Skin cancer has arrived as the most prevalent cancer type worldwide. Depending of the cell type, it can be divided into melanoma and nonmelanoma skin cancer, which includes basal and squamous cell carcinoma. Cutaneous T-cell lymphomas (CTCL) are an uncommon subtype of non-Hodgkin lymphomas with an annual incidence of 0.6 per 100,000. The patients suffering of different variants of CTCL are experiencing pain, excruciating itching, pruritus, insomnia and, in advanced stages, tumor development. Nowadays, skin-directed therapy using topical formulations with meclorethamine, steroids, carmustine, bexarotene, peldesine is successful in the first stages of the disease but with several side effects such as cutaneous hypersensitivity and atrophy, pruritus, burning pain, bone-marrow depression, and secondary malignancies.
The present project aims to contribute with valuable research for improving significantly the quality of life of these patients by developing innovative topical formulations for skin-directed therapy of CTCL with enhanced efficacy and minimized side effects. To achieve this goal, modified drug delivery systems (MDDS) containing the CTCL-specific anticancer drugs entrapped within a nanoporous biocompatible matrix, are formulated in the proper semisolid base to ensure maximum efficiency and tolerability.
Firstly the CTCL-specific chemotherapeutic drugs are confined into proper selected inorganic biocompatible matrices such as natural zeolites, natural clays, and mesoporous silica nanoparticles. Hence, the human body is protected against the aggressiveness of these anticancer drugs. Immobilization of these bioactive molecules within the selected matrices will be deeply studied in order to choose the best drug-carrier tandem for each molecule, based on the criteria of the highest amount of drug loaded versus its sustained release from its carrier. Physico-chemical properties of the therapeutic systems will be studied using modern techniques such as XRD, N2-sorption, SEM, TEM, FTIR, UV-VIS, TG/DSC.
Secondly, this project contributes to improved quality of life of these patients by incorporation of the optimal nanoporous therapeutic systems selected within semisolid formulation bases. Their formulations as gel, creams, emulgel and ointments will be studied. The in vitro dissolution tests will be performed using instrument II equipped with Enhancer cell and different types of artificial membranes and the innovative test Skin-PAMPA system used for evaluation of release rate, permeability and the rate-limiting barrier of human skin in ex vivo conditions. These results will be very helpful for selecting optimal topic formulations for in vivo testing. There are no studies in literature dealing with the evaluation of diffusion and cutaneous penetrability of these substances as a function of the semisolid base used in the formulation. More, unlike other pharmaceutical forms, for topical semisolid systems, there are no standardized methods for release and cutaneous penetration degree evaluation in ex vivo conditions. Thus, this research constitutes exhaustive information for pharmaceutical research of topical semisolid formulations and a valuable platform to support the standardization of in vitro evaluation criteria of topical semisolid formulations.
Further, in vivo evaluation of pharmaceutical performances of newly semisolid formulations will be realized on mice by means of the chemical carcinogen–induced two-stage skin tumor, a well-established animal model to study mechanisms of epithelial carcinogenesis. Very few studies are presented in the literature on mice testing of topical formulations containing mechlorethamine, carmustine, bexarotene and other CTCL-specific bioactive molecules.
Results – 2015
To immobilize selected bioactive molecules on the nanoporous silica matrices, initially, it was carried out the adsorption kinetic study from solution, the solvent, pH and concentration influences. Using the optimal conditions resulted from the study were immobilized bexarotene (BXT) and carmustine (BCNU) on both clinoptilolite forms H-CLI and Na-CLI, respectively, as well as on pure and amino-functionalized mesoporous silica MCM-41. The obtained MDDS systems were tested in vitro dissolution in simulated media. The analysis of the release kinetics of BCNU from BCNU@M, BCNU@MF, BCNU@HCLI, BCNU@NaCLI systems have been accomplished with the Matlab 7.1 software, fitting the obtained results on mathematical models: zero kinetics order, first kinetic order, Higuchi model, Korsmeyer-Peppas model.
The results revealed that the system based on mesoporous silica release the drug according to a first order kinetic where the release rate is concentration dependent .
The MDDS systems based on clinoptilolite, release differently BCNU, thus BCNU@HCLI reveal a release profile of fikian diffusion, while BCNU@NaCLI system releases the drug of a zero order kinetic.
The obtained results confirm that the silica matrix has an important role in loading and release of the active agent.
The semisolid formulations BCNUMF@MF and BCNU@HCLI have been evaluated for in vitro availability showing a release of 96.22-100.37 % of introduced drug.
The best BCNU release profiles have been obtained from emulgel, using both mesoporous silica and clinoptilolite, showing a maximum release of 28.52 % for BCNU@MF, and 22.47 % for BCNU@HCLI. The emulgel formulations have shown the best permittivity characteristics.
Results – 2016
The evaluation of the release and permittivity profiles of the antineoplastic agents through StratM® membrane showed that the semisolid formulations of C_BCNU@HCLI and E_BXT@MG01 have the best characteristics to be considered for the further in vivo studies.
The research study followed the evaluation of the acute cutaneous toxicity of the new formulations of an antineoplastic agent, through Draize acute cutaneous irritation test applied to mouses.
The research on the white mouses regarding the local tolerance of the new obtained topical formulations has been approved by the ethical commission of the “Grigore T. Popa” University of Medicine and Pharmacy.
The cutaneous administration of each product did not produce any systemic effects, the values of the hematological, biochemical and immunological parameters in the normal limits and compatible with the positive control bench treated with physiological serum.
Anatomopathological investigations do not evidence any local modification after applying only one dose of the tested product.
Results 2017
In vivo evaluation of the chemoprevention potential and therapeutic efficacy of innovative topical formulations based on MDDS was studied for the cream C_BCNU @ HCLI and respective emulgel E_BXT @ MG01.
Mice with cutaneous carcinogenesis induced by the chemical method were treated for three weeks with C-BCNU @ HCLI cream and respective BXT @ MG01 in the single daily dose compared to the reference preparation.
The topical topical administration of the two new formulations did not statistically influence the biochemical parameters of hepatic and renal function compared to control groups treated with saline or creamy base or reference preparation.
Immunohistochemical analysis of tumor tissue after the completion of the third week of topical application of new formulations with carmustine or bexarotene was performed in skin and tumor tissue samples collected from 6 tumor-bearing mice.
Treatment with C_BCNU @ HCLI had a net effect on Bcl-2 expression significantly decreasing the anti-apoptotic factor in the tumors compared to all other treatments and reduced the chance of survival of tumor cells.
Treatment with C_BCNU @ HCLI also had a net effect on Cox-2 expression, significantly diminishing this tumor progression factor by comparison with all other types of treatments and thus reducing tumor aggression.
Treatment with E_BXT @ MG01 had a net effect on Bcl-2 expression, significantly decreasing the anti-apoptotic factor in the tumors in comparison to all other treatments and reduced the chance of survival of the tumor cells.
Treatment with E_BXT @ MG01 also had a net effect on Cox-2 expression, significantly diminishing this tumor progression factor by comparison with all other treatments and thus reducing tumor aggression.
CONCLUSIONS:
- Local application of C_BCNU @ HCLI cream reduced tumor survival (measured by decreasing bcl-2 protein expression) and tumor aggressiveness (measured by cox-2 protein decrease) significantly more statistically stronger than a reference cream containing Carmustine.
- Local application of the E_BXT @ MG01 emulsifier reduced tumor survival (measured by decreasing bcl-2 protein expression) and tumor aggression (measured by cox-2 protein decrease) significantly more statistically stronger than a reference cream containing Bexarotene significantly more statistically stronger than a reference cream containing Bexarotene.