Inhibition of ezrin phosphorylation by NSC305787 attenuates procaterol-stimulated ciliary beating in airway cilia.

Ciliary beating in the airway epithelium plays an important role in preventing infection by eliminating small particles and pathogens. Stimulation of ß2 adrenergic receptor (ß2AR) increases [cAMP]i levels and strongly activates this ciliary beating. ß2AR is localized to the apical membrane of the airways by indirectly binding to ezrin, an actin-binding protein. Ezrin takes active phosphorylated and inactive dephosphorylated states at Thr-567. Previously we showed that procaterol-stimulated ciliary beating was impaired in the ezrin-knockdown mice. In this study, we examined the roles of ezrin and its phosphorylation in regulating ciliary beating by using NSC305787, an ezrin inhibitor, in normal human airway epithelial cells (NHBE). We found that NSC305787 inhibits the phosphorylation of ezrin with an IC50 of 50 µM in NHBE. Treatment with NSC305787 for 4 h or more decreased the expression of ß2AR in the cell membrane and induced vesicle- or dot-like expression of ezrin and ß2AR inside the cell. As a result, inhibition of ezrin phosphorylation by NSC305787 attenuated the effect of procaterol-induced activation of ciliary beating in both frequency and distance indices.

Cisplatin and Procaterol Combination in Gastric Cancer? Targeting Checkpoint Kinase 1 for Cancer Drug Discovery and Repurposing by an Integrated Computational and Experimental Approach.

Checkpoint kinase 1 (CHK1), a serine/threonine kinase, plays a crucial role in cell cycle arrest and is a promising therapeutic target for drug development against cancers. CHK1 coordinates cell cycle checkpoints in response to DNA damage, facilitating repair of single-strand breaks, and maintains the genome integrity in response to replication stress. In this study, we employed an integrated computational and experimental approach to drug discovery and repurposing, aiming to identify a potent CHK1 inhibitor among existing drugs. An e-pharmacophore model was developed based on the three-dimensional crystal structure of the CHK1 protein in complex with CCT245737. This model, characterized by seven key molecular features, guided the screening of a library of drugs through molecular docking. The top 10% of scored ligands were further examined, with procaterol emerging as the leading candidate. Procaterol demonstrated interaction patterns with the CHK1 active site similar to CHK1 inhibitor (CCT245737), as shown by molecular dynamics analysis. Subsequent in vitro assays, including cell proliferation, colony formation, and cell cycle analysis, were conducted on gastric adenocarcinoma cells treated with procaterol, both as a monotherapy and in combination with cisplatin. Procaterol, in synergy with cisplatin, significantly inhibited cell growth, suggesting a potentiated therapeutic effect. Thus, we propose the combined application of cisplatin and procaterol as a novel potential therapeutic strategy against human gastric cancer. The findings also highlight the relevance of CHK1 kinase as a drug target for enhancing the sensitivity of cytotoxic agents in cancer.

Development and validation of a nomogram to predict cancer-specific survival in nonsurgically treated elderly patients with prostate cancer.

Prostate Cancer (PC) is the most common male nonskin tumour in the world, and most diagnosed patients are over 65 years old. The main treatment for PC includes surgical treatment and nonsurgical treatment. Currently, for nonsurgically treated elderly patients, few studies have evaluated their prognostic factors. Our aim was to construct a nomogram that could predict cancer-specific survival (CSS) in nonsurgically treated elderly PC patients to assess their prognosis-related independent risk factors. Patient information was obtained from the Surveillance, Epidemiology and End Results (SEER) database, and our target population was nonsurgically treated PC patients who were over 65 years old. Independent risk factors were determined using both univariate and multivariate Cox regression models. A nomogram was built using a multivariate Cox regression model. The accuracy and discrimination of the prediction model were tested using the consistency index (C-index), the area under the subject operating characteristic curve (AUC), and the calibration curve. Decision curve analysis (DCA) was used to examine the potential clinical value of this model. A total of 87,831 elderly PC patients with nonsurgical treatment in 2010-2018 were included in the study and were randomly assigned to the training set (N = 61,595) and the validation set (N = 26,236). Univariate and multivariate Cox regression model analyses showed that age, race, marital status, TNM stage, chemotherapy, radiotherapy modality, PSA and GS were independent risk factors for predicting CSS in nonsurgically treated elderly PC patients. The C-index of the training set and the validation set was 0.894 (95% CI 0.888-0.900) and 0.897 (95% CI 0.887-0.907), respectively, indicating the good discrimination ability of the nomogram. The AUC and the calibration curves also show good accuracy and discriminability. We developed a new nomogram to predict CSS in elderly PC patients with nonsurgical treatment. The model is internally validated with good accuracy and reliability, as well as potential clinical value, and can be used for clinical aid in decision-making.

Two-way coupling and Kolmogorov scales on inhaler spray plume evolutions from Ventolin, ProAir, and Qvar.

Previous numerical studies of pulmonary drug delivery using metered-dose inhalers (MDIs) often neglected the momentum transfer from droplets to fluid. However, Kolmogorov length scales in MDI flows can be comparable to the droplet sizes in the orifice vicinity, and their interactions can modify the spray behaviors. This study aimed to evaluate the two-way coupling effects on spray plume evolutions compared to one-way coupling. The influences from the mass loading, droplet size, and inhaler type were also examined. Large-eddy simulation and Lagrangian approach were used to simulate the flow and droplet motions. Two-way coupled predictions appeared to provide significantly improved predictions of the aerosol behaviors close to the Ventolin orifice than one-way coupling. Increasing the applied MDI dose mass altered both the fluid and aerosol dynamics, notably bending the spray plume downward when applying a dose ten times larger. The droplet size played a key role in spray dynamics, with the plume being suppressed for 2-µm aerosols and enhanced for 20-µm aerosols. The Kolmogorov length scale ratio dp/η correlated well with the observed difference in spray plumes, with suppressed plumes when dp/η < 0.1 and enhanced plumes when dp/η > 0.1. For the three inhalers considered (Ventolin, ProAir, and Qvar), significant differences were predicted using two-way and one-way coupling despite the level and manifestation of these differences varied. Two-way coupling effects were significant for MDI sprays and should be considered in future numerical studies.

Ezrin knockdown reduces procaterol-stimulated ciliary beating without morphological changes in mouse airway cilia.

Mucociliary clearance, which is conducted by beating cilia cooperating with the surface mucous layer, is a major host defense mechanism of the airway epithelium. Ezrin, a crosslinker between membrane proteins and the actin cytoskeleton, is located in microvilli and around the basal bodies in airway ciliary cells. It is also likely that ezrin plays an important role in apical localization of ß2 adrenergic receptor (ß2AR) in airway ciliary cells. Here, we studied the physiological roles of ezrin by using trachea and airway epithelial cells prepared from ezrin-knockdown (Vil2kd/kd) mice. The trachea and airway ciliary cells of Vil2kd/kd mice presented a normal morphology and basal body orientation, suggesting that ezrin is not directly involved in development and planar cell polarity of cilia. Procaterol stimulates ciliary beating (frequency and amplitude) via ß2AR in the airway ciliary cells. In the Vil2kd/kd mice, airway ciliary beating stimulated with procaterol was partly inhibited due to the impairment of cell surface expression of ß2AR. These results suggest that ezrin regulates the beating of airway ciliary cells by promoting the apical surface localization of ß2AR. This article has an associated First Person interview with the first author of the paper.

Defining a Role for G-Protein Coupled Receptor/cAMP/CRE-Binding Protein Signaling in Hair Follicle Stem Cell Activation.

Manipulation of adrenergic signaling has been shown experimentally and clinically to affect hair follicle growth. In this study, we provide direct evidence that canonical cAMP/CRE-binding protein signaling through adrenergic receptors can regulate hair follicle stem cell (HFSC) activation and hair cycle. We found that CRE-binding protein activation is regulated through the hair cycle and coincides with HFSC activation. Both isoproterenol and procaterol, agonists of adrenergic receptors, show the capacity to activate the hair cycle in mice. Furthermore, deletion of ADRB2 receptor, which is thought to mediate sympathetic nervous system regulation of HFSCs, was sufficient to block HFSC activation. Downstream, stimulation of adenylyl cyclase with forskolin or inhibition of phosphodiesterase to increase cAMP accumulation or direct application of cAMP was each sufficient to promote HFSC activation and accelerate initiation of hair cycle. Genetic induction of a Designer Receptors Exclusively Activated by Designer Drug allele showed that G-protein coupled receptor/GαS stimulation, specifically in HFSCs, promoted the activation of the hair cycle. Finally, we provide evidence that G-protein coupled receptor/CRE-binding protein signaling can potentially act on HFSCs by promoting glycolytic metabolism, which was previously shown to stimulate HFSC activation. Together, these data provide mechanistic insights into the role of sympathetic innervation on HFSC function.

Effect of pulmonary rehabilitation with assistive use of short-acting ß2 agonist in COPD patients using long-acting bronchodilators.

Background: Assistive use of short-acting ß2 agonists (SABAs) reportedly improves exercise tolerance, activities of daily living, and health-related quality of life (HRQOL) in patients with chronic obstructive pulmonary disease (COPD). However, the effect of SABA on physical activity (PA) is unclear.Objective: This study aimed to determine whether assistive use of SABA increases PA and whether additional pulmonary rehabilitation (PR) can aid further improvement.Methods: Twelve outpatients with COPD and dyspnea during daily activities despite regular use of long-acting bronchodilators were enrolled. This study comprised a 2-week pre-intervention investigation, a 12-week investigation of SABA effects, and an 8-week investigation of the additional effects of PR. Assistive use of SABA was allowed up to 4 times per day after the pre-intervention period. PA was measured for 14 consecutive days using an accelerometer sensor. Dyspnea, exercise tolerance, and HRQOL were evaluated at entry, at 4 and 12 weeks after initiating SABA use, and after completing PR.Results: Assistive use of SABA improved breathlessness during daily activities and increased PA (p < .001). PA and HRQOL were also improved following PR (p < .001 and p = .013, respectively).Conclusions: Combined therapy of SABA and PR can increase PA and HRQOL in COPD patients.

CDK12 and PAK2 as novel therapeutic targets for human gastric cancer.

Background: Gastric cancer remains the second leading cause of cancer-related death, and the third in mortality due to lack of effective therapeutic targets for late stage cancer patients. This study aims to identify potential druggable target biomarkers as potential therapeutic options for patients with gastric cancer. Methods: Immunohistochemistry of human gastric tumor tissues was conducted to determine the expression level of cyclin-dependent kinase 12 (CDK12). Multiple in vitro and in vivo assays such as RNAi, mass spectrometry, computer docking models, kinase assays, cell xenograft NU/NU mouse models (CDXs) and patient-derived xenograft NOD/SCID mouse models (PDXs) were conducted to study the function and molecular interaction of CDK12 with p21 activated kinase 2 (PAK2), as well as to find CDK12 inhibitors as potential treatment options for human gastric cancer. Results: Here we identified that CDK12 is a driver gene in human gastric cancer growth. Mechanistically, CDK12 directly binds to and phosphorylates PAK2 at T134/T169 to activate MAPK signaling pathway. We further identified FDA approved clinical drug procaterol can serve as an effective CDK12 inhibitor, leading to dramatic restriction of cancer cell proliferation and tumor growth in human gastric cancer cells and PDXs. Conclusions: Our data highlight the potential of CDK12/PAK2 as therapeutic targets for patients with gastric cancer, and we propose procaterol treatment as a novel therapeutic strategy for human gastric cancer.

Combination of assist use of short-acting beta-2 agonists inhalation and guidance based on patient-specific restrictions in daily behavior: Impact on physical activity of Japanese patients with chronic obstructive pulmonary disease.

BACKGROUND: Assist use of inhaled short-acting beta 2 agonists (SABAs) is reportedly effective for preventing shortness of breath on exertion in chronic obstructive pulmonary disease (COPD) patients. However, it is unclear what strategy would be useful for improving physical activity in such patients. The aim is to investigate the effects of assisted use of SABA (procaterol) on physical activity in Japanese COPD patients targeting patient-specific restrictions in daily behavior. METHODS: Fourteen patients with stable COPD (age: 72.1±1.5, %FEV1: 55.6±4.5%) were asked to inhale 20 µg of procaterol 15 minutes before patient-specific daily physical activity that had been identified as limited by a questionnaire and document their usage in a diary. Physical activity was measured using a triaxial accelerometer and the results were collected every month for 2 months. In the first month, a clinician assessed whether inhalation of SABA was appropriate based on a usage diary and coached patients to conduct adequate assist use of SABA for limited physical activity. RESULTS: The strategy significantly improved the physical activity level, assessed using the values of the metabolic equivalents (METs) multiplied by physical activity endurance, at ≥3.0 METs (p<0.05), and physical activity endurance at ≥2.5 and ≥3.0 METs, (p<0.05, p<0.05, respectively). The degree of improvement of physical activity level was significantly positively correlated with the baseline %FVC and %FEV1 (p<0.05, p<0.05, respectively). CONCLUSIONS: Assist use of SABA targeting patient-specific restrictions, particularly when better lung function is still preserved, could be a useful approach for improving physical activity in patients with COPD.

Inhaled procaterol for the treatment of transient tachypnea of the newborn.

BACKGROUND: Transient tachypnea of the newborn (TTN) is a respiratory disorder that results from inadequate or delayed clearance of fetal lung fluid following delivery. At present, supportive care is generally practiced for the treatment of TTN. In this study, we focused on inhaled beta-agonists for the treatment of TTN, and the aim was to verify the efficacy and the safety of inhaled procaterol for the treatment of TTN. METHODS: Inhaled procaterol or normal saline solution was administered to infants. Respiratory rate and mixed venous carbon dioxide (PvCO2 ) were evaluated as the primary outcomes. The duration of hospitalization, duration of oxygen therapy, and changes in respiratory support were evaluated as secondary outcomes. RESULTS: Thirty-seven neonates diagnosed with TTN were randomly assigned to the procaterol group (n = 18) or the placebo group (n = 19). There were no differences in PvCO2 or respiratory rate between the two groups before and after intervention. Median duration of oxygen therapy (3 days; IQR, 3-6.5 days vs 2 days, IQR, 2-4.75 days; P = 0.13) and of hospitalization (15 days; IQR, 11.25-20 days vs 11 days, IQR, 8-15.5 days; P = 0.14) were not significantly different. CONCLUSIONS: Inhaled procaterol was not effective for the treatment of TTN.

Effect of Inhalation Flow Rate on Mass-Based Plume Geometry of Commercially Available Suspension pMDIs.

Although high-speed laser imaging is the current standard to characterize the plume angle of suspension-based pressurized metered dose inhalers (pMDIs), this method is limited by the inability to identify the drug content in a droplet and simulate inhalation flow. The Plume Induction Port Evaluator (PIPE) is a modified induction port for cascade impactors that allows for the calculation of the angle of a plume based on direct drug mass quantification rather than indirect droplet illumination under airflow conditions. The objective of this study was to investigate the use of the PIPE apparatus to evaluate the effect of airflow on the Mass Median Plume Angle (MMPA) of commercially available suspension-based pMDIs (Ventolin® HFA, ProAir® HFA, and Proventil® HFA). Deposition patterns within PIPE were log-normally distributed allowing for the calculation of the MMPA for the three suspension products. Mass-based plume angles were significantly smaller (narrower angle) when inhalation airflow was used compared to no flow conditions (reduction of MMPA was 8, 16, and 13% for Ventolin® HFA, ProAir® HFA, and Proventil® HFA, respectively). Additionally, new parameters for characterizing plume geometry were calculated (MMPA ex-actuator and plume orientation). Mass-based plume angles of the suspension-based pMDI formulations were highly reproducible and demonstrated the effect of inhalation flow rate. These results suggest that plume geometry tests should be evaluated under flow conditions which is not possible using current methodologies. Graphical Abstract ᅟ.

Simultaneous voltammetry detection of dopamine and uric acid in human serum and urine with a poly(procaterol hydrochloride) modified glassy carbon electrode.

In the present study, procaterol hydrochloride (ProH) was successfully electropolymerized onto a glass carbon electrode (GCE) with simply cyclic voltammetry scans to construct a poly(procaterol hydrochloride) (p-ProH) membrane modified electrode. Compared with the bare GCE, much higher oxidation peak current responses and better peak potentials separation could be obtained for the simultaneous oxidation of dopamine (DA) and uric acid (UA), owning to the excellent electrocatalytic ability of the p-ProH membrane. And it's based on that a square wave voltammetry (SWV) method was developed to selective and simultaneous measurement of DA and UA. Under the optimum conditions, the linear dependence of oxidation peak current on analyte concentrations were found to be 1.0-100 µmol/L and 2-100 µmol/L, giving detection limits of 0.3 µmol/L and 0.5 µmol/L for DA and UA, separately. The as prepared modified electrode shows simplicity in construction with the merits of good reproducibility, high stability, passable selectivity and nice sensitivity. Finally, the proposed p-ProH membrane modified electrode was successfully devoted to the detection of DA and UA in biological fluids such as human serum and urine with acceptable results.

Evaluation of Inhaled Procaterol for Potential Assist Use in Patients with Stable Chronic Obstructive Pulmonary Disease.

OBJECTIVES: International guidelines recommend the use of long-acting bronchodilators for the treatment of chronic obstructive pulmonary disease (COPD), but the usefulness of short-acting bronchodilator assist use for stable COPD remains uncertain. The purpose of the present study was to objectively demonstrate the effects of assist use of procaterol, a short-acting ß2-agonist, on the respiratory mechanics of stable COPD patients treated with a long-acting bronchodilator using forced oscillation technique (FOT) and conventional spirometry. We also confirmed the length of time for which procaterol assist could significantly improve the pulmonary function. METHODS: We enrolled 28 outpatients with mild to severe COPD (Global Initiative for Obstructive Lung Disease stages I-III), who had used the same long-acting bronchodilator for longer than 3 months and who were in stable condition. All measures were performed using both FOT and spirometry sequentially from 15 min to 2 h after inhalation. RESULTS: Compared to baseline, inhaled procaterol assist use modestly but significantly improved spirometric and FOT measurements within 2 h after inhalation. These significant effects continued for at least 2 h. -Significant correlations were found between parameters -measured by spirometry and those measured by FOT. CONCLUSIONS: Procaterol assist use modestly but significantly improved pulmonary function determined by spirometry and respiratory mechanics in patients with stable COPD treated with long-acting bronchodilators. Thus, inhaled procaterol has the potential for assist use for COPD.

Seihai-to (TJ-90)-Induced Activation of Airway Ciliary Beatings of Mice: Ca2+ Modulation of cAMP-Stimulated Ciliary Beatings via PDE1.

Sei-hai-to (TJ-90, Qing Fei Tang), a Chinese traditional medicine, increases ciliary beat frequency (CBF) and ciliary bend angle (CBA) mediated via cAMP (3',5'-cyclic adenosine monophosphate) accumulation modulated by Ca2+-activated phosphodiesterase 1 (PDE1A). A high concentration of TJ-90 (≥40 µg/mL) induced two types of CBF increases, a transient increase (an initial increase, followed by a decrease) and a sustained increase without any decline, while it only sustained the CBA increase. Upon inhibiting increases in intracellular Ca2+ concentration ([Ca2+]i) by 10 µM BAPTA-AM (Ca2+-chelator, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or Ca2+/calmodulin-dependent PDE1 by 8MmIBMX (a selective PDE1 inhibitor), TJ-90 (400 µg/mL) induced only the sustained CBF increase without any transient CBF increase. The two types of the CBF increase (the transient increase and the sustained increase) induced by TJ-90 (≥40 µg/mL) were mimicked by the stimulation with both procaterol (100 pM) and ionomycin (500 nM). Thus, TJ-90 stimulates small increases in the intracellular cAMP concentration ([cAMP]i) and [Ca2+]i in airway ciliary cells of mice. These small increases in [cAMP]i and [Ca2+]i cause inducing a transient CBF increase or a sustained CBF increase in an airway ciliary cells, depending on the dominant signal, Ca2+-signal, or cAMP-signal.

Evaluation of Bioequivalence Between the New Procaterol Hydrochloride Hydrate Dry Powder Inhaler and the Approved Dry Powder Inhaler in Patients With Asthma in a Randomized, Double-Blind, Double-Dummy, Crossover Comparison Study: A Phase 3 Study.

Procaterol hydrochloride hydrate (procaterol) is a ß2 -adrenergic receptor agonist that induces a strong bronchodilatory effect. The procaterol dry powder inhaler (DPI) has been frequently used in patients with bronchial asthma or chronic obstructive pulmonary disease. We evaluated the bioequivalence and safety between the new procaterol DPI (new DPI) and the approved procaterol DPI (approved DPI). This study was a randomized, double-blind, double-dummy, crossover comparison to evaluate the pharmacodynamic equivalence of the new DPI and the approved DPI in patients with bronchial asthma. Primary efficacy variables were area under the concentration-time curve (AUC) forced expiratory volume in the first second (FEV1 )/h and maximum FEV1 during the 480-minute measurement period. Patients were divided into 2 groups, New-DPI-First (n = 8) and Approved-DPI-First (n = 8), according to the investigational medical product that was administered first. Patients inhaled 20 µg of procaterol in each period. FEV1 was measured by a spirometer at predose and at 15, 30, 60, 90, 120, 180, 240, 360, and 480 minutes after each investigational medical product administration. Equivalence was evaluated by confirming that the 2-sided 90%CIs for the difference between the new and the approved DPI in means of AUC (FEV1 )/h and maximum FEV1 were within the acceptance criteria of -0.15 to 0.15 L. The difference in means of AUC (FEV1 )/h and maximum FEV1 was 0.041 L and 0.033 L, respectively, and the 90%CI was 0.004 to 0.078 L and -0.008 to 0.074 L, respectively. These CIs were both within the acceptance criteria. The new DPI was assessed as being bioequivalent to the approved DPI.

ADRB2 gene polymorphism and emphysema heterogeneity can modulate bronchodilator response in patients with emphysema.

BACKGROUND: Genetic variation in the ß2-adrenergic receptor (ADRB2) gene has been thought to have an important role in the differential response to ß2-agonist therapy for asthma. However, previous studies have shown little evidence for an association between these ADRB2 variants and the bronchial dilator response (BDR) in chronic obstructive pulmonary disease (COPD) patients. This discrepancy could be explained by differences in the distribution and heterogeneity of pulmonary emphysema in COPD patients, since emphysema distribution and heterogeneity are thought to have a role in pulmonary function in COPD patients. We hypothesized that differences in emphysema distribution and heterogeneity may have masked significant alterations of the bronchodilator response among ADRB2 genotypes in COPD patients in previous studies. METHODS: The BDR (induced by 20 µg of procaterol) was measured in 211 patients who had a smoking history of more than 10 pack/years and had undergone chest high resolution computed tomography examination. A low attenuations area (<960 Hounsfield Units) was identified and the emphysema heterogeneity index (EHI%) was calculated with a range in value from -100% to 100%. ADRB2 Arg16Gly genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: The BDR was augmented in patients with homogenous emphysema compared with those with upper-dominant emphysema. In patients carrying the AA genotype of ADRB2, the BDR was significantly increased in patients with upper-dominant emphysema, but not in patients with lower-dominant emphysema. CONCLUSION: Combination analysis of ADRB2 Arg16Gly polymorphism and EHI% may predict the effectiveness of ß2-adrenergic receptor agonist treatment in patients with COPD and emphysema.

A Modified Method for Examining the Walking Pattern and Pace of COPD Patients in a 6-min Walk Test Before and After the Inhalation of Procaterol.

Objective The 6-min walk test (6MWT) is a simple test that is used to examine the exercise tolerance and outcomes in patients with chronic obstructive pulmonary disease (COPD). Although the 6MWT is useful for assessing exercise tolerance, it is difficult to evaluate time-dependent parameters such as the walking pattern. A modified 6MWT has been devised to assess the walking pattern by calculating the number of steps per second (NSPS). This study was performed to investigate walking pattern of COPD patients in the modified 6MWT before and after a single inhalation of the short-acting ß2-agonist procaterol. Methods Nine male COPD patients participated in this study. The 6MWT was performed before and after the inhalation of procaterol hydrochloride. A digital video recording of the 6MWT was made. After the 6MWT, the number of steps walked by the subject in each 5-s period was counted manually with a hand counter while viewing the walking test on the video monitor. Results After the inhalation of procaterol, the 6-min walking distance increased significantly in comparison to baseline (p<0.01). The mean NSPS was also significantly increased after the inhalation of procaterol in comparison to baseline (p<0.01). The walking pattern was displayed on a graph of time versus NSPS, and the walking pace was shown by a graph of time versus cumulative steps. Conclusion The analysis of the COPD patients' walking test performance and their walking pattern and pace in the 6MWT may help to evaluate the effects of drug treatment.