Study On Gambogic Acid Via Topical Delivery For Targeted Therapy Of Cutaneous Melanoma

Cutaneous melanoma,a malignant tumor arising from a high-grade lesion of epidermal melanocytes,is the most deadly form among skin cancers due to its high malignancy and early metastasis.Gambogic acid（GA）is an effective ingredient derived from gamboge resin and has been confirmed to be effective against malignant melanoma.Being a multi-target anticancer active component,GA brings a prospect in the prevention and treatment of cancer with advantages of the multiple signaling pathways and its synergistic antitumor effect with other chemotherapeutic drugs.However,due to its high liver first pass effect,short blood half-life and poor stability,the bioavailability of GA through oral and intravenous administration is low.In addition,clinical trials have shown that intravenous injection of GA causes toxic side effects,such as phlebitis,nausea,heart rhythm disturbances,hepatic impairment and nephrotoxicity.Therefore,it is of great research significance and clinical value to find a new delivery mode of GA with reducing toxicity and enhancing efficacy.To this end,we studied a novel route of GA via skin delivery assisted by the combination of enhancers and low-frequency ultrasound for targeted therapy of cutaneous melanoma and reduction of systemic toxicity,and the following findings were achieved:（1）The physicochemical properties and percutaneous permeation of GA in vitro were investigated.First,a high-performance liquid chromatography quantitative analysis method for GA was established,which showed good linearity,high precision and specificity.Secondly,it was found that both the solubility and stability of GA were affected by the p H value,and its solubility in the receptor fluid increased as the p H value increased,but its stability decreased instead.The choice of a weakly acidic（p H=5.5）receptor medium under sink conditions provides an accurate quantitative analysis of GA permeability.In addition,the barrier of the stratum corneum and hydrophilic surroundings of viable skin（the epidermis without stratum corneum）were the main factors limiting GA permeation.The large molecular weight（628.75）,poor aqueous solubility（0.56±0.07μg/m L）and high log P（4.32±0.11）of GA caused it difficult to penetrate through the intact hiraless mouse skin by passive diffusion.GA dissolved in the hydrophilic solvents showed higher skin permeability than that in the lipophilic solvents,and its skin accumulation at 24 h was as high as 21.4 times that of the control group.GA could be rapidly metabolized in liver tissues,in contrast it was more stable in skin tissues.Additionally,no significant binding of GA in the stratum corneum and viable skin was found,and it mainly accumulated in the skin tissue in a free form.（2）To enhance the skin permeability of GA,the percutaneous enhancement effect and mechanism of enhancers and low-frequency ultrasound on GA were studied.The results showed that both enhancers and low-frequency ultrasound used alone could increase the amount of GA in the deeper region of skin,and the best combination of enhancers was 0.5wt%azone（AZ）and 1 wt%propylene glycol（PG）.The parameters of ultrasound application at a constant frequency（30 k Hz）with a duty cycle of 100%and intensity of 1.75 W/cm²for20 min were optimal.The combination of enhancers（AZ-PG）and low-frequency ultrasound showed a considerable enhancement of the accumulation of GA in the viable skin（viable epidermis and dermis）,which was 6.0 times that of the control group,and the enhancement effect was stronger than the use of enhancers or ultrasound alone.It provides the possibility of achieving a topical targeted treatment of cutaneous melanoma and reducing the systemic toxicity of GA by topical delivery.The combined use of enhancers and low-frequency ultrasound not only altered the structure of lipid bilayers and keratins to reduce the barrier effect of the stratum corneum but also produced diffusion channels in the skin under the cavitation effect of ultrasound,thereby promoting the skin penetration of GA.（3）The antimelanoma activity and safety of GA were evaluated.The results showed that GA was highly toxic to B16-F10 cells with an IC₅₀value of 0.45±0.09μM,and it could kill melanoma B16-F10 cells at a low concentration in vitro.B16-F10 cells（4×10⁵cells/m L）were injected intradermally into each C57BL/6J mouse to establish a melanoma model.In the absence of enhancement means,both the volume and weight of tumors after topical delivery of GA were not significantly different from those of oral administration（p>0.05）,and the antitumor effect was inferior to that of intravenous injection.However,the antitumor activity of GA via topical delivery was enhanced by the combination of enhancers and low-frequency ultrasound,which increased the concentration of GA in the melanoma tissue,leading to an increased activity of caspase-3 and apoptosis of melanoma cells.Its efficacy was significantly stronger than that of GA intravenous group and oral administration group.Moreover,intravenous injection of GA induced remarkable pathological changes in various organs in mice,whereas GA was not toxic to organs or to the skin via topical delivery.These findings indicated that topical administration of GA was effective and safe.In summary,nonclinical pharmacodynamic studies have demonstrated the ability of GA via topical delivery assisted by the combination of enhancers（AZ-PG）and ultrasound（30k Hz）for targeted therapy of melanoma,with low toxicity and stronger antimelanoma efficacy than intravenous injection and oral administration.In this paper,we studied a novel delivery route of GA,which adds new possibilities for its clinical application,and also provides new ideas and technical guidance for the development of dermal therapeutic systems of lipophilic macromolecular antitumor drugs in the future.