Safety and efficacy of oral levosimendan in people with amyotrophic lateral sclerosis (the REFALS study): a randomised, double-blind, placebo-controlled phase 3 trial.

BACKGROUND: There is an urgent unmet need for new therapies in amyotrophic lateral sclerosis. In a clinical study with healthy volunteers, levosimendan, a calcium sensitiser, was shown to improve neuromechanical efficiency and contractile function of the human diaphragm. We aimed to evaluate the safety and efficacy of oral levosimendan in people with amyotrophic lateral sclerosis, with a focus on respiratory function. METHODS: The REFALS study is a randomised, double-blind, placebo-controlled phase 3 trial at 99 amyotrophic lateral sclerosis specialist centres in 14 countries worldwide. People with amyotrophic lateral sclerosis were eligible for participation if they were at least 18 years of age and had a sitting slow vital capacity (SVC) of 60-90% predicted. Participants were randomly assigned (2:1) by interactive web-response system to receive either levosimendan or placebo. The capsules for oral administration were identical in appearance to maintain blinding of participants and investigators. The primary endpoint was the change from baseline in supine SVC at 12 weeks, assessed as the percentage of predicted normal sitting SVC. The key secondary endpoint was the combined assessment of function and survival (CAFS) up to 48 weeks. Analyses were done in the intention-to-treat population, comprising all participants who were randomly assigned. This trial is registered at ClinicalTrials.gov (NCT03505021) and has been completed. An extension study (REFALS-ES; NCT03948178) has also been completed, but will be reported separately. FINDINGS: Between June 21, 2018, and June 28, 2019, 871 people were screened for the study, of whom 496 were randomly assigned either levosimendan (n=329) or placebo (n=167). Participants were followed up between June 27, 2018 and June 26, 2020, for a median duration of 50·1 (IQR 37·5-51·1) weeks. The median duration of treatment was 47·9 (IQR 26·4-48·1) weeks. Change from baseline in supine SVC at 12 weeks was -6·73% with levosimendan and -6·99% with placebo, with no significant difference between the treatments (estimated treatment difference 0·26%, 95% CI -2·03 to 2·55, p=0·83). Similarly, at week 48, CAFS did not differ between treatment groups (least squares mean change from baseline 10·69, 95% CI -15·74 to 37·12; nominal p value=0·43). The most frequent adverse events were increased heart rate (106 [33%] of 326 receiving levosimendan vs 12 [7%] of 166 receiving placebo), fall (85 [26%] vs 48 [29%]), headache (93 [29%] vs 36 [22%]), and dyspnoea (59 [18%] vs 32 [19%]). 33 (10%) participants allocated levosimendan and 20 (12%) assigned placebo died during the trial, mainly due to respiratory failure or progression of amyotrophic lateral sclerosis. INTERPRETATION: Levosimendan was not superior to placebo in maintaining respiratory function in a broad population with amyotrophic lateral sclerosis. Although levosimendan was generally well tolerated, increased heart rate and headache occurred more frequently with levosimendan than with placebo. The possibility of a clinically relevant subgroup of responsive individuals requires further evaluation. FUNDING: Orion Corporation.

Oral levosimendan in amyotrophic lateral sclerosis: a phase II multicentre, randomised, double-blind, placebo-controlled trial.

OBJECTIVE: To evaluate the efficacy and safety of oral levosimendan in patients with amyotrophic lateral sclerosis (ALS). This phase II, randomised, double-blind, placebo-controlled, crossover, three-period study with 6 months open-label follow-up enrolled adults with ALS and sitting slow vital capacity (SVC) 60%-90 % of predicted from 11 sites in four countries. METHODS: Patients received levosimendan 1 mg daily, 1 mg two times a day or placebo during three 14-day crossover periods and levosimendan 1-2 mg daily during open-label follow-up. Primary endpoint was sitting SVC; secondary endpoints included supine SVC, ALS Functional Rating Scale-Revised (ALSFRS-R), tolerability and safety. RESULTS: Of 66 patients randomised, 59 contributed to the double-blind results and 50 entered open-label follow-up. Sitting SVC was not significantly different between the treatments. In post hoc analysis using period-wise baselines, supine SVC favoured levosimendan over placebo, estimated mean differences from baseline being -3.62% on placebo, +0.77% on levosimendan 1 mg daily (p=0.018) and +2.38% on 1 mg two times a day (p=0.001). Headache occurred in 16.7% of patients during levosimendan 1 mg daily (p=0.030), 28.6% during 1 mg two times a day (p=0.002) and 3.3% during placebo. The respective frequencies for increased heart rate were 5.1% (p=0.337), 18.5% (p=0.018) and 1.7%. No significant differences between the treatments were seen for other adverse events. CONCLUSIONS: Levosimendan did not achieve the primary endpoint of improving sitting SVC in ALS. Headache and increased heart rate were increased on levosimendan, although it was otherwise well tolerated. A phase III study to evaluate the longer term effects of oral levosimendan in ALS is ongoing.

Entacapone and prostate cancer risk in patients with Parkinson's disease.

BACKGROUND: The association between Parkinson's disease (PD) and prostate cancer, both common in elderly men, is disputable. In the STRIDE-PD study, prostate cancer developed in 9 patients (3.7%) receiving levodopa/carbidopa with entacapone, a catechol-O-methyltransferase inhibitor, versus 2 cases (0.9%) without entacapone. The current pharmacoepidemiological study aimed to determine whether entacapone increases prostate cancer incidence or mortality in PD patients and whether cumulative exposure affects these rates. METHODS: We performed a retrospective cohort study using population-wide health care registers with patient-level linkage. Prostate cancer incidence and mortality were modeled by Cox's proportional hazards models. RESULTS AND CONCLUSIONS: Use of entacapone with l-dopa/dopa decarboxylase inhibitor caused no increased risk of prostate cancer incidence (hazard ratio [HR]: 1.05; 95% confidence interval: 0.76-1.44) or mortality (0.93; 0.43-1.98). The HR for cumulative entacapone use of >360 days versus never-use was 0.82 (0.56-1.18) for prostate cancer incidence and 1.27 (0.60-2.72) for prostate cancer mortality.

Bactericidal/permeability-increasing protein in lacrimal gland and in tears of healthy subjects.

BACKGROUND: To determine the expression of bactericidal/permeability-increasing protein (BPI), a novel antimicrobial molecule, in the main lacrimal gland and its content in tears of young healthy subjects. METHODS: BPI concentration of tears was measured in 42 healthy volunteers, 13 men and 29 women, with ages ranging from 22 to 30 (mean 24.7+/-2.1) years by a time-resolved fluoroimmunoassay (TR-FIA). Immunohistochemical analysis was made to localize BPI in lacrimal gland and conjunctiva of eight autopsied subjects, two men and six women, with the age range from 44 to 87 (mean 72.3+/-14.9) years. RESULT: The mean concentration of BPI in tears was 27.8+/-29.5 microg/l, and it decreased with an increase in tear flow rate (P<0.0001). There was no statistically significant difference in BPI content of tears between the genders. BPI was immunohistochemically seen in outer basal epithelial cells of intralobular and excretory ducts, squamous and basal cells of conjunctiva as well as faintly in myoepithelial cell layer of acini. The presence of BPI in the lacrimal gland and in the tear fluid was verified by Western blotting. CONCLUSIONS: The results indicate that outer basal epithelial cells of lacrimal gland ducts contain BPI, which occurs in a relatively high concentration in tears. BPI may have a substantial antibacterial role in human tears.

Group IIA phospholipase A(2) concentration of tears in patients with ocular rosacea.

PURPOSE: To determine the concentration of group IIA phospholipase A(2) (GIIAPLA(2)) in tears of patients with ocular rosacea, and to compare it with GIIAPLA(2) concentration in tears of age-matched healthy controls. METHODS: The GIIAPLA(2) concentration in tears was measured with a time-resolved fluoroimmunoassay in 21 patients with ocular rosacea (mean age 55.6+/-9.2 years) and in 21 normal subjects (mean age 53.4+/-8.2 years). Conjunctival brush cytology was carried out and eosinophils, neutrophils, lymphocytes, squamous epithelial cells, columnar epithelial cells, metaplastic changes and goblet cells were calculated separately. RESULTS: The GIIAPLA (2) concentration in tears was statistically significantly lower in patients with ocular rosacea (31.0+/-18.4 microg/ml, p=0.0099) and, more specifically, in patients who had dry eye (25.8+/-15.1 microg/ml, p=0.0034), compared to that in normal controls. There was no correlation between the GIIAPLA (2) content of tears and the conjunctival cells collected by the brush cytology. CONCLUSION: The tears of patients with dry eye symptoms due to ocular rosacea have decreased GIIAPLA (2) content. The pathogenic importance of this finding is discussed.

Group IIA phospholipase A2 content of tears in patients with atopic blepharoconjunctivitis.

BACKGROUND: To determine the concentration of group IIA phospholipase A(2) (GIIAPLA(2)) in tears of patients with atopic blepharoconjunctivitis (ABC), and to compare it with the GIIAPLA(2) concentration of tears in age-matched healthy controls. METHODS: The diagnosis of ABC was confirmed with a positive skin prick test and the presence of atopic dermatitis in lids. Conjunctival brush cytology was taken, and the cells including eosinophils, neutrophils, lymphocytes, squamous epithelial cells, columnar epithelial cells, metaplastic changes and the goblet cells were calculated separately. The GIIAPLA(2) concentration of tears was measured with a time-resolved fluoroimmunoassay in 29 patients with ABC (mean age 36.3+/-12.7 years) and 29 normal subjects (mean age 37.0+/-12.0 years). RESULTS: The GIIAPLA(2) concentration of tears in patients with ABC was 43.8+/-33.0 microg/ml, and in normal subjects it was 67.1+/-23.3 microg/ml. The difference was statistically significant (p=0.0018). The concentration of GIIAPLA(2) of tears was lowest in the subgroup of patients with ABC and dry eye (25.8()+/-23.6 microg/ml), whereas it was only slightly decreased in patients with ABC and normal tear secretion (56.6+/-33.3 microg/ml). The difference between these two subgroups was statistically significant (p=0.011). There was no statistically significant correlation between the GIIAPLA(2) concentration of tears and the quantity of different conjunctival cells gathered by the brush cytology. However, an almost significant correlation was found between the GIIAPLA(2) concentration in tears and conjunctival eosinophils. CONCLUSIONS: The results indicate that in patients with ABC the GIIAPLA(2) content of tears was decreased, without any dependence on the quantity of different conjunctival cells.

Group IIA phospholipase A(2) content in tears of patients having photorefractive keratectomy.

PURPOSE: To study the effect of photorefractive keratectomy (PRK) on the concentration of group IIA phospholipase A(2) (GIIAPLA(2)) in tears. SETTING: Departments of Ophthalmology and Pathology, University of Turku, Turku, and Helsinki University Eye Hospital, Helsinki, Finland. METHODS: Tear samples were collected from 25 eyes of 23 patients (mean age 32.3 years +/- 8.6 [SD]) preoperatively and 2 and 7 days after PRK. The GIIAPLA(2) concentration in the tears was measured by time-resolved fluoroimmunoassay. RESULTS: The GIIAPLA(2) concentration was significantly lower and the tear fluid flow rate significantly higher 2 days after PRK than preoperatively. At 7 days, the GIIAPLA(2) concentration and the tear fluid flow-corrected excretion of GIIAPLA(2) were significantly higher than preoperatively and at 2 days. The tear flow rate was also significantly higher than preoperatively. CONCLUSIONS: The GIIAPLA(2) content in tears decreased 2 days after PRK due to dilution of the GIIAPLA(2) content during hypersecretion of reflex tears. Photorefractive keratectomy caused an increase in the tear flow rate, GIIAPLA(2) concentration, and tear fluid flow-corrected excretion of GIIAPLA(2) in tears 7 days after surgery, enhancing the protection of tears against bacterial infections.

Diurnal variation in group IIa phospholipase A2 content in tears of contact lens wearers and normal controls.

PURPOSE: To study the diurnal rhythm in group IIA phospholipase A(2) (GIIAPLA(2)) content of tears and the effect of the wearing time of soft contact lenses (CL) on the content of GIIAPLA(2 )in tears. METHODS: The GIIAPLA(2 )content of tears was measured by a time-resolved fluoroimmunoassay in 22 healthy controls at 8 a.m., noon, 4 p.m. and 8 p.m. and in 20 CL wearers at 4 p.m. 1-2 days before using CLs and after 4 h (at noon), 8 h (4 p.m.) and 12 h (8 p.m.) use of soft CLs. RESULTS: The GIIAPLA(2 )content of tears of healthy controls was 80.6+/-47.8 micro g/ml (mean+/-SD). The GIIAPLA(2 )content was lower at 8 a.m. than at noon (p=0.006) and higher at 4 p.m. than at 8 p.m. ( P=0.003). There was no statistically significant difference in the GIIAPLA(2 )content of tears between the CL wearers without CLs (69.47+/-31.2 micro g/ml) and the normal subjects (92.3+/-48.2 micro g/ml) measured at 4 p.m. Compared with healthy controls, the GIIAPLA(2) values in subjects wearing CLs were statistically significantly lower at noon ( P=0.0001) and at 4 p.m. ( P=0.0002). CONCLUSION: In normal subjects, the GIIAPLA(2) content of tears increased from 8 a.m. to noon and decreased from 4 p.m. to 8 p.m. The use of CLs for 4 h and 8 h caused a decrease in the GIIAPLA(2) content of tears. This difference was not seen at 4 p.m. the day when the CL wearers did not use CLs.

Group IIA phospholipase A2 content of basal, nonstimulated and reflex tears.

PURPOSE: To determine the concentration of group IIA phospholipase A2 (GIIAPLA(2)) in basal, nonstimulated and reflex tears of young normal subjects. METHODS: The GIIAPLA(2) content of tears was measured in left eye of 16 healthy subjects (mean age 24.5 +/- 2.2 years). Five samples were taken from each subject: nonstimulated sample; basal sample in a dark room after topical anesthesia; and reflex tear samples immediately on stimulation by bright light and breathing of the vapor of onions, and 1 and 3 minutes after the onset of tear flow. RESULTS: The GIIAPLA(2) content of reflex tears was statistically significantly lower than the GIIAPLA(2) content of basal tears, whereas there was no statistically significant difference in the GIIAPLA(2) content between nonstimulated and basal tears. CONCLUSIONS: The current results indicate that the basal and nonstimulated tears are similar in their GIIAPLA(2) content, and that the GIIAPLA( 2) content of tears decreases with stimulation, possibly due to dilution of GIIAPLA(2) content during reflectory hypersecretion of tears.

Group IIA phospholipase A2 content of tears in patients with keratoconjunctivitis sicca.

PURPOSE: To determine the concentration of group IIA phospholipase A2 (GIIAPLA(2)) in tears of patients with keratoconjunctivitis sicca (KCS) and to compare it with the GIIAPLA(2) content of tears in age-matched healthy controls. METHODS: The GIIAPLA(2)content of tears was measured with time-resolved fluoroimmunoassay in 20 patients with KCS (mean age 70.7+/-8.7 years) and in 20 normal subjects (mean age 70.2+/-9.9 years). RESULTS: The GIIAPLA(2)content of tears in patients with KCS was 75.8+/-54.2 microg/ml and in normal subjects it was 34.2+/-21.4 microg/ml. The difference was statistically significant. In patients with KCS the GIIAPLA(2) concentration slightly increased with decreasing Schirmer test values (Spearman's correlation =-0.20) CONCLUSION: The results of this study indicate that in patients with KCS the relative concentration of GIIAPLA(2) of tears was increased due to decreased aqueous production in the lacrimal glands and better preservation of the acinar cells in the central parts of the lobules of the lacrimal glands producing GIIAPLA(2).