DOI: 10.5061/dryad.jdfn2z37k
title: Data from: Intranasal administration of dantrolene increased brain concentration and duration

description: Dantrolene has been demonstrated to be neuroprotective for multiple
 neurodegenerative diseases. However, dantrolene’s limited penetration into
 the CNS hampers its effectiveness as a neuroprotective agent. Here, we
 studied whether the intranasal administration of dantrolene provided
 better penetration into the brain than the commonly used oral approach.
 C57BL/6 mice, aged 2-4 months, received a single dose of either intranasal
 or oral dantrolene (5mg/kg). Inhibition of dantrolene clearance from the
 brain was examined by co-administration with P-gp/BCRP inhibitors,
 nimodipine or elacridar. The concentration of dantrolene in the brain and
 plasma was measured at 10, 20, 30, 50, 70, 120, 150 and 180 minutes after
 administration. Separate cohorts of mice were given intranasal dantrolene
 (5mg/kg) or vehicle, 3 times/ week, for either 3 weeks or 4 months, to
 examine potential adverse side effects on olfaction and motor
 coordination, respectively. We found that Dantrolene concentrations were
 sustained in the brain after intranasal administration for 180 min, while
 concentrations fell to zero at 120 min for oral administration. Chronic
 use of intranasal dantrolene did not impair olfaction or motor function in
 these mice. Blood brain barrier pump inhibitors did not further increase
 dantrolene peak concentrations in the brain. Our results suggested that
 Intranasal administration of dantrolene is an effective route to increase
 its concentration and duration in the brain compared to the oral approach,
 without any obvious side effects on olfaction or motor function.
description: <b>Animals</b> All animal
 procedures were approved by the Institutional Animal Care and Use
 Committee (IACUC) of the University of Pennsylvania. Male and female
 C57BL/6 mice (Charles River Laboratories, Inc. Wilmington, MA), 2-4 months
 old, weighing 25-35g, were used in all experiments. Mice were kept at
 21-22°C with a 12-hour light-dark cycle with food and water ad libitum.
 All efforts were made to minimize pain and distress and the number of
 mice.   <b>Drug</b>
 <b>administration </b> For the
 pharmacokinetic studies, mice were randomly divided into two experimental
 groups; intranasal dantrolene for intranasal administration (n=40) and
 oral dantrolene delivery (n=30). The vehicle was the same formulation as
 RYANODEX<sup>R</sup>(Eagle Pharmaceuticals, Inc.), consisting
 of 125mg mannitol, 25mg polysorbate 80, 4mg povidone K12 in 20 ml of
 ddH<sub>2</sub>O and pH adjusted to 10.3. Dantrolene
 (MilliporeSigma, St Louis, MO) was diluted in the vehicle to a
 concentration of 5mg/ml. For intranasal administration, the mice were held
 firmly in one hand and a total of 1 µl of drug formulation per gram of
 body weight was delivered using a pipette. Several key steps were required
 to assure accuracy of intranasal delivery: 1) the mouse’s head was
 parallel to the floor; 2) the mouse was not able to move the head or neck;
 3) the ejected droplet was as small as possible; 4) 2-3 s was allowed
 between intranasal injections; 5) the mouse was held for 10-15 seconds
 after the delivery was finished. This procedure took about 10 min/ mouse.
 Oral administration was performed as previously described [20]. The mice
 were held as for the intranasal administration and 5µl of drug per gram of
 body weight was delivered using a gavage attached to a microliter
 syringe.   Inhibition of the BBB transport
 protein, P-glycoprotein breast cancer resistance (P-gp/BCRP), function has
 been shown to increase the brain/plasma concentration ratios of dantrolene
 (Fuchs et al, 2014). In an attempt to reduce dantrolene clearance from the
 brain, BBB pump inhibitors (nimodipine, elacridar) were given prior to
 intranasal dantrolene to a separate cohort of animals. Nimodipine and
 elacridar (Sigma, St Louis, MO) were diluted in the same vehicle as
 Ryanodex, described above, 2mg/ml and 10mg/ml respectively. A total of 1
 µl of nimodipine or elacridar per gram of body weight was delivered by
 intranasal administration 30 min before intranasal administration of
 5mg/ml dantrolene (1 µl/g of body weight). Dantrolene alone was used as
 the control. Blood and plasma dantrolene concentrations were examined 20
 min after the intranasal administration of dantrolene.  
 For
 the drug safety studies,<i> </i>the potential adverse effects
 of chronic administration of dantrolene were examined. Separate cohorts of
 mice were randomly divided into groups which received intranasal
 dantrolene (5 mg/kg) or intranasal vehicle, 3 times/week, for either 3
 weeks or 4 months, for behavioral studies as described below. There was no
 mortality in all groups. <b> </b>
 <b>Sample collection and
 euthanasia</b> At the time of euthanasia, animals
 were anesthetized with 2-4% isoflurane and blood samples (0.2 ml) were
 obtained by cardiac puncture after 10, 20, 30, 50, 70, 120, 150 and 180
 minutes of dantrolene administration. The animals were then euthanized by
 intracardiac perfusion and exsanguination with phosphate buffered saline
 to ensure that dantrolene was completely washed out of the cerebrovascular
 system before the brains were harvested. Heparin anticoagulated blood
 samples were centrifuged at 3000 rpm at 4°C for 10 minutes and the
 supernatant collected. Each brain was dissected and homogenized. All
 procedures were performed in the cold room (4°C). Both the plasma and
 brain samples were stored at -80°C and protected from light until assayed.
 Separate cohorts of mice were euthanized as above after 3 weeks or 4
 months of chronic dantrolene administration for the olfaction and motor
 function tests, respectively.   <b>High performance liquid chromatography
 (HPLC)</b> The brain tissues for HPLC analysis
 were extracted as previously described [21]. Briefly, the frozen brain
 tissue was placed into 200 µl of mixture solution (acetonitrile:
 H<sub>2</sub>O, 2:1) and homogenized, the suspensions were
 then centrifuged at 4°C at 20,000 x g for 20 min. For the plasma samples,
 200 µl of acetonitrile was added into the same volume of specimen solution
 and centrifuged. After the homogenization and centrifugation, 50 µl of
 supernatant was injected into HPLC for analysis.  An Agilent
 Hewlett Packard Model 1100 Series, high performance liquid chromatography
 (HPLC) system (Agilent Technologies, Wilmington, DE), equipped with a
 refractive index monitor, was used for quantitation of dantrolene
 concentrations in the blood and brain. Acetonitrile was used as component
 A of the mobile phase, and potassium phosphate buffer solution (pH 7.0) as
 component B. The mobile phase had a flow rate of 1.0 ml/min with a
 proportion 12% to 88% for components A and B of the mobile phase,
 respectively. Detection was performed with the UV detector at 254
 nm.   <b>Behavioral assays for
 examination of adverse side effects</b> <b>Buried food test</b> As this is
 the pilot study of administrating dantrolene via the intranasal approach
 and since chronic use will be needed for the treatment of Alzheimer’s or
 other neurodegenerative diseases, we further investigated whether the
 chronic use of intranasal dantrolene will impair the sense of smell.
 Olfaction was assessed in a separate cohort of mice after 3 weeks of
 intranasal dantrolene (5mg/kg, N=10) or vehicle (equivalent volume, N=10),
 using the buried food test[22]. Mice were randomly divided into two
 experimental groups (n=10/group). Dantrolene or vehicle was administrated
 once a day, three times a week (every other day during weekdays). After 3
 weeks of chronic administration, animals were subjected to the buried food
 test by an investigator who was blinded to the experimental groups. On day
 1, cookies (1 cookie for 2 mice) were placed into the cages and left
 overnight. Cages were observed on the second day to make sure the cookies
 were consumed. On day 2 at about 4pm, food was removed from the cages and
 the testing mice were fasted overnight, water available. On day 3, at
 about 11am, mice were acclimated to the testing room for 1 hour. Mice were
 then individually placed into a clean cage with 3cm deep of bedding with a
 cookie buried 1cm beneath the bedding in one corner. The time it took the
 mouse to retrieve the food and hold it with the front paws was manually
 recorded, for a maximum of 900 seconds.  
 <b>Rotarod test</b> As dantrolene has effects of muscle relaxant, we further
 investigated the effects of chronical use of dantrolene on motor function.
 Motor coordination was examined with a rotarod [23] in a separate cohort
 of mice that were given either intranasal dantrolene (5 mg/kg, N=10) or
 vehicle (equivalent dose, N=10), once a day, 3 times/ week, for 4 months.
 The animals received two 60s training trials on the rotarod (IITC Series
 8, Life Sciences, Woodland Hills, CA) at 9 rpm with a 30 min interval
 between trials. The mice then underwent three test trials for a maximum of
 120s at variable speed, 4-40 rpm, with a 60 min interval between trials.
 The time spent on the rotarod was recorded automatically for each
 mouse.   <b>Statistical
 Analysis</b> All data are reported as the Mean ±
 95% CI and were analyzed by multiple t-tests using the Holm-Sidak method
 or by the nonparametric unpaired Mann-Whitney test (two-tailed), as
 described in each figure legend. The significance level for all of our
 analyses was set at 95% (<i>P</i>&lt; 0.05). Animal
 numbers are listed in the figure legends and were based on the FDA
 guidelines for pharmacokinetic studies recommending at least 3-4 animals
 per time point. (Ref attached) GraphPad Prism software v6.0 (GraphPad
 Software Inc. San Diego, CA) was used for all statistical
 analyses.