INTRODUCTION
Depression is a common psychiatric disorder, and statistics clearly identify it as a major public health problem1. The major disadvantage with conventional dosage forms are many patients find it difficult to swallow (dysphagia) tablets and capsules2. Nose to brain delivery has number of advantages, such as drugs can be rapidly absorbed through the nasal mucosa, giving rapid onset of action. The olfactory region of nasal mucosa that provides a direct connection between nose and brain can be exploited for targeting central nervous system (CNS)-acting enormous range of drug molecules of neurotherapeutics, both macromolecules and low molecular weight drugs, used in conditions such as Alzheimer’s disease, depression, migraine, schizophrenia5. Both hydrophobic drugs, e.g. propranolol7 and hydrophilic drugs, e.g. vanlafaxine hydrochloride8 are absorbed by the nasal mucosa. The gellan gum is an ion sensitive polymer which forms clear gel in contact with monovalent and divalent cations. In an ion free aqueous medium, it forms double helices which at room temperature are only weakly attached to each other (van der Waals attraction). In the presence of cations some of the helices associate into aggregates and cause cross-linking of the polymer chains9. Fluoxetine hydrochloride (FXH) is a selective serotonin reuptake inhibitor as an antidepressant, is widely used to treat various types of psychiatric disorders10,11. The dose of FXH ranges from 20 to 80 mg administered as 1 to 4 capsules a day. This dose and frequency may cause enhanced drug-related side effects and may pose compliance problems12. The long half-life of fluoxetine and its active metabolite essentially preclude a withdrawal phenomenon. It is an inhibitor of cytochrome P450 (CYP)2D6 and other CYP enzymes, which increases the potential for drug interactions and also undergoes extensive hepatic first-pass metabolism13-15. The objective of the present work was to prepare in situ gel of FXH for enhanced drug absorption in brain through nasal route and patient compliance for the treatment of depression.
MATERIALS AND METHODS
Materials
(FXH) was obtained from Swapnaroop drugs and chemicals, Aurangabad. Gellan gum (Gelrite) was procured as gift samples from Signet Chemical Corporation Ltd. Hydroxypropyl methylcellulose (HPMC) were obtained as a gift sample from Colorcon, Mumbai, India. Mannitol and propyl paraben were obtained from Molychem India, Mumbai.
Methods
In the present study 32 full factorial designs (Design Expert 7.0.0.0) was selected to obtain nine formulation and to study the effect of independent variables gellan gum (X1) and HPMC (X2) on dependent variables such as viscosity, mucoadhesive strength and drug release.
Preparation of in situ gel
Gellan gum and HPMC was weighed and dispersed separately in distilled water and stirred by mechanical stirrer (Remi motors Ltd, Mumbai, India, type RQ-122) for 30 min at 90°C in a water bath and then cooled to room temperature. FXH (1% w/v) and HPMC solution was added in gellan gum solution slowly with continues stirring. Appropriate quantities of mannitol as a osmotic agent and propyl paraben were added simultaneously 16. The compositions of prepared formulations of FXH are shown in (Table 1).
Ingredients | F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 |
---|---|---|---|---|---|---|---|---|---|
Fluoxetine HCl (%) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Gellan gum (%) | 0.2 | 0.2 | 0.2 | 0.4 | 0.4 | 0.4 | 0.6 | 0.6 | 0.6 |
HPMC (%) | 0.1 | 0.15 | 0.2 | 0.1 | 0.15 | 0.2 | 0.1 | 0.15 | 0.2 |
Mannitol (%) | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
Propyl paraben (%) | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 |
Distilled water (q.s) ml | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
Gelation study
The gelation study was done by taking formulation and simulated nasal fluid SNF (1:1v/v ratio) and mixed on magnetic stirrer (Magnetic stirrer, Remi). The gelation point was determined when the magnetic bar stopped moving due to gelation17. The consistency of formed gel was checked and graded, as indicated in (Table 2).
Formulation code | Gelling capacity | Viscosity (cp) | Gel strength (sec) | Mucoadhesive strength (dynes/ cm2) | Drug content (%) | % Drug permeated | |
---|---|---|---|---|---|---|---|
Sol | Gel | ||||||
F1 | + | 33.16±0.3 | 101.43± 2.25 | 18.67±0.58 | 2135±2.65 | 98.67±0.53 | 80.52±3.2 |
F2 | + | 39.06±0.6 | 112.198± 5.86 | 24±1.0 | 2446.67±2.08 | 98.03±0.73 | 74.50± 4.5 |
F3 | + | 47.80±0.3 | 124.683±8.52 | 27.33±0.58 | 2716±3.46 | 99.28±0.29 | 70.02± 5.8 |
F4 | ++ | 54.35±0.2 | 143.39±7.52 | 31±1.0 | 2282±2.65 | 99.79±0.29 | 94.24±2.6 |
F5 | ++ | 63.67±0.5 | 164.99± 8.45 | 34±1.0 | 2515.33±0.58 | 100.16±1.10 | 85.52±5.4 |
F6 | ++ | 72.31±0.5 | 182.82± 5.23 | 42.33±1.53 | 2885.67±2.52 | 99.66±1.25 | 80.12±3.4 |
F7 | +++ | 82.75±0.3 | 197.71± 8.52 | 47.67±2.08 | 2549.67±2.08 | 101.07±0.36 | 82.18±4.5 |
F8 | +++ | 97.26±0.3 | 220.43± 6.24 | 52.33±0.58 | 2891.67±1.53 | 99.13±0.29 | 80.12±5.4 |
F9 | +++ | 107.60±0.8 | 237.15± 10.23 | 56.67±2.08 | 3109.67±2.52 | 99.80±0.25 | 75.10±5.7 |
Viscosity study
The viscosity of nasal formulation before and after gelation was determined using Brookfield Rheometer R/S-CPS +1600 (Lauda Ecoline Staredition RE-204). Each point is the average of at least three readings18.
Gel strength study
Test was performed using a gel strength apparatus modified at the laboratory. In situ gel formulation (50 g) was placed in a 100 ml measuring cylinder and gelation was induced by simulated nasal fluid. The apparatus for measuring gel strength (weight: 35 g) was then placed on the gel. The gel strength was measured by the minimal weight that pushed the apparatus 5 cm down through the gel19.
Mucoadhesive strength study
Fresh nasal mucosa was obtained from the local slaughter house (Aurangabad), was carefully removed from the nasal cavity of sheep and mounted on glass surface using adhesive tape while another mucosal section was fixed in inverted position to the cylinder. 50 mg of gel was placed on mucosal surface. The glass mounted mucosal surface with gel formulation and mucosal surface attached to cylinder were held in contact with each other for 2 min to ensure intimate contact between them. In second pan, the weights were increased until the two mucosal tissues got detached from each other. The nasal mucosa was changed for each measurement. The mucoadhesive force expressed as the detachment stress in dynes/cm2 was determined from the minimal weight that detached the mucosal tissue from surface of each formulation20.
Mucoadhesive strength (dynes/cm2) = mg/A
Where,
m = Weight required for detachment in gram,
g = Acceleration due to gravity (980 cm/s2),
A = Area of mucosa exposed.
Drug content
The drug content was determined by taking 1ml of formulation in a 50 ml volumetric flask diluted with phosphate buffer pH 6.6 and analyzed using UV-visible spectrophotometer (Shimadzu, UV-1800, Lab India) at λmax 263.80 nm.
Ex-vivo permeation study
Ex-vivo permeation study of formulations was carried out using Franz diffusion cell. Nasal mucosa was placed in diffusion cells displaying a permeation area of 0.785 cm². The receiver compartment containing phosphate buffer pH 6.6 as in range of nasal cavity pH was maintained at 37 ±0.5°C. After a pre-incubation time of 20 min, formulation equivalent to 10 mg of FXH was placed in the donor chamber containing 3 ml of artificial nasal fluid. At predetermined time points (30, 60, 120, 180 and 240 min), 1ml of sample was withdrawn from the receptor compartment, replacing with fresh medium21. The amount of drug permeated was determined using UV-visible spectrophotometer (Shimadzu, UV-1800, Lab India) at λmax 263.80 nm.
Release mechanism
To study the mechanism of drug release from prepared gelling system, the in vitro permeation data were fitted to zero order, first order, Higuchi release model, Hixson and Crowell method and Korsemeyer-Peppas model by using DD Solver software, and the model with the higher correlation coefficient was considered to be the best model22.
Histopathological study
Histopathological examination on control mucosa and F4 formulation treated mucosa was performed using a light microscope (Nikon Eclipse E600, Japan). Tissue was fixed with 10% buffered formalin (pH 7.0), routinely processed and embedded in paraffin. Sections (5μm) were cut on glass slides and stained with hematoxylin and eosin.
Stability study
The stability study as per ICH guideline on optimized formulation F4 was carried out at, temperature 40°C ± 2 ºC and humidity 75% RH ± 5% condition in stability chamber (HMG, India) for three months. The formulation was examine for pH, drug content, viscosity23.
In vivo pharmacodynamic study
IAEC, M.E.S. College of Pharmacy, Sonai approved the protocol for In vivo pharmacodynamic study. Locomotor activity test (LAT) and Forced swim test (FST) was carried out to evaluate the antidepressant effect of the optimized F4 formulation. Rats of either sex weighing 250–300 g were selected and kept under standard laboratory conditions (temperature 23-30°C) with free access to standard laboratory diet. Rats were divided randomly into three groups, each containing six animals (n= 6). Group one was treated with saline and was considered as a control. Group two was treated with oral tablets of FXH containing dose of 10 mg/kg. Group three was treated with optimized F4 formulation through nasal containing 10 mg/kg. The doses were administered without anesthesia by using simple poly-ethylene tube.
Force swim test
The tank was filled 90 % with water so they swim without touching their hind limb or tail to the bottom of the tank. On day one of experiments, rats were forced to swim for 10 min. After 24 h, rats were re-exposed to forced swim after administration of dose for 5 min and animals were judged for immobility, climbing, and swimming and experiments were performed for thrice. After experiment, the rat was removed from the tank and returned to their cage 24
Locomotor activity test
Locomotor activity was measured in the open-field test. The apparatus consisted of a square arena (200×200 cm), with a 50 cm height. The floor was divided into 30 equal squares. Animals were individually positioned in the center of the arena and the activity was measured over 5 min (numbers of square crossed). A square crossed was defined as the rat placing its four paws into the quadrant and going to the neighboring quadrant. The open field was cleaned with isopropyl alcohol solution before behavioral testing to avoid possible bias due to odors and/or residues left by rats tested earlier. Also, after each 3 animals apparatus was cleaned25.
RESULTS AND DISCUSSION
Analysis of data
Statistical model incorporating interactive and polynomial terms was utilized to evaluate the responses.
Where, Y is the dependent variable, b0 is the arithmetic mean response of the nine runs and b1 (b1, b2, b12, b11 and b22) is the estimated coefficient for the corresponding factor X1 (X1, X2, X12, X11 and X22), which represents the average results of changing one factor at a time from its low to high value. The interaction term (X1X2) depicts the changes in the response when two factors are simultaneously changed.
From above equation the positive coefficient of variable X1 and X2 indicate that, as concentration of gellan gum and HPMC increased the viscosity was also increased.
From above equation the positive coefficient of variable X1 and X2 indicate that, as concentration of gellan gum and HPMC increased the mucoadhesive strength was also increased.
From above equation the negative coefficient of combine variable X1 and X2 indicate that as concentration of gellan gum and HPMC increased the percent drug release decrease.
Response surface plot (RSP)
From RSP evaluation it was found that concentration of gellan gum and HPMC increased, mucoadhesive strength and viscosity increases. Where as in case of drug release concentration of polymer increases the permeation release decreases as shown in Figure 1. The RSP results were similar to the mathematical data.
Gelation study
In gelation study, it was observed that as concentration of gellan gum increased (0.2 to 0.6 %) gelation point increased. The formulation F1 to F3 showed less gelation point whereas F4 to F6 showed immediate gelation remain for few hours (less stiff gel). Further as concentration of gellan gum increased the immediate gelation remain for extended period (stiff gel). Gelation was assessed on a scale ranging between - and +++, as shown in (Table 2). From above results, it revealed that the HPMC did not affect the gelation phenomena, but the gellan gum plays a critical role in hydrogel formation.
Viscosity study
In viscosity study it was found that as concentration of gellan gum and HPMC increased, the viscosity goes on increasing. Formulation F1 to F9 the concentration of HPMC increased (0.1 to 0.2%) and gellan gum (0.2 to 0.6 %), the viscosity increased before and after gelation as shown in Table 2.
Gel strength
The results of all formulations had gel strength between 18.67 sec to 56.67 sec as shown in (Table 2). The formulation F1 and F2 showed the less gel strength and may not retain its integrity and erode rapidly, whereas F3 to F7 show the gel strength in range 27.33 sec to 47.67 sec, remain stable and retain its integrity. In formulation F8 and F9 showed gel strength more than 50 sec which may form stiff gel and caused discomfort to nasal mucosa. Gel strength above 50 seconds suggests undesirable stiffness of gel formulation which may lead to irritation and discomfort in drug delivery. The gel strength values ranging 25-50 sec are considered adequate 26.
Mucoadhesive strength study
Study indicates that, the concentration of HPMC increases the mucoadhesive strength goes on increasing shown in (Table 2). HPMC is a hydrophilic polymer with many polar functional groups. Upon hydration the polymeric chains of HPMC get entangled with glycoprotein chains of mucin resulting in bioadhesion 27,28.
Drug content
The drug content was in the range 98.03- 101.07 %, which revealed that FXH was uniformly dispersed in gelling system. The results are shown in (Table 2).
Ex-vivo permeation study
The results reveal that as the concentration of gellan gum (0.2 to 0.6%) and HPMC (0.1 to 0.2 %) increased, the drug release was decreased. The percentage drug permeated after 240 min from all formulations was found to be between 75.10 to 94.24%. The highest drug release was found in F4 formulation (94.24%), which was selected as optimum formulation for further study. Permeation profiles for all formulations are shown in (Figure 2). The initial rates of permeation were very rapid due to incomplete gel formation, but as the time progress the permeation rate decreases due to complete gel formation. With an increase in concentration of HPMC (0.1 to 0.2 %), the diffusion rates were found to decrease gradually. It was proposed that as the concentration of gellan gum increased, the polymer chains approached closer, and the number of interactions between the polymer chains increases which leads to a denser 3-D network structure29. Decreased in drug released might be due to the increase in polymer concentration which increases the viscosity of gel layer with longer diffusional path length, resulting in greater retardation of drug in gel.
Release mechanism
The results obtained from release kinetics it could be concluded that, the formulations (F1 to F9) exhibited n values between 0.542–0.986 indicating an anomalous or nonfickian release suggesting a coupled erosion– diffusion mechanism.
Histopathological study
Intact columnar structure of epithelial cell was observed in both nasal mucosa (treated and untreated) shown in Figure 3A. From results it was found that prepared formulation is safe to use.
In vivo pharmacodynamic study
Force swim test
The FST is most used tool for screening antidepressants activity24. The F4 administer nasal route reduced total immobility period and increase climbing and swimming behavior as compared to control, orally administer group as shown in Figure 3B. This might be due to the required amount of FXH available in brain through nasal route.
Locomotor activity study
In study, difference in the values was found in control, orally administer and F4 formulation treated group through nasal as shown in Table 3. The results revealed that animals were not hyperactive.
Stability Study
In stability study there was no change found in drug content, pH and viscosity as shown in (Table 4). From stability study results, it was concluding that prepared formulation is stable.
CONCLUSION
A 32 factorial design was used successfully to study the effect of different variables. The optimized formulation F4 containing 0.4% gellan gum and 0.1% HPMC showed highest drug release (94.24 %) through sheep nasal mucosa. In pharmacodynamic study, the in situ gel treated rats showed significant responses as compared to control group. It was concluded that, the in situ nasal formulations of FXH was effectively formulated using quality by design approach which enhanced drug absorption in brain through nasal route and patient compliance for the treatment of depression.