Molecular Aspects Implicated in Dantrolene Selectivity with Respect to Ryanodine Receptor Isoforms.

Dantrolene is an intra-cellularly acting skeletal muscle relaxant used for the treatment of the rare genetic disorder, malignant hyperthermia (MH). In most cases, MH susceptibility is caused by dysfunction of the skeletal ryanodine receptor (RyR1) harboring one of nearly 230 single-point MH mutations. The therapeutic effect of dantrolene is the result of a direct inhibitory action on the RyR1 channel, thus suppressing aberrant Ca2+ release from the sarcoplasmic reticulum. Despite the almost identical dantrolene-binding sequence exits in all three mammalian RyR isoforms, dantrolene appears to be an isoform-selective inhibitor. Whereas RyR1 and RyR3 channels are competent to bind dantrolene, the RyR2 channel, predominantly expressed in the heart, is unresponsive. However, a large body of evidence suggests that the RyR2 channel becomes sensitive to dantrolene-mediated inhibition under certain pathological conditions. Although a consistent picture of the dantrolene effect emerges from in vivo studies, in vitro results are often contradictory. Hence, our goal in this perspective is to provide the best possible clues to the molecular mechanism of dantrolene's action on RyR isoforms by identifying and discussing potential sources of conflicting results, mainly coming from cell-free experiments. Moreover, we propose that, specifically in the case of the RyR2 channel, its phosphorylation could be implicated in acquiring the channel responsiveness to dantrolene inhibition, interpreting functional findings in the structural context.

Design, synthesis, and biological activity of novel semicarbazones as potent Ryanodine receptor1 inhibitors of Alzheimer's disease.

Ryanodine receptors (RyRs) are important ligand-gated Ca2+ channels; their excessive activation leads to Ca2+ leakage in the sarcoplasmic reticulum that may cause neurological diseases. In this study, three series of novel potent RyR1 inhibitors based on dantrolene and bearing semicarbazone and imidazolyl moieties were designed and synthesized, and their biological activity was evaluated. Using a single-cell calcium imaging method, the calcium overload inhibitory activities of 26 target compounds were tested in the R614C cell line, using dantrolene as a positive control. The preliminary investigation showed that compound 12a suppressed Ca2+ release as evidenced by store overload-induced Ca2+release (SOICR) (31.5 ± 0.1%, 77.2 ± 0.1%, 93.7 ± 0.2%) at 0.1 µM, 3 µM and 10 µM, respectively. Docking simulation results showed that compound 12a could bind at the active site of the RyR1 protein. The Morris water-maze test showed that compound 12a significantly improved the cognitive behavior of AD-model mice. Further studies on the structural optimization of this series of derivatives are currently underway in our laboratory.

Evaluation of Dantrolene Granules Extemporaneously Reformulated from Capsules in a Pharmacy.

Dantrolene capsule, an effective therapeutic agent for the treatment of spasticity, is administered to children who cannot swallow the capsule after reformulation into a powder. The powdered drug can alter the specified dosage and it is also difficult to dispense the powdered formulation because of its bulky and sticky nature. To resolve these problems, we reformulated dantrolene capsules into granules using a centrifugal planetary mixer in the pharmacy. The granules containing lactose-cornstarch, D-mannitol, or microcrystalline cellulose as a diluent were examined to determine particle size distribution, flowability, drug content uniformity, and disintegration time. The granules with microcrystalline cellulose were superior to the other forms, owing to their smaller size, good drug content uniformity, and rapid disintegration. We further investigated the usability of the granules in the dispensing procedure (dividing and packing) and in the dosing process (retrieval from package) using the powders as controls. The deviation of the divided amount and loss on dosing were reduced relative to the powders. In addition, drug dissolution properties and storage stability for 12 months were the same as those of the powders. Therefore, we concluded that dantrolene granules are excellent alternatives as an extemporaneous preparation in pharmacies.

Selective acetylcholinesterase inhibitors derived from muscle relaxant dantrolene.

Dantrolene, the only therapeutic agent for malignant hyperthermia, is known to have not only a muscle relaxant effect, but also a neuroprotective effect and Alzheimer's disease improving effect. Recently, it has been reported that dantrolene has a weak inhibitory effect on acetylcholinesterase (AChE), which is a therapeutic drug target for Alzheimer's disease. Thus, we focused on developing of AChE inhibitors with benzylpiperidine/piperazine moieties that are based on the dantrolene skeleton. Several derivatives showed an inhibitory activity. Among them, ortho-nitro derivative 8c showed the most potent inhibitory activity with the IC50 value of 34.2 nM. Furthermore, Lineweaver-Burk plot analysis indicated that 8c is AChE-selective inhibitor, which shows only a weak inhibitory effect on butyrylcholinesterase (BuChE) and a non-competitive inhibition.

A Prospective Repurposing of Dantrolene as a Multitarget Agent for Alzheimer's Disease.

The orphan drug dantrolene (DAN) is the only therapeutic treatment for malignant hyperthermia (MH), a pharmacogenetic pathology affecting 0.2 over 10,000 people in the EU. It acts by inhibiting ryanodine receptors, which are responsible for calcium recruitment in striatal muscles and brain. Because of its involvement in calcium homeostasis, DAN has been successfully investigated for its potential as neuroprotecting small molecule in several animal models of Alzheimer's disease (AD). Nevertheless, its effects at a molecular level, namely on putative targets involved in neurodegeneration, are still scarcely known. Herein, we present a prospective study on repurposing of DAN involving, besides the well-known calcium antagonism, inhibition of monoamine oxidase B and acetylcholinesterase, cytoprotection from oxidative insult, and activation of carnitine/acylcarnitine carrier, as concurring biological activities responsible for neuroprotection.

Dantrolene Requires Mg2+ and ATP To Inhibit the Ryanodine Receptor.

Dantrolene is a ryanodine receptor (RyR) inhibitor, which is used to relax muscles in malignant hyperthermia syndrome. Although dantrolene binds to the RyR protein, its mechanism of action is unknown, mainly because of the controversial results showing that dantrolene inhibited Ca2+ release from intact fibers and sarcoplasmic reticulum (SR) vesicles, but failed to inhibit single RyR channel currents in bilayers. Accordingly, it was concluded that an important factor for dantrolene's action was lost during the purification procedure of RyR. Recently, Mg2+ was demonstrated to be the essential factor for dantrolene to inhibit Ca2+ release in skinned muscle fibers. The aim of the present study was to confirm these results in Ca2+ release and bilayer experiments, using SR vesicles and solubilized channels, respectively. Our Ca2+ release experiments demonstrated that the effect of dantrolene and Mg2+ was cooperative and that ATP enhanced the inhibiting effect of dantrolene. Namely, 10 µM dantrolene reduced RyR channel open probability by ∼50% in the presence of 3 mM free Mg2+ and 1 mM ATP, whereas channel activity further decreased to ∼20% of control when [ATP] was increased to 2 mM. Our data provide important complementary information that supports the direct, Mg2+-dependent mechanism of dantrolene's action and suggests that dantrolene also requires ATP to inhibit RyR.

Different stability-indicating chromatographic methods for specific determination of paracetamol, dantrolene sodium, their toxic impurities and degradation products.

A well-known analgesic (paracetamol, PAR) and skeletal muscle relaxant [dantrolene sodium (DNS)] have been analyzed without interference from their toxic impurities and degradation products. The studied PAR impurities are the genotoxic and nephrotoxic p-amino phenol (PAP) and the hepatotoxic and nephrotoxic chloroacetanilide, while 5-(4-nitrophenyl)-2-furaldehyde is reported to be a mutagenic and carcinogenic degradation product of DNS. The five studied components were determined and quantified by TLC-densitometric and RP-HPLC methods. TLC-densitometry (method 1) used TLC silica gel and chloroform-ethyl acetate-acetic acid-triethylamine (7:3:0.5:0.05, by volume) as the mobile phase with UV scanning at 230 nm, while RP-HPLC (method 2) was based on separation on a C18 column using methanol-water (55:45, v/v pH 3 with aqueous formic acid) as mobile phase at 1 mL/min and detection at 230 nm. The developed methods were used for determination and quantification of the five studied components in different laboratory-prepared mixtures. The were also applied for analysis of Dantrelax® compound capsules where no interference among the studied components with each other or from excipients was observed. The methods were validated as per International Conference on Harmonization guidelines, and they compared favorably with the reported ones.

Triboelectrification of active pharmaceutical ingredients: week acids and their salts.

The effect of salt formulation on the electrostatic property of active pharmaceutical ingredients was investigated. The electrostatic property of weak acids (carboxylic acids and amide-enole type acid) and their sodium salts was evaluated by a suction-type Faraday cage meter. Free carboxylic acids showed negative chargeability, whereas their sodium salts showed more positive chargeability than the free acids. However, no such trend was observed for amide-enole type acids.

Interaction of 2-APB, dantrolene, and TDMT with IP3R and RyR modulates ER stress-induced programmed cell death I and II in neuron-like PC12 cells: an experimental and computational investigation.

Ca(2+) is an essential second messenger, playing a fundamental role in maintaining cell viability and neuronal activity. Two specific endoplasmic reticulum calcium channels, ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) play an important role in Ca(2+) regulation. In the present study, we provided a 3D structure of RyR and IP3R by homology modeling, and we predicted their interactions with a known neuroprotective compound, 3-thiomethyl-5,6-(dimethoxyphenyl)-1,2,4-triazine (TDMT), as well as two inhibitors, dantrolene and 2-aminoethoxydiphenyl borate (2-APB). Interestingly, we found that dantrolene and 2-APB can bind to the IP3-binding domain of IP3R and RyR, while TDMT may directly block both channels by interacting with the putative resident domains in the pore. Cell culture experiments showed that these compounds could protect PC12 cells against H2O2-induced apoptosis and activate autophagic pathways. Collectively, our computational (in silico) and cell culture studies suggest that RyR and IP3R are novel and promising targets to be used against neurodegenerative diseases.

Small-molecular inhibitors of Ca²âº-induced mitochondrial permeability transition (MPT) derived from muscle relaxant dantrolene.

A structure consisting of substituted hydantoin linked to a 5-(halophenyl)furan-2-yl group via an amide bond was identified as a promising scaffold for development of low-molecular-weight therapeutic agents to treat vascular dysfunction, including ischemia/reperfusion injury. Among the compounds synthesized, 5-(3,5-dichlorophenyl)-N-{2,4-dioxo-3-[(pyridin-3-yl)methyl]imidazolidin-1-yl}-2-furamide (17) possessed the most potent inhibitory activity against Ca(2+)-induced mitochondrial swelling. The structural development, synthesis and structure-activity relationship of these compounds are described.

Radiosynthesis of [13N]dantrolene, a positron emission tomography probe for breast cancer resistant protein, using no-carrier-added [13N]ammonia.

Dantrolene (1) is a substrate for breast cancer resistant protein, which is widely distributed in the blood-brain-barrier, intestine, gall bladder, and liver. PET study with 1 labeled with a positron emitter can be used to visualize BCRP and to elucidate the effect of BCRP on the pharmacokinetics of drugs. The objective of this study was to label 1 using nitrogen-13 ((13)N, a positron emitter; half-life: 9.9min). Using no-carrier-added [(13)N]NH(3) as the labeling agent, we synthesized [(13)N]dantrolene ([(13)N]1) for the first time. The reaction of carbomyl chloride 2b with [(13)N]NH(3) gave an unsymmetrical urea [(13)N]3, followed by cyclization of [(13)N]3 to afford [(13)N]1. Due to its instability, 2b was prepared in situ by treating amine 5 with triphosgene in a ratio of 4 to 1 and used for subsequent [(13)N]ammonolysis without purification.

Comparison of the therapeutic effectiveness of a dantrolene sodium solution and a novel nanocrystalline suspension of dantrolene sodium in malignant hyperthermia normal and susceptible pigs.

BACKGROUND AND OBJECTIVE: Stopping trigger agents and prompt administration of dantrolene are the cornerstones of treatment of malignant hyperthermia. However, significant time is lost in treatment of the condition because of the cumbersome preparation and administration of the commercially available dantrolene sodium for injection. A potential improvement has become available in the form of a novel nanocrystalline dantrolene sodium suspension (DSS), which is 150 times more concentrated (50 mg ml(-1)) than the standard dantrolene sodium solution (0.33 mg ml(-1)). The aims of this study were to measure the effects of DSS on clinical and laboratory variables in malignant hyperthermia normal pigs and to compare the therapeutic management and clinical effectiveness of DSS with standard dantrolene sodium in a fulminant malignant hyperthermia crisis in susceptible pigs. The pig model is a well accepted method of studying the malignant hyperthermia crisis and is an ideal way to evaluate the variables of interest in this study. METHODS: Seven malignant hyperthermia normal and 10 malignant hyperthermia susceptible pigs were studied. Malignant hyperthermia susceptible pigs (body weight approximately 24 kg) were allocated to a dantrolene sodium group or a DSS group. After induction of anaesthesia, a 22-gauge catheter was placed in an ear vein and trigger-free anaesthesia was performed. After achieving stable conditions, administration of halothane was started with 0.1% and then 0.15%. Halothane was discontinued after the administration of 0.2% (malignant hyperthermia normal pigs) or when a fulminant malignant hyperthermia crisis was achieved (malignant hyperthermia susceptible pigs). After halothane was discontinued, FIO2 was set to 1.0, respiratory minute volume was doubled and sodium bicarbonate 2 mmol kg(-1) was administered. The time required to prepare and administer each formulation was measured. To simulate the administration of the substances under typical clinical conditions for a child weighing approximately 24 kg, dantrolene sodium (5 mg kg(-1)) or DSS (5 mg kg(-1)) was prepared and injected via the intravenous 22-gauge cannula. Bolus administrations of dantrolene sodium or DSS were repeated after 24 min. RESULTS: Arterial pH, arterial pCO2, mean arterial pressure and arterial lactate concentration remained stable during the experiment with DSS in malignant hyperthermia normal pigs. A significant decrease in cardiac index and increases in systemic vascular resistance and serum potassium concentration occurred after administration of DSS. In all malignant hyperthermia susceptible animals, the inhaled administration of halothane 0.15% led to a fulminant malignant hyperthermia crisis. The therapeutic regimens with administration of dantrolene sodium or DSS were successful in treating the malignant hyperthermia crisis in all animals. The course of the malignant hyperthermia crisis and the therapeutic effects of dantrolene sodium or DSS were comparable in the two groups. The time needed to prepare DSS for administration was significantly shorter (51 ± 9 s) compared to dantrolene sodium (860 ± 202 s). The time taken to inject DSS (4 ± 2 s) was significantly shorter than for dantrolene sodium (472 ± 51 s). CONCLUSION: The therapeutic action of DSS in a malignant hyperthermia crisis in pigs was effective and comparable to that of standard dantrolene sodium. However, preparation and administration of DSS were significantly faster, which may offer a clinically significant advantage in the treatment of a fulminant malignant hyperthermia crisis and may result in a reduction in stress for the anaesthesia team.

Speeding dantrolene preparation for treating malignant hyperthermia.

Dantrolene is known to have a low solubility in water Studies have demonstrated that it dissolves more rapidly in warm water However, the overall benefit of warming has not been measured. This study evaluated the overall time benefit of warming. The first step was to measure how long it took to warm the 10 ml plastic ampoules of sterile water from ambient temperature (20 degrees C) to 45 degrees C. We found that when immersed in a water bath held at 45 degrees C, this required a minimum of seven minutes. Therefore the ampoules used for the study were kept immersed in a water bath held at 45 degrees C for at least 10 minutes. It took an average of 82.6 seconds to draw 60 ml (six ampoules) of water into a 60 ml syringe. Squirting the water into the dantrolene vial and miring until dissolved took 102.8 seconds (SD 8.3) for the warmed water and 129.0 seconds (SD 14.6) for the ambient temperature water (P = 0.009). Redrawing the dantrolene solution into the syringe took an average of 32 seconds for the warmed water and 35.2 seconds for the ambient temperature water. After the redrawing phase the average temperature of the warmed solution was 32.2 degrees C. The average total preparation time for the warmed solution was 217.4 seconds (SD 20.0) and for the ambient temperature preparation was 243.0 seconds (SD 11.7) (P = 0.038). Thus the time difference in preparation was about 256 seconds, which is the time that could be saved in preparing the standard dose of 10 vials of dantrolene using water at 45 degrees C. However, as it required about seven minutes to initially warm the sterile ampoules, there would be a net increase, not decrease in the time required. These findings indicate that warming is of no benefit in speeding dantrolene preparation.

Radiosynthesis of [2-(11)C-carbonyl]dantrolene using [(11)C]phosgene for PET.

Automated radiosynthesis of [2-(11)C-carbonyl]dantrolene, the substrate of breast cancer resistance protein (BCRP/ABCG2), was performed for the first time through a multi-step/one-pot labeling sequence that started with ethyl 2-{2-[5-(4-nitrophenyl)furfurylidene]hydrazino}acetate and used [(11)C]phosgene as a labeling agent. After optimization of the automated synthesis conditions and parameters, [2-(11)C-carbonyl]dantrolene was obtained at a radiochemical yield of 34.0+/-8.4% (decay-corrected). The radiochemical purity was greater than 98% and the specific activity was 46.8+/-15.2GBq/micromol at the end of the synthesis.

Effects of small molecule modulators on ATP binding to skeletal ryanodine receptor.

Calcium release for muscle contraction in skeletal muscle is mediated in part by the ryanodine receptor 1, RyR1, Ca2+-channel and is strongly affected by intrinsic modulators like Ca2+, Mg2+ and ATP. We showed differential effects on ATP binding in the presence of Ca2+ or Mg2+ ions using ESR spectroscopy and a spin-labeled ATP analog, SL-ATP (Dias et al. Biochemistry 45: 9408-9415, 2006). We here report the effects of RyR1 modulators like ryanodine, caffeine and dantrolene on the ATP binding of RyR1 using the same technique. We present evidence that the exogenous effectors induce changes within RyR1 that lead to different ATP binding characteristics: In the presence of the activating modulator, caffeine, or in the presence of ryanodine, which causes a half-open state of the channel, binding of eight ATP per RyR1 was observed, even in the presence of inhibitory Ca2+, suggestive of a stable "open" channel conformation. In the presence of the inhibitory modulator dantrolene, ATP binding affinity decreased in the presence of activating Ca2+, while in the presence of inhibitory Ca2+, ATP binding affinity increased, but at the same time the number of accessible sites decreased to four, suggestive of a closed conformation of the channel. The results imply that modulation of ATP binding to RyR1 as well as the overall number of accessible ATP binding sites on the channel are crucial for regulation and are in direct correlation with the modified activity of the channel induced by pharmacological agents.

Electroreduction of the muscle relaxant drug dantrolene sodium at the mercury electrode and its determination in bulk form and pharmaceutical formulation.

The electroreduction of the muscle relaxant drug dantrolene sodium at the mercury electrode has been studied in the Britton-Robinson universal buffer of pH 2.5-11.5 containing 20% (v/v) methanol by means of dc-polarography, cyclic voltammetry and controlled-potential coulometry. Its reduction took place via three irreversible cathodic steps in solutions of pH < or =6, two steps in solutions of 6 or =10 through the consumption of 10, 8 or 4 electrons, respectively. This behavior was attributed to the reduction of NO(2) group (1st and 2nd steps at pH < or =6 or the single step at pH > or =10) and the -CH=N- double bond (3rd step at pH <10). Two polarographic procedures (direct current and differential-pulse modes) and three adsorptive cathodic stripping voltammetric procedures (linear-sweep, differential-pulse and square-wave modes) were described and successfully applied for quantification of dantrolene sodium in its bulk form and in pharmaceutical formulation (Dantrolex tablets).

The Icarus effect: the influence of diluent warming on dantrolene sodium mixing time.

Prompt administration of intravenous (i.v.) dantrolene sodium (DS) is the primary determinant of successful treatment of malignant hyperthermia (MH) syndrome. Because DS has a long reconstitution time for use in treating an MH crisis, we evaluated an alternative technique for hastening the reconstitution. Simulating real-world conditions, with equipment common to the operating room environment, we conducted a randomized, controlled, single-blind study dividing 16 DS vials into 2 equal groups: warm (41 degrees C) and ambient temperature (22 degrees C). With an i.. fluid warmer at 41 degrees C, primed with a 1-L bag of preservative-free sterile water, attached to a 60-mL syringe via a 3-way stopcock, we aspirated and injected the diluent directly into each DS vial. The Icarus effect was clearly demonstrated: warmed diluent vs ambient temperature hastened the reconstitution time for DS. The mean time to particulate-free DS solution suitable for i.v. injection with the warm diluent was 58.88 seconds compared with 93.87 seconds for the ambient temperature group (P <.001). A practical method using a reliable and safe warming device readily available to anesthetists and ubiquitous to the operating room environment speeds the time to administration of DS ultimately reducing morbidity and mortality associated with MH.

Specific inhibition of the plasmodial surface anion channel by dantrolene.

The plasmodial surface anion channel (PSAC), induced on human erythrocytes by the malaria parasite Plasmodium falciparum, is an important target for antimalarial drug development because it may contribute to parasite nutrient acquisition. However, known antagonists of this channel are quite nonspecific, inhibiting many other channels and carriers. This lack of specificity not only complicates drug development but also raises doubts about the exact role of PSAC in the well-known parasite-induced permeability changes. We recently identified a family of new PSAC antagonists structurally related to dantrolene, an antagonist of muscle Ca++ release channels. Here, we explored the mechanism of dantrolene's actions on parasite-induced permeability changes. We found that dantrolene inhibits the increased permeabilities of sorbitol, two amino acids, an organic cation, and hypoxanthine, suggesting a common pathway shared by these diverse solutes. It also produced parallel reductions in PSAC single-channel and whole-cell Cl- currents. In contrast to its effect on parasite-induced permeabilities, dantrolene had no measurable effect on five other classes of anion channels, allaying concerns of poor specificity inherent to other known antagonists. Our studies indicate that dantrolene binds PSAC at an extracellular site distinct from the pore, where it inhibits the conformational changes required for channel gating. Its affinity for this site depends on ionic strength, implicating electrostatic interactions in dantrolene binding. In addition to the potential therapeutic applications of its derivatives, dantrolene's specificity and its defined mechanism of action on PSAC make it a useful tool for transport studies of infected erythrocytes.