Predictivity of standardized and controlled permeation studies: Ex vivo - In vitro - In vivo correlation for sublingual absorption of propranolol.

In preclinical drug development, ex vivo and in vitro permeability studies are a decisive element for specifying subsequent development steps. In this context, reliability, physiological alignment and appropriate in vivo correlation are mandatory for predictivity regarding drug absorption. Especially in oromucosal drug delivery, these prerequisites are not adequately met, which hinders its progressive development and results in the continuous need for animal experiments. To address current limitations, an innovative, standardized, and controlled ex vivo permeation model was applied. It is based on Kerski diffusion cells embedded in automated sampling and coupled to mass spectrometric quantification under physiologically relevant conditions. This study aimed to evaluate the predictivity of the developed model using porcine mucosa (ex vivo) in relation to data of sublingual propranolol absorption (in vivo). In addition, the usefulness of biomimetic barriers (in vitro) as a replacement for porcine mucosa was investigated. Therefore, solubility and permeability studies considering microenvironmental conditions were conducted and achieved good predictivity (R2 = 0.997) for pH-dependent permeability. A multiple level C correlation (R2 ≥ 0.860) between obtained permeability and reported pharmacokinetic animal data (AUC, Cmax) was revealed. Furthermore, a point-to-point correlation was demonstrated for several sublingual formulations. The successful IVIVC confirms the standardized ex vivo model as a viable alternative to animal testing for estimating the in vivo absorption behavior of oromucosal pharmaceuticals.

Is rat a good model for assessment of particulate-based taste-masked formulations?

Recently there has been an increased interest to develop specialised dosage forms that are better suited to specific patient populations, such as paediatrics and geriatrics. In these patient populations the acceptability of the oral dosage form can be paramount to the products success. However, many Active Pharmaceutical Ingredients (APIs) are known to cause an aversive taste response. One way to increase the acceptability and to enhance the palatability of the formulation is to design coated taste-masked particulate-based dosage forms. The masking of poorly tasting drugs with physical barriers such as polymer coatings can be utilised to prevent the release of drug within the oral cavity, thus preventing a taste response. However, currently, there are few assessment tools and models available to test the efficiency of these particulate-based taste-masked formulations. The rat brief access taste aversion model has been shown to be useful in assessment of taste for liquid dosage forms. However, the applicability of the rat model for particulate-based taste masked formulations is yet to be assessed. It is not understood whether dissolution, solubility and thus exposure of the drug to taste receptors would be the same in rat and human. Therefore, rat saliva must be compared to human saliva to determine the likelihood that drug release would be similar within the oral cavity for both species. In this study rat saliva was characterised for parameters known to be important for drug dissolution, such as pH, buffer capacity, surface tension, and viscosity. Subsequently dissolution of model bitter tasting compounds, sildenafil citrate and efavirenz, in rat saliva was compared to dissolution in human saliva. For all parameters characterised and for the dissolution of both drugs in rat saliva, a substantial difference was observed when compared to human saliva. This discrepancy in saliva parameters and dissolution of model drugs suggests that preclinical taste evaluation of particulate-based taste-masked formulations suggests rat is not a good model for predicting taste of solid dosage forms or undissolved drug where dissolution is required. Alternative preclinical in vivo models in other species, or improved biorelevant in vitro models should be considered instead.

Tart cherry (Prunus cerasus L.) fractions inhibit biofilm formation and adherence properties of oral pathogens and enhance oral epithelial barrier function.

Dental caries, candidiasis, and periodontal disease are the most common oral infections affecting a wide range of the population worldwide. The present study investigated the effects of two tart cherry (Prunus cerasus L.) fractions on important oral pathogens, including Candida albicans, Streptococcus mutans, and Fusobacterium nucleatum, as well as on the barrier function of oral epithelial cells. Procyanidins and quercetin and its derivatives were the most important constituents found in the tart cherry fractions. Although the fractions showed poor antimicrobial activity, they inhibited biofilm formation by the three oral pathogens in a dose-dependent manner. The tart cherry fractions also attenuated the adherence of C. albicans and S. mutans to a hydroxylapatite surface as well as the adherence of F. nucleatum to oral epithelial cells. Treating oral epithelial cells with the tart cherry fractions significantly enhanced the barrier function as determined by monitoring the transepithelial electrical resistance. In conclusion, this study showed that the tart cherry fractions and their bioactive constituents could be promising antiplaque compounds by targeting biofilm formation and adherence properties of oral pathogens. Furthermore, its property of increasing the epithelial barrier function may protect against microbial invasion of the underlying connective tissue.

Rhodoptilometrin, a Crinoid-Derived Anthraquinone, Induces Cell Regeneration by Promoting Wound Healing and Oxidative Phosphorylation in Human Gingival Fibroblast Cells.

Gingival recession (GR) potentially leads to the exposure of tooth root to the oral cavity microenvironment and increases susceptibility to dental caries, dentin hypersensitivity, and other dental diseases. Even though many etiological factors were reported, the specific mechanism of GR is yet to be elucidated. Given the species richness concerning marine biodiversity, it could be a treasure trove for drug discovery. In this study, we demonstrate the effects of a marine compound, (+)-rhodoptilometrin from crinoid, on gingival cell migration, wound healing, and oxidative phosphorylation (OXPHOS). Experimental results showed that (+)-rhodoptilometrin can significantly increase wound healing, migration, and proliferation of human gingival fibroblast cells, and it does not have effects on oral mucosa fibroblast cells. In addition, (+)-rhodoptilometrin increases the gene and protein expression levels of focal adhesion kinase (FAK), fibronectin, and type I collagen, changes the intracellular distribution of FAK and F-actin, and increases OXPHOS and the expression levels of complexes I~V in the mitochondria. Based on our results, we believe that (+)-rhodoptilometrin might increase FAK expression and promote mitochondrial function to affect cell migration and promote gingival regeneration. Therefore, (+)-rhodoptilometrin may be a promising therapeutic agent for GR.

Proliferation, migration, and expression of oral-mucosal-healing-related genes by oral fibroblasts receiving low-level laser therapy after inflammatory cytokines challenge.

BACKGROUND AND OBJECTIVES: Increased expression of inflammatory cytokines in the oral cavity has been related to the etiopathogenesis of oral mucositis and to delayed oral mucosal repair. Low-level laser therapy (LLLT) stimulates proliferation and migration of gingival fibroblasts, but the effects of specific inflammatory cytokines on oral mucosal cells and the modulation of these effects by LLLT have not been fully investigated. Therefore, this study investigated the effects of LLLT on oral fibroblasts after being challenged by oral-mucositis-related inflammatory cytokines. METHODS: Human gingival fibroblasts were seeded in plain culture medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 hours. Then, cells were kept in contact with inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) in serum-free DMEM for 24 hours. After this period, cells were subjected to LLLT with a diode laser device (LaserTABLE, InGaAsP, 780 nm, 25 mW) delivering energy doses from 0.5 to 3 J/cm2 . Irradiation was repeated for 3 consecutive days. Twenty-four hours after the last irradiation, cell migration (wound-healing and transwell migration assays), cell proliferation (BrdU), gene expression of COL-I and growth factors (real-time PCR), and synthesis of COL-I (Sirius Red assay) and VEGF (ELISA) were assessed. Data were subjected to two-way ANOVA and Tukey's tests or Kruskall-Walis and Mann-Whitney tests (P < 0.05). RESULTS: The inflammatory cytokines decreased the migration capacity of gingival fibroblasts. However, a statistically significant difference was observed only for IL-6, detected by transwell assay, where 30% less cells migrated through the pores (P < 0.05) and IL-8, with an increased wound area (116%; P < 0.05), detected by the wound healing method. Cell proliferation was not affected by contact with cytokines, while growth factors and COL-I expression (approximately 80%; P < 0.05), as well as VEGF synthesis (approximately 20%; P < 0.05), were decreased after contact to all tested cytokines. The opposite was seen for total collagen synthesis. LLLT promoted an acceleration of fibroblast migration (30%; P < 0.05) and proliferation (112%; P < 0.05) when delivering 0.5 J/cm2 to the cells previously in contact with the inflammatory cytokines. Gene expression of VEGF (approximately 30%; P < 0.05), and EGF (17%; P < 0.05), was stimulated by LLLT after contact with TNF-α and IL-6. CONCLUSION: LLLT can counteract the negative effects of high concentrations of inflammatory cytokines, especially IL-6 and IL-8 on gingival fibroblast functions directly related to the wound-healing process. Lasers Surg. Med. 48:1006-1014, 2016. © 2016 Wiley Periodicals, Inc.

Percepção dos pacientes em uso de enxaguatórios bucais: óleos essenciais e cloreto de cetilperidíneo / Perception of patients with use mouthwashes: essential oils and cetylpyridinium chloride

Objetivo: Avaliar a percepção dos pacientes quanto às sensações após o uso de enxagua­tórios à base de óleos essenciais e àlcool e à base de cloreto de cetilperidíneo. Métodos: Foram constituídos 2 grupos (n= 15) que realizaram 2 bochechos diários durante 3 semanas: G 1 - enxaguatório a base de óleos essenciais e álcool, em bochechos de 30 segundos; G2 - enxaguatório à base de cloreto de cetilperidínio, em bochechos de 60 segundos. Ao final de 3 semanas, os participantes responderam a um questionário sobre desconforto, alteração de paladar, sensibi­lidade dentária e ressecamento da mucosa oral. Resultados: O desconforto foi de 93,3% para G1 e de 26,6% em G2, sendo o ardor o mais relatado (86,6% em G1 e 26,6% em G2). Não foi relatada sensibilidade ou alteração dentária nos grupos avaliados. O ressecamento da mucosa ocorreu em 66,6% no G 1 e 33,3% no G2. A alteração de paladar ocorreu somente em 13,3% do G1 com diminuição da sensibilidade e gosto metálico. Conclusão: A utilização contínua de enxaguatórios com óleos essenciais e álcool causou maior desconforto quando comparado ao cloreto de cetilperidínio.

Objective: Evaluate the perceptions of patients about the effects of the use of mouthwash based on essential oils and alcohol -based and cetylpyridinium chloride. Methods: 2 groups ( n = 15) who underwent 2 mouthwash daily for 3 weeks were formed: G1 - a mouthwash based on essential oils and alcohol in mouthwash for 30 seconds; G2 - the base of mouthwash cetil­ peridínio chloride in mouthwashes 60 seconds. At the end of 3 weeks, participants answered a questionnaire on discomfort , change in taste, tooth sensitivity and dryness of the oral mucosa. Results: The discomfort was 93.3 % for G1 and 26.6% in G2, with the ardor as reported (86.6% in G1 and 26.6 % in G2) . It was reported tooth sensitivity or change in study groups. The dryness of the mucosa occurred in 66.6 % in G1 and 33.3% in G2. The taste change occurred in only 13.3% of G 1 with decreased sensitivity and metallic taste . Conclusion: The continuous use of mouthwashes with essential oils and alcohol caused greater discomfort when compared to cetilperidínio chloride.

A randomized, double-blind, placebo-controlled, crossover study to evaluate the subjective abuse potential and cognitive effects of nabiximols oromucosal spray in subjects with a history of recreational cannabis use.

OBJECTIVE: This study aimed to evaluate the abuse potential and cognitive effects of nabiximols (Sativex, GW Pharma Ltd. Salisbury, UK), an oromucosal spray primarily containing delta­9­tetrahydrocannabinol (THC) and cannabidiol (CBD). METHODS: This was a single­dose, randomized, double­blind, crossover study comparing nabiximols (4, 8, and 16 consecutive sprays: 10.8, 21.6, and 43.2 mg THC, respectively) with dronabinol 20 and 40 mg (synthetic THC: Marinol, Solvay Pharmaceuticals, Brussels, Belgium) and matching placebos in 23 recreational cannabis users. Subjective and cognitive/psychomotor measures were administered over 24 h post­dose. RESULTS: Dronabinol was significantly different from placebo on abuse potential measures, thereby confirming study validity. Nabiximols 10.8 mg was not significantly different from placebo on primary measures but was different on some secondary measures. Nabiximols 21.6 mg was significantly greater than placebo on some primary/secondary measures, whereas nabiximols 43.2 mg showed significant effects on most measures. Nabiximols 10.8 mg was significantly lower than dronabinol doses on most measures ( p < 0.05). Dronabinol 20 mg effects were numerically higher than nabiximols 21.6 mg but were statistically significant only for some measures. Dronabinol 40 mg and nabiximols 43.2 mg were generally not statistically different. CONCLUSIONS: Both dronabinol and nabiximols had significant abuse potential compared with placebo at higher doses. Nabiximols showed similar or slightly less abuse potential compared with dronabinol. Therefore, the abuse potential of nabiximols should be no higher than that of dronabinol.

CD36 involvement in orosensory detection of dietary lipids, spontaneous fat preference, and digestive secretions.

Rats and mice exhibit a spontaneous attraction for lipids. Such a behavior raises the possibility that an orosensory system is responsible for the detection of dietary lipids. The fatty acid transporter CD36 appears to be a plausible candidate for this function since it has a high affinity for long-chain fatty acids (LCFAs) and is found in lingual papillae in the rat. To explore this hypothesis further, experiments were conducted in rats and in wild-type and CD36-null mice. In mice, RT-PCR experiments with primers specific for candidate lipid-binding proteins revealed that only CD36 expression was restricted to lingual papillae although absent from the palatal papillae. Immunostaining studies showed a distribution of CD36 along the apical side of circumvallate taste bud cells. CD36 gene inactivation fully abolished the preference for LCFA-enriched solutions and solid diet observed in wild-type mice. Furthermore, in rats and wild-type mice with an esophageal ligation, deposition of unsaturated LCFAs onto the tongue led to a rapid and sustained rise in flux and protein content of pancreatobiliary secretions. These findings demonstrate that CD36 is involved in oral LCFA detection and raise the possibility that an alteration in the lingual fat perception may be linked to feeding dysregulation.

Histomorphometric study of the healing of human oral mucosa after gingivoplasty and low-level laser therapy.

BACKGROUND AND OBJECTIVES: The aim of the present study was to analyze the effects of diode laser irradiation on the healing of human oral mucosa. MATERIALS AND METHODS: After gingivoplasty, the right hemi-arch (test group) of 16 patients was irradiated with a diode laser. The left side (control group) was not irradiated. Incisional biopsies were performed on both sides at 7, 14, 21, and 60 days after surgery and morphometrically analyzed by light microscopy. RESULTS: Epithelium width ranged from 260.6 to 393.5 microm. Volume densities of basal (20.2%), prickle cell (55.6%), and cornified (24.2%) layers remained stable. The peak number of neutrophils were 6 cells/mm(2) and the mononuclear cells were 44 cells/mm(2). Collagen fibers (80%) and fibroblasts (14%) occupied the main volume of connective tissue. The one-way ANOVA and the paired Student's t-test were used for statistical analysis (P < 0.05). CONCLUSION: Low-level laser therapy did not accelerate the healing of oral mucosa after gingivoplasty.

A plethysmographic method for demonstrating the response specificity of the oral vascular bed.

In this paper, we describe a new method for measuring the oral plethysmogram, and we assess its sensitivity and specificity under differing psychological stimulation. Finger and palate pulse amplitudes and blood pressure were monitored while individuals (N = 13) performed several tasks: mental arithmetic, nausea imagery, fear imagery, and anger imagery. Pulse pressure, having a major effect on pulse amplitude, was partialed out in analyses. Palate pulse amplitude increased significantly in response to the degree to which the individual felt irritated, judged, nauseated, or angry. In contrast, finger pulse amplitude changed significantly only in the arithmetic task and, unlike the palate, showed a decreased amplitude with increased irritation and being judged. Results indicate that the oral plethysmogram can serve as a reliable measure of oral mucosal vasomotor reactivity and that it has a different pattern of response specificity than does the finger.

Effects of anaesthesia of human oral structures on masticatory performance and food particle size distribution.

Ten persons with intact dentitions performed a series of 6 masticatory tests, employing 5, 10, 20, 40, 80 and 160 strokes to chew standard quantities of peanuts on their preferred chewing side for each of three test conditions: (1) before anaesthesia: (2) after maxillary anaesthesia; and, (3) after maxillary and mandibular anaesthesia (unilateral). A seventh test employing 20 strokes was also repeated on the non-anaesthetized contralateral side. The chewed food was sieved through 5, 10, 20, 40, 80 and 100 mesh screens. The percentage of the ratio of the volume of peanuts that passed through the sieve and the total volume of recovered food provided the performance score for the given sieve. The performance scores increased significantly with the number of stokes and dropped markedly after anaesthesia. The maximum reduction of 19.6% in the mean masticatory performance and 46% in the mean masticatory efficiency occurred after unilateral anaesthesia at 10 mesh particle size in the 20-stroke test. An average of 40 strokes was required after unilateral anaesthesia to achieve almost the same performance achieved with 20 strokes before anaesthesia. The regression slopes, derived from the 5, 10 and 20 mesh particle distributions showed that coarse particles were ground more rapidly than fine particles before anaesthesia. This preferential comminution became less evident after maxillary anaesthesia and was least evident after unilateral anaesthesia. The regression slope for the control peaked at 10 strokes as compared to 20 strokes after unilateral anaesthesia. Thus peripheral sensory impairment affects masticatory efficiency in dentate persons.

The acute effects of smokeless tobacco on transport and barrier function of buccal mucosa.

The acute effects of smokeless tobacco (ST) on buccal mucosal transport and barrier function were studied by means of in vivo and in vitro techniques. In humans, in vivo exposure to 0.5 g ST transiently increased the transmural electrical potential difference (PD). However, despite continued exposure, PD returned to baseline within 20 min. The mechanisms for these changes were explored by use of dog buccal mucosa mounted in Ussing chambers. Luminal exposure to a Ringer-extract of ST (EOST) increased PD and short-circuit current (Isc) and decreased electrical resistance (R), with changes reversible upon removal of EOST from the bath. Further, radioisotopic fluxes showed that the increase in Isc in EOST-exposed tissues was accompanied by increased absorption of electrolytes (Na, Cl, and other ions), and the decrease in R was accompanied by increased permeability to mannitol. Light microscopy of tissues exposed to EOST showed no morphological changes after exposure to 0.5 g of ST, but after exposure to 1.5-2.5 g of ST, dilated intercellular spaces were identified. Contact of aqueous media with ST led to the release of electrolytes and other soluble compounds into solution. To determine the effect of electrolyte release on buccal function, we exposed mucosae luminally to a solution with ion composition and/or osmolality similar to EOST or to one with an EOST previously dialyzed against Ringer. Solutions with similar ion composition and/or osmolarity changed PD, Isc, and R in a manner similar to EOST, while dialyzed-EOST had no effect. In addition, luminal nicotine produced effects different from EOST, decreasing PD and Isc and increasing R.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of salivary epidermal growth factor in the maintenance of physicochemical characteristics of oral and gastric mucosal mucus coat.

The involvement of salivary epidermal growth factor (EGF) in the maintenance of oral and gastric mucosal mucus coat dimension and chemical characteristics was investigated using sialoadenectomized rats. Examination of the oral and gastric mucosal surface by phase contrast microscopy and Alcian blue uptake revealed that deprivation of salivary EGF caused a 31-36% reduction in mucus coat thickness and a 38-43% reduction in adherent mucin content. Chemical analyses indicated that the mucus coat of sialoadenectomized group exhibited a 21-28% increase in protein and a 67% decrease in covalently bound fatty acids, a 30% decrease in carbohydrates, and a 32-37% decrease in lipids. Sialoadenectomy also evoked changes in the chemical composition of mucus glycoprotein component of oral and gastric mucus coat reflected in the lower content of sulfate (25-26%), associated lipids (24-25%), and covalently bound fatty acids (67-75%). Intragastric supplementation of EGF had no effect on the physicochemical changes caused by sialoadenectomy in the oral mucosal mucus coat, while nearly complete restoration to normal characteristics occurred in the gastric mucosal mucus coat. The results suggest that salivary EGF is essential for the maintenance of mucus coat dimension and quality needed in the protection of alimentary tract epithelium.