Self-emulsifying drug delivery systems: Design of a novel vaginal delivery system for curcumin.

AIM: The aim of this study was to develop a vaginal self-emulsifying delivery system for curcumin being capable of spreading, of permeating the mucus gel layer and of protecting the drug being incorporated in oily nanodroplets towards mucus interactions and immobilization. METHODS: The emulsifying properties of curcumin loaded SEDDS containing 30% Cremophor RH40, 20% Capmul PG-8, 30% Captex 300, 10% DMSO and 10% tetraglycol (SEDD formulation A) as well as 25% PEG 200, 35% Cremophor RH40, 20% Captex 355, 10% Caprylic acid and 10% Tween 80 (SEDD formulation B) after diluting 1+2 with artificial vaginal fluid were characterized regarding droplet size and zeta potential. Collagen swelling test was used to examine the irritation potential of SEDDS. Additionally to mucus binding studies, permeation studies in the mucus were performed. Furthermore, spreading potential of the novel developed formulations was compared with a commercial available o/w cream (non-ionic hydrophilic cream) on vaginal mucosa. RESULTS: SEDDS displayed a mean droplet size between 38 and 141nm and a zeta potential of -0.3 to -1.6mV. The collagen swelling test indicated no significant irritation potential of both formulations over 24h. An immediate interaction of unformulated curcumin with the mucus was determined, whereas both SEDDS facilitated drug permeation through the mucus layer. Formulation B showed a 2.2-fold improved transport ratio of curcumin compared to SEDD formulation A. In comparison to the vaginal cream, SEDD formulation A and B were able to spread over the vaginal mucosa and cover the tissue to a 17.8- and 14.8-fold higher extent, respectively. CONCLUSION: According to these results, SEDDS seems to be a promising tool for vaginal application.

EVI2B, a gene lying in an intron of the neurofibromatosis type 1 (NF1) gene, is as the NF1 gene involved in differentiation of melanocytes and keratinocytes and is overexpressed in cells derived from NF1 neurofibromas.

The EVI2B gene is one of three genes embedded in intron 27b of the neurofibromatosis type 1 (NF1; M. Recklinghausen) gene, which are transcribed in the direction opposite that of the NF1 gene. The function of EVI2B and its relation to NF1 symptoms is unknown. Here, the amounts of NF1 and EVI2B mRNA were investigated in detail in cells involved in NF1 manifestations as café-au-lait macules and neurofibromas. These investigations showed that aside from the NF1 gene, EVI2B is involved in melanocyte and keratinocyte differentiation. Whereas in NF1 melanocytes from café-au-lait macules, EVI2B expression was not altered, in fibroblast-like cells derived from neurofibromas, an increased level of EVI2B mRNA was found. We investigated whether this increase was attributable to an influence of NF1 gene expression on the expression of the EVI2B gene, as suggested by the fact that the EVI2B primary transcript is antisense to the NF1 primary transcript. Investigations of cells derived from patients with different amounts of NF1 pre-mRNA showed no correlation between the amount of NF1 pre-mRNA and the increased level of EVI2B mRNA.

Post-transcriptional regulation of neurofibromin level in cultured human melanocytes in response to growth factors.

Among the symptoms that characterize neurofibromatosis type 1 (NF1) are pigmentation anomalies such as cafe au lait spots. It has been suggested that the reduction of the neurofibromin level in the epidermis of NF1 patients is responsible for the observed signs such as altered melanogenesis and altered density of melanocytes. Our studies show that in cultured normal human melanocytes, the neurofibromin level can be varied in vitro over a wide range by using different culture conditions. The influence of factors that control differentiation and proliferation of melanocytes on neurofibromin levels was studied. Immunoprecipitation followed by western blotting showed a 3- to 4-fold increase of neurofibromin after stimulation by PMA or bFGF, respectively, and a 1.5-fold increase in cells stimulated with steel factor. The increase of neurofibromin was not paralleled by a higher NF1 mRNA level as proved by northern blotting. Pulse-chase experiments with 35S-labeled melanocytes revealed an approximately 3-fold increase in the half-life of neurofibromin in bFGF- or PMA-stimulated cells compared to controls. These results indicate that the neurofibromin level of cultured melanocytes can be regulated by a mechanism independent of NF1 gene transcription and translation, which might influence the degradation rate of the protein.

Reduced neurofibromin content but normal GAP activity in a patient with neurofibromatosis type 1 caused by a five base pair duplication in exon 12b of the NF1 gene.

We screened a total of 87 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exons 11, 12a, and 12b of the NF1 gene using temperature gradient gel electrophoresis (TGGE). A novel mutation (1998insCCTCT) was found in exon 12b. The 5-bp duplication comprising nucleotides 1994 to 1998 is predicted to lead to a truncated protein product lacking three quarters of its C-terminal sequence including the entire GTPase-activating protein-(GAP)-related domain. This mutation is associated with a reduction by 50% of the detectable amount of neurofibromin found in this patient. Despite the reduced level of neurofibromin cellular GAP activity was normal, which suggests that defects in other functions of the neurofibromin molecule may be important in the pathogenesis of NF1.

On unequal allelic expression of the neurofibromin gene in neurofibromatosis type 1.

The autosomal dominantly inherited disease neurofibromatosis type 1 (NF1) is caused by mutations of a large gene comprising 59 exons, which code for a protein with 2818 amino acids called neurofibromin. Employing an expressed polymorphic site in exon 5 of the neurofibromin gene, the expression of its alleles was analysed quantitatively by scanning radioactive RT-PCR fragments of this exon prepared from the RNA of fibroblast cell cultures from 15 NF1 patients and of white blood cells from one NF1 patient. Thirteen of the RNA preparations yielded unequal amounts of the allelic messages. The deviations of the expression ratios (A2:A1) from 1.0 ranged from -0.9 to +25.8. The allelic messages were equally represented in the RNA preparations from five informative healthy donors. Apart from fibroblasts this phenomenon could also be detected in keratinocytes, melanocytes from normally pigmented skin and melanocytes from a café-au-lait spot of one patient. Only one of three patients affected by stop mutations exhibited unequal allelic expression. When nuclear RNA from 10 of the 13 patients was examined, equal amounts of the primary transcripts were found (average ratio A2/A1: 1.08 +/- 0.07 S.E.M.), indicating that unequal expression on the level of mRNA was not caused by mutations affecting transcriptional regulation. The ratio of the amount of neurofibromin to that of p120 GAP did not seem to be correlated with the extent of unequal allelic expression.

Ras-GTP regulation is not altered in cultured melanocytes with reduced levels of neurofibromin derived from patients with neurofibromatosis 1 (NF1).

As derivatives of the neural crest, epidermal melanocytes are supposed to be clinically affected by NF1 gene defects. The NF1 gene shares sequence homology with the p120 GTPase activating protein (p120-GAP) and neurofibromin has been shown to participate in Ras-regulation. By immunoprecipitation and Western blotting, neurofibromin was found to be expressed in melanocytes from the unaffected skin and café au lait macules of NF1 patients, but the intensity of the neurofibromin band was decreased compared to control cultures. The Ras-GTP/Ras-GDP ratios of NF1 derived melanocyte cultures were comparable to those derived from healthy donors. Furthermore, the total GAP-activity of cell lysates was not altered in NF1 melanocyte cultures compared to controls. However, lysates of proliferating melanocytes, both from NF1 patients and from healthy donors, showed an about 2-fold higher GAP-activity than poorly growing cells. Neurofibromin contributed approximately one third of total GAP-activity, in both control and NF1 melanocytes, indicating that it is not the major regulator of Ras in these cells. These results suggest that the function of neurofibromin in melanocytes is not limited to regulation of Ras activity.