Ex vivo permeation characteristics of venlafaxine through sheep nasal mucosa

Ex vivo permeation characteristics of venlafaxine through sheep nasal mucosa

European Journal of Pharmaceutical Sciences 48 (2013) 195–201 Contents lists available at SciVerse ScienceDirect European Journal of Pharmaceutical ...

527KB Sizes 6 Downloads 45 Views

European Journal of Pharmaceutical Sciences 48 (2013) 195–201

Contents lists available at SciVerse ScienceDirect

European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Ex vivo permeation characteristics of venlafaxine through sheep nasal mucosa Swati Pund ⇑,1, Ganesh Rasve 1, Ganesh Borade Department of Pharmaceutics, STES’s Sinhgad Institute of Pharmacy, Pune, India

a r t i c l e

i n f o

Article history: Received 18 August 2012 Received in revised form 15 October 2012 Accepted 29 October 2012 Available online 16 November 2012 Keywords: Venlafaxine Ex vivo permeation In situ gel

a b s t r a c t Venlafaxine, a dual acting antidepressant is a new therapeutic option for chronic depression. Depression is a common mental disorder associated with the abnormalities in neuronal transport in the brain. Since the nose-to-brain pathway has been indicated for delivering drugs to the brain, we analyzed the transport of venlafaxine through sheep nasal mucosa. Transmucosal permeation kinetics of venlafaxine were examined using sheep nasal mucosa mounted onto static vertical Franz diffusion cells. Nasal mucosa was treated with venlafaxine in situ gel (100 ll; 1% w/v) for 7 h. Amount of venlafaxine diffused through mucosa was measured using validated RP-HPLC method. After the completion of the study histopathological investigation of mucosa was carried out. Ex vivo studies through sheep nasal mucosa showed sustained diffusion of venlafaxine with 66.5% permeation in 7 h. Transnasal transport of venlafaxine followed a non-Fickian diffusion process. Permeability coefficient and steady state flux were found to be 21.11  103 cm h1 and 21.118 lg cm2 h1 respectively. Cumulative amount permeated through mucosa at 7 h was found to be 664.8 lg through an area of 3.14 cm2. Total recovery of venlafaxine at the end of the permeation study was 87.3% of initial dose distributed (i) at the mucosal surface (208.4 lg; 20.8%) and (ii) through mucosa (664.8 lg; 66.5%). Histopathological examinations showed no significant adverse effects confirming that the barrier function of nasal mucosa remains unaffected even after treatment with venlafaxine in situ gel. Permeation through sheep nasal mucosa using in situ gel demonstrated a harmless nasal delivery of venlafaxine, providing new dimension to the treatment of chronic depression. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Depression is the most common major mental illnesses associated with the high mortality rate. About 121 million people worldwide are affected by depression. Symptoms of the depression are commonly observed in the age category of 15–44 years for both the sexes (Mathers and Loncar, 2006). Medication noncompliance is a major factor resulting in failure of antidepressant therapy. Efficacy of antidepressant drugs depends mainly upon the continued presence of drug at the site of action (brain). Therefore, a sustained release antidepressant delivery is required to maintain the steady state levels of antidepressant in the brain, which is limited in case of conventional oral as well as parenteral therapy (Kilts, 2003). Selective serotonin reuptake inhibitors have dominated the antidepressant market since the late 1980. However, interest is increased in new antidepressants having broader mechanism of action with improved efficacy and less adverse effects (Gutierrez et al., 2003).

⇑ Corresponding author. Address: Department of Pharmaceutics, STES’s Sinhgad Institute of Pharmacy, Narhe (Ambegaon), Pune 411 041, Maharashtra, India. Tel.: +91 20 66831801; fax: +91 20 66831816. E-mail address: [email protected] (S. Pund). 1 These authors contributed equally to this work. 0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2012.10.029

Venlafaxine (VLF) is the first line, dual acting antidepressant and is more effective than selective serotonin reuptake inhibitors. It inhibits central serotonin and norepinephrine neuronal reuptake and has proven efficacy in the treatment of depression and anxiety disorders. VLF is effective in the resistant depression and is superior to selective serotonin reuptake inhibitors in preventing the recurrence of depression (Gutierrez et al., 2003). VLF has short half-life (4–5 h), hence needs frequent administration to maintain a blood levels in therapeutic window (Troy et al., 1995). In addition, hydrophilic nature of VLF limits its blood brain barrier permeability, resulting in poor antidepressant action in the brain. Although VLF is well absorbed from the gastro-intestinal tract it undergoes extensive metabolism in the liver (Karlsson et al., 2010). The oral use of VLF is also associated with a number of predictable adverse effects like tachycardia, increased blood pressure, fatigue, headache, dizziness, sexual dysfunction, and dry mouth etc. (Stahl et al., 2005). Dose dependant elevation of creatinine kinase has also been reported in the patients suffering from VLF toxicity (Singh et al., 2008). Hence to control the plasma concentration within an acceptable range and antidepressant action in the brain, a well defined dosage form is needed. Though the oral drug delivery is most accepted and convenient, is thought to be non-ideal for most of the antidepressants (Singh

196

S. Pund et al. / European Journal of Pharmaceutical Sciences 48 (2013) 195–201

et al., 2008). In the last few decades the nasal cavity has also been exploited for systemic delivery of small molecular weight drugs, especially where a rapid onset of action is required (Illum, 2012). In recent decades, the nasal mucosa has become an established administration site for brain targeting as well as for systemic drug delivery and a desirable alternative to the parenteral medication since it is amenable to self-medication and virtually painless. It does not require sterile technique, does not contribute to the biohazardous waste and the risk of accidental sticks is not a concern (Costantino et al., 2007). From a pharmacokinetic standpoint, intranasal administration circumvents first-pass metabolism and drug absorption is rapid due to the existence of a rich vasculature and a highly permeable structure within the nasal membranes. These characteristic features provide faster onset of action as compared to peroral administration. All these advantages may help maximize the patient compliance (Costantino et al., 2007). Hence intranasal delivery could be especially important in the management chronic diseases and for the drugs having extensive first pass metabolism. Even though a number of challenges are still to be overcome, especially with respect to toxicity, the potential of nasal drug delivery, including the ability to target drug across mucosal permeation, still remains to be explored. In addition, the physical and chemical parameters of drugs and vehicles (e.g. Permeation characteristics, partition coefficient, molecular weight, drug concentration, and pH), nasal mucociliary clearance, and nasal enhancers (e.g., microspheres, liposomes, and gels) must be taken into account in nasal drug absorption (Samson et al., 2012). The current research concerns the intranasal delivery of VLF using in situ gel which provides an alternative portal for VLF that would result in better therapeutic efficacy and reduced toxicity. Recently reported study on VLF nanoparticles by Haque et al. (2012) supports the hypothesis of VLF for nasal delivery. The main objective of current study was to demonstrate the feasibility of transmucosal permeation of VLF through sheep nasal mucosa and to study the permeation kinetics and toxicity of VLF to the nasal mucosa. 2. Material and methods 2.1. Materials Venlafaxine hydrochloride (VLF) was a gift from Ranbaxy Laboratories Ltd., Gurgaon, India. PluronicÒ F127 (Poloxamer 407, BASF, Mumbai) and Methocel A4M (Methyl cellulose, Colorcon Asia Pvt. Ltd. Mumbai) were used as excipients and were obtained from indicated sources. All other ingredients and reagents were of analytical grade and were used as received. 2.2. Methods 2.2.1. Preparation of VLF in situ gel and VLF simple solution The VLF in situ gel containing VLF; 1% w/v, Methocel A4M; 1% w/ v and PluronicÒ F127; 17% w/v was prepared by cold method (Schmolka, 1972). Accurately weighed amount of Methocel A4M was added in half of the desired volume of hot distilled water at 70 °C. The mixture was cooled to 20 °C with continuous stirring to obtain homogenous solution. This dispersion was further cooled to 4 °C and the required quantity of PluronicÒ F127 was added slowly with continuous stirring. Volume was adjusted and the solution was stored at 4 °C overnight to obtain clear solution. To the resultant solution accurately weighed VLF was added and mixed well. Benzalkonium chloride solution (50% v/v) was added as a preservative to get 0.006% v/v concentration in the formulation (Zaki et al., 2007). The formulation was stored in refrigerator

prior to evaluation. For comparison of ex vivo permeation profile, a simple aqueous solution of VLF without any excipients was prepared (VLF content: 1% w/v). 2.2.2. In vitro characterization of VLF in situ gel 2.2.2.1. Measurement of sol–gel transition temperature (Tsol–gel) and gelation time. The Tsol–gel of the formulation was determined by test tube inversion method. VLF in situ gel (2 ml) was transferred to a test tube and sealed with parafilm. This test tube was placed in the constant temperature water bath. The temperature of water bath was increased in the increments of 2 °C and left to equilibrate at each new temperature. However, in the region of Tsol–gel temperature was raised slowly in the increments of 0.5 °C. The formulation was examined for gelation which was said to have occurred when the meniscus would no longer move upon tilting through 90°. Gelation time was determined from time required to form the gel when the solution was maintained at 35 ± 1 °C. Measurements were done in triplicate (Gilbert et al., 1987). 2.2.2.2. Rheological behavior. Viscosity was measured on Brookfield Viscometer (DV-II+ Pro, Brookfield Engineering Labs. Inc., Middleboro, USA) equipped with helipath stand and T bar spindle. Viscosity measurements were made at variable temperature and variable shear rate (Asasutjarit et al., 2011; Fawaz et al., 2004). For temperature dependency study, formulation was subjected to shear rate of 50 and 100 rpm from 20 to 44 °C. During this testing the temperature was raised gradually by 2 °C and viscosity of the sample was measured after attaining the set temperature. Measurements were done in triplicate. Steady shear sweep test was carried out by measuring the viscosity at constant temperature of 20 °C and 34 °C but varying the rotation speed of spindle from 5 to 150 rpm (Asasutjarit et al., 2011). 2.2.2.3. Mucoadhesive strength. The mucoadhesive potential of the developed formulation was determined by measuring the force required to detach the formulation from nasal mucosal tissue using a modified method described by Majithiya et al. (2006). Mucoadhesive strength was measured for VLF in situ gel and VLF in situ gel without Methocel A4M and was expressed in dynes/cm2. 2.2.2.4. Estimation of VLF content and the analytical procedure. VLF content of the in situ gel as well as VLF permeated through mucosa during permeation studies were analyzed by validated RP-HPLC method after suitably diluting with the mobile phase. The HPLC system consisted of Pump (Jasco PU-2080 plus, Intelligent LC pump, Japan) with a Interface (Jasco LC-Net II/ADC, Japan) connected to Detector (Jasco UV-2075 plus, Intelligent UV–VIS detector, Japan). The chromatographic separation was performed using an isocratic elution. The mobile phase consisted of a mixture of methanol and monobasic potassium dihydrogen phosphate buffer (0.05 M) (70:30) and delivered at a flow rate of 1 ml/min. The separation was carried out at 20 °C, on a reversed phase HiQ Sil C8 column (250  4.6 mm, 5 lm particle size). An injection volume of 20 lL was used. Detections were carried out at 225 nm. 2.2.3. Release of VLF from nasal in situ gels and analysis of release data VLF release studies were carried out in USP Dissolution test apparatus Type II (TDT 06T, Electrolab, India) (Ugwoke et al., 2000; Zaki et al., 2007). A dialysis bag (M. Wt. cutoff: 12,000– 14,000, Himedia, India) was filled with 1 g VLF in situ gel formulation equivalent to 10 mg of VLF. Test was carried out at 35 ± 1 °C in 500 ml of Simulated Nasal Electrolyte Solution (SNES) as a dissolution medium using agitation speed 50 rpm (n = 6). The SNES was composed of 7.45 mg/ml NaCl, 1.29 mg/ml KCl and 0.32 mg/ml CaCl22H2O and pH adjusted to 5.5. Aliquot (1 ml) of the dissolution medium was withdrawn at time intervals of 15, 30, 45, 60,

S. Pund et al. / European Journal of Pharmaceutical Sciences 48 (2013) 195–201

90, and 120 min and was replaced with 1 ml of fresh SNES. All the dissolution samples filtered through 0.22 l MilliporeÒ (Polyvinylidine difluoride, PVDF) filter were analyzed immediately after completion of the test by validated RP-HPLC as described above. The amount of VLF was determined from a previously constructed calibration curve. The dissolution data obtained was analyzed by simple but useful empirical Korsmeyer–Peppas power law equation,

M t =M1 ¼ Kt n

ð1Þ

where Mt and M1 are the amount of drug released at time t and at infinity. Mt/M1 represent the fraction of VLF released at time t. K is the constant and depends on structural and geometric characteristics of drug/polymer system or the device. The diffusional exponent of drug release ‘n’ indicates the type of release mechanism during the dissolution process. 2.2.4. Ex vivo permeation of VLF from in situ gel and simple solution 2.2.4.1. Preparation of sheep nasal mucosa and experimental setup. The freshly excised sheep nasal mucosa, except septum part was collected from a local slaughter house. The superior nasal membrane was identified and separated from the nasal cavity and made free from adhered tissues. Maintaining the viability of the excised nasal tissues during the experimental period is crucial. Therefore, within 10 min of the killing of the animal, the mucosa was carefully removed, then immediately immersed in ice-cold phosphate buffer saline pH 6.4 for 15 min and was aerated. Ex vivo permeation of VLF though the sheep nasal mucosa was carried out using vertical static jacketed Franz diffusion cell (Samson et al., 2012). Mucosal specimen having thickness 0.12 cm and effective surface area 3.14 cm2 was used for permeation study (Rathnam et al., 2008). Within 30 min of removal, the excised nasal mucosa was mounted in the diffusion cell with mucosal surface facing the donor chamber and serosal side facing the receptor chamber. Both sides of the nasal mucosa were filled with the SNES solution and bubbled. Following a preincubation period of approximately 10 min, used in order to reach 34 °C and electrophysiological equilibrium, permeability experiments were started by replacing the media with 15 ml fresh prewarmed (35 °C) gassed SNES. The temperature of the receptor chamber was controlled at 35 ± 1 °C using constant circulation water bath. Mucosa was allowed to stabilize for 15 min prior to loading of the test sample. The solution in the receptor chamber was stirred continuously using teflon coated magnetic bar at constant rate, in such a way that the nasal membrane surface just flushes the SNES (Basu and Bandyopadhyay, 2010; Karasulu et al., 2008; Majithiya et al., 2006). 2.2.4.2. Intranasal VLF delivery. Ex vivo permeation of VLF though the sheep nasal mucosa was investigated after topical exposure of 7 h (Basu and Bandyopadhyay, 2010; Karasulu et al., 2008). The surface of nasal mucosa was treated with 100 ll of VLF in situ gel (equivalent to 1 mg VLF) by placing sample in donor chamber. Aliquots of SNES (0.5 ml) from receptor chamber were withdrawn every 60 min interval for 7 h. Same volume of fresh SNES maintained at same temperature was replenished in the receptor chamber after every sampling to maintain the volume. The aliquots of the receptor chamber taken during permeation study were stored at 20 °C until analyzed for VLF content. Blank samples were run simultaneously throughout the experiment (Park et al., 2003; Samson et al., 2012). After 7 h permeation experiment, the mucosal surface was washed and sonicated for 30 s with 2 ml of SNES to recover the unabsorbed VLF from the surface. For comparison purpose, ex vivo permeation was also carried out for 100 ll of 1% w/v VLF simple aqueous solution without any excipients for 3.5 h.

197

2.2.5. Analysis of VLF nasal mucosal permeation data The ex vivo nasal mucosal permeation profile of VLF was fitted to the non-steady state solution to Fick’s second law (Eq. (2)) for a single layer membrane to determine the permeability characteristics of VLF. ( ) 1   X D Q T ¼ C d  ðKLÞ 2 t  1=6  2=p2 ð1Þn =n2  exp D=L2  n2  p2  t ð2Þ L n¼1 where Qt is the cumulative quantity of VLF absorbed through the mucosa as a function of time (Samson et al., 2012). Cd is the initial total donor chamber concentration of VLF (1 mg). Steady state flux (Jss, lg cm2 h1) was calculated as the amount of VLF passing across 1 cm2 of the permeation membrane per unit time.

J ss ¼ DQ t =Dt  S

ð3Þ

where DQt/S is the cumulative drug permeation per unit of mucosal surface area (lg cm2), t is time expressed in h. Jss was calculated by plotting the cumulative amount of VLF (lg) permeated per unit area against time (h) and slope of the linear portion of the curve was considered as steady state flux. Apparent permeability coefficient (Papp, cm h1) and steady state diffusion coefficient (D) were calculated according to the following equations:

Papp ¼ J ss =C d

ð4Þ

D ¼ Papp  L=K

ð5Þ

K is the partition coefficient of the VLF (log P), and L is the diffusion path length (Heda et al., 2010; Park et al., 2003; Samson et al., 2012). 2.2.6. Assessment of local toxicity on nasal mucosa Histopathology was carried out after 7 h treatment of nasal mucosa with VLF in order to determine the pathological changes occurring in cell morphology and tissue organization. Studies were performed on treated and untreated mucosa with the formulation. Normal mucosa was considered as negative control and mucosa treated with 100 ll; 37% v/v nitric acid for 2 h, was considered as positive control. Mucosal preparations after treatment were fixed in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin. Sections were analyzed by pathologist blinded to the experimental conditions (Giannola et al., 2007; Samson et al., 2012). 3. Results and discussion Permeation kinetics of VLF through sheep nasal mucosa were studied for exploring the potential of VLF for nose to brain delivery in the treatment of chronic depression. VLF is amongst the first line drugs used in the treatment of depression. However, efficacy of VLF relies upon its continued presence at the site of action over a prolonged period of time. The peak-valley pattern of plasma VLF concentrations observed with oral administration often causes adverse events at maxima and loss of therapeutic effect at minima, which leads to intolerability (Haque et al., 2012; Kilts, 2003). This fact provides a strong rationale for designing more effective intranasal dosage forms capable of directly transporting the VLF to the brain to overcome the disadvantages associated with oral therapy. 3.1. Preliminary experiments and composition of in situ gel For nasal formulations, viscosity enhancers may be necessary in order to prevent drainage of the formulation. However, a simple boost in viscosity of the nasal formulation is disadvantageous

S. Pund et al. / European Journal of Pharmaceutical Sciences 48 (2013) 195–201

3.2. In vitro characterization of VLF in situ gels 3.2.1. Tsol–gel Thermoreversible polymer based liquid formulation that provide in situ gelling property in nasal cavity was designed to delay clearance of the formulations from the nasal cavity. The gelation temperature from 25 °C to 37 °C is suitable and desirable. The gelation temperature below 25 °C, indicates that the gel might be formed at room temperature leading to difficulty in manufacturing, handling, and administering. The gelation temperature above 37 °C indicates liquid state of formulation at body temperature, resulting in rapid and early nasal clearance (Majithiya et al., 2006). As the temperature of the nasal cavity is 34 °C, we aimed to prepare a composition with gelation temperature less than 34 °C. The VLF in situ gel exhibited gelation temperature 33.4 ± 1.63 °C (n = 3, mean ± SD). This indicates that the formulation will get gelled at physiological temperature (34 °C). 3.2.2. Gelation time Mucociliary clearance mechanism rapidly removes the applied dosage forms from the absorption site and is a problem in nasal drug delivery. Mucociliary clearance involves the combined actions of the mucus layer and the cilia, and is an important factor in the physiological defence of the respiratory tract against inhaled hazardous particles. In physiological conditions, mucus is transported at a rate of 5 mm/min and its transit time in human nasal cavity is reported to be 15–20 min (Pires et al., 2009). Generally, conventional nasal formulations such as liquid drops or sprays are rapidly cleared from the nose, and residence times in man of 12–15 min have been described. The residence time of a liquid formulation can be improved by increasing its viscosity. However, viscous solutions are difficult to administer as drops or sprays. Powder formu-

lations show longer nasal residence times than solutions but require special manufacturing techniques, and delivery devices for administration and accurate dosing (Zaki et al., 2007). Therefore, the purpose of this study was to prepare and characterize a formulation that will be able to deliver a unique dose of VLF in the nasal cavity and achieve a controlled release thereafter. Gelation time for the developed nasal in situ gelling formulation at 34 °C was found to be 3.0 ± 0.25 min (n = 3, Mean ± SD). This indicates the rapid gelling of the formulation before it could get cleared off from the site of absorption. 3.2.3. Rheological behavior Rheological behavior was studied by subjecting the VLF in situ gel to variable temperature at a shear rate of 50 and 100 rpm in order to observe the gelling capacity. Influence of temperature on viscosity of in situ gel of VLF is shown in Fig. 1a. The graph clearly depicts the thermosensitive nature of poloxamer gel. The formulation showed low viscosity at 20 °C, consequently, it could be administered into the nose, at ease without being stored under refrigeration. Viscosity increased as the temperature was increased from 20 °C to 34 °C. The increase in viscosity is indicative of conversion of sol to gel. This conversion was rapid between 32 and 34 °C. However, no significant rise in viscosity was observed beyond 34 °C at both the shear rates. Comparatively lower values of viscosity at high shear rate (100 rpm) shows shear thinning property of gel. Fig. 1b clearly indicates the exponential decline in viscosity with increasing shear rate. High viscosity at low shear rate could prolong the contact time at the site of administration. 3.2.4. Mucoadhesive strength Use of polymers with strong bioadhesive capacities can significantly limit the total clearance of the formulation from the nasal

a

22500 20500 50 rpm 18500 100 rpm

Viscosity (cps)

due to inconvenience associated with delivering high viscosity formulations with ease and consistency. A nasal mucoadhesive in situ gel appears very attractive alternative since this formulation is fluid-like prior to nasal administration and can thus easily be instilled as a drop allowing accurate dosing of drug. Nasal in situ gel will gel after instillation and will prevent drainage and increase in the retention time of the dosage form at the mucus membranes (Cai et al., 2011). The long residence time of the formulation increases the statistical probability for sufficient drug permeation to take place. PluronicÒ F127 (Poloxamer 407) has excellent thermo-sensitive gelling properties. It is one of the most studied member of family of poloxamers. Poloxamer form structures with micellar or gel-like features in a concentration and temperature dependent way. As the temperature is increased, polymer desolvation and subsequently micellization occur forming more closely packed viscous gel. The driving forces for the micellization and the accompanied expulsion of the hydrating water from the core of the micelles are the conformational changes in the orientation of the methyl groups in the side chains of the polymer (Majithiya et al., 2006; Zaki et al., 2007). Because of their biocompatibility and low toxicity, poloxamer-based gels have the potential of great utility in drug delivery. Poloxamer 407 has been studied as a formulation adjuvant in a number of applications like ocular, nasal, periodontal, vaginal, rectal, transdermal and subcutaneous drug delivery (Rehman et al., 2011). Methocel A4M (Methyl cellulose) was added to the poloxamer in situ gel to slow down the release of VLF and also to improve the residence time of the formulation at the nasal mucosa by mucoadhesion. Methocel is a bioadhesive as well as viscocity modifier (El-Kamel, 2002; Zhou and Donovan, 1996). With several preliminary trials, concentration of PluronicÒ F127 and Methocel A4M were finalized as 17 and 1% w/v respectively based on gelation temperature and mucoadhesive strength.

16500 14500 12500 10500 8500 6500 18

b

20

22

24

26

28 30 32 34 Temperature (°C)

36

38

40

42

44

80000 20°C

65000

Viscosity (cps)

198

34°C 50000

35000 20000 5000 0

25

50

75

100

125

150

shear rate (rpm)

Fig. 1. Plots showing a. Influence of temperature on viscosity of VLF in situ gel formulation at variable shear rate; 50 rpm and 100 rpm (n = 3). b. Influence of shear rate on viscocity of VLF in situ gel formulation at 20 °C and 34 °C (n = 3).

199

S. Pund et al. / European Journal of Pharmaceutical Sciences 48 (2013) 195–201

cavity. An optimal system for nasal drug delivery would therefore be fluid enough for easy administration yet would not undergo rapid initial clearance, and would have sufficient interaction with the mucosal surface to continue to limit clearance for extended time periods. Residence time of any formulation in nasal cavity depends on the mucoadhesive strength of polymers. The polymers with many hydrophilic functional groups set up electrostatic and hydrophobic interactions and hydrogen bond with the underlying surface. Zhou and Donovan (1996) studied several mucoadhesive polymers and rated methylcellulose as the best bioadhesive polymer for nasal use. Mucoadhesive strength of VLF in situ gel and VLF in situ gel without Methocel A4M was found to be 4119.72 and 2964.97 dynes/cm2 respectively. Addition of Methocel A4M has significantly increased the mucoadhesion by 39%. 3.2.5. VLF content Content of VLF in in situ gel was determined by validated RPHPLC method and was found to be 99.63 ± 0.47% of the added amount (n = 3, Mean ± SD). 3.3. In vitro dissolution In vitro VLF release from VLF in situ gel (1 g gel containing 10 mg VLF) time from dialysis bag, using USP type II apparatus as a function of is shown in Fig. 2. The dissolution results data was analyzed by simple and useful Korsmeyer–Peppas power law equation (Eq. (1)). The mathematical modeling described the release behavior of VLF from a dissolving gel. The correlation coefficient value R2 was found to be >0.99 indicating goodness of fit of the data in the Korsmeyer–Peppas equation. When n is equal to 0.5, the fraction of drug released is proportional to the square root of time (Higuchi kinetics) and the drug release is solely diffusion controlled (Fickian diffusion kinetics). If n = 1, indicates drug release is swelling controlled (zero-order kinetics), while if 0.5 < n < 1 indicates anomalous transport and superposition of both phenomenon (non-Fickian kinetic) (Zaki et al., 2007). In many applications of controlled drug delivery, a constant drug release rate or zero-order delivery is desired. Fickian release, which is the usual diffusioncontrolled release from drug delivery systems, has a rate of drug release that decreases as a function of time, due to a decrease in the concentration gradient. Zero-order release is observed when the rate of dissolution of the polymer matrix solely controls the drug release rate. The results of the in vitro dissolution study revealed the non-Fickian (n = 0.538) or anomalous behavior of release of VLF from the in situ gel. This indicates that the dissolution of the gel controlled the VLF release. The decrease in the diffusion rate of drug with time due to decrease in the concentration gradient can be due to gel dissolution.

3.4. Intranasal VLF delivery Although human nasal mucosa would be the ideal substrate for nasal permeation studies, its limited availability has made investigators to use suitable alternative. Gardiner et al. (1996), showed that the sinus anatomy (including the placement of nasal cavity, turbinates, frontal and maxillary sinuses) in sheep is comparable to humans. Histology of the sheep’s nasal mucosa is also identical to that of humans (Illum, 1996). Ex vivo permeation of VLF through sheep nasal mucosa was investigated (n = 3). VLF permeability from in situ gel and simple solution of VLF through the sheep nasal mucosa as function of time of mucosal exposure is shown in Fig. 3. Comparative permeation of VLF from in situ gel and simple solution clearly shows permeation of VLF. Approximately 90% of VLF got permeated from the simple solution in 3 h whereas, 39.5% VLF permeated from in situ gel. This indicates considerable retardation of diffusion of VLF by the gel matrix and prolongation of diffusion process. The profile first showed an exponential increase in permeation followed by a linear trend corresponding to unsteady and steady state conditions. The results of the VLF permeation from in situ gel and the recovery of VLF from mucosa and receptor chamber were used for further processing. Sustained delivery of VLF; 66.50% at 7 h of permeation was observed. After 7 h of permeation, the amount recovered from the mucosal surface and the receptor chamber accounted for 20.8% (208.4 lg) and 66.5% (664.8 lg) of the initial dose applied (Cd, 1 mg), respectively. The total amount recovered from the permeation experiment was 87.3%. To be successfully delivered through the nasal route, drug candidates should have adequate permeability. Physicochemical parameters are of prime importance in the choice of drugs as the candidates for nasal delivery. In the present study, the permeability parameters of VLF through the nasal mucosa calculated using Eqs. (3)–(5) are listed in Table 1. Linear regression analysis of pseudo steady-state diffusion data allowed calculation of the steadystate flux (Jss). The apparent permeability coefficient (Papp), steady state flux (Jss) and the steady state diffusion coefficient (D) of VLF through the mucosa were found to be 21.11 ± 0.9  103 cm h1, 21.118 ± 2.6 lg cm2 h1 and 58.93 ± 0.41  104 cm2 h1, respectively. These parameters derived from in situ gel as well as 90% VLF permeation in 3 h from simple solution suggest significant transport of VLF through nasal mucosa. It has been reported that the drugs cross the nasal epithelial membrane either by transcellular route, exploiting simple concentration gradients, receptor med-

300

VLF Permeated (µg.cm-2)

250

Cumulative amount of VLF released from vin situ gel (µg)

10000 8000 6000 4000

200

150

100

VLF in situ gel 50

VLF simple solution

2000 0

0

0

0

15

30

45

60 75 Time (min)

90

105

120

Fig. 2. In vitro VLF release as a function of time from VLF in situ gel (1 g gel containing 10 mg VLF) from dialysis bag, using USP type II apparatus (n = 6. Error bars indicate SD).

1

2

3

4

5

6

7

Time (h) Fig. 3. Ex vivo permeation of VLF as a function of time from in situ gel (100 ll, 1% w/ v) and simple solution of VLF without any excipients (100 ll, 1% w/v) through sheep nasal mucosa, mounted onto a vertical Franz diffusion cell (n = 3. Error bars indicate SD).

200

S. Pund et al. / European Journal of Pharmaceutical Sciences 48 (2013) 195–201

Table 1 Ex vivo permeation characteristics of VLF through sheep nasal mucosa (Initial concentration of VLF in the in situ gel in the donor compartment, Cd = 1 mg and the effective surface area = 3.14 cm2). (n = 3 ± SD). JSS (lg cm2 h1) 21.118 ± 2.6

Papp (cm h1)  103

D (cm2 h1)  104

Cumulative VLF permeated at 7 h (lg cm2)

21.11 ± 0.9

58.93 ± 0.41

211.72 ± 4.297

Fig. 4. Histopathological sections of nasal mucosa (magnification 100) (a) negative control: untreated mucosa, (b) positive control: mucosa treated with nitric acid (37% v/v) for 2 h, and (c) test specimen: mucosa treated with in situ gel (100 ll) containing VLF (1% v/v) for 7 h.

iated transport or vesicular transport mechanism or by paracellular route though the tight junction between the cells. Transcellular transport can be mediated by transporters that exist in the nasal mucosa, including organic cation transporters and amino acids transporters. In contrast, paracellular route is involved in the transport of small polar drugs and it takes place between adjacent epithelial cells through hydrophilic porous and tight junctions. Tight junctions are dynamic structures localized between the cells, which open and close accordingly to (in) activation of signaling mechanisms (Pires et al., 2009). Polar drugs with molecular weight less than 1000 Da generally pass the membrane by paracellular route. The pKa of VLF is 9.4 (Bonnet et al., 2010) which indicates that it will be ionized at physiological pH (5.0–6.5 pH of nasal mucosa) and hence polar. Molecular weight and lipophilicity of drugs also have a great impact in the rate and extent of nasal absorption. Though VLF has pKa 9.4, its instrinsic lipophilicity (log P = 2.8) and small mol wt. (313.87) must be contributing for the greater permeability. It can also be concluded that, for polar drugs, partition coefficient is the major factor influencing the permeability through nasal mucosa (Pires et al., 2009). 3.5. Local toxicity of VLF in situ gel The safety of intranasal formulations is of crucial importance for medications for chronic therapy. The tissue damage of VLF in situ gel was evaluated by paraffin section stained with hematoxylin and eosin. Histopathology was performed to observe the integrity of mucosa and irritation or toxicity caused to nasal mucosa by formulation, if any. Histological sections of treated and untreated nasal mucosa were found similar in tissue architecture after 7 h exposure (Fig. 4). In normal nasal mucosa (untreated) (negative control), the structure of the mucosa was well preserved (Fig. 4a). The surface pseudo epithelium displayed normal characteristics. After treatment with nitric acid (positive control), marked alterations in the surface pseudo epithelium were visible; the epithelial lining was completely distracted and they showed lose cohesive, sometimes detached and often vacuolated structure (Fig. 4b). After treatment with the VLF in situ gel (test), no marked alteration was observed as compared to negative control from the histological structure (Fig. 4c). There was also no any evidence of hemorrhage, necrosis and ulceration in VLF treated and untreated nasal mucosa.

4. Conclusion This study gives the evidence for nasal transport of VLF which can be exploited further for the treatment of depression. The nasal route minimizes the risk of systemic adverse events. Histopathology study showed no evidence of any noticeable histological effects, confirming the safety of nasal VLF delivery. Nasal delivery of VLF using mucoadhesive in situ gel is a promising and safe approach for improving the bioavailability and targeting the brain, for the treatment of depression.

References Asasutjarit, R., Thanasanchokpibull, S., Fuongfuchat, A., Veeranondha, S., 2011. Optimization and evaluation of thermoresponsive diclofenac sodium ophthalmic in situ gels. Int. J. Pharm. 411, 128–135. Basu, S., Bandyopadhyay, A.K., 2010. Development and characterization of mucoadhesive in situ nasal gel of midazolam prepared with Ficus carica mucilage. AAPS PharmSciTechnol. 11, 1223–1231. Bonnet, U., Bingmann, D., Wiltfang, J., Scherbaum, N., Wiemann, M., 2010. Modulatory effects of neuropsychopharmaca on intracellular pH of hippocampal neurones in vitro. Br. J. Pharmacol. 159 (2), 474–483. Cai, Z., Song, X., Sun, F., Yang, Z., Hou, S., Liu, Z., 2011. Formulation and evaluation of in situ gelling systems for intranasal administration of gastrodin. AAPS PharmSciTechnol. 12 (4), 1102–1109. Costantino, H.R., Illum, L., Brandt, G., Johnson, P.H., Quay, S.C., 2007. Intranasal delivery: physicochemical and therapeutic aspects. Int. J. Pharm. 337, 1–24. El-Kamel, A.H., 2002. In vitro and in vivo evaluation of Pluronic F127-based ocular delivery system for timolol maleate. Int. J. Pharm. 241, 47–55. Fawaz, F., Koffi, A., Guyot, M., Millet, P., 2004. Comparative in vitro–in vivo study of two quinine rectal gel formulations. Int. J. Pharm. 280, 151–162. Gardiner, Q., Oluwole, M., Tan, L., White, P.S., 1996. An animal model for training in endoscopic nasal and sinus surgery. J. Laryngol. Otol. 110, 425–428. Giannola, L.I., Caro, V.D., Giandalia, G., Siragusa, M.G., Tripodo, C., Florena, A.M., Campisi, G., 2007. Release of naltrexone on buccal mucosa: permeation studies, histological aspects and matrix system design. Eur. J. Pharm. Biopharm. 67, 425–433. Gilbert, J.C., Richardson, J.L., Davies, M.C., Palin, K.J., Hadgraft, J., 1987. The effect of solutes and polymers on the gelation properties of Pluronic F-127 solutions for controlled drug delivery. J. Controlled Release 5, 113–118. Gutierrez, A.M., Stimmel, L.G., Aiso, Y.J., 2003. Venlafaxine: a 2003 update. Clin. Ther. 25, 2138–2154. Haque, S., Md, S., Fazil, M., Kumar, M., Sahni, J.K., Ali, J., Baboota, S., 2012. Venlafaxine loaded chitosan NPs for brain targeting: pharmacokinetic and pharmacodynamic evaluation. Carbohydr. Polym. 89, 72–79. Heda, A.A., Sonawane, A.R., Naranje, G.H., Somani, V.G., Puranik, P.K., 2010. Development and in vitro evaluation of betahistine adhesive-type transdermal delivery system. Topical J. Pharm. Res. 9 (6), 516–524. Illum, L., 1996. Nasal delivery. The use of animal models to predict performance in man. J. Drug Targeting 3, 427–442.

S. Pund et al. / European Journal of Pharmaceutical Sciences 48 (2013) 195–201 Illum, L., 2012. Nasal drug delivery—recent developments and future prospects. J. Controlled Release 161, 254–263. Karasulu, H.Y., Sanal, Z.E., Sözer, S., Güneri, T., Ertan, G., 2008. Permeation studies of indomethacin from different emulsions for nasal delivery and their possible anti-inflammatory effects. AAPS PharmSciTechnol. 9 (2), 342–348. Karlsson, L., Schmitt, U., Josefsson, M., Carlsson, B., Ahlner, J., Bengtsson, F., Kugelberg, F.C., Hiemke, C., 2010. Blood–brain barrier penetration of the enantiomers of venlafaxine and its metabolites in mice lacking P-glycoprotein. Eur. Neuropsychopharmacol. 20, 632–640. Kilts, C.D., 2003. Potential new drug delivery system for antidepressants: an overview. J. Clin. Psychiatry 64, 31–33. Majithiya, R.J., Ghosh, P.K., Umrethia, M.L., Murthy, R.S.R., 2006. Thermoreversiblemucoadhesive gel for nasal delivery of sumatriptan. AAPS PharmSciTechnol. 7(3) Article 67, E1–E7. Mathers, C.D., Loncar, D., 2006. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med. 3 (11), 2011–2030. Park, Y.J., Yong, C.S., Kim, H.M., Rhee, J.D., Oh, Y.K., Kim, C.K., Choi, H.G., 2003. Effect of sodium chloride on the release, absorption and safety of diclofenac sodium delivered by poloxamer gel. Int. J. Pharm. 263, 105–111. Pires, A., Fortuna, A., Alves, G., Falcão, A., 2009. Intranasal drug delivery: how, why and what for? J. Pharm. Pharm. Sci. 12 (3), 288–311. Rathnam, G., Narayanan, N., Ilavarasan, R., 2008. Carbopol-based gels for nasal delivery of progesterone. AAPS PharmSciTechnol. 9 (4), E1–E9. Rehman, T., Tavelin, S., Gröbner, G., 2011. Chitosan in situ gelation for improved drug loading and retention in poloxamer 407 gels. Int. J. Pharm. 409, 19–29.

201

Samson, G., Calera, A.G., Girod, S.D., Faure, F., Decullier, E., Paintaud, G., Vignault, C., Scoazec, J.Y., Pivot, C., Plauchu, H., Pirot, F., 2012. Ex vivo study of bevacizumab transport through porcine nasal mucosa. Eur. J. Pharm. Biopharm. 80, 465–469. Schmolka, I.R., 1972. Artificial skin. I. Preparation and properties of Pluronic F-127 gels for the treatment of burns. J. Biomed. Mater. Res. 6, 571–582. Singh, G., Ghosh, B., Kaushalkumar, D., Somsekhar, V., 2008. Screening of venlafaxine hydrochloride for transdermal delivery: passive diffusion and iontophoresis. AAPS PharmSciTechnol. 9 (3), 791–797. Stahl, S.M., Grady, M.M., Moret, C., Briley, M., 2005. SNRIs: their pharmacology, clinical efficacy and tolerability in comparison with other classes of antidepressants. CNS Spectrosc. 10, 732–747. Troy, S.M., Parekar, V.D., Fruncillo, R.J., Chiang, S.T., 1995. The pharmacokinetics of venlafaxine when given in a twice daily regimen. J. Clin. Pharmacol. 35, 404– 409. Ugwoke, M.I., Kaufmann, G., Verbeke, N., Kinget, R., 2000. Intranasal bioavailability of apomorphine from carboxymethylcellulose-based drug delivery systems. Int. J. Pharm. 202, 125–131. Zaki, N.M., Awad, G.A., Mortada, N.D., Abd El Hady, S.S., 2007. Enhanced bioavailability of metoclopramide HCl by intranasal administration of a mucoadhesive in situ gel with modulated rheological and mucociliary transport properties. Eur. J. Pharm. Sci. 32, 296–307. Zhou, M., Donovan, M.D., 1996. Intranasal mucociliary clearance of putative bioadhesive polymer gels. Int. J. Pharm. 135, 115–125.