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.

Animals

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.

Drug administration

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^R(Eagle Pharmaceuticals, Inc.), consisting of 125mg mannitol, 25mg polysorbate 80, 4mg povidone K12 in 20 ml of ddH₂O 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, 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.

Sample collection and euthanasia

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.

High performance liquid chromatography (HPLC)

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: H₂O, 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.

Behavioral assays for examination of adverse side effects

Buried food test

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.

Rotarod test

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.

Statistical Analysis

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% (P< 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.