Preparations Of Water-soluble Curcuminoids And Their Anti-metastasis Effect On Lung Cancer

In China, both the incidence and the mortality of lung cancer are the highest among malignant tumors. During the past several decades, though much progress has been made in the therapeutic measures in tumor treatment, e.g. surgical treatment, radiotherapy and chemotherapy, the 5-year survival rate of lung cancer patients has not been greatly improved. The recurrence and metastasis of lung cancer is still the big obstacle to the clinical therapeutic efficacy of the clinical treatment of patients with lung cancer.The previous studies have demonstrated that angiogenesis induced by tumor is responsible for the tumor growth and metastasis. In the past few years, studies on the angiogenesis inhibitors have attracted the attention of many oncologists, and hence several dozens of angiogenesis inhibitors have come into clinical trial stage overseas. However, studies on the tumor angiogenesis in our country are still at the exploratory stage.The main aspects of the studies on the tumor angiogenesis in our country include the following:1. Subjects are the clinical pathological specimens. The relationship of tumor angiogenesis with the degrees of malignancy, tumor metastasis, and clinical prognosis is observed by approaches such as immunohistochemistry.2. The molecular mechanisms of the well-studied anti-angiogenesis inhibitors (such as endo statin) by oversea researchers are investigated by methods such as animal and cytological experiments. Besides, the new ways for the synthesis of the angiogenesis inhibitors are investigated for the purpose of their development.3. The effects of active components of traditional Chinese medicinal herbs on the anti-angiogenesis are observed to attempt to screen the new angiogenesis inhibitors. The number of researchers engaged in this field of study is still small in our country.So far, there are few angiogenesis inhibitors having come into clinical study in our country.Traditional Chinese medicinal herbs have been practiced in our country for several thousands of years, resulting in rich clinical experience. Due to the multiple species of traditional Chinese medicinal herbs and their rich resource, and particularly the mild toxic or side effects, an increasing number of oversea researchers are attracted to the pharmacological effects and the mechanisms of actions of the traditional Chinese medicinal herbs.Curcuminoids, the major active component of curcuma of the curcuma genus, has effects on anti-tumor, anti-inflammation, anti-oxydation, and lowering blood fat, which has been largely reported overseas. It is reported that curcuminoids have been listed as the new medicine to be developed for tumor prevention in the United States of America.The previous studies have demonstrated that curcuminoids have definite effects on anti-angiogenesis and are most likely to be developed as a new type of angiogenesis inhibitors. Curcuma, one of the traditional Chinese medical herbs, is distributed widely and is inexpensive, so development of the new pharmacological activities of curcuminoids, the anti-tumor components of curcuma is of great value in clinical application.Curcuminoids, insoluble in water, are not feasible in administration by injection, so oral administration is usually employed in the current animal experiments and even clinical trials. However, some researchers of the related literatures thought that curcumin could be hardly absorbed but easily degradated in digestive tracts after the oral administration, resulting in very low bioavailability.The property of insolubility of curcuminoids not only greatly restrains the current experimental study and the administration pattern, but also significantly reduces its applied value. Therefore, the attempt to enhance the water solubility of curcuminoids to alternate its administration pattern and improve its bioavailability is one of the key problems to be urgently solved in the current studies of curcuminoids.In the field of pharmaceutics, methods for the enhancement of the water solubility ofdrugs include mainly the following:1. Decrease of grain size2. Preparation of prodrugs3. Utilization of salification (turning it into its muriaficus or methanesulfonate)4. Utilization of organ-dissolvant (e.g. alcohol)5. Utilization of surface active agent (e.g. F68)6. Utilization cyclodextrins or their derivants to prepare water soluble inclusion compoundsThe technique of cyclodextrins inclusion is one of the ideal methods for the solution of the problems of insolubility of drugs. Cyclodextrins and their derivants, often called molecular capsules in pharmaceutics, are a kind of substances with unique molecular structures and can enclose the water-insoluble guest molecules in the barrel-like molecular structures to form reversible inclusion compounds, resulting in enhancement of the water insolubility and stability of the guest molecules.Hydroxypropyl-β-cyclodextrin (HP-B-CD), the derivant of β-cyclodextrin, is increasingly applied in the field of pharmaceutics and regarded as one of the pharmaceutical adjuvants with bright future for medicinal use in that it is highly water soluble and has little toxic and side effects such as hemocytolysis and local stimulus Some researchers of the related literatures have reported that the inclusion compounds fed with it can significantly enhance drug's solubility and stability but decrease its toxic and side effects such as the local stimulus by forming inclusion compounds with HP-β-CD.In our study, using HP-β-CD as the inclusion agent, we prepared the water-soluble agent, curcuminoid-cyclodextrin inclusion compounds. We conducted a preliminary analysis of the physical and chemical properties of the inclusion compound, such as the water insolubility, stability, and toxic and side effects. In order to investigate the feasibility in the development of the inclusion compound into the preparation for anti-tumor metastasis by injection administration, we employed the lung cancer cell line with high incidence of metastasis as the subject to investigate the effect of the inclusion compound onanti-tumor metastasis. Besides, we also observed the effect of curcuminoids on the synthesis of pro-angiogenesis factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor-C (VEGF-C), the pro-lymphoangiogenesis factor in tumor cells for the purpose of further investigation of the anti-angiogenesis and anti-lymphoangiogenesis mechanisms of curcuminoids in anti-tumor metastasis.The contents of our study are as follows:1. Preparation of the water-soluble agent by cyclodextrin inclusion technique(1) Preparation and identification of curcuminoids-cyclodextrin inclusion compounds(2) Analysis of the inclusion effect of HP-β-CD on curcuminoids(3) Effects of cyclodextrin inclusion technique on the enhancement of the solubility of curcuminoids by phase-dissolubility method(4) Analysis of the stability of curcuminoid-cyclodextrin inclusion solution2. Acute toxicity testing of the curcuminoids-cyclodextrin inclusion compound3. Experimental study of the effects of curcuminoids-cyclodextrin inclusion compounds on anti-tumor metastasis(1) Analysis of the effect of water-soluble curcuminoids on anti-tumor metastasis by transplantation tumor model(2) Analysis of the inhibitive effect of water-soluble curcuminoids on the angiogenesis induced by tumor cells coated in alginate-pearl.(3) Analysis of the effect of water-soluble curcuminoids on the migration and invasion of endothelial cells and tumor cells by Boyden camerula method(4) Observation of the effect of water-soluble curcuminoids on pro-angiogenesis factors such as MMP-2, MMP-9, VEGF and bFGF and pro-lymphoangiogenesis factors such as VEGF-C synthesized by tumor cells by cell culture methods.Main results:1. Phase-dissolubility method: Al style, the phase dissolubility curve of curcumin-1,indicated the production of the inclusion compound at 1 : 1 (molar ratio) by curcumin-1 and HP-beta-CD whereas Ap style, the phase dissolubility curve of curcumin-3, indicated that the inclusion compound at 1 I 2 could be produced after the production of the inclusion compound at 1 : 1 by curcumin-3 and HP-beta-CD (at the concentration of HP-beta-CD: 0.625% or so).2. Effect of cyclodextrin inclusion technique on the enhancement of solubility of curcumin-1 and curcumin-3: HP-β -CD solution at the concentration of 5% could enhance the water solubility of curcumin-1 and curcumin-3 by 173.6 and 702.8 folds, respectively, resulting in water-soluble drugs.3. Stability of drugs: The main factors affecting the stability of curcumin-1 and curcumin-3 are oxidation degradation and photo degeneration (and heat maybe). After inclusion of the two curcuminoids by HP-β-CD, no significant change was found in the instability of these two curcuminoids. However, under the light-proof, tightly closed, and hypothermal conditions, the two curcuminoids and their cyclodextrin inclusion compounds were of satisfactory stability, e.g. no significant decrease of the curcuminoid contents in 40% alcohol/water solution was found within 3 month.4. Acute toxicity testing: All the indexes of acute toxicity of curcumin-cyclodextrin inclusion compound was similar to those of the negative control drug HP-β-CD, suggesting that there was little toxic and side effects of curcumin after inclusion by cyclodextrin.5. Transplantation tumor in mice: Curcumin-cyclodextrin inclusion compound had significant inhibitory effect on the proliferation of pulmonary and lymphatic metastases, but not on their incidences. In addition, curcumin-cyclodextrin inclusion compound had significant inhibitory effect on the angiogenesis on the surface and in the tissues of the body of the pulmonary adenocarcinoma transplanted in mice.6. Alginate-pearl coating tumor cells method: Curcumin-cyclodextrin inclusion compound had significant inhibitory effect on the angiogenesis induced by tumor cells, which might be one of the patterns in anti-metastasis.7. Cell culture: Curcumin-cyclodextrin inclusion compound or curcumin-3-cyclodextrin might exert their effects on anti-tumor metastasis through the inhibition of the migration capability of endothelial cells and the expression and secretion of pro-angiogenesis factors such as MMP-2, VEGF and bFGF, resulting in inhibition of the tumor angiogenesis and the migration capability of tumor cells.Conclusion:In our study, we prepared two water-soluble inclusion compounds of curcuminoids, curcumin-1 and curcumin-3 inclusion compounds, using HP- β -CD by cyclodextrin inclusion technique and investigated the inhibitory effect of curcuminoid-cyclodextrin inclusion compounds on tumor metastasis and the possible molecular mechanisms by employing murine model of transplanted pulmonary adenocarcinoma accompanied by spontaneous metastasis. Our study demonstrated that the water-soluble curcuminoids prepared by us had little toxic and side effects and could significantly inhibit the spontaneous angiogenic and lymphatic metastasis of the pulmonary adenocarcinoma transplanted in mice.Our study indicated that effecting both of angiogenesis and tumor cells itselves at the same time may be one of the main mechanisms of anti-metastasis of water-soluble inclusion compound of curcumin on the pulmonary adenocarcinoma transplanted in mice:1. To inhibit angiogenesis induced by tumor: inhibiting both of the pro-angiogenesis factors such as MMP-2, VEGF and bFGF produced in tumor cells and the migration of endothelial cells.2. To inhibit the migration and invasion of tumor cells.