Estudio de la permeación ex vivo e in vivo de antidepresivos tricíclicos en tejido biológico. Estudio de la actividad anestésica y analgésica

La tesis doctoral consta de 3 artículos desarrollados a lo largo de varios años. El primer trabajo tiene el título de “Transdermal delivery of imipramine and doxepin from newly oil-in-water nanoemulsions for an analgesic and anti-allodynic activity: Development, characterization and in vivo evaluation”. Los antidepresivos y más concretamente los antidepresivos tricíclicos por su mayor eficacia y actividad analgésica han sido objeto de numerosos estudios. En este primer artículo, hemos estudiado la actividad analgésica y anestésica de dos antidepresivos tricíclicos (imipramina y doxepina). Con el fin de investigar formulaciones analgésicas de aplicación cutánea, hemos desarrollado una nanoemulsión compuesta por propilenglicol, Transcutol® , agua, Labrasol®, Plurol Oleico®, isostearato de isoestearilo, ácido oleico y D-limoneno. La concentración final de imipramina o doxepina en el sistema de la nanoemulsión fue del 3% (p/p) En este trabajo, las nanoemulsiones han sido caracterizadas en función de su pH, viscosPERMEATION STUDY EX VIVO AND IN VIVO OF TRICYCLIC ANTIDEPRESSANT IN BIOLOGICAL TISSUES. STUDY OF ANESTHETIC AND ANALGESIC ACTIVITY This study is based on three papers published between 2013 and 2014. The first paper has the title: “Transdermal delivery of imipramine and doxepin from newly oil-in-water nanoemulsions for an analgesic and anti-allodynic activity: Development, characterization and in vivo evaluation”. We study the local analgesic and anesthetic properties of nanoemulsions with imipramine and doxepin. The concentration of these tricyclic antidepressants in the nanoemulsion system was 3% (w/w). The composition of optimized nanoemulsion is 45% of aqueous phase, 40% mixture of surfactants and 15% oil phase. The second study was published in 2014 in the European Journal of Pharmaceutical Sciences and has the title:”An improved cryopreservation method for porcine buccal mucosa in ex vivo drug permeation studies using Franz diffusion cells”. We study a cryopreservation method for porcine buccal mucosa based in the mixture of phosphate buffer saline (PBS) with 10% DMSO and 4% albumin as cryoprotective agents. The porcine buccal mucosa has been proposed as a substitute for the buccal mucosa barrier on ex vivo permeability studies avoiding unnecessary sacrifice of animals. We used a Franz diffusion cell system and HPLC as detection method. The freezing effects on drug permeability parameters were evaluated. Equally histological studies were performed. Furthermore, the use of the parameter transmucosal water loss (TMWL) as an indicator of the buccal mucosa integrity was evaluated just as transepidermal water loss (TEWL) is utilized for skin integrity. The results showed no difference between fresh and frozen mucosal flux, permeability coefficient or lag time. However, statistical significant difference in TMWL between fresh and frozen mucosa was observed. The last paper:” Transbuccal delivery of doxepin: Studies on permeation and histological investigation” 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. Finally, a histological evaluation of the buccal mucosa was performed to test potential damage due to the permeation phenomenon. 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

Carprofen Permeation Test through Porcine Ex Vivo Mucous Membranes and Ophthalmic Tissues for Tolerability Assessments: Validation and Histological Study

Carprofen (CP), a non-steroidal anti-inflammatory drug (NSAID), is profusely used in veterinary medicine for its analgesic and anti-inflammatory activity. Some undesirable effects are associated with its systemic administration. Alternative local routes are especially useful to facilitate its administration in animals. The main aim of this paper is to validate the suitability of ex vivo permeation experiments of CP with porcine mucous membranes (buccal, sublingual and vaginal) and ophthalmic tissues (cornea, sclera and conjunctiva) intended to be representative of naïve in vivo conditions. Chromatographic analysis of CP in membrane-permeated samples and drug-retained have been validated following standard bioanalytical guidelines. Then, recovery levels of drugs in tissue samples were assessed with aqueous phosphate buffered saline (PBS) buffer to preserve the histological integrity. Finally, as a proof of concept, a series of CP permeation tests in vertical Franz diffusion cells has been performed to evaluate permeation flux and permeability constants in all tissues, followed by a histological study for critical evaluation. Furthermore, synthetic tissue retention-like samples were prepared to verify the value of this experimental study. Results show linear relationships with good determination coefficient (R2 > 0.998 and R2 > 0.999) in the range of 0.78 to 6.25 mg/mL and 3.125 mg/mL to 100 mg/mL, respectively. Low limits of quantification around 0.40 µg/mL were allowed to follow permeation levels until a minimum of 0.40% of the locally-applied dose. This method showed a good accuracy and precision with values lower than 2%. After the recovery technique, reproducible values below 30% were achieved in all tissues, suggesting it is a non-damaging method with low efficiency that requires the use of further solvents to enhance the extraction percentages. After permeation and histology tests, no relevant peak interferences were detected, and no cell or tissue damage was found in any tissue. In conclusion, results demonstrate the suitability of this test to quantify the distribution of CP with good histological tolerability

A Regenerative Endodontic Approach in Mature Ferret Teeth Using Rodent Preameloblast-conditioned Medium

Background: This study evaluated the effectiveness of a regenerative endodontic approach to regenerate the pulp tissue in mature teeth of ferret. The presence of odontoblast-like cells in the newly-formed tissue of teeth treated with or without preameloblast-conditioned medium was evaluated based on morphological criteria. Materials and methods: Twenty-four canines from six ferrets were treated. The pulp was removed, and the apical foramen was enlarged. After inducing the formation of a blood clot, a collagen sponge with or without preameloblast-conditioned medium was placed underneath the cementoenamel junction. The samples were analyzed at the eighth week of follow-up. Results: Vascularized connective tissue was observed in 50% of teeth, without differences between groups. The tissue occupied the apical third of the root canals. Odontoblast-like cells were not observed in any group. Conclusion: Revitalization of mature teeth is possible, at least in the apical third of the root canal. Further experimental research is needed to produce more reliable outcomes

Ex Vivo Permeation of Carprofen Vehiculated by PLGA Nanoparticles through Porcine Mucous Membranes and Ophthalmic Tissues

(1) Background: Carprofen (CP), 2-(6-chlorocarbazole) propionic acid, is used as an anti-inflammatory, analgesic and anti-pyretic agent and it belongs to the family of non-steroidal anti-inflammatory drugs (NSAIDs). CP has some adverse reactions in systemic administration; for this reason, topical administration with CP nanoparticles (CP-NPs) can be an optimal alternative. The main objective of this work is the investigation of ex vivo permeation of CP through different types of porcine mucous membranes (buccal, sublingual and vaginal) and ophthalmic tissues (cornea, sclera and conjunctiva) to compare the influence of CP-NPs formulation over a CP solution (CP-Solution). (2) Methods: The ex vivo permeation profiles were evaluated using Franz diffusion cells. Furthermore, in vivo studies were performed to verify that the formulations did not affect the cell structure and to establish the amount retained (Qr) in the tissues. (3) Results: Permeation of CP-NPs is more effective in terms of drug retention in almost all tissues (with the exception of sclera and sublingual). In vivo studies show that neither of the two formulations affects tissue structure, so both formulations are safe. (4) Conclusions: It was concluded that CP-NPs may be a useful tool for the topical treatment of local inflammation in veterinary and human medicine

Swine as the Animal Model for Testing New Formulations of Anti-Inflammatory Drugs: Carprofen Pharmacokinetics and Bioavailability of the Intramuscular Route

Abstract Carprofen (CP) is a non-steroidal anti-inflammatory drug (NSAID) frequently used to treat respiratory diseases in numerous small animals, but also in large species. CP is a formidable candidate for further therapeutic research of human inflammatory diseases using the pig as an animal model. However, CP administration in swine is very uncommon and respective pharmacokinetics/bioavailability studies are scarce. A simultaneous population pharmacokinetic analysis after CP intravenous and intramuscular administrations in pigs has shown high extent and rate of absorption and a similar distribution profile with respect to man and other mammals. However, clearance and half-life values found in swine suggest a slower elimination process than that observed in man and some other animal species. Although not reported in other species, liver and kidney concentrations achieved at 48 h post-intramuscular administration in pigs were ten times lower than those found in plasma. Simulations pointed to 4 mg/kg every 24 h as the best dosage regimen to achieve similar therapeutic levels to those observed in other animal species. All these findings support the use of pig as an animal model to study the anti-inflammatory effects of CP in humans