Improved Bioavailability of Montelukast through a Novel Oral Mucoadhesive Film in Humans and Mice

The leukotriene receptor antagonist Montelukast (MTK) is an approved medication for the treatment of asthma and allergic rhinitis. The existing marketed tablet forms of MTK exhibit inconsistent uptake and bioavailability, which partially explains the presence of a significant proportion of MTK low- and non-responders in the population. Besides that, tablets are suboptimal formulations for patients suffering from dysphagia, for example, seen in patients with neurodegenerative diseases such as Alzheimer’s disease, a disease with increasing interest in repurposing of MTK. This, and the need for an improved bioavailability, triggered us to reformulate MTK. Our aim was to develop a mucoadhesive MTK film with good safety and improved pharmacological features, i.e., an improved bioavailability profile in humans as well as in a mouse model of Alzheimer’s disease. We tested dissolution of the MTK mucoadhesive film and assessed pharmacoexposure and kinetics after acute and chronic oral application in mice. Furthermore, we performed a Phase I analysis in humans, which included a comparison with the marketed tablet form as well as a quantitative analysis of the MTK levels in the cerebrospinal fluid. The novel MTK film demonstrated significantly improved bioavailability compared to the marketed tablet in the clinical Phase 1a study. Furthermore, there were measurable amounts of MTK present in the cerebrospinal fluid (CSF). In mice, MTK was detected in serum and CSF after acute and chronic exposure in a dose-dependent manner. The mucoadhesive film of MTK represents a promising alternative for the tablet delivery. The oral film might lower the non-responder rate in patients with asthma and might be an interesting product for repurposing of MTK in other diseases. As we demonstrate Blood-Brain-Barrier (BBB) penetrance in a preclinical model, as well as in a clinical study, the oral film of MTK might find its use as a therapeutic for acute and chronic neurodegenerative diseases such as dementias and stroke.


Introduction
Montelukast (MTK) is a leukotriene receptor antagonist commonly used to treat patients suffering from chronic asthma as well as seasonal allergies and allergic rhinitis. MTK binds to the cysteinyl-leukotriene receptor 1 and to the leukotriene receptor GPR17 with high affinity and selectivity, thereby blocking the leukotriene signaling pathway 1,2 . It reduces leukotriene-mediated respiratory inflammation reaction such as vasoconstriction and relieves asthma symptoms 3 .
Currently, MTK is marketed under the brand name Singulair® and in several generic products in oral tablet and other pharmaceutical forms such as oral granules. The available forms present a number of limitations such as inconsistent solubility, uptake, and bioavailability, for several reasons. Although MTK is freely soluble in water, its solubility increases significantly above pH 7.5 and drastically reduces under acidic conditions normally found in the gastrointestinal tract, in particular in the stomach 4 . This has led to relatively slow and inconsistent absorption into the blood stream, with maximum concentrations occurring between 2-4 hours following consumption, thereby limiting its use to chronic applications rather than for rapid acute treatment. Uptake and bioavailability of MTK is further determined by pharmacogenetics (for review see 5 ). For example, more than 20% of the population is not responding to MTK with a clinical benefit 6 . Among the various genetic reasons are variations in the SLCO2B1 gene coding for the organic anion transporting OATP2B1, which has been associated with altered absorption of MTK 7 . Uptake of MTK was further modified by the intake of citrus juice 8 . Besides the physicochemical and genetic basis for the insufficient uptake and bioavailability of MTK in its current tablet form, a further drawback is the inadequateness of the MTK tablets for patients suffering from dysphagia such as elderly patient, for patients with dementias, and for patients that require intubation or ventilation. In summary, the tablet form of MTK represent a number of limitations, and in consequence, there is an increasing interest in developing improved strategies for the delivery of MTK aiming for increased bioavailability 9 .
Oral mucoadhesive application has several advantages, such as the relatively easy and convenient accessibility of the oral cavity, the limiting of first-pass effects and degradation during the gastro-intestinal transit and the rapid drug absorption due to high blood circulation in the mucosa (as reviewed in 10,11 ). In general, buccal films are seen as a promising alternative application route for drugs as they combine several features, like better patient compliance (no swallowing and the possibility to modify the taste) and increased bioavailability (reviewed in 12 ).
We developed a novel MTK oral mucoadhesive film to circumvent the limitations of MTK in its tablet form, tested its stability and dissolution, and performed a Phase I bioavailability and safety study in healthy volunteers, where we assessed MTK pharmacokinetics in serum and cerebrospinal fluid. Furthermore, we wanted to investigate the possibility of repurposing MTK in form of the novel oral film for Alzheimer's Disease (AD). To test its suitability for a pre-clinical efficacy study of MTK in AD we assessed pharmacokinetics and tested the effects of acute and chronic application on pharmacoexposure in an animal model of AD, the 5xFAD mouse model.

Film Preparation
The mucoadhesive films were produced by a solvent-casting technique. For that, excipients and active pharmacological ingredient (API) in a range from 0 -30.00 % (w/w) were mixed and dissolved/suspended in water. The resulting wet blend was spread to a thickness less than 1 mm onto a release liner. The wet film was dried in an oven for 60-75 minutes at 65°C. The dried film sheet was cut into film strips containing 10 mg of MTK. The composition of the film is depicted in table 1.

In vitro dissolution test
Dissolution studies were carried out in a paddle type apparatus at 50 rpm and 37°C. Dissolution of MTK tablet, MTK mucoadhesive film and pre-solubilized MTK oral film was assessed in 900 mL of phosphate based saliva buffer (pH 6.8), For the dissolution test of pre-solubilized MTK film, a single film unit was mixed with 2 mL of saliva buffer pH 6.8 for 5 min and directly injected into the dissolution chamber. Aliquots of the release medium (8 mL) were collected at the following time points: 2.5, 5, 7.5, 10, 15, 20, 30, 45 minutes and analyzed by HPLC UV at 255 nm as specify in the USP.

Mechanical properties
To determine potential impact of cold temperature during transport films containing 10 mg MTK were placed at -20°C for 15 and 30 days, respectively and were compared to a control product unexposed to cold temperature. The mechanical properties were evaluated by testing 3 films for folding endurance, and 8 films for elongation and tensile strength. A Mecmesin Multitest 1-d force testing system (SN 09-1004-11), including a Mecmesin Advanced Force Gauge 50N (SN 09-0071-11), was used to perform the tests. The flexibility of a film was assessed by repeatedly folding the film at the same place until cracking or breakage. The number of times the film was folded without breaking was recorded as the folding endurance value. A film strip was fixed at half-length on the bottom grip of the instrument, and then folded 10 times in both directions.
Elongation at break was measured by stretching the film to its maximum deformation until it torn apart. The test was carried out by affixing a film by its ends to the grips of a Mecmesin Multitest 1-d instrument and stretched at a constant speed of 10 mm/min until it cracked or broke. The length of the film section was measured before and after stretching. Elongation is defined as the ratio between the increase in film length as result of stretching, and the initial length of the film, expressed as a percentage of the initial length. Tensile strength was determined simultaneously with the elongation test and is defined as the maximum force applied to the film area being stretched. All tests were performed in a room with controlled temperature and humidity.

Clinical Study -Subjects and Study Design
To determine the bioequivalence of MTK 10 mg mucoadhesive film (test drug -treatment A) and Singulair® 10

Animals
Acute exposure experiment: Two-month old C57BL6 female mice (n=10) were treated with a single dose of MTK mucoadhesive film. One group (n= 5) received a dose of 3,3 mg/kg/d of MTK and another group (n=5) received 10 mg/kg/d of MTK. The respective films were punched out of an initial film into circular films using punch pliers with 3 mm diameter, which results in an area of 7 mm 2 per film. Based on the area of the film punch we had calculated the different concentrations of MTK in the initial film to reach 3.3 mg/kg/d and 10 mg/kg/d respectively in a 25 g mouse with one 7mm 2 film. For oral treatment mice were hold in the mouse grip and the film was placed on the oral mucosa using riffled forceps with a 0.5 diameter at the tip.
To assess serum levels of MTK blood samples were collected one, three and seven hours after the beginning of treatment ( Fig 4A) For statistical analysis of the animal data, Prism 8 software (GraphPad) was used. The data were tested for normal distribution with the Kolmogorov-Smirnov test. For comparison of two groups an unpaired t test was performed, whereas for comparison of more than two groups, one-way analysis of variance (ANOVA) was used with Tukey's or Bonferroni's multiple comparison test as a post-hoc test. The data were depicted as mean and standard error of the mean (SEM) or standard deviation (SD) with a 95% confidence interval as indicated in the respective figure legends. P values of p < 0.0001 and p < 0.001 were considered extremely significant (**** or ***), p < 0.01 very significant (**) and p < 0.05 significant (*).

Composition, preparation, physical and mechanical properties of the MTK mucoadhesive film
Besides the active pharmacological ingredient, the general composition of the film blend includes 60.0 -85.0 %w/w wet of water as solvent and 5.0 -15.0 %w/w wet mucoadhesive polymer to form the film. Furthermore, it includes gum to modify viscosity, stabilizers to prevent degradation, plasticizers to tune mechanical properties, permeation enhancers to increase absorption and colorant and flavor for patient compliance (table 1).
The film was prepared using a solvent-casting technique, where excipients and active pharmacological ingredient (API) were mixed, dissolved in water and spread out to a thin layer that was dried and then cut into strips with a concentration of 10 mg of MTK per strip.
To assess the impact of exposure to freezing conditions on the mechanical properties of 10 mg MTK film, films were tested for folding endurance, elongation and tensile strength at T 0 as well as after 15 and 30 days at -20°C (table 2). Results of folding endurance testing showed that films from all three conditions can be folded at least 10 times without tearing and therefore meet the acceptance criteria of the folding test. A one-way ANOVA analysis was performed to compare the elongation mean values of the three conditions. At a α =0.05 significance level the elongation data from films exposed to freezing conditions are not significantly different to the initial product (T 0 ). A similar statistical analysis was applied to the tensile strength data and also revealed no significant differences between the three stability conditions.
These results show that the 10 mg MTK film has stable mechanical properties under freezing conditions for at least 30 days. Therefore, the integrity and quality of the film product are maintained in case to exposure to low temperatures during air transport of the films from the manufacturing site to the location where the clinical study would take place.

Dissolution
Release of MTK from films was compared to Singulair® tablets using a USP grade dissolution apparatus (Fig 1, table 3). In general, the dissolution experiments were conducted using a 10 mg dosage unit of either film or tablet. In these experiments, MTK release from coated tablets is compared to that from the mucoadhesive film and pre-solubilized film. The use of a "presolubilized" form of film simulated the condition in which the film, after applied in the oral mucosa, starts to slowly dissolve in saliva and is swallowed.
With MTK-film 80% of the API was released after approximately 6 minutes, while with the tablets an equivalent release was only achieved after 10 minutes. This highlights the rapid oral disintegration advantage of the film based platform. However, the most significant improvement using the film technology is observed when comparing the tablet to the pre-solubilized MTK-film.
This experiment is particularly informative as it is a direct comparison of how API is released from swallowed MTK-tablets versus swallowed dissolved oral films under the same environmental conditions. This is important, as we cannot exclude that some patients, instead of placing the film on the mouth mucosa, might swallow the film. As a result, the pre-solubilized MTK-film reaches 80% released API in only approximately 1 minute, whereas the tablet reaches 80% released API after 10 minutes. This clearly demonstrates how the orally pre-solubilized MTK-film platform releases API more quickly than the MTK-tablet dosage in the stomach.

Improved MTK bioavailability in human plasma and CSF in MTK mucoadhesive film
We have performed a phase 1 clinical study in healthy human subjects to determine the pharmacokinetics of MTK administered in form of an oral film product compared to the marketed reference product Singulair® which both contained 10 mg MTK free base (Fig 2). Enrolled subjects had a mean age of 44 years and a mean BMI of 26 kg/m 2 (table 4). Pharmacokinetic parameters of MTK in plasma were collected for each subject individually and are summarized in The area under the concentration-time curve (AUCt), measured from timepoint zero until the last sampling timepoint at 24 h, was significantly higher after the film (AUCt = 3673 ng*h/ml) than after the tablet (AUCt = 2409 ng*h/ml). The calculated film to tablet geometric mean ratio was 152.46% demonstrating a significantly higher extent of absorption after the film compared to the tablet. Similar results were obtained for the AUCinf, measured from timepoint zero to infinity, calculated as AUCt + C last /λ, where C last is the last measurable concentration of MTK. The ratio of film to tablet was 153.15 %, again demonstrating significant higher extent of absorption of the film. Mean pharmacokinetic plasma profiles of MTK for both products over time are shown in figure 3. The geometric mean of Cmax was 338 ng/ml after treatment with Singulair® and 554 ng/ml after treatment with the film (Fig 3). Tmax was reached at 3.63 hrs after Singulair® and at 2.63 hrs after the film (Fig 3). These results indicate that the MTK film has approximately 1.5 times the Cmax and AUC (area under the curve) values compared to the Singulair® reference.
This indicates that using equivalent API loading, the MTK mucoadhesive film exhibits significantly improved bioavailability. Furthermore, treatment with the film had a one hour earlier Tmax than the tablet.
Leukotriene blockers (i.e., leukotriene receptor antagonists and leukotriene synthesis inhibitors) can function to improve cognitive impairment by reducing the neuroinflammatory response within the brain (reviewed in [17][18][19] ). Leukotriene blockers, such as MTK, must therefore cross the bloodbrain barrier and accumulate in the CSF. To investigate the ability of MTK to cross of the bloodbrain barrier in humans, CSF levels of MTK were measured 3 and 7 hours after dosing with the MTK mucoadhesive film. Measurable amounts, ranging from 3.2 -4.7 ng/ml of MTK in the CSF were detected at both time points (table 6). A comparison of the mean concentrations of MTK in the CSF three hours (3.6 ng/ml) and seven hours (4.2 ng/ml) after application reveals a slower and different pharmacokinetic profile in the CSF than in the serum, where Cmax was reached after approximately 2.7 hours after application. Although, compared to serum levels the concentration of MTK in the CSF is lower, but could be more stable over time. As the mean concentration is slightly higher after seven hours than after three hours we might not have reached Cmax after seven hours. However, further studies with longer periods of observation are needed to monitor the temporal profile of MTK pharmacokinetics in the CSF in more detail.

Clinical safety
Six mild adverse effects (AEs), including somnolence, headache, pruritus, mechanical urticaria and back pain, were experienced by the subjects after taking MTK mucoadhesive film (table 7), four of which (pruritus, mechanical urticaria, headache and backpain) were classified as unrelated or unlikely related to drug (table 8). The mild AEs that were classified as possibly related to the drug were somnolence reported by two subjects. No AEs were reported by the subjects after taking the reference product (table 7). No serious adverse events were reported during the conduct of this study (table 8).

Pharmacoexposure and -kinetics of MTK in WT and in 5xFAD mice
As outlined in the introduction, there is a current interest in repurposing of MTK for the treatment of patients with neurodegenerative diseases such as dementia patients 17,20,21 , and of COVID-19 patients 22,23 . Both show dysphagia, and therefore, a mucoadhesive film might have certain advantages. Especially for repurposing of MTK in neurodegenerative diseases, preclinical evidence of efficacy in rodent models is typically required. Therefore, we tested the MTK buccal mucoadhesive film in the context of the present study for its pharmacoexposure and -kinetic profile in mice, healthy WT mice and 5-FAD mice, a mouse model of genetic AD. We first briefly assessed the serum pharmacokinetics of the buccal MTK film in 2 months old female C57bl6 wildtype (WT) mice. In a short-term pharmacokinetic experiment, WT animals received one dose of film ( Fig 4A) with either 3.3 mg/kg/d (referred to as low dose) or 10 mg/kg/d (referred to as high dose) of MTK ( Fig 4B). Regardless of the dose, the highest MTK concentration in serum was detected 1 hour post-dosing, whereas measurements 3 and 7 hours after the film application showed decreased MTK serum concentrations with time ( Fig 5A). The animals receiving the high dose treatment showed, in general, higher MTK serum concentrations ( Fig   5A). Next, we analyzed MTK serum concentration in 5 months old 5xFAD animals after chronic exposure. For that, animals were treated daily for 13 weeks with vehicle, low or high dose of MTK (Fig 4C), and serum MTK levels were assessed 7 hours after the last dose ( Fig 5B). MTK serum levels were dose-dependent, with animals in the higher dose treatment group showing significantly higher serum concentrations compared to the animals in the lower dose treatment group (low dose: 103.7 ± 15.51 ng/ml, high dose 360.9± 67.5 ng/ml) ( Fig 5B). As expected, in the vehicle treated group no MTK was detected in the serum ( Fig 5B). Interestingly, MTK serum levels after chronic exposure were significantly higher in comparison with the serum concentrations determined 7-hours after a single dose administration (acute pharmacoexposure) ( Fig 5C).
As a proof of concept we also analyzed MTK concentration in the CSF after chronic exposure and detected higher levels in the high-dose group than in the low dose group and no MTK in the vehicle group (Fig 5D), demonstrating again the ability of MTK to cross the BBB.

Safety and general health in mice
General health status of the animals was monitored during the chronic exposure study by weekly bodyweight assessment. Mean bodyweight was stable through the experiment (Fig 6A), with individual bodyweight fluctuating less than approximately 10 % of the initial bodyweight from week to week (Fig 6B). Mean bodyweight did not significantly differ between treatment groups ( Fig 6C). Furthermore, the fecal motility of the animals was monitored for 20 minutes. There was no significant difference in the number of fecal boli between the groups (Fig 6D) showing no effect of the new formulation of MTK on fecal motility in 5xFAD mice. In summary, we conclude that prolonged treatment with low and high dose of MTK did not negatively affect general health of mice and no adverse events have occurred in mice.

Discussion
Montelukast is an approved anti-allergic and anti-asthmatic drug which is commercialized only

Availability of data and material
The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest
Justin Conway, Erick Gonzales-Labrada, Rodolphe Obeid, Horst Zerbe, Nadine Paiement are from IntelgenX, who has filed patent applications on the MTK film and its use. Ludwig Aigner is consultant at IntelgenX.

Funding
The research leading to these results was supported by the Austrian Science Funds (FWF) Project P 31362-B34, and through a PhD fellowship for Johanna Michael provided by IntelgenX.  c  h  a  e  l  ,  J  .  ,  M  a  r  s  c  h  a  l  l  i  n  g  e  r  ,  J  .  &  A  i  g  n  e  r  ,  L  .  T  h  e  l  e  u  k  o  t  r  i  e  n  e  s  i  g  n  a  l  i  n  g  p  a  t  h  w  a  y  :  a  d  r  u  g  g  a  b  l  e  t  a  r  g  e  t  i  n  A  l  z  h  e  i  m  e  r  '  s  d  i  s  e  a  s  e  .   D  r  u  g  D  i  s  c  o  v  T  o  d  a  y   2  4  ,  5  0  5  -5  1  6  ,  d  o  i  :  1  0  .  1  0  1  6  /  j  .  d  r  u  d  i  s  .  2  0  1  8  .  0  9  .  0  0  8  (  2  0  1  9  )  .  1  8  G  e  l  o  s  a  ,  P  .  ,  C  o  l  a  z  z  o  ,  F  .  ,  T  r  e  m  o  l  i  ,  E  .  ,  S  i  r  o  n  i  ,  L  .  &  C  a  s  t  i  g  l  i  o  n  i  ,  L  .  C  y  s  t  e  i  n  y  l  L  e  u  k  o  t  r  i  e  n  e  s  a  s  P  o  t  e  n  t  i  a  l  P  h  a  r  m  a  c  o  l  o  g  i  c  a  l  T  a  r  g  e  t  s  f  o  r  C  e  r  e  b  r  a  l  D  i  s  e  a  s  e  s  .   M  e  d  i  a  t  o  r  s  o  f  i  n  f  l  a  m  m  a  t  i  o  n   2  0  1  7  ,  3  4  5  4  2 I  m  p  r  o  v  e  m  e  n  t  o  f  f  i  b  e  r  c  o  n  n  e  c  t  i  v  i  t  y  a  n  d  f  u  n  c  t  i  o  n  a  l  r  e  c  o  v  e  r  y  a  f  t  e  r  s  t  r  o  k  e  b  y  m  o  n  t  e  l  u  k  a  s  t  ,  a  n  a  v  a  i  l  a  b  l  e  a  n  d  s  a  f  e  a  n  t  i  -a  s  t  h  m  a  t  i  c  d  r  u  g  .   P  h  a  r  m  a  c  o  l  o  g  i  c  a  l  r  e  s  e  a  r  c  h   1  4  2  ,  2  2  3  -2  3 M  o  n  t  e  l  u  k  a  s  t  p  o  t  e  n  t  i  a  t  e  s  t  h  e  a  n  t  i  c  o  n  v  u  l  s  a  n  t  e  f  f  e  c  t  o  f  p  h  e  n  o  b  a  r  b  i  t  a  l  i  n  m  i  c      On the last day of treatment mice were perfused and blood and CSF samples were collected approximately seven hours after the last application of MTK.  Bodyweight of the mice was assessed on a weekly basis for 13 weeks as a parameter of health.

References
(A) Mean bodyweight did not significantly differ between the weeks. (B) Individual bodyweigths did fluctuate less than +/-10 % each week. (C) Bodyweights did not significantly differ between groups. (D) Fecal motility did not differ significantly between all groups. Data are shown as mean +/-SEM. One-way ANOVA with bonferroni post hoc test or students t-test were performed. Pvalue <0.05 were considered significant.  and tensile strentgh of the 10 mg MTK film exposed to normal (T 0 ) and freezing conditions (T 15 days and 30 days). Results are shown as the average of three -eigth films (mean with SD).