The interest of drug delivery via the skin is progressively increasing due to its convenience and affordability. However, the skin has an effective barrier function which hinders drugs to penetrate. This can be overcome, using topical formulations, which contain vehicles optimized for penetration into skin or permeation through skin into underlying tissues or blood stream. In development of transdermal drugs, ex-vivo skin models are frequently used for permeability studies. However, these models may suffer from limited reproducibility, due to their biological variability, and are related to ethical issues. This has led to development of in-vitro skin models. These new skin models need to be verified by comparison to the existing models before applied in drug studies. In this project, penetration and permeation of proteins and small molecules was studied using an ex-vivo pig skin model. The effect of topical formulations and penetration enhancers was also investigated. The results showed a significant increase in penetration and permeation in the absence of stratum corneum, proving its barrier effect. While no significant differences could be seen regarding molecule size in the presence of stratum corneum (intact skin model), there was significantly less permeation measured for large protein molecules tested than for smaller tested compounds in the absence of the stratum corneum (damaged skin model). The permeation and penetration were slightly increased in presence of the penetration enhancing limonene oil. / Intresset för läkemedel som tas upp genom huden ökar ständigt eftersom administreringen är både bekväm och lättillgänglig. Däremot har huden en effektiv barriärfunktion, vilket hindrar penetration av läkemedlen. För att lösa detta problem kan läkemedlen presenteras i olika formuleringar, vilka innehåller transportmedel anpassade för att öka penetrationen in i huden eller permeationen genom huden, till underliggande vävnad samt blodomloppet. Vid utvecklandet av läkemedel som ska tas upp genom huden används ofta ex-vivo hudmodeller i penetrations- och permeationsstudier. Dessa modeller kan emellertid ha otillräcklig reproducerbarhet, på grund av biologisk variation, samt är kopplade till etiska problem. Detta har lett till utvecklandet av hudmodeller av in-vitro typ. Dessa nya hudmodeller måste verifieras genom jämförelse med existerande modeller, innan de används i läkemedelsstudier. I det här projektet studerades penetration och permeation av proteiner och små molekyler i en ex-vivo hudmodell från gris. Effekten av olika formuleringar och penetrationsförstärkare undersöktes också. Resultaten visade en signifikant ökning av penetration och permeation vid avsaknad av stratum corneum, vilket påvisar dess barriärfunktion. Samtidigt som ingen märkbar skillnad gällande molekylstorlek kunde urskiljas vid närvaro av stratum corneum (intakt hudmodell), var permeationen av större molekyler tydligt minskad i jämförelse med små molekyler vid avsaknad av stratum corneum (skadad hudmodell). Både penetrationen och permeationen var svagt förhöjda vid närvaro av penetrationsförstärkaren limonen.

BERTOLINI, WAGNER L. H. M. A influência do D-limoneno como promotor de absorção do ácido 5-aminolevulínico para Terapia Fotodinâmica do câncer de pele: avaliação in vitro e in vivo da permeação e retenção cutâneas. 2009 118f. Tese (Doutorado). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Preto Universidade de São Paulo, Ribeirão Preto, 2009. O câncer é a segunda doença principal do planeta, muito próximo de se tornar a mais incidente. Os tratamentos tradicionais do câncer, tais como cirurgia, radioterapia e quimioterapia apresentam severos efeitos colaterais ao paciente devido à citotoxicidade que podem causar às células normais, além das cancerosas. Objetivando minimizar estes efeitos indesejáveis pesquisadores de diversas áreas afins vislumbram novas técnicas, novos tipos e formas de tratamentos que apresentem um melhor perfil terapêutico; que possam agir de forma mais seletiva contra as células cancerosas, minorando os efeitos indesejáveis em relação às células saudáveis. Dentre as técnicas pesquisadas destaca-se a Terapia Fotodinâmica (TFD). Esta é uma técnica de tratamento nova e promissora. A técnica do tratamento consiste em aplicar, no tecido alvo, substâncias fotossensibilizantes, posteriormente ativadas com luz de comprimentos de onda específicos, com a finalidade de produzir destruição celular, por meio da ação de produtos citotóxicos fotoativados. Destaca-se a seletividade apresentada pela técnica, que deve ser reconhecida como uma das vantagens entre as técnicas empregadas no tratamento do câncer. O presente trabalho objetiva verificar a influência do D-limoneno como promotor de permeação do ácido 5-aminolevulínico (5-ALA) em aplicação tópica. Isto visa aumentar a permeação do 5-ALA quando aplicado topicamente. O 5-ALA é convertido a protoporfirina-IX (PpIX) pela via do ciclo Heme. Esta é um potente agente fotossensibilizador endógeno. O interesse no uso deste promotor foi aumentar a taxa de penetração do 5-ALA, possibilitando a permeação de maiores quantidades do fármaco em questão. Para isto foram realizados estudos de permeação in vitro do 5-ALA, em concentração de 1% (p/p) utilizando-se formulações (emulsões O/A) com diferentes concentrações do promotor D-limoneno (0, 5, 10, 20 e 30%), (p/p). Observou-se que o fluxo in vitro do 5-ALA através da pele de orelha de porco aumentou para todas as formulações empregadas quando comparado ao controle, sem D-limoneno, para um período de estudo de 12h. A retenção no estrato córneo e na [epiderme + derme] apresentou aumento significativo, evidenciando, assim, um efeito eficiente do D-limoneno como promotor. Experimentos in vivo foram conduzidos em camundongos objetivando a análise do efeito da formulação na produção e acúmulo de PpIX na pele. Observou-se que o D-limoneno aumentou significantemente a quantidade de PpIX extraída da pele. Os resultados in vitro e in vivo mostraram o potencial do D-limoneno como promotor de absorção cutânea para o 5-ALA para a TFD tópica do cancer de pele. / BERTOLINI, WAGNER L. H. M. D-limonene influence in the cutaneous penetration enhancer for 5-aminolevulinic acid in photodynamic therapy of skin cancer: in vitro and in vivo skin permeation and retention studies. 2009 118f. Thesis (Doctoral). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Preto Universidade de São Paulo, Ribeirão Preto, 2009. The topical administration of 5-ALA has been distinguished on skin cancer photodynamic therapy (PDT) because of its efficiency in the treatment of tumors and the reduced phototoxic collateral effects. However, this effectiveness is limited by its low penetration in the skin. A proposal to optimize the 5-ALA penetration in the skin is the use of cutaneous penetration enhancers, which seek to alter the cutaneous barrier for many bioactive molecules. The aim of this work was the pharmaceutical development of formulations containing D-limonene as a cutaneous penetration enhancer, seeking the increase of the cutaneous penetration of 5-ALA for skin cancer PDT. The in vitro flux of 5-ALA present in 1% (w/w) from different formulations containing D-limonene (20 e 30% w/w) was significantly increased after 12 hours of experiment, in comparison with formulations without D-limonene, mainly for the formulations containing 20% and 30% of D-limonene with 1% of 5-ALA (1,45 and 2,01 times, respectively). The stratum corneum (SC) and [epidermis + dermis] without SC retention were also significantly increased, showing an enhancer effect of the promoter. In addition, in vivo experiments were accomplished in mice, with the purpose of analyzing the formulation effect on the PpIX production and accumulation in the skin. It was observed that the penetration enhancer\'s presence significantly increased the amount of PpIX extracted from the skin. Both in vitro and in vivo results showed the potentiality of the formulations containing D-limonene as a cutaneous penetration enhancer for 5-ALA delivery on the skin cancer PDT.

Στην παρούσα διατριβή, μελετήθηκε η χρήση ελαστικών λιποσωμάτων στη διαδερμική χορήγηση υδατοδιαλυτών ουσιών και πραγματοποιήθηκαν επίσης μελέτες προ-μορφοποίησης αντιϋπερτασικών βιοδραστικών ενώσεων για διαδερμική χορήγηση. Αρχικά, πραγματοποιήθηκαν μελέτες της φυσιολογίας του δέρματος με τεχνικές μικροσκοπίας και μέτρησης της απώλειας ύδατος μέσω της επιδερμίδας (TEWL) με σκοπό να διαπιστωθεί η λειτουργία του φραγμού της κεράτινης στοιβάδας, καθώς και η ορθότητα χρήσης των σχετικών τεχνικών προετοιμασίας των δειγμάτων δέρματος που χρησιμοποιήθηκαν. Η οπτική μικροσκοπία ειδικά μας έδωσε πληροφορίες ως προς τη δομή της επιδερμίδας και ως προς τον πιθανό μηχανισμό μεταφοράς ουσιών μέσω της χρήσης λιποσωμάτων. Στη συνέχεια πραγματοποιήθηκε μελέτη και σύγκριση των διαφόρων φυσικοχημικών χαρακτηριστικών ελαστικών λιποσωμάτων που μπορεί να χρησιμοποιηθούν ως πρώτοι δείκτες για πρόγνωση της διαδερμικής απορρόφησης (in vivo) των υδρόφιλων βιοδραστικών ενώσεων. Ως μοντέλα υδρόφιλων βιοδραστικών ενώσεων χρησιμοποιήθηκαν οι φθορίζουσες χρωστικές καλσεΐνη και καρβοξυφλουορεσκεΐνη. Όλες οι λιποσωμικές διασπορές (τόσο τα συμβατικά λιποσώματα-CLs, όσο και τα ελαστικά λιποσώματα τύπου transfersome-TRs και τύπου invasomes-INVs) χαρακτηρίστηκαν ως προς τα εξής φυσικοχημικά χαρακτηριστικά: Την κατανομή μεγέθους, το ζ-δυναμικό, το σφαιρικό σχήμα και τη μορφολογία τους, την ικανότητα εγκλωβισμού υδρόφιλων ουσιών, τη σταθερότητά τους (ως προς τα μορφολογικά χαρακτηριστικά, τη διασπορά μεγέθους και το φορτίο επιφανείας, και τη συγκράτηση της εγκλωβισμένης σε αυτά ουσίας) σε σχέση με το χρόνο, την ελαστικότητα και τέλος ως προς τη δυνατότητα διαπέρασης εγκλωβισμένων σε αυτές υδρόφιλων ουσιών μέσω ανθρώπινου δέρματος in vitro. Το μέγεθος των λιποσωμάτων ήταν παρόμοιο και αρκετά ομοιογενές, ενώ η προσθήκη διαφορετικών τύπων ενισχυτικών διαπέρασης στα ελαστικά λιποσώματα, στα συνήθη εύρη συγκεντρώσεων που χρησιμοποιούνται, δεν επηρεάζει καθόλου το επιφανειακό φορτίο των παραχθέντων λιποσωμάτων. Αύξηση του μεγέθους και αστάθεια ως προς τη συγκράτηση της εγκλωβισμένης ουσίας παρατηρήθηκε στην περίπτωση προσθήκης μεγαλύτερης ποσότητας ενισχυτικού διαπέρασης, η οποία αλλάζει και τα φυσικοχημικά χαρακτηριστικά των λιποσωμάτων. Τα TRs που περιείχαν χολικό νάτριο ήταν πιο ελαστικά από τα αντίστοιχα με Tween 80, τα οποία είχαν συγκριτικά χαμηλές τιμές ελαστικότητας (<40mg/s∙cm2). Οι υψηλότερες τιμές ελαστικότητας από όλους τους τύπους ελαστικών λιποσωμάτων που παρασκευάστηκαν στα πλαίσια αυτής της διατριβής, παρατηρήθηκε στην περίπτωση των INVs με 1% w/w PE και 1% w/w LIM. Επιπλέον, τα περισσότερα λιποσώματα τύπου INVs είχαν υψηλότερες τιμές ελαστικότητας σε σχέση με τα αντίστοιχα χωρίς τερπένια (INVs αναφοράς), εκτός από την περίπτωση των INVs με citral και cineol, όπου η ελαστικότητα σημείωσε μείωση σε σχέση με τα INVs χωρίς τερπένια. Οι διάφοροι τύποι λιποσωμάτων φαίνεται να μεταβάλλουν τη διαπέραση της χρωστικής με την εξής σειρά: Διάλυμα<CLs<TRs<<INVs. H διαπερατότητα (permeability), η ροή (flux) και ο λόγος προσαύξησης (ER) δείχνουν ότι τα συμβατικά λιποσώματα αύξησαν ελάχιστα τη ροή της καλσεΐνης, ενώ τα TRs και INVs κατά 1.8 και 7.2 φορές, αντίστοιχα. Τα αποτελέσματα αυτά επιβεβαιώνουν το γεγονός ότι τα συμβατικά λιποσώματα είναι ανεπαρκή συστήματα μεταφοράς υδρόφιλων μορίων μέσω του δέρματος, ενώ παράλληλα δείχνουν ότι τα ελαστικά λιποσώματα με τη μεγαλύτερη τιμή ελαστικότητας είναι πιο αποτελεσματικά στη μεταφορά της ουσίας μέσω του δέρματος. Σε επόμενα πειράματα μελετήθηκε η δυνατότητα διείσδυσης τόσο υδρόφιλων όσο και λιπόφιλων μορίων στις στοιβάδες του δέρματος, σε μια προσπάθεια να διερευνηθεί ο μηχανισμός αύξησης της διαδερμικής διαπέρασης των υδρόφιλων χρωστικών από τους διάφορους τύπους ελαστικών λιποσωμάτων. Ως μοντέλο υδρόφιλης ουσίας χρησιμοποιήθηκε η φθορίζουσα χρωστική καλσεΐνη, ενώ η λιπιδική ροδαμίνη χρησιμοποιήθηκε ως μοντέλο λιπόφιλης ουσίας (που δεν φεύγει από τα λιποσώματα και ουσιαστικά μας δείχνει το βάθος εις το οποίο εισχωρούν μέσα στο δέρμα τα αντίστοιχα είδη λιποσωμάτων). Τα λιποσώματα τύπου TR φαίνεται ότι επάγουν τη διαπέραση με το να βοηθούν τη χρωστική να προχωρά (υπό μορφή λιποσωμάτων που την εγκλωβίζουν) σε βαθύτερα στρώματα της κεράτινης στιβάδας σε σχέση με τα συμβατικά λιποσώματα όπου σε μεγάλο ποσοστό διαρρηγνύονται και απελευθερώνουν την υδρόφιλη ουσία η οποία στη συνέχεια διέρχεται μόνη της, ως ελεύθερο μόριο, στις στιβάδες του χορίου. Αντίθετα, τα λιποσώματα τύπου INV αποδείχτηκε ότι εισδύουν σε βαθύτερες στιβάδες της επιδερμίδας, φθάνοντας σε αρκετά υψηλά ποσοστά σε στιβάδες του χορίου, παρασύροντας μαζί τους και τα μόρια του εγκλωβισμένου σε αυτά υδρόφιλου φαρμάκου. Τέλος, μελετήθηκε η δυνατότητα μορφοποίησης ενός πειραματικού φαρμάκου με ανάλογη δομή γνωστού φαρμάκου προκειμένου για διαδερμική αντιϋπερτασική θεραπεία. Αρχικά, μελετήσαμε το πειραματικό φάρμακο ως προς τις φυσικοχημικές του ιδιότητες (διαλυτότητα σε διάφορα μέσα, σύνδεση με πρωτεΐνες) και στη συνέχεια μελετήσαμε τη δυνατότητα χορήγησης μιας φαρμακοτεχνικής μορφής μέσω του δέρματος σε κατάλληλο σύστημα διαλυτών. Αφού βρέθηκαν οι κατάλληλες συνθήκες, χρησιμοποιήθηκαν ενισχυτικά διαπέρασης στη φαρμακοτεχνική μορφή για να επιταχυνθεί η διαδερμική απορρόφηση του φαρμάκου, όσο είναι δυνατόν, και να ευρεθεί η βέλτιστη φαρμακοτεχνική μορφή. Τέλος, πραγματοποιήθηκαν πειράματα σύγκρισης μεταξύ του πειραματικού φαρμάκου και γνωστού αντιϋπερτασικού (Los) ως προς τις φυσικοχημικές ιδιότητες και την ικανότητα μεταφοράς τους διαδερμικά. Τα αποτελέσματα έδειξαν ότι το ΠΦ παρουσίασε μεγαλύτερη διαπέραση υπό μορφή ουδέτερου μορίου (σε σχέση με την αντίστοιχη του άλατος με TFA (όπως φάνηκε από τα αρχικά πειράματα αυτής της σειράς) ή του μετά Καλίου άλατος (όπως προκύπτει μετά από σύγκριση των σχετικών τιμών Ροής και Συντελεστή Διαπερατότητας (P). Συγκρίνοντας λοιπόν τις τιμές διαπερατότητα και ροής διαμέσου ανθρώπινης επιδερμίδας με τις αντίστοιχες τιμές του φαρμάκου Los θα πρέπει να λάβουμε υπ’ όψη και τις τιμές για το ουδέτερο μόριο. Η διαπέραση του ουδέτερου μορίου του ΠΦ είναι σημαντικά χαμηλότερη από την αντίστοιχη του μετά καλίου άλατος της los. / In this study, we investigated the use of elastic liposomes in transdermal delivery of hydrophilic substances and we studied the possibility of formulating a new antihypertensive drug for transdermal delivery. Primarily, morphological studies of human skin were conducted by the use of optical microscopy and transepidermal water loss measurements (TEWL), in order to assess the barrier function of stratum corneum (SC), and the integrity of the techniques used in this study. By optical microscopy, in particular, data concerning the skin structure and the possible mechanism by which substances can be delivered through / into the skin, were obtained. Secondly, we studied and compared various physicochemical characteristics of two different types of elastic liposomes, which could help us predict the transdermal absorption (in vivo) of hydrophilic molecules. Fluorescent markers calcein and carboxyfluorescein were used as hydrophilic model drugs. All liposomal dispersions (conventional liposomes CLs, and elastic liposomes i.e. transfersomes TRs and invasomes INVs) were evaluated in means the following physicochemical properties: size distribution, z-potential, stability upon storage, morphology by cryo-electron microscopy and membrane elasticity. Moreover, their ability to encapsulate and also to retain aqueous soluble markers, was investigated. Finally, the permeation of calcein through human skin was tested and compared by use of elastic and conventional rigid liposomes. The mean diameter was found relatively homogenous, similar for most liposomal dispersions, while the addition of different penetration enhancers during the preparation did not influence z-potential. Increase of size average and instability – as far as the retention of the encapsulated substance is concerned – was observed at higher concentrations of penetration enhancers used. Sodium cholate containing TRs were found more elastic compared to Tween 80 containing TRs, which showed relatively low elasticity values (<40mg/s∙cm2). The highest elasticity values among all types of elastic liposomes prepared, were found in the case of INVs with 1%w/w PE and 1% w/w LIM. Furthermore, most INVs showed higher elasticity values compared to the ones without terpenes (control INVs), except from citral and cineol INVs, where elasticity decreased compared to the control ones. It appears that different types of liposomes can alter fluorescent permeation in the following order: Solution<CLs<TRs<<INVs. Permeability, flux and enhancement ratio (ER) show that conventional liposomes increased slightly calcein flux, while TRs and INVs 1.8 and 7.2 times respectively. These findings confirm the fact that CLs are inefficient drug delivery systems for water soluble molecules through human skin, and show that elastic liposomes having the highest elasticity value are more efficient in delivering CF transdermally. In further experiments, the penetration of both hydrophilic and lipophilic molecules in human skin was studied, in order to investigate the mechanism of action by which liposomes could enhance the drug delivery in skin. Fluorescent marker calcein was used as a hydrophilic drug model, while lipid rhodamine-PE was used as a lipophilic model (it shows us how deeply the liposomes penetrate into the skin). Liposomes TRs seem to enhance skin permeation by helping the marker to penetrate (through liposomes) in deeper stratum corneum layers while conventional liposomes the hydrophilic substance which penetrates the skin layers as a free molecule. In contrast, INV liposomes were proved to be able to penetrate in deeper skin layers, and deliver relatively high amounts of the encapsulated substance. In the end, experiments with a new molecule with similar structure to losartan were conducted. This study aimed at discovering a new formulation for this experimental drug in order to be used as a transdermal antihypertensive. First, we studied the physicochemical properties (solubility in PBS, BSA etc, and protein binding). Secondly, we investigated the possibility of preparing a formulation to be used transdermally. After establishing the techniques and drug properties we used penetration enhancers in order to increase the transdermal absorption as possible and help find the right skin formulation. Last, we compared the experimental drug and losartan in terms of physicochemical properties and their ability to be transported through human skin. The results showed that the experimental drug had better permeation rate as a neutral molecule compared to TFA salt, (as shown in preliminary experiments) or K+ (as shown after comparing P and flux values). Therefore, the values given for the neutral molecule should be taken into consideration when comparing the two drugs. (losartan and experimental drug). The permeation of the neutral molecule nevertheless is significantly lower than the one for losartan.

BERTOLINI, WAGNER L. H. M. A influência do D-limoneno como promotor de absorção do ácido 5-aminolevulínico para Terapia Fotodinâmica do câncer de pele: avaliação in vitro e in vivo da permeação e retenção cutâneas. 2009 118f. Tese (Doutorado). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Preto Universidade de São Paulo, Ribeirão Preto, 2009. O câncer é a segunda doença principal do planeta, muito próximo de se tornar a mais incidente. Os tratamentos tradicionais do câncer, tais como cirurgia, radioterapia e quimioterapia apresentam severos efeitos colaterais ao paciente devido à citotoxicidade que podem causar às células normais, além das cancerosas. Objetivando minimizar estes efeitos indesejáveis pesquisadores de diversas áreas afins vislumbram novas técnicas, novos tipos e formas de tratamentos que apresentem um melhor perfil terapêutico; que possam agir de forma mais seletiva contra as células cancerosas, minorando os efeitos indesejáveis em relação às células saudáveis. Dentre as técnicas pesquisadas destaca-se a Terapia Fotodinâmica (TFD). Esta é uma técnica de tratamento nova e promissora. A técnica do tratamento consiste em aplicar, no tecido alvo, substâncias fotossensibilizantes, posteriormente ativadas com luz de comprimentos de onda específicos, com a finalidade de produzir destruição celular, por meio da ação de produtos citotóxicos fotoativados. Destaca-se a seletividade apresentada pela técnica, que deve ser reconhecida como uma das vantagens entre as técnicas empregadas no tratamento do câncer. O presente trabalho objetiva verificar a influência do D-limoneno como promotor de permeação do ácido 5-aminolevulínico (5-ALA) em aplicação tópica. Isto visa aumentar a permeação do 5-ALA quando aplicado topicamente. O 5-ALA é convertido a protoporfirina-IX (PpIX) pela via do ciclo Heme. Esta é um potente agente fotossensibilizador endógeno. O interesse no uso deste promotor foi aumentar a taxa de penetração do 5-ALA, possibilitando a permeação de maiores quantidades do fármaco em questão. Para isto foram realizados estudos de permeação in vitro do 5-ALA, em concentração de 1% (p/p) utilizando-se formulações (emulsões O/A) com diferentes concentrações do promotor D-limoneno (0, 5, 10, 20 e 30%), (p/p). Observou-se que o fluxo in vitro do 5-ALA através da pele de orelha de porco aumentou para todas as formulações empregadas quando comparado ao controle, sem D-limoneno, para um período de estudo de 12h. A retenção no estrato córneo e na [epiderme + derme] apresentou aumento significativo, evidenciando, assim, um efeito eficiente do D-limoneno como promotor. Experimentos in vivo foram conduzidos em camundongos objetivando a análise do efeito da formulação na produção e acúmulo de PpIX na pele. Observou-se que o D-limoneno aumentou significantemente a quantidade de PpIX extraída da pele. Os resultados in vitro e in vivo mostraram o potencial do D-limoneno como promotor de absorção cutânea para o 5-ALA para a TFD tópica do cancer de pele. / BERTOLINI, WAGNER L. H. M. D-limonene influence in the cutaneous penetration enhancer for 5-aminolevulinic acid in photodynamic therapy of skin cancer: in vitro and in vivo skin permeation and retention studies. 2009 118f. Thesis (Doctoral). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Preto Universidade de São Paulo, Ribeirão Preto, 2009. The topical administration of 5-ALA has been distinguished on skin cancer photodynamic therapy (PDT) because of its efficiency in the treatment of tumors and the reduced phototoxic collateral effects. However, this effectiveness is limited by its low penetration in the skin. A proposal to optimize the 5-ALA penetration in the skin is the use of cutaneous penetration enhancers, which seek to alter the cutaneous barrier for many bioactive molecules. The aim of this work was the pharmaceutical development of formulations containing D-limonene as a cutaneous penetration enhancer, seeking the increase of the cutaneous penetration of 5-ALA for skin cancer PDT. The in vitro flux of 5-ALA present in 1% (w/w) from different formulations containing D-limonene (20 e 30% w/w) was significantly increased after 12 hours of experiment, in comparison with formulations without D-limonene, mainly for the formulations containing 20% and 30% of D-limonene with 1% of 5-ALA (1,45 and 2,01 times, respectively). The stratum corneum (SC) and [epidermis + dermis] without SC retention were also significantly increased, showing an enhancer effect of the promoter. In addition, in vivo experiments were accomplished in mice, with the purpose of analyzing the formulation effect on the PpIX production and accumulation in the skin. It was observed that the penetration enhancer\'s presence significantly increased the amount of PpIX extracted from the skin. Both in vitro and in vivo results showed the potentiality of the formulations containing D-limonene as a cutaneous penetration enhancer for 5-ALA delivery on the skin cancer PDT.

This work deals with the transmittance of a family of non-steroidal anti-inflammatory drugs across biological membranes in combination with the use of hyaluronan. Hyaluronic acid (hyaluronan, HYA) is a linear polysaccharide formed from disacharide units containing N-acetyl-D-glucosamine and glucuronic acid. HYA is present in almost all biological fluids and tissues, so there is an assumption that could affect the penetration of substances through the skin. Standards were prepared by anti-inflammatory drugs in admixture with various concentrations hyaluronan and subsequently measured transmittance of such substances through the skin. Standards were applied to the skin obtained from pig auricle. The drug content was determined by HPLC.