±1.10?mg/L, P<0.001). Low‐HDL subjects had increased body mass index (BMI) and waist, and elevated insulin and triglyceride levels. Adhesion molecules and hsCRP increased according to the number of the features of the MetS.

Conclusions. The presence of the MetS in subjects with familial low HDL‐cholesterol aggravates the low‐grade inflammation and endothelial activation, and ultimately may add to the higher susceptibility for atherosclerotic disease in these individuals.     

Drug interaction between St John's wort and zolpidem in healthy subjects

What is known and objective: St John’s wort (SJW, Hypericum perforatum) is one of the most commonly used herbal antidepressants for treatment of mild to moderate depression. SJW enhances CYP3A4 activity and alters the pharmacokinetics of CYP3A4 substrates. This study investigated the effect of SJW on the pharmacokinetics of zolpidem in healthy subjects. Methods: A controlled, open‐label, non‐randomized, fixed‐dose schedule design was used. Fourteen healthy male subjects received a single 10 mg oral dose of zolpidem followed by SJW administration (300 mg orally, three times a day) for 14 days; the last dose of SJW was coadministered with a single dose of zolpidem. Blood samples were obtained over a 24‐h period after zolpidem administration. Pharmacokinetic data for zolpidem alone and in combination with SJW were analysed by high‐performance liquid chromatography. Results: After repeated administration of SJW, the mean values of AUC and C_max for zolpidem significantly decreased (380·3 ± 181·4 vs. 265·4 ± 134·2 ng h/mL, P = 0·001; 83·1 ± 30·1 vs. 55·1 ± 24·8 ng/mL, P = 0·000 respectively) and the mean value of CL/F for zolpidem significantly increased (38·4 ± 31·5 vs. 56·9 ± 57·2 mL/min, P = 0·040). However, in three subjects, the AUC showed a small increase after SJW treatment. What is new and conclusion: The effect of SJW on the pharmacokinetics of zolpidem has not previously been reported. Repeated administration of SJW decreases the plasma concentration of zolpidem, probably by enhancing CYP3A4 activity. Given the wide inter‐subject variability observed, for personalized medicine, advice on the use of the combination should be individualized, based on the circumstances of the patient.     

Acute effects of right ventricular apical pacing on left atrial remodeling and function

Background : The acute effects of right ventricular apical (RVA) pacing on left atrial (LA) function in patients with normal ejection fraction are not clear. Methods : A total of 94 patients (age 68.1 ± 11.1 years, 26 men) with implanted RVA‐based dual‐chamber pacemakers were recruited into this study. Patients who were pacemaker‐dependent, in persistent atrial fibrillation or left ventricular ejection fraction <45% were excluded. Echocardiography (iE33, Philips, Andover, MA, USA) was performed during intrinsic ventricular conduction (V‐sense) and RVA pacing (V‐pace) with 15 minutes between switching modes. The total maximal LA volume (LAV_max), preatrial contraction volume (LAV_pre), and minimal volume (LAV_min) were assessed by area‐length method. Peak systolic, early diastolic, and peak late diastolic (atrial contractile) velocity (Sm‐la, Em‐la, and Am‐la) and strain (?s‐la, ?e‐la, and ?a‐la) were measured by color‐coded tissue Doppler imaging (TDI) in four mid‐LA walls at apical four‐ and two‐chamber views. Results : During V‐pace, LA volumes increased significantly compared with V‐sense (LAV_max: 52.0 ± 18.8 vs 55.2 ± 21.1 mL, P = 0.005; LAV_pre: 39.8 ± 16.4 vs 41.3 ± 16.6 mL, P = 0.014; LAV_min: 27.4 ± 14.0 vs 29.1 ± 15.1 mL, P = 0.001). TDI parameters showed significant reduction in Sm‐la and Em‐la. Furthermore, ?s‐la, ?e‐la, and ?a‐la decreased significantly, especially in patients with preexisting diastolic dysfunction (all P < 0.01). Conclusions: RVA pacing acutely induced LA enlargement and impaired atrial contractility. Patients with preexisting diastolic dysfunction may be more vulnerable to develop LA dysfunction and remodeling after acute RVA pacing. (PACE 2011;XX:1–7)     

Smoking behaviour modulates pharmacokinetics of orally administered clopidogrel

Background and objectives: Clopidogrel is an important antiplatelet drug that is effective in preventing thrombotic events, especially for patients undergoing percutaneous coronary intervention. The therapeutic usefulness of clopidogrel has been limited by documented inter‐individual heterogeneity in platelet inhibition, which may be attributable to known clopidogrel pharmacokinetic variability. The objective of this study was to assess the influence of smoking cigarettes and abnormal body weight on the pharmacokinetics of clopidogrel. Methods: Seventy‐six healthy adult male volunteers were selected randomly. Each subject received a single 75 mg oral dose of clopidogrel after overnight fast. Clopidogrel carboxylate plasma levels were measured and non‐compartmental analysis was used to determine peak plasma concentration (C_max), time to peak plasma concentration (T_max), elimination half‐life (t_1/2e), and area under the curve (AUC_0→∞). Results: One‐third of volunteers were smokers (n = 27) and one‐half had abnormal body weight (n = 39). Smokers had lower AUC_0→∞ (smokers: 6·24 ± 2·32 μg/h/mL vs. non‐smokers: 8·93 ± 3·80 μg/h/mL, P < 0·001) and shorter half‐life (smokers: 5·46 ± 2·99 vs. non‐smokers: 8·43 ± 4·26, P = 0·001). Smoking behaviour had no influence on C_max (P = 0·3) and T_max (P = 0·7). There was no statistically significant difference in C_max, AUC_0→∞, T_max and t_1/2e between volunteers with abnormal body weight and normal body weight. However the difference in body weight of the two groups was relatively narrow (mean ± SE; 26·93 ± 0·16 vs. 23·11 ± 0·27). In general, the pharmacokinetic parameters were characterized by considerable inter‐individual differences (C_max = 3·09 ± 0·99 μg/mL, CV = 32%), (T_max =0·76 ± 0·24 h, CV = 31·6%), (AUC_0→∞ = 7·98 ± 3·58 μg/h/mL, CV = 44·8%), and (t_1/2e = 7·38 ± 4·10 h, CV = 55·6%). Conclusion: Smoking is a significant factor affecting the pharmacokinetics of clopidogrel, following administration of a single 75 mg dose in healthy young volunteers. The study supports smoking‐cessation recommendations. Further studies are required to evaluate the influence of smoking and body weight on the pharmacokinetics of the active metabolite of clopidogrel and on the clinical effects of any differences observed.     

Effect of polymerized-type I collagen in knee osteoarthritis. II. In vivo study

Background Polymerized‐Type I Collagen (Polymerized‐Collagen) is an anti‐inflammatory and a tissue regenerator biodrug. The aim of the study was to evaluate the efficacy and safety of intra‐articular injections of Polymerized‐Collagen in patients with knee osteoarthritis (OA). Methods and design Patients (n = 53) were treated with 12 intra‐articular injections of 2 mL of Polymerized‐Collagen (n = 27) or 2 mL of placebo (n = 26) during 6 months. Follow up period was 6 months. The primary endpoints included Western Ontario and McMaster University Osteoarthritis Index, Lequesne index, and pain intensity on a visual analogue scale (VAS). Secondary outcomes were patient global score, investigator global score and drug evaluation. Clinical improvement was determined if the decrease in pain exceeds 20 mm on a VAS and patients achieved at least 20% of improvement from baseline. Urinary levels of C‐terminal crosslinking telopeptide of collagen type II (CTXII) and serum high‐sensitivity C‐reactive protein (hsCRP) were determined by enzyme immunoassays. Statistical analysis was performed by intention to treat. Results Polymerized‐Collagen was safe and well tolerated. Patients had a statistically significant improvement (P < 0·05) from baseline vs. Polymerized‐Collagen and vs. placebo at 6 months in: Lequesne Index (13·1 ± 0·5 vs. 7·1 ± 0·7 vs. 9·6 ± 0·8; P = 0·027), WOMAC (9·0 ± 0·5 vs. 4·0 ± 0·6 vs. 5·80 ± 0·8; P = 0·032), patient VAS (60·0 ± 2·6 vs. 20·6 ± 2·4 vs. 36·1 ± 4·5; P = 0·003), physician VAS (49·8 ± 1·9 vs. 16·8 ± 2·9 vs. 29·8 ± 2·9; P = 0·002), patient global score (1·08 ± 0·1 vs. 2·7 ± 0·1 vs. 1·9 ± 0·2; P = 0·028) and analgesic usage (30·1 ± 9·4 vs. 11·0 ± 3·4 vs. 17·9 ± 4·9; P = 0·001). This improvement was persistent during the follow up. A threefold increase in CTXII was determined in placebo group. No differences were found on hs CRP and incidence of adverse events between groups. Conclusion Polymerized‐Collagen is safe and effective in the treatment of knee OA.     

Improvement in right ventricular systolic function after cardiac resynchronization therapy correlates with left ventricular reverse remodeling

Background: Cardiac resynchronization therapy (CRT) improves left ventricular (LV) systolic function in heart failure (HF). However, the effects of CRT on right ventricular (RV) systolic function are not fully understood. Objective: We aimed to determine echocardiographic correlates of improvement in RV systolic function after CRT. Methods: Fifty‐four patients (61.9 ± 10.5 years; 43 men; LV ejection fraction 24.6 ± 4.0%; QRS duration > 120 ms) with HF were enrolled. Standard echocardiography, strain rate (SR), and tissue Doppler imaging were performed in all patients before and 6 months after CRT. Pulsed‐wave TDI‐derived systolic indices of RV included systolic (RV_S) and isovolumic velocity (RV_IVV) and isovolumic acceleration (RV_IVA). Response to CRT was defined as decline in LV end‐systolic volume (LVESV) ≥ 10%. Results: When indices of RV systolic function were assessed between responders and nonresponders, in responders (38 patients, 70.4%) RV end‐diastolic diameters (RVD1–3), mid‐RV strain, and mid‐RV SR improved significantly (P < 0.01, for all). RV_S (10.77 ± 4.29 vs 12.62 ± 4.10 cm/sec, P = 0.005), RV_IVV (14.71 ± 5.88 vs 18.52 ± 6.62 cm/sec, P < 0.001), and RV_IVA (1.69 ± 0.70 vs 2.39 ± 0.77 m/sec², P < 0.001) significantly increased among responders. There was no significant change in these parameters among nonresponders. Pearson's analyses revealed moderate positive correlations between reduction of LVESV and ΔRV_IVV (r = 0.467, P = 0.001) and ΔRV_IVA (r = 0.473, P = 0.001), respectively. Conclusions: RV diameters and systolic indices after CRT improved only in the responder group. Improvement in RV systolic performance after CRT is correlated with the reduction of LVESV. (PACE 2011; 34:200–207)     

Association of circulating insulin‐like growth factor 1 with hepatocellular carcinoma: one cross‐sectional correlation study

Deregulation of insulin‐like growth factor‐1 (IGF‐1) has been implicated in the pathogenesis of several malignancies. This study aimed to investigate the association of changes in circulating IGF‐1 with hepatocellular carcinoma (HCC). The radioimmunoassay was used to analyze serum IGF‐1 levels of 65 HCC patients and 165 healthy subjects. Serum IGF‐1 levels were significantly decreased in the HCC patients as compared with the healthy subjects (158.46±105.07 vs. 247.63±149.96 ng/mL, P<0.001). Furthermore, insulin resistance was significantly higher in the HCC patients than the healthy subjects (P=0.027). In addition, the significant correlations of serum IGF‐1 levels with age and insulin resistance in the healthy subjects were not noted in the HCC patients. Intriguingly, individuals with hepatitis C virus (HCV), not hepatitis B virus, had remarkably decreased IGF‐1 levels in both groups of the HCC patients and healthy subjects. Moreover, in the HCV subgroup, serum IGF‐1 levels were significantly reduced in the HCC patients than the healthy subjects (113.14±71.28 vs. 172.42±74.02 ng/mL, P=0.003). In conclusion, decreased serum IGF‐1 levels were associated with HCC and the decrease was remarkably noted in those patients concomitant with chronic hepatitis C. J. Clin. Lab. Anal. 24:195–200, 2010. © 2009 Wiley‐Liss, Inc.     

Plasma levels of matrix metalloproteinase‐8 in patients with carotid atherosclerosis

Matrix metalloproteinases (MMPs) are involved in the remodeling of the extracellular matrix (ECM) in the arterial wall during atherogenesis. Collagens are the most abundant proteins in the ECM. MMP‐8 is expressed by cells associated with the development of the atherosclerotic plaque. It cleaves collagen type I three times more potently than two other interstitial collagenases MMP‐1 and MMP‐13. The aim of this study was to investigate whether plasma MMP‐8 values are associated with occurrence of carotid plaque (CP) and possible correlations with clinical and biochemical parameters in carotid atherosclerosis (CA) patients. Total plasma MMP‐8 levels were quantified by ELISA in 63 patients with ultrasonographic evidence of CP presence and 12 controls. Plasma MMP‐8 values were significantly higher in patients with CA compared with controls (median 23.36 ng/ml vs. 13.02 ng/ml, P<0.001) but they did not differ significantly according to gender, smoking and hypertensive status, associated diseases, and use of statins. Statistically significant positive correlations were observed between MMP‐8 plasma values and C reactive protein (r=0.41, P=0.001), urea (r=0.50, P<0.001), aspartate transaminase (r=0.48, P=0.001), and creatinine levels (r=0.38, P=0.006). These results suggest association of MMP‐8 plasma levels with occurrence of CP and correlation with certain biochemical markers. J. Clin. Lab. Anal. 24:246–251, 2010. © 2010 Wiley‐Liss, Inc.     

Acute hemodynamic effects of right and left ventricular lead positions during the implantation of cardiac resynchronization therapy defibrillators

Background: To evaluate the acute hemodynamic effects of different right (RV) and left ventricular (LV) pacing sites in patients undergoing the implantation of a cardiac resynchronization therapy defibrillator (CRT‐D). Methods: Stroke volume index (SVI), assessed via pulse contour analysis, and dp/dt max, obtained in the abdominal aorta, were analyzed in 21 patients with New York Heart Association class III heart failure and left bundle branch block (mean ejection fraction of 24 ± 6%), scheduled for CRT‐D implantation under general anesthesia. We compared the hemodynamic effects of RV apical (A), RV septal (B), and biventricular pacing using the worst (lowest SVI; C) and best (highest SVI; D) coronary sinus lead positions. Results: Mean arterial pressure, SVI, and dp/dt max did not differ significantly between RV apical and septal pacing. Dp/dt max and SVI increased significantly during biventricular pacing (dp/dt max: B, 588 ± 160 mmHg/s; C, 651 ± 218 mmHg/s, P = 0.03 vs B; D, 690 ± 220 mmHg/s, P = 0.02 vs C; SVI: B, 33.6 ± 5.5 mL/m², C, 34.8 ± 6.1 mL/m², P = 0.08 vs B, D 36.0 ± 6.0 mL/m², P < 0.001 vs C). The best hemodynamic response was associated with lateral or inferior lead positions in 15 patients. Other LV lead positions were most effective in six patients. Conclusions: The optimal LV lead position varies significantly among patients and should be individually determined during CRT‐D implantation. The impact of the RV stimulation site in patients with intraventricular conduction delay, undergoing CRT‐D implantation, has to be investigated in further studies. (PACE 2011; 34:1537–1543)     

Cloning and paratope analysis of an antibody fragment, a rational approach for the design of a PAI-1 inhibitor

Summary. This study reports the cloning, characterization and paratope analysis of the plasminogen activator inhibitor‐1 (PAI‐1) neutralizing single‐chain variable fragment 56A7C10 (scFv‐56A7C10). ScFv‐56A7C10‐wt exhibits a similar affinity (K_A = 1.01 ± 0.3 × 10⁹ m ^?1) and PAI‐1 inhibitory capacity (90 ± 6% PAI‐1 inhibition at a 16‐fold molar excess and IC₅₀ = 44 ± 14 ng mL^?1) as MA‐56A7C10 (K_A = 1.43 ± 0.4 × 10⁹ m ^?1, 90 ± 2% PAI‐1 inhibition at a 16‐fold molar excess and IC₅₀ = 122 ± 26 ng mL^?1). Subsequently, alanine scanning of the six complementarity determining regions (CDRs) was performed and the scFv‐56A7C10‐mutants (n = 26) were analyzed for their PAI‐1 binding and PAI‐1 inhibitory properties. Mutation of the residues Y32 and V33 in the CDR1 of the heavy chain (HCDR1) and the residues R98, H99, W100 or F100a (HCDR3) resulted in reduced PAI‐1 inhibitory capacities (IC₅₀ ≥ 418 ng mL^?1), confirmed by reduced affinities (14‐, 17‐, 7‐, 9‐ and 16‐fold reduced, respectively, vs. scFv‐56A7C10‐wt). In the light chain, mutation of the residues W50 (LCDR2), H91, Y92, D93, or W96 (LCDR3) resulted in reduced PAI‐1 inhibitory properties (IC₅₀ ≥ 160 ng mL^?1) and decreased affinities (i.e. 4‐, 9‐, 3‐, 3‐ and 2‐fold reduced affinity, respectively, vs. scFv‐56A7C10‐wt). Furthermore, an overlapping peptide scan confirmed the importance of the HCDR3 region. These data, combined with a three‐dimensional model of scFv‐56A7C10, reveal the molecular and structural properties of the paratope and contribute to the rational design of PAI‐1 neutralizing compounds.     

Anatomic and electrophysiological differences between chronic and paroxysmal forms of common atrial flutter and comparison with controls:

Whether chronic typical atrial flutter differs from paroxysmal atrial flutter regarding electrophysiological properties of reentry pathways and cardiac function remains unknown. If so, can remodeling due to long duration of persistently rapid atrial or ventricular rates explain these changes? The aim of the study was to compare RA local conduction velocities and heart function parameters between three groups: (1) chronic atrial flutter, (2) paroxysmal atrial flutter, and (3) controls. The study evaluated 52 patients undergoing radiofrequency ablation for typical atrial flutter. There were 35 patients with chronic atrial flutter (62.7 ± 14 years) and 17 patients with paroxysmal atrial flutter (62.7 ± 10 years). Underlying structural heart disease was present in 20 (57%) of 35 chronic atrial flutter patients and in 7 (41%) of 17 paroxysmal atrial flutter patients (P = 0.1). Chronic atrial flutter duration was 10.9 ± 17 months and paroxysmal atrial flutter duration was 8.5 ± 10 (P = 0.06). RA conduction velocity measurements were carried out before ablation during sinus rhythm under pacing (600‐ms cycle length) with a 12‐pole steerable catheter positioned in the high lateral RA (poles 11‐12 [H6]), mid‐lateral RA (poles 9‐10 [H5]), and along the inferior vena caval tricuspid isthmus (poles 7‐8 [H4]; 5‐6 [H3]; 3‐4 [H2]) with its distal electrode pair at the coronary sinus origin (pole 1‐2 [H1]). Counter‐clockwise RA conduction velocities were assessed from H6 to H1 and clockwise RA conduction velocities from H1 to H6. After successful ablation, RA and LA areas, LV volumes, LVEF, inferior vena caval tricuspid annulus, and coronary sinus tricuspid annulus (septal isthmus) lengths were measured by two‐dimensional echocardiography. The control group included 12 patients without structural heart disease, referred for electrophysiological evaluation of AVN reentry. Counter‐clockwise RA conduction velocities at the inferior vena caval tricuspid isthmus were lower in chronic atrial flutter than in paroxysmal atrial flutter (H4, 1.19 ± 0.4 vs 1.89 ± 1 m/s, P = 0.0051; H3, 1.14 ± 0.4 vs 1.6 ± 0.7 m/s, P = 0.0015; H2, 1.16 ± 0.4 vs 1.53 ± 0.5 m/s, P < 0.0056 and H1, 1.2 ± 0.4 vs 1.5 ± 0.4 m/s, P = 0.03, respectively). Counter‐clockwise RA conduction velocities were identical at the high and mid‐lateral RA. Counter‐clockwise caval isthmus RA conduction velocities from H3 to H1 were significantly different between chronic atrial flutter and controls (H3, 1.14 ± 0.4 vs 1.7 ± 0.3 m/s, P = 0.0014; H2, 1.16 ± 0.4 vs 1.83 ± 0.4 m/s, P < 0.0001 and H1, 1.2 ± 0.4 vs 1.94 ± 0.4 m/s, P < 0.0001, respectively). A difference was found regarding clockwise isthmus RA conduction velocities between the two groups of atrial flutter and controls but not between chronic atrial flutter and paroxysmal atrial flutter. Respectively, chronic atrial flutter had greater RA and LA areas (24.5 ± 5 vs 13 ± 2 cm ² ; P < 0.0001 and 23 ± 5 vs 16 ± 3 cm ² , P < 0.0001), LV end‐systolic and end‐diastolic volumes (50 ± 25 vs 32 ± 13 cm ³ , P = 0.0084 and 112 ± 40 vs 85 ± 25 cm ³ , P = 0.01), septal isthmus length (21 ± 3 vs 13 ± 2 mm, P < 0.0001), and inferior vena caval tricuspid isthmus length (39 ± 6 vs 23 ± 5 mm; P < 0.0001). Chronic common atrial flutter is characterized by more prolonged counter‐clockwise conduction times and larger anatomic conduction pathways than the paroxysmal form, the causal relationship between electrophysiological and anatomic characteristics remains to be demonstrated.     

Increased intraatrial conduction abnormality assessed by P-wave signal-averaged electrocardiogram in patients with Brugada syndrome

Background: Atrial fibrillation (AF) is observed in patients with Brugada syndrome (BS), especially those showing coved‐type electrocardiogram (ECG) pattern. Using P‐wave signal‐averaged ECG (P‐SAE), we investigated whether increased intraatrial conduction abnormality contributed to AF generation in BS patients. Methods: Twenty BS patients and 20 age‐ and gender‐matched healthy controls were enrolled. At the P‐SAE recording, 12 of the 20 BS patients showed coved‐type (C‐BS) and eight showed saddleback‐type (S‐BS). The total duration (Ad) and root mean square voltage for the terminal 20 ms (LP₂₀) of the filtered P wave were measured. P‐wave dispersion (P‐disp) was defined as the difference between the maximum and minimum, measured from 16 precordial recording sites. Results: BS patients had a significantly longer Ad (128.2 ± 7.6 vs 116.3 ± 8.2 ms, P < 0.0001), lower LP₂₀ (2.6 ± 0.9 vs 3.4 ± 0.8 μV, P < 0.01), and greater P‐disp (15.5 ± 7.0 vs 7.4 ± 3.2 ms, P < 0.0001) than the controls. C‐BS patients had significantly longer Ad (131.0 ± 7.2 vs 124.1 ± 6.8 ms, P < 0.05) and lower LP₂₀ (2.2 ± 0.6 vs 3.2 ± 1.0 μV, P < 0.05) than S‐BS patients. All C‐BS patients and only three S‐BS patients had atrial late potential (100% vs 38%, P < 0.01). Conclusion: Intraatrial conduction delay and its heterogeneity may exist in BS patients, especially those showing coved‐type ECG patterns. These atrial electrical abnormalities could be a substrate for atrial reentrant tachycardia such as AF. (PACE 2011; 34:1138–1146)     

Determination of glycyrrhetic acid in human plasma by HPLC-MS method and investigation of its pharmacokinetics

Objective: To develop a high performance liquid chromatography mass spectrometry (HPLC‐MS) method for the determination of the glycyrrhetic acid (GA) in human plasma and for the investigation of its pharmacokinetics after the oral administration of 150 mg diammonium glycyrrhizinate test and reference capsule formulations. Methods: The GA in plasma was extracted with ethyl acetate, separated on a C₁₈ column with a mobile phase of methanol (5 mmol/L ammonium acetate)–water (85 : 15, V/V) and analysed using a MS detector. Ursolic acid (UA) was used as internal standard. The target ions were m/z 469·5 for GA and m/z 455·6 for UA, the fragment voltages were 200 V and 100 V for GA and UA respectively. Results: The calibration curve was linear over the range of 0·5–200 ng/mL (r = 0·9974). The limit of quantification for GA in plasma was 0·5 ng/mL, the recovery was 76·0–80·0%, and the inter‐ and intra‐day relative standard deviations (RSD) were <12%. The pharmacokinetic parameters of GA after a single dose of 150 mg diammonium glycyrrhizinate test and reference were as follows: the half life (t_1/2) 9·65 ± 3·54 h and 9·46 ± 2·85 h, the time to peak concentration (T_max) 10·95 ± 1·32 h and 11·00 ± 1·30 h, the peak concentration (C_max) 95·57 ± 43·06 ng/mL and 103·89 ± 49·24 ng/mL; the area under time‐concentration curve (AUC_0–48 and AUC_0–∞) 1281·84 ± 527·11 ng·h/mL and 1367·74 ± 563·27 ng·h/mL, 1314·32 ± 566·40 ng·h/mL and 1396·97 ± 630·06 ng·h/mL. The relative bioavailability of diammonium glycyrrhizinate capsule was 98·88 ± 12·98%. Conclusion: The assay was sensitive, accurate and convenient, and can be used for the determination of GA in human plasma. Comparison of the bioavailability and pharmacokinetic profile of GA indicated that the test and reference capsules were bioequivalent.     

Permanent Pacing in Patients with Chagas' Disease

Background :Chagas' disease is an endemic disease in most Latin American countries. The cardiomyopathy associated with this condition often requires permanent pacing due to bradycardia. The aim of this study was to compare the indications for pacemaker implantation, intraoperative measurements, and long‐term follow‐up of patients with Chagas' cardiomyopathy (ChCM) and ischemic cardiomyopathy (ICM) referred for pacemaker implantation. Methods : Retrospective study including consecutive patients with ChCM (Group 1) and ICM (Group 2), who underwent pacemaker implantation in a single center. Results : We analyzed 360 patients. Patients in Group 1 were younger (66.29 ± 7.01 vs 75.3 ± 7.11 years; P = 0.0001) and more often male (72% vs 60%; P = 0.05). Sinus node dysfunction (SND) was more prevalent in Group 1 (70% vs 52%; P = 0.03). Atrioventricular block was less prevalent in Group 1 (30% vs 48%; P = 0.04). No significant differences were found with respect to left ventricular ejection fraction (54.2 ± 9.1 vs 53.4 ± 8.2%; P = NS) and baseline QRS duration (119 ± 34 vs 108 ± 29 ms; P = NS). Right bundle branch block was more frequent in Group 1 (44% vs 12%; P = 0.0001), and left bundle branch block in Group 2 (6% vs 22%; P = 0.0001). Implantation time was longer in Group 1 (39 ± 19 vs 29 ± 13 minutes; P = 0.001) and was with higher atrial and ventricular pacing thresholds (1.4 ± 0.8 vs 1.0 ± 0.5 V; P = 0.001 and 1.2 ± 0.8 vs 0.6 ± 0.8 V; P = 0.001, respectively). During a follow‐up of 42.8 ± 13.6 months, Group 1 had a higher incidence of new atrial fibrillation (34% vs 25.5%; P = 0.001), and there was a nonsignificant trend toward more displacements of the ventricular lead (6% vs 3.5%; P = 0.3). There were no deaths during the follow‐up. Conclusions : ChCM patients receiving pacemakers are younger and more frequently have SND compared to those with ICM. Pacemaker implant is longer in patients with ChCM disease and is with higher pacing thresholds. The incidence of new atrial fibrillation during the follow‐up is significantly higher in patients with ChCM. (PACE 2012;35:1494‐1497)     

Evaluation by Cardiopulmonary Exercise Test of DDDR Versus DDD Pacing

In eight patients (age 62 ± 6 years) a DDDR pacemaker was implanted for sick sinus syndrome (three cases) or second- and third-degree AV block (five cases). In five subjects chronotropic incompetence (maximal heart rate on effort < 110 beats/min) was present before implantation. One month after implantation the patients were randomized to DDDR or DDD pacing for 3 weeks each, with subsequent crossover, and at the end of each period a symptom limited Cardiopulmonary exercise test (25 watts/2 min) was performed and the patients were requested to fill a symptoms questionnaire. Results: DDDR pacing, compared to DDD, was associated with higher maximal heart rates (127 ± 20 vs 110 ± 27 beats/min, P < 0.02), higher (VO₂ max (25.4 ± 6.1 vs 21.5 ± 7.8 mL/kg/per min, P < 0.03) and higher VO₂ at the anaerobic threshold (20.3 ± 5.0 vs 15.8 ± 4.9 mL/kg per min, P < 0.03), without significant differences in mean exercise time (526 ± 193 vs 472 ± 216 sec, NS). The increase in VO₂ max obtained in DDDR versus DDD was significantly related to the increase in maximal heart rate (r = 0.72, P < 0.05) and the increase in VO₂ at the anaerobic threshold obtained in DDDR versus DDD was related to the increase in heart rate at the anaerobic threshold (r = 0.81, P < 0.02). In patients with chronotropic incompetence the improvement obtained in DDDR versus DDD was even more significant (VO₂ max = 22.7 ± 5.9 vs 16.1 ± 4.4 mL/kg per min, P < 0.03; VO₂ at the anaerobic threshold = 18.4 ± 5.1 vs 13.2 ± 2.8 mL/kg per min, P < 0.05; exercise time = 438 ± 132 vs 352 ± 150 sec, P < 0.02). In the population as a whole, no significant differences were found relative to subjective symptoms, meanwhile in patients with chronotropic incompetence a better subjective tolerance was apparent with DDDR than with DDD pacing. In conclusion, DDDR pacing induces a significant improvement of exercice capacity, in comparison to DDD pacing, related to the ability to reach higher heart rates during exercise. This phenomenon is particulary evident in patients with chronotropic incompetence in whom DDDR pacing also is subjectively better tolerated.