Intranasal Clonazepam Mucoadhesive Microspheres: Factorial Designing and Primary Evaluation.
Shaji Jessy, Poddar Aditi.
Clonazepam is a drug of choice for various Central Nervous System (CNS) disorders. Out of that, it is commonly used for the treatment of status epileptics. The drug however does not reach the CNS in an efficient manner. The nasal route could be a possible solution to the problem, as many evidences have been reported for nose to brain targeting. In the present work, the main objective was to prepare blank microspheres of gelatin and chitosan as per 33 randomized full factorial design. The design was used to study the effect of the selected variables on the average particle size. ANOVA, response surface curve, desirability plots were done to treat the design so as to arrive at a particular model and to find out the crucial factors affecting the response. Based on average particle size, 9 formulations were subjected to drug loading. The loaded microspheres were characterized with respect to entrapment efficiency, swellability, mucoadhesion, in-vitro, ex-vivo release studies.
ANOVA showed that average particle size was best fitted to a response surface linear model. SEM depicted a smooth surface. Also the other characteristics; like maximum entrapment was found to be 54.23±0.5% and swellability were 0.987±0.9. Both in-vitro and ex-vivo studies showed almost 100% release in a period of 120 min, thus indicating towards its use in prompt drug delivery systems via the nasal route.
Conventionally, the nasal route has been used for the delivery of drugs in the treatment of local diseases. However in last decade the importance of the nasal cavity as potential route for drug delivery has been recognised. This interest arises from the different possible advantages presented by the nasal cavity, such as; the epithelium being very vascularized and with a relatively large surface area available for drug absorption, the porous endothelial basement membrane, the direct transport of absorbed drugs into the systemic circulation thereby avoiding the first- pass effect present in peroral administration, the lower enzymatic activity compared with the alternative both to parentral and oral routes (Illum L, 2003 ).
Clonazepam (CZ) is a benzodiazepine derivative and is widely used in disorders like status epileptics, myoclonus and associated abnormal movements, panic attacks, psychiatric disorders. Presently, CZ is marketed as tablets and injectables. Though CZ is well absorbed following oral administration, it is extensively metabolized in the liver. The principle metabolite being 7- aminoclonazepam; which probably has little or no antiepileptic activity (Parfitt K, 1999). In addition to this, the tablets and injectables release the drug in peripheral circulation, with subsequent limited uptake by the Blood Brain Barrier (BBB). Moreover, these routes may become inconvenient or impractical depending upon the patient condition. Hence, there arises a need for an alternate route of administration. The nasal route provides a good solution to the aforementioned problem ( Vyas TK. et al, 2006).
Limitation of nasal drug delivery is the mucociliary clearance that determines a limited time residence for absorption within the nasal cavity. One strategy for increasing drug absorption is to prevent the rapid clearance of the delivery system from the nasal mucosa and thereby prolong the contact between the nasal mucosa and the formulation. A system should be designed so as to provide rapid transport to drug across nasal mucosa and longer residence time in nasal cavity (Behl CR. et al, 1998). Mucoadhesive microspheres can be and are being effectively used as a delivery medium via the nasal route. Microspheres of a wide variety of polymers have been reported, out of which starch microspheres were the first example of mucoadhesive microparticulate nasal delivery system (Illum L, 2001).
Gelatin A (GL) is an acid hydrolytic product of collagen, matrix material. It has its applicability as a biodegradable matrix material. It has the capacity to form a uniform smooth film, thus leading to effective entrapment of actives (Benita S. 10th Ed.)(Rowe KC. et al, 2006). Chitosan (CH) is a linear polysaccharide produced by a process of deacetylation from chitin, is one such material that has good mucoadhesive properties. It may be a good option in nasal delivery as it binds to the basal mucosal membrane with an increased retention time and it is a good absorption enhancer. Furthermore CH is an excipient able to enhance the dissolution rate of low water- soluble drugs (Ravi Kumar MNV. et al, 2004) (Giunchedi P. et al, 2002). When used in combination, the resulting delivery system would have the advantages of both the polymers.
The objective of this work was to optimize the processing variables for the preparation of gelatin- chitosan (GL-CH) mucoadhesive microspheres of Clonazepam so as to obtain rapid onset of action, to characterize it and to evaluate its performance with respect to drug release.
MATERIALS AND METHODS
Clonazepam, USP was a gift sample from IPCA Laboratories, Mumbai; Chitosan (Degree of Deacetylation – 85%) was graciously provided by Healer’s Neutraceuticals, Chennai. All other chemicals and solvents were of analytical reagent grade and were locally procured.
Preparation of Microspheres (Dandagi PM. et al, 2007)
The study design:
There are many process variables which can have a substantial influence on characteristics of the formed microspheres. It is not easy to quantify the effect of variables individually or in combination. The effect of 3 variables: (1) glutaraldehyde volume, (2) surfactant volume, (3) CH amount per 250mg of GL, on the average particle size of microspheres was studied using a factorial design. For the purpose of experimental design, blank microspeheres were initially prepared and the batches were statistically tested on the basis of average particle size. Out of these experimental batches, 9 batches were further selected for evaluation like entrapment of drug, swellabiltiy, surface morphology, in-vitro release studies and ex-vivo studies
A technique of 33 factorial design considering 3 factors at 3 different levels affecting the average particle size were considered. A factorial design evaluating 3 factors at all combinations for each factor would result in a full factorial design consisting of 33 = 27 runs. The addition of centre points grants for detection of nonlinearity in the responses. One centre point was added, thus there were 27+1= 28 runs. F test was used to compare the variance among the treatment that in turn indicates the variance of individuals among the specific treatments.
The experimental method:
The method was divided into two parts: (a) Blank microspheres and (b) Clonazepam Loaded microspheres.
(a) Blank microspheres (BM)
GL-CH microspheres were prepared using the emulsion cross- linking method to obtain a w / o emulsion. Three independent formulation variables at 3 different levels viz: low, medium, high were considered. Values of the selected variables at different levels are shown in Table 1. All other factors, namely, GL amount (250 mg), rpm (1500), strength of gluteraldehyde (25%) and strength of surfactant (SLS) (0.01%w/v) were kept constant. The varied amount of CH, as indicated in table 1, was taken with a fixed amount of GL (250 mg). CH was weighed and made into a solution using 2 mL of 2%v/v acetic acid. GL was soaked in 1m
L water and then dissolved at an elevated temperature of 550C. These two solutions were mixed in a magnetic stirrer which was maintained at a temperature of 45º C. The pH of the solution was determined with the help of a pH paper (S. D. Fine Chemicals, Ltd, India) This was then added to paraffin oil (30mL) (heavy: light= 1:1) by means of a spatula which in turn was kept under high stirring using a Remi mechanical stirrer at 1500 rpm. After 15 minutes of stirring, surfactant was added in a varied volume (0.5, 0.75, 1 mL) per batch as indicated in table 1. Further 15 minutes later glutaraldehyde saturated toluene (gst) was added dropwise as a cross- linking agent. The volume of gst was also varied (3, 4.5, 6 mL) as per the experimental design format. The stirring was discontinued after 2 hr. The microspheres were separated from oil by decantation and subjected to continuous washing with iso propyl alcohol (IPA), till no traces of oil remained, which was assessed microscopically. The microspheres so obtained were air dried at room temperature. All the experiments were carried out at controlled illumination as the drug is light sensitive.
(b) Clonazepam Loaded microspheres (LM)
Procedure similar to above was followed on the 9 selected formulations. Only in the initial stage, CZ (50 mg) was suspended in 0.5 ml of co- solvent Polyethylene glycol 400 (PEG 400) and then added to the GL-CH mix under constant stirring using a magnetic stirrer. This was slowly added to oil under same stirring as used earlier. The rest of the procedure followed was on the lines of preparation of blank microspheres.
|A (gst volume) [mL]||3||4.5||6|
|B (surfactant volume) [mL]||0.5||0.75||1|
|C (amount of Chitosan) [mg]||50||75||100|
Table 1: Coded values in 33 factorial design.
Percent yield (Gavini E. et al, 2006)
The percent yield was calculated as the weight percentage of the final product (both BM and LM) after drying, with respect to the initial total amount of GL and CH in case of blank microspheres; and including the amount of CZ added in case of loaded microspheres.
Percent yield= (Practical yield/ Theoretical Yield) X 100 (1)
Statistical Approach to Designing (Mathew ST. et al, 2007)
A commercially available software program was used (Design Expert, Version 7.0.2, Stat-Ease Inc, Minneapolis, MN). The experimental design chosen was Response Surface, 3- factor, 3- level factorial; 28 formulations were formulated. Run order was kept in the randomize mode to protect against the effects of time related variables and also to satisfy the statistical requirement of independence of observations.
Analysis of variance (ANOVA) and all statistical analyses were also performed using the same software. Calculation of the effects was performed and half- normal plots, response surface plots were plotted. Also, ANOVA was used to treat the data, and for proper model selection. The F value was checked to see whether it was within the desired limits. The F value was calculated by comparing the treatment variance with the error variance.
Particle Size Analysis (Sinko P, 5th Ed)
The particle size was determined by measuring 100 particles per batch using an optical microscope (Metzer OPTIK) under 450 X (eye piece: 10X and objective lense: 45 X). This was used as the parameter for selection of batches for further studies. The results was a mean of average of triplicate experiments. The blank microspheres batch selected were subjected to loading and further characterizations.
Surface morphology of the microspheres (BM and LM) (Jameela SR. et al, 1998) was studied by using a Scanning Electron Microscope (JSM- 5400, JEOL Ltd, Tokyo, Japan). The microspheres were placed on one side of an adhesive stub, and the stub was then coated with conductive gold with twin coater (JES- 550) attached to the instrument. The prepared sample was scanned at 0.5kV.
Entrapment Efficiency (Patil SB. et al, 2006)
To calculate the entrapment efficiency of CZ into the microspheres, a weighed quantity of microspheres were crushed and suspended in methanol in screw capped vials and shaken using a mechanical shaker for 24 hours to extract the drug from microspheres. After 24 hours, the mix was filtered under vacuum and the filtrate was subjected to spectroscopic analysis (V- 550, Jasco, Japan) at 308 nm for CZ content against methanol as blank. Corresponding CZ concentrations in the samples were calculated from the calibration plot generated by regression of the data taken in triplicate.
Swelling Index (Patil SB. et al, 2006)
The swelling indices of LM were determined by immersing preweighed (AB 104- S/FACT, Mettler Toledo, Germany) dried microspheres in10 ml of Saline Phosphate Buffer pH 7.4 (PBS) at a temperature of 37±2º C. After 24 hours, the sample was removed, blotted with a piece of paper towel to absorb excess water on surface and then reweighed. The difference in weight before and after soaking was found out. The swelling index of LM was calculated from the following expression:
QS= (WS-WD)/WD (2)
Where WS was the weight of the swollen LM and WD was the weight of dried LM. This test was performed in triplicate.
Mucoadhesion (Wei HJ. et al, 2007)
Male Sprague Dawley rats were used for this purpose. The intestinal tissue was removed within 2 hours of dissection.The tissue cleaned well with PBS till the mucous was free of food and faecal matter. Of this a 3 cm piece was taken from the upper part of the intestine. This was spread on a polyethylene support (a tube of 2 cm diameter, cut longitudinally at its centre) and was held in position with the help of pins. Polyethylene support was in turn attached to a rubber tube (3.5 cm diameter, longitudinally cut like the polyethylene support). 30 mg of accurately weighed microspheres were spread uniformly on the ileum. The tissue was placed in a dessicator maintained at 80% relative humidity and room temperature for 20 minute to allow the microspheres to hydrate and interact with the mucous and to prevent drying of the mucous. After 20 minute, the tissue was removed from the dessicator and washed with distilled water at a rate of 1 mL/min. the washings were collected in a tared beaker which was kept weighed in its empty state (W1). After collecting the washing in oven to constant weight at 80OC, the beaker was dried and weighed again (W2). The amount of the microspheres washed away was obtained from the difference in weight between W1 and W2.
MS=(W2-W1)/W2 X 100 (3)
The data collection was performed in triplicate.
In-vitro drug Release ((Gavini E. et al, 2006)
LM batches were subjected to in-vitro drug release studies. The performances of these batches were compared against dissolution of CZ alone. This was conducted with USP dissolution apparatus 2(TDT-08L, Electrolab, India). Accurately weighed 10 mg microspheres and 1mg pure drug, respectively, were tested in 500 ml of saline phosphate buffer pH 7.4, to assure the desired level of sink conditions. The rotational speed was set at 100 rpm and the temperature was 370C. Samples (5 ml) were withdrawn at predetermined time points (3, 6, 10, 15, 25, 35, 50, 70, 90 & 120 min), and for each withdrawal the volume was replaced with fresh medium at the same temperature. Samples were filtered through 0.45µ Millipore filter paper and assayed spectrophotometrically(V- 550, Jasco, Japan)for CZ at 308 nm. All the in-vitro tests were performed in triplicate.
Ex-vivo studies (Rao R. et al, 1989)
Sheep nasal mucosa was obtained from an authorized abattoir in Mumbai. The time from slaughter to removal of the nose was 5 min maximum. Within 60 min of removal, the mucosa was cut and mounted on the bottom of a cylindrical plastic support consisting of a tube (height 1.85 cm, diameter 2.3 cm)connected to a dive shaft of the dissolution apparatus as shown in Figure 1. The mucosa was clamped to the support by a plastic and then microspheres (10 mg) or CZ (1mg) was uniformly spread out on the surface of the mucosa (Figure 1). The system was then introduced into the vessel containing 500 mL of PBS, keeping the nasal mucosa just in contact with the surface. The shaft speed was 50 rpm. Sample (5mL) was withdrawn at time intervals of 3, 6, 10, 15, 25, 35, 50, 70, 90 & 120 min, and for each withdrawal the volume was replaced with fresh medium at the same temperature. Samples were filtered through 0.45µ Millipore filter paper and assayed spectrophotometrically(V- 550, Jasco, Japan)for CZ at 308 nm. All the ex-vivo tests were performed in triplicate.
Fig. 1: Modified dissolution apparatus for evaluation of ex-vivo permeation of
CZ- loaded microspheres (Gavini E. et al, 2004).
RESULTS AND DISCUSSION
Preparation of Microspheres
The method used was emulsion- cross-linking method. This method employed depended upon the polymer selection; that were GL and CH. The polymers being biodegradable were preferred over non-biodegradable polymers. In this method, the aqueous polymer mix entrapped the CZ during the course of stirring. In this case, there were many aspects to be noted. Firstly, an anionic surfactant (SLS) was used, so that it not only helped in emulsifying and producing uniform droplet, but also formed an anionic covering over the cationic polymer blend (Benita S. 10th Ed.). Secondly, PEG 400 was a solvent for CZ. But PEG 400 had limited compatibility with GL so the amount of PEG 400 was kept within limits so as to form a uniform dispersion with CZ without causing coagulation of GL. This helped in better entrapment into the hydrophilic matrix. Thirdly, the gst addition was slow and gradual. Sufficient time was given for the droplet size distribution within a period of not more than 30 min depending upon various parameters of the systems. An important feature of this technique was that the polymer droplets were converted to swollen particle of same size. This can be explained as glutaraldehyde reacts with the Lysine ε – NH2 group of GL forming bonds similar to those in the formation of Schiff base ( Fraenkal – Conrat H. et al, 1947) ; and in case of CH, the free pendant amine groups of CH polymer interact with the aldehydic group of the glutaraldehyde to form stable imine bonds( Oryton AC. et al, 1999). It was interesting to note that both the cross – linking reactions takes place at a pH range 5 – 7 and the pH of the solution was found to be 6 thus favoring the reaction. The presence of toluene in the aqueous solution of glutaraldehyde helps in proper incorporation of cross-linking agent into the oil, in comparison to solution of glutaraldehyde in water alone (Mathew ST. et al, 2007 ). IPA was used to remove oil wad was found to remove oil efficiently without changing the morphology of the microspheres and preventing aggregation.
The percent yield ranged from 65.1% to 75.4%.
The effect of different factors on the percent yield of microspheres was not clear, possibly as a result of the improper recovery of microspheres.
Loss of microspheres may be due to the finer particles being washed off during the process of decantation from the oil.
Particle size analysis
The various combinations in which the process variables were varied are represented in Table2.
Table 2: Design Matrix and Average (Avg.) Particle Size
*Average of 3 determinations.
Figure 2 presents the Size Distribution for BM 27, taken as a representative batch.
Fig. 2: Size Distribution for BM 27.
ANOVA was carried put using the Partial sum of squares- Type III method. The model had a F value of 30.63, the most significant factors in the model being gst volume and surfactant volume. Though the third factor also contributed towards average particle size, its contribution was less than the other two. ANOVA (Table 3) for particle size showed that the data could be best fitted in response surface linear model. Thus showing a linear relationship between the evaluated factors. The polynomial equation obtained was.
Average particle size= 80.06-7.67XA- 5.39XB-3.54XC (4)
Table 3: ANOVA for Response Surface Linear Model
Analysis of variance table [Partial sum of squares - Type III]
According to the analysis, the F- value has only 0.01% chance that its higher value is because of noise. The Predicted R-Squared of 0.7117 is in reasonable agreement with the Adjusted R-Squared of 0.7670.
Normal plot of residuals show that the variables influencing average particle size lie away from the centre (Figure 3).
Fig. 3: Normal Plot of Residuals.
A Response surface plot was plotted taking into consideration the effect of two factors, gst vlume and surfactant volume on the average particle, as these factors had a greater contribution in controlling the particle size. (Figure 4) It could be seen that there existed a linear relatonship between the factors and the response.
Fig. 4: Response Surface Plot .
In addition to this, the Desirability Plot was also made (Figure 5) .The numbers on the graph indicate the desirability values. Higher the values, the better are chances of getting the needed particle size. The target was the minimum avereage particle size achieved, i.e. 61 µm. Hence the plot sheds light on how the parameters could be varied to get the desired particle size.Also, as the value on the graph was increased, the average particle size went more close to the deired value.
Fig. 5: Desirability Plot
It was observed that the increase in CH amount reduced the overall particle size of the BM. Thus the batches providing the smaller range of average particle size were chosen, subjected to CZ loading, followed by further characterizations.Table 5 shows which BM batches were selected for loading and proceeded to corresponding LM batches.
Table 5: Selected BM batches for Loading.
|Selected Batches||Loaded Batch Code|
|BM 27||LM 1|
|BM 26||LM 2|
|BM 24||LM 3|
|BM 23||LM 4|
|BM 21||LM 5|
|BM 18||LM 6|
|BM 12||LM 8|
|BM 9||LM 9|
The particle size didn’t show much variation after and before loading.
Figure 6 shows the SEM surface morphology BM 27 (a) and LM 1 (b). The SEM images indicated towards a smooth, nonporous and spherical microspheres. Some of the particles appeared to be in aggregates, but without evidence of any collapsing particles. Also, the blank microspheres had a relatively smoother surface in comparison to the loaded microspheres(Miglani DS, 2002). Thus it could be seen that loading modified the surface characteristics of the microspheres. The loaded microspheres didn’t show the presence of free CZ on the surface. Many a times, it is difficult to obtain microspheres of smooth surface in case of natural polymers, due to variabilities like molecular weight and other properties of polymers(Mathew ST. et al, 2006). The microspheres had a smooth appearance might be due to usage of light paraffin alongwith heavy paraffin. This reduces the viscosity and hence the drag on the microspheres during preparation.
Fig. 6 : Scanning electron images of batch BM 27 (a) and LM 1 (b).
The entrapment improved from the LM 1 to LM 4 as the amount of CH increased, thus attributing the contribution of CH in improving the entapment of drug due to its solubility enhancing properties(Sinha VR. et al, 2004). PEG 400 also contributed in increasing the entrapment of CZ by the process of partial solvation.To determine the entrapment efficeincy an organic solvent was employed taking into consideration the poor solubility of CZ.Similar procedure has ben reported in many of the papers(Gavini E. et al, 2006) The data is tabulated in Table 6.
The swelling ability of CH is less in neutral pH a compared to acidic pH. Hence the formulation containing more of CH showed lower swellability in PBS that is the decrease was evident from LM 1 to LM 9 . In addition to this, the GL content was more in formulaton containing less amount of CH, this contributed better swelling in such batches. This information is reflected in Table 6.
The mucoadhesion property also varied with the CH amount. Attributing to the mucoadhesive nature of the ploymer, the higher the content of CH, better was the mucoadhesion(Sinha VR. et al, 2004). Thus there was a gradient decrease in mucoadhesion from LM 1to LM 9. The data is displayed in Table 6.
Table 6: Physical Characterizations of the Loaded Microsphere
|Batch Code||Average Entrapment
|Average Swelling Index#||Average Mucoadhesion#
|LM 1||54.23 ± 0.5||0.881 ± 0.8||89.56 ± 0.4|
|LM 2||53.58 ± 0.7||0.868 ± 2.3||89.23 ± 1.7|
|LM 3||51.29 ± 2.1||0.918 ± 0.3||88.7 ± 2.2|
|LM 4||48.83 ± 1.8||0.859 ± 1.2||86.38 ± 1.2|
|LM 5||43.41 ± 0.9||0.948 ± 0.5||85.92 ± 0.8|
|LM 6||44.6 ± 0.6||0.961 ± 1.1||84.81 ± 1.5|
|LM 7||42.5 ± 0.4||0.957 ± 1.5||85.13 ± 1.8|
|LM 8||41.76 ± 1.3||0.974 ± 0.6||83.8 ± 2.2|
|LM 9||38.9 ± 1.5||0.987 ± 0.9||82.61 ± 0.5|
# Values expressed as Mean±SD, n= 3
In-vitro release studies
Figure 7 shows the in-vitro release profiles obtained from the drug loaded microspheres, compared to the dissolution profile of the drug alone. The rate of dissolution of CZ powder was significantly lesser (approximately less than 50% of the drug dissolved in 120 min). The loading of CZ into the GL-CH microspheres led to an improvement of its dissolution rate. The increase in rate in dissolution was remarkable,from about 90- 100% of release drug was achieved in 120 min from the microspheres.
PBS was employed taking into considearation the fact that intranasal solutions have to be isotonic in nature.
This could be attributed to the polymer used. GL has no rate limiting property also, CH is already known for its properties of dissolution rate enhancer of poorly water soluble drugs(Gavini E. et al, 2006). The improvement of dissolution might also be contrbuted to the incorporation of the cosolvent which causes partial solubilization and uniform distribution of drug in the polymer blend prior to its addition into the paraffin during the preparation of microspheres.
This could be a useful characteristic for the nasal delivery of a drug of low or no solubility such as CZ (Gavini E. et al, 2006).
Fig. 7: In-vitro release studies of pure drug and the CZ loaded microspheres.
Ex- vivo studies
Figure 8 represents the CZ release profile after it permeates through the sheep nasal mucosa. The release profile shows CZ permeates quickly through mucosa into the dissolution medium. As in the case of in-vitro studies, the formultion showed improved release properties. This might be, owing to the presence of CH, surfactant and co- solvent. The batch containing the highest amount of CH showed the best release profile.
For a preliminary study of a nasal formulation the ex- vivo model used offers advantages like use of an official apparatus, rapid performance of test, and simple analytical procedure(Gavini E.et al, 2004).
Fig. 8: Ex-vivo release studies of pure drug and the CZ loaded microspheres.
Thus, using the technique of experimental design,plain GL-CH microsphers were successfully prepared. The batches with the best possible particle size of 61µm could be loaded with satisfactory entrapment taking into consideration the poor solubility of the drug. Also the other characterizations were obtained as desired. The release studies indicated complete release in a short period of time, hence hinting towards its use in case of acute seizure attacks, via the nasal route.Further, their potential to improve CZ bioavailability through the nasal route could be established by in - vivo evaluaton of CZ loaded microspheres in animals or human.
Shaji Jessy*, Poddar Aditi.
Affiliation : Prin. K. M. Kundnani College of Pharmacy,
23, Jote Joy Bldg., Rambhau Salgaonkar Marg, Cuffe Parade,
Colaba, Mumbai- 400005, Maharashtra.
Behl, CR, Pimplaskar, HK, Sileno, AP, De Meirelers, J, Romeo, V.(1998) Effects of Physicochemical Properties and other factors on Systemic Nasal Delivery. Adv. Drug Deli. Rev., 28, 89- 116.
Benita, S. Microencapsulation: Methods and Industrial Applications. 10th Ed. Vol 73. New York: Marcel Dekker Inc, 31- 7.
Dandagi, PM, Mastiholimath, VS, Gadad, AP, Iliger, SR. (2007) Mucoadhesive microspeheres of Propranolol Hydrochloride for Nasal Delivery. Indian Journal of Pharmaceutical sciences, 69, 402- 7.
Gavini, E, Hegge, AB, Rassu, G, Sanna, V, Tasta, C, Pirisino, G, Karlsen, J, Giunchedi, P.(2006) Nasal administration of Carbamazepine using Chitosan microspheres: In-vitro/ in-vivo studies. Int. J. Pharm., 307, 9- 15.
Gavini, E, Rasu, G, Sanna, V, Cossu, M, Giunchedi, P.(2004) Mucoadhesive microspheres for nasal administration of an antiemetic drug, metoclopramide: in-vitro/ ex-vivo studies. J. Pharm. Pharmacol., 57, 287- 94.
Giunchedi, P, Juliano, C, Gavini, E, Cossu, M, Soventi, M.(2002) Formulation and In-vivo evaluation of Chlorhexidine buccal tablets prepared using drug loaded chitosan microspheres. Eur. J. Pharm. Biopharm., 53, 233- 39.
Fraenkal – Conrat, H, Brandon, BA, and Olcott, HS. (1947) The Reaction of Formaldehyde Proteins: Cross – linking of Amino Groups with Phemol, Imidazol or Indole Groups.J. Biol. Chem., 174, 827 – 43.
Illum, L. Nasal drug delivery- possibilities, problems and solutions.(2003) J, Control, Rel., 87, 187- 98.
Illum, L, Jorgensoen, H, Bisgaard, H, Krogsgaard, O, Rossing, N.(2001) Bioadhesive microspheres as a potential nasal drug delivery system. Int. J. Pharm., 222, 109-119.
Jameela, SR, Kumary, TV, Lal, AV, Jayakrishnan, A.(1998) Progesterone-loaded chitosan microspheres: a long acting biodegradable controlled delivery system. J. Control. Rel., 53, 17-24.
Mathew, ST, Gayathri, Devi, S, Sandhya, KV.(2007) Formulaton and Evaluation of Ketorolac Tromethamine-loaded Albumin Microspheres for Potential Intramuscular administration. AAPS Pharm. SciTech. , 8, E1-E9.
Miglani, DS. Preparation and evaluation of controlled release oral dosage forms of glipizide and Nifedipine.(2002) Ph.D. thesis, University Institute of Pharmaceutical Sciences, Punjab University, Chandigarh..
Oryton, AC, Monteiro Jr. and Airoldi, C. (1999). Some Studies on Crosslinking Chitosan – Glutaraldehyde interaction in a homogenous system. Int. J. Pharm., 26, 119 – 28.
Parfitt K,. Martindale: The Complete Drug Reference. 32nd Ed. London: Pharmaceutical Press; 1999, 343- 344.
Patil, SB, Murthy, RSR.(2006) Preparation and In-vitro Evaluation of Mucoadhesive Chitosan Microspheres of Amlodipine Besylate for Nasal Administraton. Indian Journal of Pharmaceutical Sciences, 68, 64-7 .
Rao, R, Buri, P.(1989) A novel in- situ method to test polymers and coated microspheres for bioadhesion. Int. J. Pharm., 52, 265- 70.
Ravi, Kumar, MNV, Muzzarelli, RAA, Muzarelli, C, Sashivva, H, Domb, AJ.(2004) Chitosan Chemistry and Pharmaacuetical Perspectives. Chem.Rev. 104, 6067- 84.
Rowe, KC, Shesky, PJ, Owen, SC.(2006) Handbook of Pharmacutical Excipients. 5th Ed. Great Britain: Pharmaceutical Press and the American Pharmacists Associaton,.295- 298.
Sinha, VR, Singla, AK, Wadhawan, S, Kaushik, R, Kumria, R, Bansal, K, Dhawan, S. (2004) Chitosan microspheres as a potential carrier for drugs. Int. J. Pharm., 274, 1-33.
Sinko P.(2006) Martin’s Physical Pharmacy an Pharmaceutical Sciences. 5th Ed. Philadelphia: Lippincott Williams and Wilkin, 533- 560.
Vyas, TK, Babbar, AK, Sharma, RK, Singh, S, Misra, A.(2006) Intranasal Mucoadhesive Microemulsion of Clonazepam: Preliminary Studies on Brain Targeting. J. Pharm. Sci., 95, 570- 80.
Wei, HJ, Yang, HH, Chen, CH, Wei, WW, Chen, SC, Lai, PH, Chan, Y, Sung, HW.(2007) Gelatin Microspheres encapsulated with a Nonpeptide angiogenic agent, ginsenosideRg, for intramyocardial injection in a rat model with infarcted myocardium. J. Control. Rel.,120, 27- 34.
Copyright Priory Lodge Education Ltd. 2009
First Published September 2009