Caution for psychiatrists: malignant hyperthermia risks with the anesthetic agent succinylcholine (Suxamethonium) during electroconvulsive therapy.

BACKGROUND: Malignant hyperthermia is a potentially lethal condition triggered by specific anesthetic drugs, especially a depolarizing muscle relaxant of succinylcholine (Suxamethonium). Despite the frequent use of succinylcholine with electroconvulsive therapy (ECT), there has been no reported case of potentially lethal malignant hyperthermia following ECT. In addition, the time interval between the administration of succinylcholine and the onset of malignant hyperthermia has not been outlined in the context of ECT. CASE PRESENTATION: We present the case of a 79-year-old woman suffering from severe depression, who experienced severe malignant hyperthermia due to succinylcholine administration during an ECT session. She presented with a high fever of 40.2 °C, tachycardia of 140/min, hypertension with a blood pressure exceeding 200 mmHg, significant muscle rigidity, and impaired consciousness. These symptoms emerged two hours after ECT, which occurred in a psychiatric ward rather than an operating room, and reached their peak in less than 24 h. She was given 60 mg of dantrolene, which quickly reduced the muscular rigidity. Subsequently, she received two additional doses of 20 mg and 60 mg of dantrolene, which brought her fever down to 36.2 °C and completely eased her muscle rigidity within two days after ECT. CONCLUSIONS: This is the first reported case of potentially lethal malignant hyperthermia after ECT. In addition, it highlights the delayed onset of malignant hyperthermia following an ECT procedure, emphasizing the necessity for psychiatrists to recognize its onset even after the treatment. In the light of potentially lethal consequences of malignant hyperthermia, it is critically important for psychiatrists to closely monitor both intraoperative and postoperative patient's vital signs and characteristic physical presentations, promptly identify any symptomatic emergence, and treat it immediately with dantrolene.

The novel rapid formulation of intravenous dantrolene (NPJ5008) versus standard dantrolene (Dantrium®): A clinical part-randomised phase 1 study in healthy volunteers.

BACKGROUND: Delays in treating anaesthesia-induced malignant hyperthermia increase risks of complications and death. NPJ5008 is a novel formulation of the indicated treatment, dantrolene sodium, developed to shorten preparation and administration times compared with the reference formulation Dantrium®. The two formulations have been compared preclinically. OBJECTIVES: Assess bioequivalence of overall dantrolene (free acid) exposure of NPJ5008 versus Dantrium® and ascertain similarities in their pharmacokinetics and safety/tolerability profiles. Evaluate preparation/administration time savings for the new formulation. DESIGN: Part 1 of this open-label trial in humans was a 1 : 1 randomised crossover study; part 2 was a single-arm study. Trial pharmacy data and laboratory simulations assessed preparation/administration step timings. SETTING: Single clinical centre in the UK, April to July 2021. PARTICIPANTS: Twenty-one healthy male and female individuals. INTERVENTIONS: Part 1: single intravenous 60 mg dose of NPJ5008 or Dantrium®, sequentially. Part 2: single intravenous 120 mg dose of NPJ5008. Simulation: five vials per formulation using paediatric and adult cannulas. MAIN OUTCOME MEASURES: Overall drug exposure to last measurable concentration (AUC 0 to last ) and extrapolated to infinity (AUC 0 to ∞ ) were primary endpoints. Other pharmacokinetic, clinical and muscle-function parameters, and adverse events, were monitored. RESULTS: Adjusted geometric mean ratios of NPJ5008 versus Dantrium® were 90.24 and 90.44% for AUC 0 to last and AUC 0 to ∞ , respectively, with the 90% confidence intervals (CI) within the 80 to 125% acceptance interval, establishing bioequivalence. No new safety issues emerged: any adverse events were of a similar magnitude across treatments and related to pharmacological properties of dantrolene. Pharmacy and simulation data revealed that every step in preparation and administration was 26 to 69% faster for NPJ5008 than Dantrium®. CONCLUSION: NPJ5008 showed comparable pharmacokinetic and safety profiles to Dantrium®, while reducing dantrolene dose preparation/administration times, potentially reducing patient complications/healthcare resourcing in malignant hyperthermia. TRIAL REGISTRATION: EudraCT Number: 2020-005719-35, MHRA approval.

Cardioprotective Effect of Polymyxin-B and Dantrolene Combination on Isoproterenol-Induced Hypertrophic Cardiomyopathy in Rats, via Attenuation of Calmodulin-Dependent Protein Kinase II.

Hypertrophic cardiomyopathy is a major cause of mortality worldwide. In this study, we hypothesized that the combination of Dantrolene and Polymyxin-B will provide cardioprotective action against isoproterenol-induced hypertrophic cardiomyopathy via attenuation of Calmodulin-dependent protein kinase II (CaMKII). Hypertrophic cardiomyopathy was induced in rats by subcutaneous administration of isoproterenol (5 mg/kg) for 14 days. Simultaneously, animals were treated with Polymyxin-B per se, Dantrolene per se, and Dantrolene and Polymyxin-B combination for 14 days. Hemodynamic parameters, biochemical parameters, and histological analysis were performed. Administration of isoproterenol for 14 days resulted in severe myocardial damage, characterized by cardiac hypertrophy and increase serum CK-MB, CK-Nac, LDH, AST, and ALT levels. It also caused alteration in electrocardiogram and blood pressure. A significant increase in CaMKII was observed in heart homogenate. Treatment with the Polymyxin-B and Dantrolene combination significantly ameliorated cardiac hypertrophy, biochemical parameters, ECG parameters, and heart histopathology. Further, significant attenuation in CaMKII levels was observed. The effect of the combination was more than per se treatment. Results of the current study showed that the combination of Polymyxin-B and Dantrolene prevented the development of isoproterenol-induced hypertrophic cardiomyopathy in rats via attenuation of the CaMKII.

A carvedilol analogue, VK-II-86, prevents hypokalaemia-induced ventricular arrhythmia through novel multi-channel effects.

BACKGROUND AND PURPOSE: QT prolongation and intracellular Ca2+ loading with diastolic Ca2+ release via ryanodine receptors (RyR2) are the predominant mechanisms underlying hypokalaemia-induced ventricular arrhythmia. We investigated the antiarrhythmic actions of two RyR2 inhibitors: dantrolene and VK-II-86, a carvedilol analogue lacking antagonist activity at ß-adrenoceptors, in hypokalaemia. EXPERIMENTAL APPROACH: Surface ECG and ventricular action potentials (APs) were recorded from whole-heart murine Langendorff preparations. Ventricular arrhythmia incidence was compared in hearts perfused with low [K+ ], and those pretreated with dantrolene or VK-II-86. Whole-cell patch clamping was used in murine and canine ventricular cardiomyocytes to study effects of dantrolene and VK-II-86 on AP parameters in low [K+ ] and effects of VK-II-86 on the inward rectifier current (IK1 ), late sodium current (INa_L ) and the L-type Ca2+ current (ICa ). Effects of VK-II-86 on IKr were investigated in transfected HEK-293 cells. A fluorogenic probe quantified the effects of VK-II-86 on oxidative stress in hypokalaemia. KEY RESULTS: Dantrolene reduced the incidence of ventricular arrhythmias induced by low [K+ ] in explanted murine hearts by 94%, whereas VK-II-86 prevented all arrhythmias. VK-II-86 prevented hypokalaemia-induced AP prolongation and depolarization but did not alter AP parameters in normokalaemia. Hypokalaemia was associated with decreased IK1 and IKr , and increased INa-L , and ICa . VK-II-86 prevented all hypokalaemia-induced changes in ion channel activity and oxidative stress. CONCLUSIONS AND IMPLICATIONS: VK-II-86 prevents hypokalaemia-induced arrhythmogenesis by normalizing calcium homeostasis and repolarization reserve. VK-II-86 may provide an effective treatment in hypokalaemia and other arrhythmias caused by delayed repolarization or Ca2+ overload.

A phase Ib/IIa clinical trial of dantrolene sodium in patients with Wolfram syndrome.

BACKGROUNDWolfram syndrome is a rare ER disorder characterized by insulin-dependent diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Although there is no treatment for Wolfram syndrome, preclinical studies in cell and rodent models suggest that therapeutic strategies targeting ER calcium homeostasis, including dantrolene sodium, may be beneficial.METHODSBased on results from preclinical studies on dantrolene sodium and ongoing longitudinal studies, we assembled what we believe is the first-ever clinical trial in pediatric and adult Wolfram syndrome patients with an open-label phase Ib/IIa trial design. The primary objective was to assess the safety and tolerability of dantrolene sodium in adult and pediatric Wolfram syndrome patients. Secondary objectives were to evaluate the efficacy of dantrolene sodium on residual pancreatic ß cell functions, visual acuity, quality-of-life measures related to vision, and neurological functions.RESULTSDantrolene sodium was well tolerated by Wolfram syndrome patients. Overall, ß cell functions were not significantly improved, but there was a significant correlation between baseline ß cell functions and change in ß cell responsiveness (R2, P = 0.004) after 6-month dantrolene therapy. Visual acuity and neurological functions were not improved by 6-month dantrolene sodium. Markers of inflammatory cytokines and oxidative stress, such as IFN-γ, IL-1ß, TNF-α, and isoprostane, were elevated in subjects.CONCLUSIONThis study justifies further investigation into using dantrolene sodium and other small molecules targeting the ER for treatment of Wolfram syndrome.TRIAL REGISTRATIONClinicalTrials.gov identifier NCT02829268FUNDINGNIH/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (DK112921, DK113487, DK020579), NIH/National Center for Advancing Translational Sciences (NCATS) (TR002065, TR000448), NIH training grant (F30DK111070), Silberman Fund, Ellie White Foundation, Snow Foundation, Unravel Wolfram Syndrome Fund, Stowe Fund, Eye Hope Foundation, Feiock Fund, Washington University Institute of Clinical and Translational Sciences grant UL1TR002345 from NIH/NCATS, Bursky Center for Human Immunology & Immunotherapy Programs.

Dantrolene Potentiates the Antineoplastic Effect of Sorafenib in Hepatocellular Carcinoma via Targeting Ca+2/PI3K Signaling Pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is the 6th prevalent cancer and the 4th leading cause of cancer-related deaths all over the world. A major challenge for sorafenib, the standard chemotherapeutic agent in HCC treatment, is the chemo-resistance. OBJECTIVE: This study was conducted to evaluate the role of dantrolene as a possible antineoplastic agent in HCC, and in chemo-sensitization of sorafenib via targeting Ca+2/PI3K pathway. METHODS: HCC was induced in rats using a single dose of diethylnitrosamine (DENA) (200 mg/kg, ip), followed by phenobarbital sodium (0.05%) in drinking water for 18 weeks. At the end of the 18th week, rats were allocated into 4 groups (10 rats/each), one group was left without treatment (DENA group) and the other three groups were treated with either sorafenib, dantrolene, or their combination for further 4 weeks. One day after the last injection, serum and liver tissues were collected. Liver tissue p53, VEGF, MMP-9, Cyclin D1, PI3K, and, serum AFP were assessed using immunoassay. Hepatic and serum Ca+2 were also computed. Furthermore, Ki-67 was assessed immunohistochemically. RESULTS: Dantrolene exhibited synergistic effect when used in combination with sorafenib compared to either drug alone (p <0.05) through decreasing p53, VEGF, MMP-9, Cyclin D1, and Ki-67. In addition, dantrolene was evidenced to have an inhibitory effect on Ca+2/PI3K pathway that mediates its antineoplastic action when used alone or in combination with sorafenib. CONCLUSION: Dantrolene exerted antineoplastic effect as well as augmented sorafenib antineoplastic activity via the intervention of Ca+2/PI3K pathway, manifested by ameliorating angiogenesis, apoptosis, proliferation and metastasis.

Dietary Caffeine Synergizes Adverse Peripheral and Central Responses to Anesthesia in Malignant Hyperthermia Susceptible Mice.

Ryanodine receptor (RYR) mutations confer stress-triggered malignant hyperthermia (MH) susceptibility. Dietary caffeine (CAF) is the most commonly consumed psychoactive compound by humans. CAF-triggered Ca2+ release and its influences on skeletal muscle contractility are widely used as experimental tools to study RYR function/dysfunction and diagnose MH susceptibility. We hypothesize that dietary CAF achieving blood levels measured in human plasma exacerbates the penetrance of RYR1 MH susceptibility mutations triggered by gaseous anesthetic, affecting both central and peripheral adverse responses. Heterozygous R163C-RYR1 (HET) MH susceptible mice are used to investigate the influences of dietary CAF on both peripheral and central responses before and after induction of halothane (HAL) maintenance anesthesia under experimental conditions that maintain normal core body temperature. HET mice receiving CAF (plasma CAF 893 ng/ml) have significantly shorter times to respiratory arrest compared with wild type, without altering blood chemistry or displaying hyperthermia or muscle rigor. Intraperitoneal bolus dantrolene before HAL prolongs time to respiratory arrest. A pilot electrographic study using subcutaneous electrodes reveals that dietary CAF does not alter baseline electroencephalogram (EEG) total power, but significantly shortens delay to isoelectric EEG, which precedes respiratory and cardiac arrest. CAF ± HAL are studied on RYR1 single-channel currents and HET myotubes to define molecular mechanisms of gene-by-environment synergism. Strong pharmacological synergism between CAF and HAL is demonstrated in both single-channel and myotube preparations. Central and peripheral nervous systems mediate adverse responses to HAL in a HET model of MH susceptibility exposed to dietary CAF, a modifiable lifestyle factor that may mitigate risks of acute and chronic diseases associated with RYR1 mutations. SIGNIFICANCE STATEMENT: Dietary caffeine at a human-relevant dose synergizes adverse peripheral and central responses to anesthesia in malignant hyperthermia susceptible mice. Synergism of these drugs can be attributed to their actions at ryanodine receptors.

A Novel Dantrolene Sodium-Loaded Mixed Micelle Containing a Small Amount of Cremophor EL: Characterization, Stability, Safety and Pharmacokinetics.

Dantrolene sodium (DS) is the only drug specifically used for the treatment of malignant hyperthermia. Nevertheless, its clinical application is significantly restricted due to its aqueous insolubility and the limited formulations available in clinical practice. In order to solve these problems, a novel mixed micelle composed of phospholipid and Cremophor EL was designed and evaluated. The mixed micelle was prepared using a stirring- ultrasonic method. The Dynamic Light Scattering (DLS) results showed that the micelle was small in size (12.14 nm) and narrowly distributed (PdI = 0.073). Transmission Electron Microscopy (TEM) images showed that the micelle was homogeneous and spherical. The stability study indicated that the system was stable for storage and dilution with distilled water, while the safety testing showed that the micelle was safe for intravenous administration with low hemolysis rates and low allergic reaction rates. In the pharmaceutics study, the Cmax and AUC0-t of the DS-loaded micelle were 4- and 4.5- folds higher than that of the DS. Therefore, the constructed phospholipid-Cremophor EL mixed micelle is a promising drug delivery system for DS.

Identification of enzymes responsible for dantrolene metabolism in the human liver: A clue to uncover the cause of liver injury.

Dantrolene is used for malignant hyperthermia during anesthesia, and it sometimes causes severe liver injury as a side effect. Dantrolene is metabolized to acetylaminodantrolene, which is formed via the reduction of dantrolene to aminodantrolene and subsequent acetylation. Formation of hydroxylamine during the metabolic process may be associated with liver injury. We identified the enzymes responsible for dantrolene metabolism in humans to elucidate the mechanism of liver injury. Dantrolene reductase activity was not detected in human liver microsomes, but it was detected in cytosol. Formation was increased in the presence of N1-methylnicotineamide, which is an electron donor to aldehyde oxidase 1 (AOX1). Potent inhibitors of AOX1 and a correlation study with a marker of AOX1 activity, namely phthalazine oxidase activity, in a panel of 28 human liver cytosol samples supported the role of AOX1 in dantrolene reduction. Acetylaminodantrolene formation from aminodantrolene was highly detected in recombinant N-acetyltransferase (NAT) 2 rather than NAT1. A glutathione trapping assay revealed the formation of hydroxylamine via an AOX1-dependent reduction of dantrolene but not via hydroxylation of aminodantrolene. In conclusion, we found that AOX1 and NAT2 were responsible for dantrolene metabolism in humans and that AOX1-dependent metabolism determines dantrolene-induced liver injury.

Succinylcholine for Emergency Airway Rescue in Class B Ambulatory Facilities: The Society for Ambulatory Anesthesia Position Statement.

Procedures in class B ambulatory facilities are performed exclusively with oral or IV sedative-hypnotics and/or analgesics. These facilities typically do not stock dantrolene because no known triggers of malignant hyperthermia (ie, inhaled anesthetics and succinylcholine) are available. This article argues that, in the absence of succinylcholine, the morbidity and mortality from laryngospasm can be significant, indeed, higher than the unlikely scenario of succinylcholine-triggered malignant hyperthermia. The Society for Ambulatory Anesthesia (SAMBA) position statement for the use of succinylcholine for emergency airway management is presented.

Neuromuscular conditions associated with malignant hyperthermia in paediatric patients: A 25-year retrospective study.

Malignant Hyperthermia (MH) is a rare pharmacogenetic syndrome that can be fatal and the risk of MH in non RYR1-related disorders is unknown. We conducted a retrospective study to determine the prevalence of neuromuscular disorders among patients with MH at our centre. Patients who were admitted to the Hospital for Sick Children during the study period of January 1, 1990 to April 1, 2015 with a CK level > 8000 IU/L, or who received dantrolene, or who had a clinical diagnosis of MH were included. Medical records of 166 patients who met the inclusion criteria were reviewed and 13 patients were identified with MH-like reactions. Nine patients were classified as having true MH after review of the anaesthesia record and genetic testing results were available for 7 patients, 5 of whom had mutations in RYR1. Of the four patients who had severe reactions to anaesthesia but did not meet the criteria for true MH, two had Duchenne muscular dystrophy (DMD). In this retrospective study over 25 years, RYR1 mutations were the most common cause of MH in our cohort, and of these, one third had an underlying neuromuscular diagnosis. Genetic testing of RYR1 is indicated for all patients with MH, and anaesthetic precautions should be considered for any child with symptoms of neuromuscular disease.

Dantrolene for cerebral vasospasm after subarachnoid haemorrhage: a randomised double blind placebo-controlled safety trial.

BACKGROUND: Dantrolene is neuroprotective in animal models and may attenuate cerebral vasospasm (cVSP) in human aneurysmal subarachnoid haemorrhage (aSAH). We evaluated safety, feasibility and tolerability of intravenous dantrolene (IV-D) in patients with aSAH. METHODS: In this single-centre, randomised, double blind, placebo-controlled trial, 31 patients with aSAH were randomised to IV-D 1.25 mg every 6 h for 7 days (n=16) or equiosmolar free water/5% mannitol (placebo; n=15). Primary safety end points were incidence of hyponatraemia (sNa≤132 mmol/L) and liver toxicity (proportion of patients alanine transaminase, aspartate aminotransferase and AlkPhos >5× upper-limit-of-normal). Secondary end points included tolerability, systemic hypotension and intracranial hypertension. Efficacy was explored for clinical/radiological cVSP, delayed cerebral ischaemia (DCI), and 3-month functional outcomes. Quantitative analyses of angiograms and daily transcranial Doppler (TCD) were performed. RESULTS: Between IV-D versus placebo, no differences were observed in the primary outcomes (hyponatremia 44% vs 67% (p=0.29); liver toxicity 6% vs 0% (p=1.0)). Three patients in the IV-D versus two in the placebo group had severe adverse events possibly attributable to infusion and reached stop criteria: one IV-D patient developed liver toxicity; two patients in each group developed brain oedema requiring osmotherapy. The majority of adverse events were not related to infusion (17 vs 5 (RR 2.2; 95% CI 0.7 to 6.7; p=0.16) in IV-D vs placebo). No differences in any categorical cVSP outcomes, DCI, 3-month outcomes or quantitative angiogram and TCD analyses were seen in this small safety trial not powered to detect efficacy. CONCLUSIONS: In this small trial, IV-D after aSAH was feasible, tolerable and safe. TRIAL REGISTRATION NUMBER: http://clinicaltrials.gov NCT01024972.

Pharyngeal spasticity due to dantrolene.

WHAT IS KNOWN AND OBJECTIVE: Dantrolene can be combined with baclofen to better treat spasticity, but may cause muscular weakness and dysphagia. We instead describe a pharyngeal spasm due to dantrolene. CASE SUMMARY: A 12-year-old male received dantrolene 3 mg/kg/day in adjunct to baclofen 2 mg/kg/day, to improve spasticity. After 5 days of full-dose dantrolene, his dysphagia worsened and he developed pharyngeal spasm. Dantrolene was suspected for an adverse reaction and removed. The patient subsequently improved. WHAT IS NEW AND CONCLUSION: Causality analysis determined a probable relationship between dantrolene and pharyngeal spasm. This may be due to direct muscle contraction by dantrolene, an effect seen previously in vitro.

Malignant hyperthermia in Canada: characteristics of index anesthetics in 129 malignant hyperthermia susceptible probands.

BACKGROUND: Between 1992 and 2011, 373 Canadian individuals with adverse anesthetic reaction were referred to the Malignant Hyperthermia Unit in Toronto, Ontario, Canada for malignant hyperthermia (MH) diagnostic testing. We analyzed the epidemiologic characteristics of the index adverse anesthetics for those probands who were confirmed to be MH susceptible. METHODS: One hundred twenty-nine proband survivors of adverse anesthetic reactions, whose MH susceptible status was confirmed by caffeine-halothane contracture testing were selected. Individuals were excluded if the index anesthetic record was not available for review. Data regarding demographics, clinical signs, laboratory findings, treatment, and complications were retrospectively compiled and analyzed. A Fisher exact test and χ test were applied to compare categorical variables. The Wilcoxon rank-sum test was applied with continuous variables. RESULTS: Young males (61.2%) dominated among selected patients. Seventeen of 129 (13.2%) patients had prior unremarkable anesthesia. Anesthetic triggers were volatile-only (n = 58), succinylcholine-only (n = 20), or both volatile and succinylcholine (n = 51). Eight (6.2%) cases occurred in the postanesthetic care unit. There were no reactions after discharge from the postanesthetic care unit. The most frequent clinical signs were hyperthermia (66.7%), sinus tachycardia (62.0%), and hypercarbia (51.9%). Complications occurred in 20.1% of patients, the most common complication being renal dysfunction. When 20 or more minutes between the first adverse sign and dantrolene treatment elapsed, complication rates increased to ≥30%. CONCLUSIONS: This is the first Canadian study in 3 decades to report nationwide data on MH epidemiology. Features that differ from earlier reports include a 15.5% incidence of reactions triggered by succinylcholine alone and lower complication rates. In agreement with previously published studies, we confirmed in this independent dataset that increased complication rates were associated with an increased time interval between the first adverse clinical sign and dantrolene treatment. This underscores the need for early diagnosis and rapid dantrolene access and administration in anesthetizing locations using either succinylcholine or volatile anesthetic drugs.

Safety of low-dose oral dantrolene sodium on hepatic function.

OBJECTIVE: To investigate the incidence of hepatobiliary dysfunction after administration of low-dose dantrolene sodium. DESIGN: A retrospective survey of medical records. SETTING: One secondary and 2 tertiary hospitals. PARTICIPANTS: Patients (N=243; 144 men, 27 children; mean age ± SD, 47.8 ± 19.7y) who were administered dantrolene at a daily dose of 12.5 to 400mg for more than 4 weeks. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Liver function test (LFT) results, including serum total bilirubin, aspartate transaminase, alanine transaminase, and alkaline phosphatase, were recorded before and at least 1 month after the initial dose of dantrolene. In cases of treatment cessation, the reason was investigated. Significantly elevated LFT levels were defined as ≥ to 2 times the upper limit of the normal range. RESULTS: Treatment duration was 268.0 ± 428.5 days with a daily dose of 65.2 ± 44.7 mg. At the end of the investigation, 95 patients (39.1%) had been lost to follow-up, and 105 (43.2%) had stopped treatment. The reasons for cessation were improved spasticity (42.9%), no effect of the medication (27.6%), weakness (6.7%), and other medical problems (5.7%). Patients with weaknesses did not have elevated LFT values. A 32-year-old man with head injuries and multiple trauma developed hepatic dysfunction 82 days after the initial dose and 43 days after a dose increment to 400mg/d. Other patients did not experience significant LFT abnormalities. CONCLUSIONS: One case of hepatic dysfunction was recorded in 243 cases after at least 4 weeks of low-dose oral dantrolene administration. Low-dose dantrolene can be used safely with meticulous clinical and laboratory monitoring.