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See all Premium member Presentation Transcript Mucoadhesive drug delivery system : Mucoadhesive drug delivery system By Arvind Gohel M.Pharm(pharmaceutics) Outline : Outline In Vivo Techniques for Studying the Oral Mucosal Absorption. In Vitro Methods for Measuring Permeability of the Oral Mucosa. Nasal drug delivery system. Pulmonary drug delivery system. In Vivo Techniques for Studying the Oral Mucosal Absorption. : In Vivo Techniques for Studying the Oral Mucosal Absorption. knowledge of the absorption characteristics of the drug are important since, in both cases, it needs to be taken up by the oral mucosa. In the case of local delivery , knowledge of how long the drug remains in the mucosa following uptake is an important. In the case of systemic delivery the amount of drug crossing the oral mucosa and entering the blood is an important. A. ANIMAL TECHNIQUES Animal models such as the dog, cat , rabbit, hamster, rat, and sheep have been used to determine the oral mucosal absorption characteristics of drugs. The methodologies used can broadly be categorized into Application of dosage forms Absorption cells Perfusion cells. Application of dosage forms : Application of dosage forms Several different dosage forms have been applied to oral tissues of various animal species for the purpose of assessing the oral mucosal absorption characteristics of drugs. These include patches , bioadhesive tablets, non-bioadhesive tablets , adhesive film, ointments, bonded compacts, aqueous solutions and gels. The absorption characteristics of the drug were assessed either by measurement of the amount of drug reaching the blood or by removal and determination of the amount of drug remaining in the delivery system. Absorption Cells Absorption cells are defined as those techniques which restrict known volumes of an aqueous test solution to a defined area of oral mucosa. The cell can be open or closed to the oral environment, but in either case the test solution within the cell is protected from salivary secretions and therefore does not change in volume. An additional criterion which defines this type of cells is that the test solution is not stirred during an experiment. The simplest example of a method that uses an absorption cell is that described by Siegel and co-worker. To restrict the test solution to a defined surface area of oral mucosa (ventral surface of the rat tongue) Siegel and co-workers used a rubber O-ring with an internal diameter of 2.64 mm which was fixed to the mucosa using a cyanoacrylate adhesive. At the beginning of each experiment, the opening in the O-ring was filled with a small quantity (10 µL) of buffered test solution and the absorption characteristics of the organic solutes under investigation were determined by taking plasma samples and samples of the test solution in the O-ring. Investigators at the Ciba-Geigy Corporation, confined 1 mL of test solution on the buccal mucosa of anesthetized beagle dogs using a glass cell with an internal surface area of 1.5 cm2. The dog was positioned on a surgical table, the mouth was braced open and the tongue taped to the upper jaw. The cell was placed on the inner central buccal surface and retained in position using either silicone grease or by mechanical clamping for 4 hours. The extent of drug absorption was assessed by taking plasma samples and in order to quantify the total amount absorbed, at the end of the experiment the drug solution was aspirated from the cell and analyzed for drug content . Quadros briefly described a method involving a "cup" which exposed a surface area of Q.2 cm2 to investigate the absorption characteristics. Slide 5: The cup allowed 1 mL of drug solution to remain in contact with the buccal mucosa for 4 hours. The absorption characteristics of the drug were determined by blood samples. A closed cell made from stainless steel was designed by Ritschel et al . The cell was placed on the buccal mucosa of an anesthetized dog and maintained in position with two alligator clamps (Figure 1). Two holes were included in the top of the donor compartment; one for administration or sampling of the solution (on the outer perimeter) and the other for air ventilation (in the center). The inner surface of the cell exposed the test solution to 1.0 cm2 of buccal mucosa. To use the cell 0.5 mL of drug solution was added via the peripheral hole using a glass syringe with a blunt needle. After a set exposure period (e.g., 30 minutes) the solution was aspirated and the cell rinsed twice with 0.5 mL normal saline. The solutions were then combined and analyzed to determine the amount of drug that was administered. Blood samples were taken to determine the absorption characteristics of the drug under test. Oh and Ritschel also designed an apparatus (Figure 2) for retention of solutions on the buccal mucosa of the rabbit [17, 18]. Rabbits were anesthetized and the mouth cavity of the rabbit was cleaned with a cotton ball. The absorption cell (A) was placed in the buccal cavity of the rabbit and firmly attached to the buccal mucosa by tightening the knob (D) of the apparatus. 200 µL of drug solution was administered through the upper opening of the absorption cell (B) using a 250 µL micro syringe. After 1 hour the buccal cell was removed and the mouth cavity cleaned with a cotton wool ball. Drug absorption was measured by blood sampling. The dimensions of the cell were:12 mm 10, 14 mm 00, height 5 mm, capacity 300 µL and surface area 113 mm2. Kurosaki et al [29] prepared a closed absorption cell for use on various mucosa of the anesthetized hamster from the plastic body of a disposable needle for injection (Figure 3). The steel needle was cut off and two polyethylene tubes were fixed in position (Figure 3). The central tube was used as the inlet hole and allowed test solution to be entered into the cell, while the peripheral tube acted as a vent to allow air to escape during addition of the test solution. The cell was fixed to the surface of the oral mucosa with cyanoacrylate tissue cement. Drug absorption was monitored by periodically collecting blood. The dimensions of the cell are given in Figure 3. Slide 6: Kellaway [13, 14] developed a small cell machined out of a small block of Perspex (Figure 4) which comprised two concentric sealed chambers. The inner chamber (0.6 cm diameter, 0.486 cm3 volume) contained the drug solution, while the outer chamber was used to apply a vacuum which maintained the cell in position on the buccal mucosa of anesthetized cats. The cell exposed an area of 0.33 cm2 for the duration of an experiment (2 hours). Temporary association of the apparatus with the tissue caused no visible damage during a 2 hour application period. The inner chamber was equipped with two pipes to allow solution to be introduced into the inner compartment. To do this a known concentration of drug solution was injected through the inlet pipe until excess. drained from the outlet tube. The absorption characteristics of the drug under test were determined by blood sampling. Introduced a known volume of solution (0.5 mL) containing a known amount of drug into the cell at time t = O. By taking aliquots of the solution at defined time intervals and analyzing for drug concentration in those samples by HPLC . Perfusion Cells Perfusion cell techniques are characterized by the restriction of known volumes of an aqueous test solution to a defined area of oral mucosa using cells that are closed to the oral environment. The test solution is therefore protected from salivary secretions and do not change in either volume or pH during an experiment. In contrast to absorption cells the test solution is (i) well stirred and (ii) continually perfused across the mucosal surface throughout the duration of an experiment. The absorption characteristics of the drug can be determined either by monitoring drug loss from the perfusing solution or by..,measurement of blood levels. Veillard et al developed the perfusion cell shown in Figure 7 which was made from a medical grade silicone polymer. The cell had a volume of 0.075 cm3 and exposed an area of 0.25 cm2. It was attached to the buccal mucosa of anesthetized dogs using cyanoacrylate cement. A syringe pump was used to force aqueous drug solution Slide 7: Aqueous drug solution was perfused through the device for 30 minutes at a rate of I mg/mLl5 minutes and collected in I mL fractions. Blood samples were taken and used to determine the absorption characteristics of the drug . A glass perfusion cell (Figure 8) was developed by Yamahara et al . A flat faced, non irritating, biocompatible bioadhesive polymer a-ring was attached to the glass cell which allowed the cell to easily adhere to and be readily removed from the oral mucosa. Drug solution was maintained in a reservoir thermostatted to 37°C and recirculated at a rate of 2 mL minute by a peristaltic pump. Anesthetized male beagle dogs were used in the experiment with up to four cells being attached on a single dog at one time (Figure 10). A 2 or 3 hour perfusion time was employed and drug loss was monitored by either taking samples from the reservoir and replacing with fresh solvent or by taking blood samples. Weaver et al [2] reported the use of a Teflon@ cell in anesthetized dogs which was held in place against the surface of the buccal mucosa by the use of forceps and a rigid backing held against the cheek. The cell has an area of 2.8 cm2 and two 19 gauge needles provide inlet and outlet ports which allow the aqueous drug solution to be flushed across the mucosa. Forceps were used to retain the cell in position on the buccal mucosa with the beaks being held firmly. Initially normal saline should be pumped through the cell at a rate of 0.5 mL/minute and the effluent collected to confirm mass balance. After a minimum of 15 minutes of leak-tree operation the saline should be replaced with drug solution and the flow rate reduced to 0.2 mL/minute. The cell effluent should be collected throughout the treatment period and may be analyzed for drug content. B.HUMAN TECHNIQUES : B.HUMAN TECHNIQUES Accurate measurement of the oral mucosal absorption characteristics of drugs in man is needed for the rational design of a delivery system. Several methods are used include The buccal absorption test Disc methods perfusion cells. Buccal Absorption Test The buccal absorption test was introduced by Beckett and Triggs in 1967. The method involves controlled swirling of a buffered drug solution of known concentration around the oral cavity by movement of the cheeks and tongue approximately once per second. After a known period of time the solution is expelled and the subject rinses their mouth with an aliquot of either distilled water or an appropriate buffer solution. The drug solution and rinse are then combined, adjusted to volume and analyzed for drug content. The difference between the amount of drug contained in the original solution and the amount recovered is assumed to be the amount of drug lost into the oral cavity mucosa during the test period. Drug loss is usually expressed as a percentage of the original amount entered. If the percentage loss over a fixed time period is measured this provides information on the relative permeability properties of drugs under different condition. Advantages It is a simple to perform, non-invasive. Both the rate and extent of drug loss from the oral cavity from aqueous solutions can be measured, Slide 9: Disadvantage The amount of intact drug that reaches the systemic circulation remains unknown as only loss from the oral cavity is monitored. Differences in the absorption characteristics of the different membranes that line the oral cavity cannot be determined as absorption simultaneously takes place over all mucosal surfaces. The determination of the kinetics of absorption is time consuming because not only has each time point to be determined on separate occasions but also a waiting period must be observed to ensure that at the beginning of each experiment the oral mucosa is void of drug. Disc Methods Absorption across a defined oral cavity mucosa can be studied. Kaaber used a gravimetric method for measuring the uptake or loss of water using a small filter paper disc applied to the mucosal surface. Schurr and Ziegler used a Polytef disc with a diameter of approximately 3.5 cm and height of 1cm. The disc had a central circular depression depth of 4 mm which left an elevated rim. A water-soaked filter paper disc was placed in the depression and a known amount of drug spread onto the filter paper. Once the drug had dissolved the device was placed on the surface of buccal mucosa. Pimlott and Addy [81] placed a drug soaked filter paper disc of known dimensions onto a defined oral mucosal surface and maintained it in place for 5 minutes. After removal a nonimpregnated disc was used to wipe the oral mucosa, the discs combined, analyzed for drug content and the extent of drug disappearance calculated. Slide 10: Perfusion Cells Three perfusion cells have been used in human volunteers. All have utilized the buccal membrane as the platform for perfusion. (a). Barsuhn et al Barsuhn et al constructed a pliable cell made of a hydrophilic vinyl polysiloxane polymer which had an internal volume of 1 mL and allowed a 1.8 cm area of buccal membrane to be perfused (Figure 13). Before placement of the perfusion cell , the membrane of each subject was rinsed with sterile saline solution applied with a cotton gauze. Cells were placed visually. Perfusing drug solutions were maintained at 37°C and recirculated at 10 mL/minute using a reciprocating piston pump. During a 42 minute perfusion time, 0.05 mL samples were removed from the stirred reservoir every 7 minutes and analyzed for drug content. Absorption of the drug was measured from the rate of disappearance of drug from the reservoir. (b). Rathbone Rathbone reported an improved buccal perfusion cell design. this perfusion cell was constructed from an inflexible material such as nylon or Teflon. (c). Kurosaki et al Here in addition to investigating the buccal membrane as the platform for perfusion these authors also studied the absorption characteristics of drugs across the dorsum of the tongue, ventral surface of the tongue, labial mucosa and floor of the mouth of human volunteers. The cells had inner diameters of 13 or 16 mm were clamped on the human oral mucosa and 5 mL of test solution was recirculated at a flow rate of 0.8 mL/minute using a peristaltic pump. The absorption characteristics of the drugs were measured by following their disappearance from the perfusing drug solution. In Vitro Methods for Measuring Permeability of the Oral Mucosa : In Vitro Methods for Measuring Permeability of the Oral Mucosa In vitro permeability studies are more suitable for studying mechanism of drug transport across oral mucosa because the tissue is isolated from whole animal thus restricting the number of variable that need to be controlled. The conditions of the experiment, such as temperature, pH, osmolarity, etc. can be easily controlled. The permeability of the tissue is measured by the drug concentration on the receiver side, which by analogy, is equivalent to drug that is available to the circulation. ANIMAL MODELS Since human oral mucosa is not widely available, animal oral mucosa is routinely used for in vitro studies. The most commonly used animals are dogs, rabbits, pigs, Rhesus monkeys, guinea pigs, rats and hamsters. Animals are sacrificed ,Oral mucosa is removed from the oral cavity and placed in ice-cold buffer solution. The connective tissue is removed. The cleaned oral mucosa is then placed in ice-cold buffer before mounting in the diffusion cell. Another method used for tissue preparation are chemical , thermal and enzymatic method. A. Experimental Details : A. Experimental Details A diagram of a typical plastic side-by-side diffusion cell is shown in Figure below. There are three components in the system, the donor and receiver chambers and the oral mucosa sandwiched in the middle. The donor chamber refers to the side with high drug concentration which mimics the site of drug delivery whereas the receiver chamber mimics the general circulation. The epithelial side of the oral mucosa usually faces the donor chamber except when back diffusion is examined to explore the permeability symmetry of the tissue. In this case, the epithelial side faces the receiver chamber. in both donor and receiver chambers are agitated by magnetic bars or gas bubbles to reduce the stagnant diffusion layer. Samples are taken periodically from the receiver chamber and replaced by fresh buffer solution. A variety of diffusion cells such as the Franz, small volume diffusion cells ,homemade modified Ussing chambers are used. B. Permeability Coefficient Measurement For most drugs, absorption via the oral mucosa is a passive diffusion process and can be described by Fick's first law J = (DKp /h)Cd where J = the flux of the drug absorption. D = the diffusion coefficient of the drug inside the oral mucosa. Kp = the partition coefficient between the oral mucosa and the buffer solution. h = the effective path length of the oral mucosa across which the drug must traverse CD = drug concentration in the donor chamber. Slide 13: It is impractical to measure D, Kp and h. However, the permeability coefficient, P, which is defined as: P = DKp /h In a typical diffusion experiment, the amount of drug (A) moving across the tissue at time t is determined by measuring drug concentration on the receiver side. The surface area of the tissue (A) and the initial drug concentration in the donor chamber are known and remain constant during the course of the experiment. Thus, the flux is: J = PCd = A /St The amount of drug transported at time t is: A = PSCdt The total amount of the drug (AT) on the donor side is: At = VdCd where Vd = the volume of the donor chamber, The percent of drug transported (T%) at time t is: T% = A /At =( PS /Vd)t A plot of T% versus t will give a slope of PS/Vd and from this slope ,the permeability coefficient can be determined. You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
Mucoadhesive drug delivery system AG1 arvindgohel Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 1768 Category: Education License: All Rights Reserved Like it (3) Dislike it (0) Added: January 21, 2009 This Presentation is Public Favorites: 1 Presentation Description No description available. Comments Posting comment... By: sumanjali.d (8 month(s) ago) dear sir, presentation is very and informative, so kindly send this to my email i.d anjali2327@gmail.com Saving..... Post Reply Close Saving..... Edit Comment Close By: rapa63 (15 month(s) ago) respected sir,please send me a copy of this presentation to me,i will be very thankfull to you. my email id is rapa63@gmail.com Saving..... Post Reply Close Saving..... Edit Comment Close By: monali_86 (15 month(s) ago) Dear sir ,Please send me a copy of this presentation to my email mkparbhane@gmail.com Saving..... Post Reply Close Saving..... Edit Comment Close By: hareesha (23 month(s) ago) sir this ppt was use full for my academic can you send this to my mail id harishahari2000@gmail.com , Saving..... Post Reply Close Saving..... Edit Comment Close By: aups (23 month(s) ago) Dear sir I am a M.pharm pharmaceutics student from bangalore . I really impressed by your ppt file and it is highly informative and it will be healpful for me .... sir please send me a copy to my mail ID pragatimpharm@yahoo.co.in Saving..... Post Reply Close Saving..... Edit Comment Close loading.... See all Premium member Presentation Transcript Mucoadhesive drug delivery system : Mucoadhesive drug delivery system By Arvind Gohel M.Pharm(pharmaceutics) Outline : Outline In Vivo Techniques for Studying the Oral Mucosal Absorption. In Vitro Methods for Measuring Permeability of the Oral Mucosa. Nasal drug delivery system. Pulmonary drug delivery system. In Vivo Techniques for Studying the Oral Mucosal Absorption. : In Vivo Techniques for Studying the Oral Mucosal Absorption. knowledge of the absorption characteristics of the drug are important since, in both cases, it needs to be taken up by the oral mucosa. In the case of local delivery , knowledge of how long the drug remains in the mucosa following uptake is an important. In the case of systemic delivery the amount of drug crossing the oral mucosa and entering the blood is an important. A. ANIMAL TECHNIQUES Animal models such as the dog, cat , rabbit, hamster, rat, and sheep have been used to determine the oral mucosal absorption characteristics of drugs. The methodologies used can broadly be categorized into Application of dosage forms Absorption cells Perfusion cells. Application of dosage forms : Application of dosage forms Several different dosage forms have been applied to oral tissues of various animal species for the purpose of assessing the oral mucosal absorption characteristics of drugs. These include patches , bioadhesive tablets, non-bioadhesive tablets , adhesive film, ointments, bonded compacts, aqueous solutions and gels. The absorption characteristics of the drug were assessed either by measurement of the amount of drug reaching the blood or by removal and determination of the amount of drug remaining in the delivery system. Absorption Cells Absorption cells are defined as those techniques which restrict known volumes of an aqueous test solution to a defined area of oral mucosa. The cell can be open or closed to the oral environment, but in either case the test solution within the cell is protected from salivary secretions and therefore does not change in volume. An additional criterion which defines this type of cells is that the test solution is not stirred during an experiment. The simplest example of a method that uses an absorption cell is that described by Siegel and co-worker. To restrict the test solution to a defined surface area of oral mucosa (ventral surface of the rat tongue) Siegel and co-workers used a rubber O-ring with an internal diameter of 2.64 mm which was fixed to the mucosa using a cyanoacrylate adhesive. At the beginning of each experiment, the opening in the O-ring was filled with a small quantity (10 µL) of buffered test solution and the absorption characteristics of the organic solutes under investigation were determined by taking plasma samples and samples of the test solution in the O-ring. Investigators at the Ciba-Geigy Corporation, confined 1 mL of test solution on the buccal mucosa of anesthetized beagle dogs using a glass cell with an internal surface area of 1.5 cm2. The dog was positioned on a surgical table, the mouth was braced open and the tongue taped to the upper jaw. The cell was placed on the inner central buccal surface and retained in position using either silicone grease or by mechanical clamping for 4 hours. The extent of drug absorption was assessed by taking plasma samples and in order to quantify the total amount absorbed, at the end of the experiment the drug solution was aspirated from the cell and analyzed for drug content . Quadros briefly described a method involving a "cup" which exposed a surface area of Q.2 cm2 to investigate the absorption characteristics. Slide 5: The cup allowed 1 mL of drug solution to remain in contact with the buccal mucosa for 4 hours. The absorption characteristics of the drug were determined by blood samples. A closed cell made from stainless steel was designed by Ritschel et al . The cell was placed on the buccal mucosa of an anesthetized dog and maintained in position with two alligator clamps (Figure 1). Two holes were included in the top of the donor compartment; one for administration or sampling of the solution (on the outer perimeter) and the other for air ventilation (in the center). The inner surface of the cell exposed the test solution to 1.0 cm2 of buccal mucosa. To use the cell 0.5 mL of drug solution was added via the peripheral hole using a glass syringe with a blunt needle. After a set exposure period (e.g., 30 minutes) the solution was aspirated and the cell rinsed twice with 0.5 mL normal saline. The solutions were then combined and analyzed to determine the amount of drug that was administered. Blood samples were taken to determine the absorption characteristics of the drug under test. Oh and Ritschel also designed an apparatus (Figure 2) for retention of solutions on the buccal mucosa of the rabbit [17, 18]. Rabbits were anesthetized and the mouth cavity of the rabbit was cleaned with a cotton ball. The absorption cell (A) was placed in the buccal cavity of the rabbit and firmly attached to the buccal mucosa by tightening the knob (D) of the apparatus. 200 µL of drug solution was administered through the upper opening of the absorption cell (B) using a 250 µL micro syringe. After 1 hour the buccal cell was removed and the mouth cavity cleaned with a cotton wool ball. Drug absorption was measured by blood sampling. The dimensions of the cell were:12 mm 10, 14 mm 00, height 5 mm, capacity 300 µL and surface area 113 mm2. Kurosaki et al [29] prepared a closed absorption cell for use on various mucosa of the anesthetized hamster from the plastic body of a disposable needle for injection (Figure 3). The steel needle was cut off and two polyethylene tubes were fixed in position (Figure 3). The central tube was used as the inlet hole and allowed test solution to be entered into the cell, while the peripheral tube acted as a vent to allow air to escape during addition of the test solution. The cell was fixed to the surface of the oral mucosa with cyanoacrylate tissue cement. Drug absorption was monitored by periodically collecting blood. The dimensions of the cell are given in Figure 3. Slide 6: Kellaway [13, 14] developed a small cell machined out of a small block of Perspex (Figure 4) which comprised two concentric sealed chambers. The inner chamber (0.6 cm diameter, 0.486 cm3 volume) contained the drug solution, while the outer chamber was used to apply a vacuum which maintained the cell in position on the buccal mucosa of anesthetized cats. The cell exposed an area of 0.33 cm2 for the duration of an experiment (2 hours). Temporary association of the apparatus with the tissue caused no visible damage during a 2 hour application period. The inner chamber was equipped with two pipes to allow solution to be introduced into the inner compartment. To do this a known concentration of drug solution was injected through the inlet pipe until excess. drained from the outlet tube. The absorption characteristics of the drug under test were determined by blood sampling. Introduced a known volume of solution (0.5 mL) containing a known amount of drug into the cell at time t = O. By taking aliquots of the solution at defined time intervals and analyzing for drug concentration in those samples by HPLC . Perfusion Cells Perfusion cell techniques are characterized by the restriction of known volumes of an aqueous test solution to a defined area of oral mucosa using cells that are closed to the oral environment. The test solution is therefore protected from salivary secretions and do not change in either volume or pH during an experiment. In contrast to absorption cells the test solution is (i) well stirred and (ii) continually perfused across the mucosal surface throughout the duration of an experiment. The absorption characteristics of the drug can be determined either by monitoring drug loss from the perfusing solution or by..,measurement of blood levels. Veillard et al developed the perfusion cell shown in Figure 7 which was made from a medical grade silicone polymer. The cell had a volume of 0.075 cm3 and exposed an area of 0.25 cm2. It was attached to the buccal mucosa of anesthetized dogs using cyanoacrylate cement. A syringe pump was used to force aqueous drug solution Slide 7: Aqueous drug solution was perfused through the device for 30 minutes at a rate of I mg/mLl5 minutes and collected in I mL fractions. Blood samples were taken and used to determine the absorption characteristics of the drug . A glass perfusion cell (Figure 8) was developed by Yamahara et al . A flat faced, non irritating, biocompatible bioadhesive polymer a-ring was attached to the glass cell which allowed the cell to easily adhere to and be readily removed from the oral mucosa. Drug solution was maintained in a reservoir thermostatted to 37°C and recirculated at a rate of 2 mL minute by a peristaltic pump. Anesthetized male beagle dogs were used in the experiment with up to four cells being attached on a single dog at one time (Figure 10). A 2 or 3 hour perfusion time was employed and drug loss was monitored by either taking samples from the reservoir and replacing with fresh solvent or by taking blood samples. Weaver et al [2] reported the use of a Teflon@ cell in anesthetized dogs which was held in place against the surface of the buccal mucosa by the use of forceps and a rigid backing held against the cheek. The cell has an area of 2.8 cm2 and two 19 gauge needles provide inlet and outlet ports which allow the aqueous drug solution to be flushed across the mucosa. Forceps were used to retain the cell in position on the buccal mucosa with the beaks being held firmly. Initially normal saline should be pumped through the cell at a rate of 0.5 mL/minute and the effluent collected to confirm mass balance. After a minimum of 15 minutes of leak-tree operation the saline should be replaced with drug solution and the flow rate reduced to 0.2 mL/minute. The cell effluent should be collected throughout the treatment period and may be analyzed for drug content. B.HUMAN TECHNIQUES : B.HUMAN TECHNIQUES Accurate measurement of the oral mucosal absorption characteristics of drugs in man is needed for the rational design of a delivery system. Several methods are used include The buccal absorption test Disc methods perfusion cells. Buccal Absorption Test The buccal absorption test was introduced by Beckett and Triggs in 1967. The method involves controlled swirling of a buffered drug solution of known concentration around the oral cavity by movement of the cheeks and tongue approximately once per second. After a known period of time the solution is expelled and the subject rinses their mouth with an aliquot of either distilled water or an appropriate buffer solution. The drug solution and rinse are then combined, adjusted to volume and analyzed for drug content. The difference between the amount of drug contained in the original solution and the amount recovered is assumed to be the amount of drug lost into the oral cavity mucosa during the test period. Drug loss is usually expressed as a percentage of the original amount entered. If the percentage loss over a fixed time period is measured this provides information on the relative permeability properties of drugs under different condition. Advantages It is a simple to perform, non-invasive. Both the rate and extent of drug loss from the oral cavity from aqueous solutions can be measured, Slide 9: Disadvantage The amount of intact drug that reaches the systemic circulation remains unknown as only loss from the oral cavity is monitored. Differences in the absorption characteristics of the different membranes that line the oral cavity cannot be determined as absorption simultaneously takes place over all mucosal surfaces. The determination of the kinetics of absorption is time consuming because not only has each time point to be determined on separate occasions but also a waiting period must be observed to ensure that at the beginning of each experiment the oral mucosa is void of drug. Disc Methods Absorption across a defined oral cavity mucosa can be studied. Kaaber used a gravimetric method for measuring the uptake or loss of water using a small filter paper disc applied to the mucosal surface. Schurr and Ziegler used a Polytef disc with a diameter of approximately 3.5 cm and height of 1cm. The disc had a central circular depression depth of 4 mm which left an elevated rim. A water-soaked filter paper disc was placed in the depression and a known amount of drug spread onto the filter paper. Once the drug had dissolved the device was placed on the surface of buccal mucosa. Pimlott and Addy [81] placed a drug soaked filter paper disc of known dimensions onto a defined oral mucosal surface and maintained it in place for 5 minutes. After removal a nonimpregnated disc was used to wipe the oral mucosa, the discs combined, analyzed for drug content and the extent of drug disappearance calculated. Slide 10: Perfusion Cells Three perfusion cells have been used in human volunteers. All have utilized the buccal membrane as the platform for perfusion. (a). Barsuhn et al Barsuhn et al constructed a pliable cell made of a hydrophilic vinyl polysiloxane polymer which had an internal volume of 1 mL and allowed a 1.8 cm area of buccal membrane to be perfused (Figure 13). Before placement of the perfusion cell , the membrane of each subject was rinsed with sterile saline solution applied with a cotton gauze. Cells were placed visually. Perfusing drug solutions were maintained at 37°C and recirculated at 10 mL/minute using a reciprocating piston pump. During a 42 minute perfusion time, 0.05 mL samples were removed from the stirred reservoir every 7 minutes and analyzed for drug content. Absorption of the drug was measured from the rate of disappearance of drug from the reservoir. (b). Rathbone Rathbone reported an improved buccal perfusion cell design. this perfusion cell was constructed from an inflexible material such as nylon or Teflon. (c). Kurosaki et al Here in addition to investigating the buccal membrane as the platform for perfusion these authors also studied the absorption characteristics of drugs across the dorsum of the tongue, ventral surface of the tongue, labial mucosa and floor of the mouth of human volunteers. The cells had inner diameters of 13 or 16 mm were clamped on the human oral mucosa and 5 mL of test solution was recirculated at a flow rate of 0.8 mL/minute using a peristaltic pump. The absorption characteristics of the drugs were measured by following their disappearance from the perfusing drug solution. In Vitro Methods for Measuring Permeability of the Oral Mucosa : In Vitro Methods for Measuring Permeability of the Oral Mucosa In vitro permeability studies are more suitable for studying mechanism of drug transport across oral mucosa because the tissue is isolated from whole animal thus restricting the number of variable that need to be controlled. The conditions of the experiment, such as temperature, pH, osmolarity, etc. can be easily controlled. The permeability of the tissue is measured by the drug concentration on the receiver side, which by analogy, is equivalent to drug that is available to the circulation. ANIMAL MODELS Since human oral mucosa is not widely available, animal oral mucosa is routinely used for in vitro studies. The most commonly used animals are dogs, rabbits, pigs, Rhesus monkeys, guinea pigs, rats and hamsters. Animals are sacrificed ,Oral mucosa is removed from the oral cavity and placed in ice-cold buffer solution. The connective tissue is removed. The cleaned oral mucosa is then placed in ice-cold buffer before mounting in the diffusion cell. Another method used for tissue preparation are chemical , thermal and enzymatic method. A. Experimental Details : A. Experimental Details A diagram of a typical plastic side-by-side diffusion cell is shown in Figure below. There are three components in the system, the donor and receiver chambers and the oral mucosa sandwiched in the middle. The donor chamber refers to the side with high drug concentration which mimics the site of drug delivery whereas the receiver chamber mimics the general circulation. The epithelial side of the oral mucosa usually faces the donor chamber except when back diffusion is examined to explore the permeability symmetry of the tissue. In this case, the epithelial side faces the receiver chamber. in both donor and receiver chambers are agitated by magnetic bars or gas bubbles to reduce the stagnant diffusion layer. Samples are taken periodically from the receiver chamber and replaced by fresh buffer solution. A variety of diffusion cells such as the Franz, small volume diffusion cells ,homemade modified Ussing chambers are used. B. Permeability Coefficient Measurement For most drugs, absorption via the oral mucosa is a passive diffusion process and can be described by Fick's first law J = (DKp /h)Cd where J = the flux of the drug absorption. D = the diffusion coefficient of the drug inside the oral mucosa. Kp = the partition coefficient between the oral mucosa and the buffer solution. h = the effective path length of the oral mucosa across which the drug must traverse CD = drug concentration in the donor chamber. Slide 13: It is impractical to measure D, Kp and h. However, the permeability coefficient, P, which is defined as: P = DKp /h In a typical diffusion experiment, the amount of drug (A) moving across the tissue at time t is determined by measuring drug concentration on the receiver side. The surface area of the tissue (A) and the initial drug concentration in the donor chamber are known and remain constant during the course of the experiment. Thus, the flux is: J = PCd = A /St The amount of drug transported at time t is: A = PSCdt The total amount of the drug (AT) on the donor side is: At = VdCd where Vd = the volume of the donor chamber, The percent of drug transported (T%) at time t is: T% = A /At =( PS /Vd)t A plot of T% versus t will give a slope of PS/Vd and from this slope ,the permeability coefficient can be determined.