The use of liposomes functionalized with the NFL-TBS.40-63 peptide as a targeting agent to cross the in vitro blood-brain barrier and target glioblastoma cells.

Glioblastoma is the most common and aggressive brain tumor. Current treatments do not allow to cure the patients. This is partly due to the blood-brain barrier (BBB), which limits the delivery of drugs to the pathological site. To overcome this, we developed liposomes functionalized with a neurofilament-derived peptide, NFL-TBS.40-63 (NFL), known for its highly selective targeting of glioblastoma cells. First, in vitro BBB model was developed to check whether the NFL can also promote barrier crossing in addition to its active targeting capacity. Permeability experiments showed that the NFL peptide was able to cross the BBB. Moreover, when the BBB was in a pathological situation, i.e., an in vitro blood-brain tumor barrier (BBTB), the passage of the NFL peptide was greater while maintaining its glioblastoma targeting capacity. When the NFL peptide was associated to liposomes, it enhanced their ability to be internalized into glioblastoma cells after passage through the BBTB, compared to liposomes without NFL. The cellular uptake of liposomes was limited in the endothelial cell monolayer in comparison to the glioblastoma one. These data indicated that the NFL peptide is a promising cell-penetrating peptide tool when combined with drug delivery systems for the treatment of glioblastoma.

Glioblastoma-targeted, local and sustained drug delivery system based on an unconventional lipid nanocapsule hydrogel.

The objective of this work was to develop an implantable therapeutic hydrogel that will ensure continuity in treatment between surgery and radiochemotherapy for patients with glioblastoma (GBM). A hydrogel of self-associated gemcitabine-loaded lipid nanocapsules (LNC) has shown therapeutic efficacy in vivo in murine GBM resection models. To improve the targeting of GBM cells, the NFL-TBS.40-63 peptide (NFL), was associated with LNC. The LNC-based hydrogels were formulated with the NFL. The peptide was totally and instantaneously adsorbed at the LNC surface, without modifying the hydrogel mechanical properties, and remained adsorbed to the LNC surface after the hydrogel dissolution. In vitro studies on GBM cell lines showed a faster internalization of the LNC and enhanced cytotoxicity, in the presence of NFL. Finally, in vivo studies in the murine GBM resection model proved that the gemcitabine-loaded LNC with adsorbed NFL could target the non-resected GBM cells and significantly delay or even inhibit the apparition of recurrences.

Formulation of benznidazole-lipid nanocapsules: Drug release, permeability, biocompatibility, and stability studies.

Benznidazole, a poorly soluble in water drug, is the first-line medication for the treatment of Chagas disease, but long treatment periods at high dosages cause several adverse effects with insufficient activity in the chronic phase. According to these facts, there is a serious need for novel benznidazole formulations for improving the chemotherapy of Chagas disease. Thus, this work aimed to incorporate benznidazole into lipid nanocapsules for improving its solubility, dissolution rate in different media, and permeability. Lipid nanocapsules were prepared by the phase inversion technique and were fully characterized. Three formulations were obtained with a diameter of 30, 50, and 100 nm and monomodal size distribution with a low polydispersity index and almost neutral zeta potential. Drug encapsulation efficiency was between 83 and 92 % and the drug loading was between 0.66 and 1.04 %. Loaded formulations were stable under storage for one year at 4 °C. Lipid nanocapsules were found to protect benznidazole in simulated gastric fluid and provide a sustained release platform for the drug in a simulated intestinal fluid containing pancreatic enzymes. The small size and the almost neutral surface charge of these lipid nanocarriers improved their penetration through mucus and such formulations showed a reduced chemical interaction with gastric mucin glycoproteins. LNCs. The incorporation of benznidazole in lipid nanocapsules improved the drug permeability across intestinal epithelium by 10-fold compared with the non-encapsulated drug while the exposure of the cell monolayers to these nanoformulations did not affect the integrity of the epithelium.

Physicochemical Characterization of Ferrocifen Lipid Nanocapsules: Customized Drug Delivery Systems Guided by the Molecular Structure.

Ferrocifens, lipophilic organometallic complexes, comprise a biologically active redox motif [ferrocenyl-ene-p-phenol] which confers very interesting cytotoxic properties to this family. However, because of their highly lipophilic nature, a formulation stage is required before being administered in vivo. In recent decades, ferrocifen lipid nanocapsules (LNCs) have been successfully formulated and have demonstrated anticancer activity on multidrug-resistant cancers in several mice and rat models (glioblastoma, breast cancer, and metastatic melanoma). A recent family of ferrocifens (succinimidoalkyl-ferrociphenols, including P722) appears to be most efficacious on several resistant cancer cell lines, with IC50 values in the nanomolar range together with promising in vivo results on murine ovarian cancer models. As LNCs are composed of an oily core (caprylic/capric triglycerides), modulation of the succinimido-ferrociphenol lipophilicity could be a valuable approach toward improving the drug loading in LNCs. As the drug loading of the diphenol P722 in LNCs was low, it was structurally modified to increase its lipophilicity and thereby the payload in LNCs. Chemical modification led to a series of five succinimido-ferrocifens. Results confirmed that these slight structural modifications led to increased drug loading in LNCs for all ferrocifens, with no reduction of their cytotoxicity on the SKOV3 ovarian cancer cell line. Interestingly, encapsulation of two of the ferrocifens, diester P769 and monophenolic ester (E)-P998, led to the formation of a gel. This was unprecedented behavior, a phenomenon that could be rationalized in terms of the positioning of ferrocifens in LNCs as shown by the decrease of interfacial tension measurements at the water/oil interface. Moreover, these results highlighted the importance of obtaining a gel of this particular motif, in which the acetylated phenolic ring and the succinimidoalkyl moieties are mutually cis relative to the central double bond. Promising perspectives to use these ferrocifen-loaded LNCs to treat glioblastoma could be readily envisaged by local application of the gel in the cavity after tumor resection.

Tailoring PEGylated nanoparticle surface modulates inflammatory response in vascular endothelial cells.

Polymer nanoparticles (NPs) are extensively studied as drug delivery systems for various therapeutic indications, including drug and imaging agent delivery to the brain. Despite intensive research, their toxicological profile has yet to be fully characterized. In particular, the more subtle effects of nanomaterials on inflammatory processes have scarcely been investigated. Surface properties of NPs are amongst parameters governing interactions between living cells and NPs. They could considerably influence the toxicity and inflammatory response of the cells exposed to NPs. Polymeric NPs investigated here present a core-shell structure. The core is constituted of hydrophobic poly(lactic acid) (PLA) block and the surface is composed of a shell of hydrophilic block of polyethylene glycol (PEG). The effect of PEG chain length coating on the expression of genes involved in the inflammation response was investigated in two vascular endothelial cell lines (bEnd.3 and HUVEC) by qPCR. Moreover, ROS generation following NP uptake was evaluated. PEGylated NPs induce a mild and transient activation of inflammatory cytokine and chemokine genes. However, differences in PEG chain length did not show any significant effect on cytokine and chemokine gene expression and PEGylated NPs did not trigger ROS generation. The present results could contribute significantly to a deeper understanding of nanomaterial interactions and toxicity with vascular endothelial cells, guiding scientists in material coating choices.

Polymer-free hydrogel made of lipid nanocapsules, as a local drug delivery platform.

Nanoparticle-loaded hydrogels are attractive pharmaceutical drug delivery systems that combine the advantages of both hydrogel (local administration and/or sustained drug release) and nanoparticle (stealthiness, targeting and decreased toxicity). The design of nanoparticle-loaded hydrogels is largely conventional, consisting of the dispersion of nanoparticles in a natural or synthetic polymer matrix to form a gel network. Novel nanoparticle-loaded hydrogels architecture could provide advantages in terms of innovation and application. We focused on the development of lipid nanocapsule (LNC)-based hydrogels without the use of a polymer matrix as a platform for drug delivery. Cytidine was modified by grafting palmitoyl chains (CytC16) and the new entity was added during the LNC phase-inversion formulation process allowing spontaneous gelation. Positioned at the oil/water interface, CytC16 acts as a crosslinking agent between LNCs. Association of the LNCs in a three-dimensional network led to the formation of polymer-free hydrogels. The viscoelastic properties of the LNC-based hydrogels depended on the LNC concentration and CytC16 loading but were not affected by the LNC size distribution. The LNC and drug-release profiles were controlled by the mechanical properties of the LNC-based hydrogels (slower release profiles correlated with higher viscoelasticity). Finally, the subcutaneous administration of LNC-based hydrogels led to classic inflammatory reactions of the foreign body-reaction type due to the endogenous character of CytC16, shown by cellular viability assays. New-generation nanoparticle-loaded hydrogels (LNC-based polymer-free hydrogels) show promise as implants for pharmaceutical applications. Once LNC release is completed, no gel matrix remains at the injection site, minimizing the additional toxicity due to the persistence of polymeric implants. Sustained drug-release profiles can be controlled by the mechanical properties of the hydrogels and could be tailor-made, depending on the therapeutic strategy chosen.

Characterization of Biological Material Adsorption to the Surface of Nanoparticles without a Prior Separation Step: a Case Study of Glioblastoma-Targeting Peptide and Lipid Nanocapsules.

PURPOSE: Current preclinical therapeutic strategies involving nanomedicine require increasingly sophisticated nanosystems and the characterization of the complexity of such nanoassemblies is becoming a major issue. Accurate characterization is often the factor that can accelerate the translational approaches of nanomedicines and their pharmaceutical development to reach the clinic faster. We conducted a case study involving the adsorption of the NFL-TBS.40-63 (NFL) peptide (derived from neurofilaments) to the surface of lipid nanocapsules (LNCs) (a combined nanosystem used to target glioblastoma cells) to develop an analytical approach combining the separation and the quantification in a single step, leading to the characterization of the proportion of free peptide and thus the proportion of peptide adsorbed to the lipid nanocapsule surface. METHODS: LNC suspensions, NFL peptide solution and LNC/NFL peptide mixtures were characterized using a Size-Exclusion Chromatography method (with a chromatographic apparatus). In addition, this method was compared to centrifugal-filtration devices, currently used in literature for this case study. RESULTS: Combining the steps for separation and characterization in one single sequence improved the accuracy and robustness of the data and led to reproducible results. Moreover the data deviation observed for the centrifugal-filtration devices demonstrated the limits for this increasingly used characterization approach, explained by the poor separation quality and highlighting the importance for the method optimization. The high potential of the technique was shown, proving that H-bond and/or electrostatic interactions mediate adsorption of the NFL peptide to the surface of LNCs. CONCLUSIONS: Used only as a characterization tool, the process using chromatographic apparatus is less time and solvent consuming than classical Size-Exclusion Chromatography columns only used for separation. It could be a promising tool for the scientific community for characterizing the interactions of other combinations of nanosystems and active biological agents.

Local Delivery and Glioblastoma: Why Not Combining Sustained Release and Targeting?

Glioblastoma is one of the most aggressive brain tumors and is associated with a very low overall median survival despite the current treatment. The standard of care used in clinic is the Stupp's protocol which consists of a maximal resection of the tumor when possible, followed by radio and chemotherapy using temozolomide. However, in most cases, glioblastoma cells infiltrate healthy tissues and lead to fatal recurrences. There are a lot of hurdles to overcome in the development of new therapeutic strategies such as tumor heterogeneity, cell infiltration, alkylating agent resistance, physiological barriers, etc., and few treatments are on the market today. One of them is particularly appealing because it is a local therapy, which does not bring additional invasiveness since tumor resection is included in the gold standard treatment. They are implants: the Gliadel® wafers, which are deposited post-surgery. Nevertheless, in addition to presenting important undesirable effects, it does not bring any major benefit in the therapy despite the strategy being particularly attractive. The purpose of this review is to provide an overview of recent advances in the development of innovative therapeutic strategies for glioblastoma using an implant-type approach. The combination of this local strategy with effective targeting of the tumor microenvironment as a whole, also developed in this review, may be of interest to alleviate some of the obstacles encountered in the treatment of glioblastoma.

Residual Solvents in Nanomedicine and Lipid-Based Drug Delivery Systems: a Case Study to Better Understand Processes.

PURPOSE: Complexities surrounding the manufacture and quality control of nanomedicines become increasingly apparent. This research article offers a case study to investigate how, at the laboratory scale, various stages of liposome and nanoparticle synthesis affect the amount of residual solvent found in the formulations. The objective is to bring insights on the reliability of each of these processes to provide final products which meet regulatory standards and facilitate identifying possible bottleneck early during the development process. METHODS: The residual solvent at various stages of preparation and purification was measured by headspace gas chromatography. Liposomes were prepared by two different methods with and without solvent. Polymer nanoparticles prepared via nanoprecipitation and purified by ultrafiltration were studied. The effects of purification by size exclusion chromatography and dialysis were also investigated. RESULTS: The complete removal of residual solvent requires processes which go beyond usual preparation methods. CONCLUSIONS: This work might prove valuable as a reference for scientists of different fields to compare their own practices and streamline the translation of nanomedicines into efficacious and safe drug products.

Behavior of poly(d,l-lactic-co-glycolic acid) (PLGA)-based droplets falling into a complex extraction medium simulating the prilling process.

HYPOTHESIS: Prilling process is one of advanced techniques for manufacturing microspheres of controlled and uniform size. In this process, homogenous polymer droplets fall into an extraction medium. The aim of this study was to identify the key parameters influencing the behavior of PLGA polymer-based droplets falling into a complex extraction medium, to select appropriate conditions for prilling. EXPERIMENTS: Polymer solutions and extraction media were characterized by determining their viscosity, density and surface tension. A simple model simulating the prilling process was developed to study droplet behavior. Particle shape and velocity at the air-liquid interface and during sedimentation in the container were analyzed step by step. The correlations between the variables studied were visualized by principal component analysis (PCA). FINDINGS: Droplet deformation at the interface greatly affected the recovery and final particle shape. It depended on the viscosity ratio of polymer solution/extraction medium. The particle shape recovery depended on the viscosity and density of extraction media and polymer solutions. The solidification speed is also an important parameter. In media which the solvent diffused slowly, particles were able to relax and recover their shape, however, they can also deform during sedimentation and collision with the bottom of the cuvette.

Self-assembled hyaluronan nanocapsules for the intracellular delivery of anticancer drugs.

Preparation of sophisticated delivery systems for nanomedicine applications generally involve multi-step procedures using organic solvents. In this study, we have developed a simple self-assembling process to prepare docetaxel-loaded hyaluronic acid (HA) nanocapsules by using a self-emulsification process without the need of organic solvents, heat or high shear forces. These nanocapsules, which comprise an oily core and a shell consisting of an assembly of surfactants and hydrophobically modified HA, have a mean size of 130 nm, a zeta potential of -20 mV, and exhibit high docetaxel encapsulation efficiency. The nanocapsules exhibited an adequate stability in plasma. Furthermore, in vitro studies performed using A549 lung cancer cells, showed effective intracellular delivery of docetaxel. On the other hand, blank nanocapsules showed very low cytotoxicity. Overall, these results highlight the potential of self-emulsifying HA nanocapsules for intracellular drug delivery.

Nanoencapsulated deltamethrin as synergistic agent potentiates insecticide effect of indoxacarb through an unusual neuronal calcium-dependent mechanism.

The use of neurotoxic chemical insecticides has led to consequences against the environment, insect resistances and side-effects on non-target organisms. In this context, we developed a novel strategy to optimize insecticide efficacy while reducing doses. It is based on nanoencapsulation of a pyrethroid insecticide, deltamethrin, used as synergistic agent, combined with a non-encapsulated oxadiazine (indoxacarb). In this case, the synergistic agent is used to increase insecticide efficacy by activation of calcium-dependant intracellular signaling pathways involved in the regulation of the membrane target of insecticides. In contrast to permethrin (pyrethroid type I), we report that deltamethrin (pyrethroid type II) produces an increase in intracellular calcium concentration in insect neurons through the reverse Na/Ca exchanger. The resulting intracellular calcium rise rendered voltage-gated sodium channels more sensitive to lower concentration of the indoxacarb metabolite DCJW. Based on these findings, in vivo studies were performed on the cockroach Periplaneta americana and mortality rates were measured at 24 h, 48 h and 72 h after treatments. Comparative studies of the toxicity between indoxacarb alone and indoxacarb combined with deltamethrin or nanoencapsulated deltamethrin (LNC-deltamethrin), indicated that LNC-deltamethrin potentiated the effect of indoxacarb. We also demonstrated that nanoencapsulation protected deltamethrin from esterase-induced enzymatic degradation and led to optimize indoxacarb efficacy while reducing doses. Moreover, our results clearly showed the benefit of using LNC-deltamethrin rather than piperonyl butoxide and deltamethrin in combination commonly used in formulation. This innovative strategy offers promise for increasing insecticide efficacy while reducing both doses and side effects on non-target organisms.

Drug combination using an injectable nanomedicine hydrogel for glioblastoma treatment.

Lauroyl-gemcitabine lipid nanocapsules (GemC12-LNC) hydrogel, administered intratumorally or perisurgically in the tumor resection cavity, increases animal survival in several orthotopic GBM models. We hypothesized that GemC12-LNC can be used as nanodelivery platform for other drugs, to obtain a combined local therapeutic approach for GBM. Paclitaxel (PTX) was selected as a model molecule and PTX-GemC12-LNC formulation was evaluated in terms of physicochemical and mechanical properties. The PTX-GemC12-LNC hydrogel stability and drug release were evaluated over time showing no significant differences compared to GemC12-LNC. The drug combination was evaluated on several GBM cell lines showing increased cytotoxic activity compared to the original formulation and synergy between PTX and GemC12. Our results suggest that GemC12-LNC hydrogel can be used as nanodelivery platform for dual drug delivery to encapsulate active agents with different mechanisms of action to achieve a better antitumor efficacy against GBM or other solid tumors.

Evaluation of lauroyl-gemcitabine-loaded hydrogel efficacy in glioblastoma rat models.

AIM: Anticancer drug-loaded hydrogels are a promising strategy for the local treatment of incurable brain tumors such as glioblastoma (GBM). Recently, we demonstrated the efficacy of lauroyl-gemcitabine lipid nanocapsule hydrogel (GemC12-LNC) in a U-87 MG xenograft orthotopic mouse model. In this study, we developed a reliable and reproducible surgical procedure to resect orthotopic GBM tumors in rats. GemC12-LNC hydrogel integrity was tested after brain administration in rats and its anti-tumor efficacy was tested on a 9L syngeneic orthotopic model. RESULTS: We demonstrated that LNC integrity is maintained at least for one week after local administration of GemC12-LNC. GemC12-LNC was able to delay the formation of recurrences in 9L tumor-bearing resected rats, demonstrating the efficacy of this nanomedicine hydrogel in this preclinical model. CONCLUSION: Our results confirm that GemC12-LNC, a hydrogel uniquely formed by a nanocarrier and a cytotoxic drug, could be a promising and safe delivery tool for the local treatment of operable GBM tumors.

Gemcitabine and glioblastoma: challenges and current perspectives.

Gemcitabine is a nucleoside analog currently used for the treatment of various solid tumors as a single agent or in combination with other chemotherapeutic drugs. Its use against highly aggressive brain tumors (glioblastoma) has been evaluated in preclinical and clinical trials leading to controversial results. Gemcitabine can inhibit DNA chain elongation, is a potent radiosensitizer and it can enhance antitumor immune activity, but it also presents some drawbacks (e.g., short half-life, side effects, chemoresistance). The aim of this review is to discuss the challenges related to the use of gemcitabine for glioblastoma and to report recent studies that suggest overcoming these obstacles opening new perspectives for its use in the field (e.g., gemcitabine derivatives and/or nanomedicines).

Recent advances in nanocarrier-loaded gels: Which drug delivery technologies against which diseases?

The combination of pharmaceutical technologies can be a wise choice for developing innovative therapeutic strategies. The association of nanocarriers and gels provides new therapeutic possibilities due to the combined properties of the two technologies. Gels support the nanocarriers, localize their administration to the target tissue, and sustain their release. In addition to the properties afforded by the gel, nanocarriers can provide additional drug sustained release or different pharmacokinetic and biodistribution profiles than those from nanocarriers administered by the conventional route to improve the drug therapeutic index. This review focuses on recent (over the last ten years) in vivo data showing the advances and advantages of using nanocarrier-loaded gels. Liposomes, micelles, liquid and solid lipid nanocapsules, polymeric nanoparticles, dendrimers, and fullerenes are all nanotechnologies which have been recently assessed for medical applications, such as cancer therapy, the treatment of cutaneous and infectious diseases, anesthesia, the administration of antidepressants, and the treatment of unexpected diseases, such as alopecia.

Synergistic interactions between antimicrobial peptides derived from plectasin and lipid nanocapsules containing monolaurin as a cosurfactant against Staphylococcus aureus.

Development of effective antibacterial agents for the treatment of infections caused by Gram-positive bacteria resistant to existing antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA), is an area of intensive research. In this work, the antibacterial efficacy of two antimicrobial peptides derived from plectasin, AP114 and AP138, used alone and in combination with monolaurin-lipid nanocapsules (ML-LNCs) was evaluated. Several interesting findings emerged from the present study. First, ML-LNCs and both plectasin derivatives showed potent activity against all 14 tested strains of S. aureus, independent of their resistance phenotype. Both peptides displayed a considerable adsorption (33%-62%) onto ML-LNCs without having an important impact on the particle properties such as size. The combinations of peptide with ML-LNC displayed synergistic effect against S. aureus, as confirmed by two methods: checkerboard and time-kill assays. This synergistic interaction enables a dose reduction and consequently decreases the risk of toxicity and has the potential of minimizing the development of resistance. Together, these results suggest that ML-LNCs loaded with a plectasin derivative may be a very promising drug delivery system for further development as a novel antibacterial agent against S. aureus, including MRSA.

Stealth nanocarriers based sterosomes using PEG post-insertion process.

Sterosomes (STEs), a new and promising non-phospholipidic liposome platform based on palmitic acid (PA) and cholesterol (Chol) mixtures, need to have polyethylene glycol (PEG) chains grafted to their surface in order to obtain long-circulating nanocarriers in the blood stream. A post-insertion method was chosen to achieve this modification. The post-insertion process of PEG-modified distearoylphosphoethanolamine (DSPE-PEG) was monitored using the zeta potential value of STEs. Various conditions including PEG chain length and the DSPE-PEG/PA-Chol ratio, were explored. Zeta potential of STEs changed from about -40mV for non-modified STEs to values close to 0mV by the end of the process, i.e. for PEG-modified STEs. The kinetics of DSPE-PEG insertion and the stability of the resulting PEG-modified STEs were not considerably influenced, within the investigated range, by changes in PEG chain lengths and in DSPE-PEG/PA-Chol proportion. The post-insertion of PEG chains reduced in vitro complement activation as well as in vitro macrophage uptake compared to the non-modified STEs. Moreover, longer blood circulation time in mice was established for PEG-modified STEs intravenously injected compared to non-modified STEs. These results establish that post-insertion process of PEG chains to STEs is a promising strategy for developing long-term circulating drug delivery nanocarriers.

Perfluorocarbon-Loaded Lipid Nanocapsules to Assess the Dependence of U87-Human Glioblastoma Tumor pO2 on In Vitro Expansion Conditions.

Growing tumor cell lines, such as U87-MG glioma cells, under mild hypoxia (3% O2) leads to a ca. 40% reduction in growth rate once implanted in the brain of nude mice, as compared to normoxia (21% O2) grown cells, wherein the former over-express HIF-1 and VEGF-A. Despite developing differently, the tumors have similar: blood perfusion, oxygen consumption, and vascular surface area parameters, whereas the number of blood vessels is nearly doubled in the tumor arising from normoxia cultured cells. Interestingly, tumor oxygen tension, measured using 19F-oximetry, showed that the normoxia grown cells led to tumors characterized by mild hypoxic environment (approximately 4%) conditions, whilst the hypoxia grown cells led to tumors characterized by physioxic environment (approximately 6%) conditions. This reversal in oxygen concentration may be responsible for the apparent paradoxical growth profiles.

Formulation and characterization of a 0.1% rapamycin cream for the treatment of Tuberous Sclerosis Complex-related angiofibromas.

Medicines for the treatment of rare diseases frequently do not attract the interest of the pharmaceutical industry, and hospital pharmacists are thus often requested by physicians to prepare personalized medicines. Tuberous Sclerosis Complex (TSC) is a rare disease that causes disfiguring lesions named facial angiofibromas. Various topical formulations of rapamycin (=sirolimus) have been proved effective in treating these changes in small case series. The present study provides for the first time characterization of a 0.1% rapamycin cream formulation presenting good rapamycin solubilisation. The first step of the formulation is solubilisation of rapamycin in Transcutol(®), and the second step is the incorporation of the mixture in an oil-in-water cream. A HPLC stability-indicating method was developed. Rapamycin concentration in the cream was tested by HPLC and confirmed that it remained above 95% of the initial concentration for at least 85days, without characteristic degradation peaks. The preparation met European Pharmacopoeia microbial specifications throughout storage in aluminum tubes, including when patient use was simulated. Odour, appearance and colour of the preparation were assessed and no change was evidenced during storage. The rheological properties of the cream also remained stable throughout storage. To conclude, we report preparation of a novel cream formulation presenting satisfactory rapamycin solubilisation for the treatment of TSC cutaneous manifestations, with stability data. The cream is currently being used by our patients. Efficacy and tolerance will be reported later.