Excised Porcine Buccal Mucosa Research Articles

Evaluation of pilocarpine in situ forming buccal gels as potential formulations for the management of xerostomia

Buccally administrated pilocarpine (PIL) can be a unique formulation approach for the management of xerostomia. In this way the severity of systemically induced side effects can be effectively controlled, while maintaining a high level of therapeutic efficacy. The aim of the present study was to evaluate the sol–gel and mucoadhesive properties of an in situ thermoresponsive hydrogel designed for PIL's buccal administration. For this purpose, poloxamer 407 (P407) and 188 (P188) were used as in situ gel forming agents, while xanthan gum (XG) was added as a mucoadhesion enhancer. Implementation of a Box-Behnken experimental design, combined with multi-linear regression (MLR) fitting, unraveled the role of formulation factors on several critical product attributes (such as gel transition temperature, gelation time, solution viscosity and gel viscosity). Artificial neural networks (ANNs) showed excellent generalizing ability, with verification experiments showing good agreement between the ANN- predicted and the experimentally-obtained results. The optimum sol-gel formulations were sprayable at room/storage temperature, while forming a strong gel at temperatures prevailing in the buccal cavity. In vitro dissolution profiles of all optimum formulations exhibited a sustained release pattern (releasing almost 80% of the total dose in 360 min) characterized by an initial rapid release followed by a steady state plateau. The evaluation of drug release kinetics, by applying different release kinetic models, suggested that the drug release from the in situ gels predominantly follows Fick's law and is primarily diffusion-controlled. The in vitro studies were complemented with ex vivo studies, performed on a freshly excised porcine buccal mucosa, showing good mucoadhesion properties with approximately 27% of the total drug permeating the buccal porcine mucosa, while no damage to the epithelium was observed based on the histological evaluation.

Buccal mucosal application of dissolvable microneedle patch containing photosensitizer provides effective localized delivery and phototherapy against oral carcinoma

The effectiveness of phototherapy using photosensitizers is limited by the challenges in their delivery at the site of irradiation. Here, we demonstrate the localized application of a photosensitizer-loaded microneedle patch for effective photodynamic and photothermal therapy in oral carcinoma. Indocyanine green (ICG) was studied as a photosensitizer for its effect on oral carcinoma, FaDu cells. Different parameters including concentration, near-infrared (NIR) laser irradiation intensity and irradiation time were optimized while measuring temperature increase and reactive oxygen species (ROS) generation in FaDu cells. A dissolvable microneedle (DMN) patch made of sodium carboxymethyl cellulose and sodium alginate was fabricated by the micromolding technique. DMN showed sufficient mechanical strength for insertion in the excised porcine buccal mucosa. DMN dissolved within 30 s in phosphate buffer and 30 min in the excised buccal mucosa. Confocal microscopy studies revealed DMN penetration up to a depth of 300 µm within the buccal mucosa. ICG-DMN applied on the back of the rat was found to be localized at the application site before and after irradiation using an 808 nm NIR laser. ICG-DMN was applied on the FaDu xenografted tumor model in athymic nude mice. The localized temperature increase and ROS generation significantly (P < 0.05) decreased the tumor volume after ICG-DMN application compared with the control group. In conclusion, DMN can be developed for the localized administration of photosensitizers for phototherapy in oral carcinoma.

Controlled Delivery of Apomorphine Through Buccal Mucosa, Towards a Noninvasive Administration Method in Parkinson's Disease: A Preclinical Mechanistic Study

Apomorphine (APO), a potent treatment for Parkinson's disease, is only administered parenterally either as intermittent injections or as an infusion. This is due to extensive hepatic “first pass” metabolism. Prolonged delivery through buccal mucosa may be potential substitute for parenteral infusions. To investigate this concept of buccal mucosal delivery, permeability ex vivo studies were performed through excised porcine buccal mucosa by utilizing Ussing diffusion chamber. Permeability rates were assessed for APO from simulated saliva medium at pH 7.4 as well as with utilization of different permeability modifying methods. Lowering the pH to 5.9 decreased permeability rate six-fold, while addition of ethanol : propylene glycol solution elevated it four-fold. Addition of nano-scale lipospheres to the donor compartment delayed the accumulation of APO at the receiver side, prolongating the lag-time from one to approx. three hours. These findings were strengthened by results obtained with co-administration of permeability markers (standards) atenolol and metoprolol. Simulation of the obtained permeability rates to in vivo setup in human showed therapeutically relevant plasma levels when using the outcomes of the current study. These findings verify the novel concept of APO prolonged release buccal administration as a noninvasive substitute for parenteral infusions in treating Parkinson's disease.

A preliminary study for the development and optimization by experimental design of an in vitro method for prediction of drug buccal absorption

The work was aimed at developing an in vitro method able to provide rapid and reliable evaluation of drug absorption through buccal mucosa. Absorption simulator apparatus endowed with an artificial membrane was purposely developed by experimental design. The apparent permeation coefficient (Papp) through excised porcine buccal mucosa of naproxen, selected as model drug, was the target value to obtain with the artificial membrane. The multivariate approach enabled systematic evaluation of the effect on the response (Papp) of simultaneous variations of the variables (kind of lipid components for support impregnation and relative amounts). A screening phase followed by a response-surface study allowed optimization of the lipid-mixture composition to obtain the desired Papp value, and definition of a design space where all mixture components combinations fulfilled the desired target at a fixed probability level. The method offers a useful tool for a quick screening in the early stages of drug discovery and/or in preformulation studies, improving efficiency and chance of success in the development of buccal delivery systems. Further studies with other model drugs are planned to confirm the buccal absorption predictive capacity of the developed membrane.

Buccal patches of atenolol formulated using fenugreek (Trigonella foenum-graecum L.) seed mucilage.

The use of mucoadhesive natural polymers in designing mucoadhesive patch systems has received much attention. The study involved the development and evaluation of buccal patches of atenolol using fenugreek (Trigonella foenum-graecum L.) seed mucilage with hydroxylpropyl methyl cellulose (HPMC K4M) and a backing membrane (ethyl cellulose 5% w/v). These atenolol-releasing buccal patches were prepared using a solvent casting technique. The buccal patches prepared were evaluated for average weight, thickness, drug content, folding endurance and moisture content. Ex vivo mucoadhesive strength, force of adhesion and bonding strength were determined using porcine buccal mucosa. The mucosal permeation of atenolol through the porcine buccal mucosa was carried out using a Franz diffusion cell in phosphate buffer saline, pH 6.8. These buccal patches were also characterized by SEM and FTIR spectroscopy. The average weight, thickness, drug content, folding endurance and moisture content of these atenolol-releasing buccal patches were found satisfactory for all the patches. Amongst all, the F-4 buccal patch showed maximum mucoadhesive strength (31.12 ±1.86 g), force of adhesion (30.53 × 10-2 N) and bond strength (1748.89 N/m2). Ex vivo atenolol permeation from the buccal patches showed drug permeation across the excised porcine buccal mucosa over 12 h. The F-4 buccal patch showed maximum permeation flux (29.12 μg/cm2/h). The developed atenolol-releasing buccal patches can be beneficial over the conventional drug delivery systems to decrease the dosing frequency and enhance patient compliance.

In vitro permeability of silver nanoparticles through porcine oromucosal membrane

Silver nanoparticles (AgNPs) can come in contact with human oral mucosa due to their wide use in food industry and hygiene devices. We evaluate transmucosal absorption of 19nm AgNPs using excised porcine buccal mucosa applied on Franz diffusion cells. Two donor solutions were used: one containing AgNPs (0.5g/L) and one derived from the ultrafiltration of the former and containing only Ag in its soluble form. Experiments were carried out separately for 4h. Silver flux permeation was demonstrated through oral mucosa, showing similar values for AgNPs (6.8±4.5ngcm−2h−1) and Ag ions (5.2±4.3ngcm−2h−1). Our study demonstrates that silver can permeate the oromucosal barrier and that absorption is substantially due to Ag ions, since no permeation difference was found using the two solutions. Mucosal absorption has to be considered in further risk assessment studies.

Transbuccal delivery of doxepin: Studies on permeation and histological investigation

According to previous studies reporting the anesthetic/analgesic action of oral topical doxepin administration, this study evaluated a model of buccal permeation to determine the depth of delivery of doxepin into excised porcine buccal mucosa following topical application of a saturated aqueous doxepin solution. Buccal mucosa permeation studies were performed using Franz diffusion cells. Cumulative amounts of doxepin permeated were plotted as a function of time. Kinetic permeation parameters as flux (Js), lag time (Tl) and permeability coefficient (Kp) were calculated. Theoretical human plasmatic steady-state doxepin concentration and drug retained in the tissue were also determined in order to evaluate its potential therapeutic use, central or peripheral. Finally, a histological evaluation of the buccal mucosa was performed to test potential damage due to the permeation phenomenon. Obtained results showed a poor aqueous doxepin permeation through buccal mucosa membrane (median parameters Js=34.79μg/h, Kp=0.49×10−3cm/h and Tl=2.8h). Predicted doxepin plasma concentrations would reach 46ng/mL, far from the required to have central nervous system activity as tricyclic agent. However, median doxepin amount remaining in the mucosa membrane was 0.24μg/cm2/μg tissue, which evidenced a reservoir function of the buccal mucosa. Histologically, no structural damage was observed in the tissues. This study lays the foundation for further research within this area with a view to potentially adopting alternative strategies for enhanced buccal absorption of doxepin in clinical practice.

Evaluation of a physiological in vitro system to study the transport of nanoparticles through the buccal mucosa

A buccal physiological in vitro testing system for the evaluation of the permeability, the transport route and toxic effects of nanoparticles was developed. Carboxyl polystyrene (CP, 20 nm, 200 nm) and amine modified polystyrene (AP, 200 nm) particles were used as reference particles and characterized in biological media. The permeability through excised porcine buccal mucosa was investigated with Franz diffusion cells. To evaluate the transport route, particle uptake into oral H376 cells was recorded and the cell damage was measured. All particles immediately formed aggregates once dispersed in saliva. 20 nm CP particles permeated the mucus layer and penetrated into the stratum superficiale of the top third region of the epithelium by the transcellular route. The positively-charged 200 nm AP particles permeated the mucus-layer and penetrated into deeper regions of the tissue. By decreasing the temperature to 4°C, particle uptake was inhibited for 20 nm CP and 200 nm AP particles. 200 nm CP particles interacted with the mucus, formed agglomerates and did not penetrate into the epithelium. It can be concluded that the presented system serves as a valuable tool to evaluate the behavior of nanoparticles in the buccal mucosa.

In Vitro Evaluation of Various Buccal Permeation Enhancing Systems for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide)

Buccal administration of pituitary adenylate cyclase-activating polypeptide (PACAP) could represent a new possibility for the treatment of type 2 diabetes. In this study the effect of various buccal permeation enhancers on PACAP and FD-4 was evaluated. The permeation-enhancing properties of the well-established permeation enhancers sodium deoxycholate (Na DOC) and cetrimide on the permeation of PACAP were investigated on freshly excised porcine buccal mucosa in Ussing chambers. Furthermore, the effect of chitosan and that of chitosan-4-thiobutylamidine conjugate (chitosan-TBA) optionally in combination with reduced glutathione (GSH) on the permeation of PACAP across the buccal mucosa was studied. The apparent permeability coefficient (P(app)) of PACAP in buffer only was 5.7 +/- 3.1x10(-8) cm/s. In the presence of 5% (m/v) Na DOC, the enhancement of the permeation was 18.6-fold, whereas due to the addition of 5% (m/v) cetrimide an enhancement ratio of 46.5 was obtained. In the presence of the chitosan-TBA conjugate (1%), a 38.9-fold increased permeation was achieved, whereas unmodified chitosan (1%) did not show any effect. The combination of chitosan-TBA conjugate (1%) with GSH (2%) led to an increase in P(app) up to 441.7 +/- 89.9x10(-8) cm/s, which represents a 77.5-fold improvement. The P(app) of GSH per se was only 1.0 +/- 0.2x10(-9) cm/s, showing that GSH remains concentrated on the surface of the buccal mucosa. Results were confirmed by additional permeation studies performed with FD-4 used as hydrophilic macromolecular test compound. Based on their permeation-enhancing properties, chitosan-TBA conjugates represent a promising tool for the buccal administration of peptide drugs, e.g., PACAP.

Ex Vivo Permeability Characteristics of Porcine Buccal Mucosa to Drugs with Various Polarity

The aim of this study was to analyze characteristics of the barrier function of excised porcine buccal mucosa to the test compounds, estradiol, propranolol HCI, melatonin, and mannitol with a wide range of partition coefficient values. The permeability of melatonin was measured through frozen, stored, and fresh porcine buccal mucosa to examine the impact of storage conditions on the permeability of porcine buccal mucosa. The results demonstrated that the ex vivo permeability of the porcine buccal mucosa was greater for more lipophilic solutes, which was consistent with a series of molecules transported by passive transepithelial diffusion. The melatonin permeation profiles through frozen, stored, and fresh mucosa illustrated that damage was incurred by the freezing process of the mucosal tissue, leading to loss of the barrier function and thereby an increased permeation coefficient. It can be observed that the influence of compound lipophilicity on the association of the compounds with buccal mucosa was clear. The relationship between permeation coefficient and Log P values for the four compounds investigated demonstrated a proportional relationship, further confirming the importance of the lipophilicity of a compound to permeate the buccal mucosa. These results showed that the ex vivo porcine buccal mucosa model is a suitable tool to screen oral mucosal permeability.

The inhibitory effect of glutathione on buccal enzymatic degradation of therapeutic peptides (leu-enkephalin, luteinizing hormone-releasing hormone and pituitary adenylate cyclase activating peptide)

The purpose of this study was to evaluate the potential of glutathione (GSH) as an auxiliary agent for buccal drug delivery systems to protect therapeutic peptides from enzymatic degradation. Stability of the model peptide drugs leu-enkephalin (Leu-Enke), luteinizing hormone-releasing hormone (LHRH) and pituitary adenylate cyclase activating peptide (PACAP) in the absence and presence of GSH on degradation by buccal enzymes was investigated. Inhibition studies were performed with isolated aminopeptidase N representing the most abundant peptidase on the buccal mucosa and on excised intact buccal mucosa from pigs. Enzymatic degradation of peptides was quantified by HPLC. The absorption of GSH across freshly excised porcine buccal mucosa was studied using Ussing type diffusion chambers. The proteolytic activity of isolated aminopeptidase N was significantly inhibited by the addition of 2% (m/v) GSH. Without the addition of enzyme inhibitors Leu-Enke was completely degraded by aminopeptidase N, whereas 34.8 ± 27.2% of LHRH and 74.2 ± 12.0% of PACAP remained intact. In contrast, by the addition of 2% (m/v) GSH 33.2 ± 2.2% of Leu-Enke, 88.6 ± 12.8% of LHRH and 97.0 ± 6.2% of PACAP remained stable after 5 h of incubation. Additionally, it was shown that only 0.2 ± 0.0% of GSH was able to permeate the buccal mucosa within 5 h, demonstrating that GSH remains concentrated at the site of drug absorption. After incubation for 5 h on the buccal mucosa 0.8 ± 0.2% of Leu-Enke, 73.2 ± 6.6% of LHRH and 82.9 ± 8.8% of PACAP remained stable. In contrast, in the presence of 2% (m/v) GSH 103.1 ± 12.7% of Leu-Enke, 98.5% ± 5.4% of LHRH and 97.2 ± 4.2% of PACAP were stable. According to these results GSH seems to represent a promising inhibitor for the buccal administration of peptide drugs.

Peptide Washout and Permeability from Glyceryl Monooleate Buccal Delivery Systems

ABSTRACTSimultaneous evaluation of the permeation and washout of a peptide from the mucoadhesive liquid crystalline phases of glyceryl monooleate (GMO) has been investigated using a donor compartment flow-through diffusion cell. [d-Ala2, d-Leu5]enkephalin (DADLE) was incorporated into the cubic and lamellar liquid crystalline phases of GMO and applied to excised porcine buccal mucosa mounted in the donor compartment flow-through cell. Phosphate-buffered saline pH 7.4 (PBS) was pumped across the upper surface of the liquid crystalline phases to mimic salivary flow. The steady-state fluxes of DADLE and GMO from the cubic phase were significantly greater than that from the lamellar phase (P<0.01). There was no statistical difference between the amounts of DADLE and GMO washed out from the lamellar and cubic phases (P>0.05). The donor compartment flow-through diffusion cell was found to be a useful tool to evaluate the impact of salivary washout on mucoadhesive oral mucosal delivery systems.

Buccal permeation of [ d-Ala 2, d-Leu 5]enkephalin from liquid crystalline phases of glyceryl monooleate

The ex vivo buccal permeability of a [ d-Ala 2, d-Leu 5]enkephalin (DADLE) and glyceryl monooleate (GMO) was examined from the cubic and lamellar liquid crystalline phases of GMO and aqueous phosphate-buffered saline (pH 7.4, PBS) solution across excised porcine buccal mucosa mounted in a Franz cell. GMO was released in vitro from the liquid crystalline phases indicating the erosion of the liquid crystal matrices. GMO released from the liquid crystalline matrices permeated the porcine buccal mucosa with fluxes of 0.10±0.03 and 0.07±0.00%/cm 2 per h for the cubic and lamellar phases, respectively. The flux of DADLE (1.21±0.32 and 1.15±0.11%/cm 2 per h for the cubic and lamellar phases, respectively) from the liquid crystalline phases was significantly enhanced by the GMO compared with PBS solution (0.43±0.08%/cm 2 per h) during the initial permeation phase ( t<3 h). Our results suggest that the cubic and lamellar liquid crystalline phases can be considered as promising buccal drug carriers for peptide drugs as well as acting as permeation enhancers.