Pharmacokinetics Of Tumor-blood Vessel-targeting Microemulsions Loaded With Doxorubicin

One of main research directions of biological treatment of tumor is to inhibit angiogenesis of tumor and to cut off nutrition of tumor. Vessel endothelial growth factor (VEGF) is the main enhancing factor of tumor angiogenesis. Accordingly, VEGF and its receptors have become promising inhibitors of tumor angiogenesis. The expression of VEGF and VEGFR2(KDR)are relatively high in tumor tissues. VEGF is expressed in the tumor cell and KDR is expressed in the endothelial cell of tumor blood vessels. Thus, it is easier for KDR antibody to attack tumor blood vessels, and it may become a stronger inhibitor of tumor angiogenesis.Doxorubicin (DXR) is one of the important anticancer drugs, but it can induce irreversible damage to the heart. Long circulating microemulsion is one type of drug carriers with thermodynamic stability. It is between 10-100 nm in diameter and can circulate in the blood for a long time. Because vascular permeability of tumor and inflammatory lesions is relatively high, long circulating microemulsions will be accumulated in tumor and inflammatory tissues, which can raise the therapeutic effects of the drugs delivered by microemulsions and reduce its adverse reactions at the same time, especially cardiac toxicity. In recent years, our research group connected KDR to the surface of long circulating microemulsions to develop tumor-blood vessel-targeting long circulating microemulsion loaded with doxorubicin. The results of animal test showed that tumor-blood vessel-targeting long circulating microemulsion can significantly increase anti-tumor effects of doxorubicin, while significantly reduce its acute toxicity.This study is to determine plasma the doxorubicin concentration -time curve and biodistribution curves of tumor-vessel-targeting long circulating microemulsion to explain the mechanisms of the pharmacological action of tumor-vessel-targeting long circulating microemulsions loaded with doxorubicin.Methods:1. HPLC analysis of doxorubicin2. Preparation of biological sample3. Standard curve and recovery4. Recoveries and precision5.To determine the plasma DXR concentration-time curve of long circulating microemulsion -loaded doxorubicin modified with KDR antibody6. To study of the biodistribution of the tumor-blood vessel-targeting microemulsions loaded with doxorubicin.Results:1. The distribution in the heart of doxorubicin carried by long-circulating microemulsion and tumor-blood vessel-targeting long circulating microemulsion was far less than free doxorubicin.2. The distribution in the lung of doxorubicin carried by long circulating microemulsion and tumor-blood vessel-targeting microemulsion had little difference with free doxorubicin.3. The distribution in the liver of doxorubicin carried by long circulating microemulsion and tumor-blood vessel-targeting long circulating microemulsion was far less than free doxorubicin.4. The distribution in the spleen of doxorubicin carried by long-circulating microemulsion and tumor-blood vessel-targeting long-circulating microemulsion was far less than free doxorubicin.5. The distribution in the kidney of doxorubicin carried by long circulating microemulsion and tumor-blood vessel-targeting microemulsion was also less than free doxorubicin.Discussion:The results of our test showed that long circulating microemulsion loaded with doxorubicin and tumor-blood vessel-targeting long circulating microemulsion loaded with doxorubicin can significantly prolong the cycle time of doxorubicin in the blood, while the distribution of doxorubicin in the heart, liver, spleen and kidney was significantly lower than free doxorubicin. However, long circulating microemulsion loaded with doxorubicin and tumor-blood vessel-targeting long circulating microemulsion loaded with doxorubicin had little effect on the distribution of the lung.1. To prolong the cycle time of doxorubicin in the bloodThe distribution of long circulating microemulsion loaded with doxorubicin and tumor-blood vessel-targeting long circulating microemulsion loaded with doxorubicin is related with the permeability of capillary wall in vivo. Blood capillaries is the most widely distributed blood vessels , vessel wall is thinnest, diameter of blood vessels is smallest, generally only 1-2 RBC can pass through it. Its vessel wall mainly is composed by a layer of endothelial cells and a thin layer of connective tissue outside. It is also often can be seen flat and excrescent cells attached to the outside wall of the capillary, called pericytes. The nature of the cells remains unclear. Some people speculate pericytes has effect of contraction, the diameter of capillary can be controlled, but not yet confirmed. There are experiments show that endothelial cells stimulated by certain chemical substances or mechanical irritation, it can shrink itself to change the diameter size. The average inner diameter of capillary is about 8μm, its length is 0.2-4 mm, as they are inter-connected into network, covered entirely the whole body, total cross-sectional area of the capillary is several hundred times more than aorta. Usually, there are only small part of blood capillary to open in turn. Because the capillarity blood vessel wall is thin, and permeability is higher , oxygen and nutrients in the blood enter the tissues through the capillary wall, carbon dioxide and the metabolite also can enter the blood through the capillary wall, thus completes exchange of gases and the material interchange between the blood and tissues. According to the observation of electron microscope, the capillary endothelium in kidneys and other organs has many, which is more advantageous to the material penetration , but the blood cell and the plasma protein are almost impossible. Permeability of glomerular filtration membrane is decided by two barriers: they are mechanical barrier– filter eyelets and the electrical barrier. The granularity scope of long circulating microemulsion loaded with doxorubicin and tumor-blood vessel-targeting long circulating microemulsion loaded with doxorubicin is 20-50nm, therefore they are all unable to go through the glomerular filtration membrane to excrete. In addition, glomerular filtration membrane also has the electrical barrier. Filtration membrane containing many layers of material with negative charge, mainly for the glycoprotein. These negatively charged material repel negative charged plasma protein to limit ttheir filterstion. In the circumstances of renal pathology, The negatively charged glycoprotein on the filteration membrane decrease or disappear, which can cause the filtration of negatively charged plasma protein increased significantly than normal filtration, thus appears the albuminuria. Doxorubicin with positive electricity enable its excretion by the kidneys. However, in our long circulating microemulsion loaded with doxorubicin and tumor-blood vessel-targeting long circulating microemulsion loaded with doxorubicin, the electric charge that doxorubicin complex with oleic acid has already be neutralized, which is not benefit for the renal excretion, this aslo one of factors of our long circulating microemulsion. Long circulating microemulsion loaded with doxorubicin and tumor-blood vessel-targeting long circulating microemulsion loaded with doxorubicin can circulate for a long time in the blood, which mainly determined by PEG ( polyethylene glycol) on the surface of microemulsion. Long circulating technology was originally from bionics perspective. Though our long circulating microemulsion loaded with doxorubicin and tumor-blood vessel-targeting long circulating microemulsion loaded with doxorubicin can circulate for a long time in the blood, but how to achieve the optimal prescription of pending further study.2. Distribution in the bodyThe mechanism of PEG drug vector prolong the time of blood circulation is not yet fully understood. And now it is condidered that it is related with two following factors:(1) hypothesis of steric hindrance: PEG-PE is a linear polymer, and it present partial excurrent helical conformation in the surface of liposome. (2) increase the hydrophilia of membrane surface: PEG-PE has a long polar groups which can increase the hydrophilia of drug carriers surface, thus increase the energy barrier of phagocytosis and katogene of reticuloendothelial system, and effectively provent the opsonification of the drug vector surface and blood albumin, and reduce the affinity fuction of drug carrier for the reticuloendothelial system.Doxorubicin is one of the important anticancer drugs, but its main problem is to cause irreversible damage to the heart, hence its clinical application are largely limited. As there is a long circulating microemulsion loaded with doxorubicin in the blood of lung tissue, which lead to that measured contents of doxorubicin is higher than that of true value , as the results of this experiment: It seems that long-circulating microemulsion and tumor-blood vessel-targeting microemulsion have nothing with the distribution of doxorubicin in the lung. As to how to measure actual contents of doxorubicin distribution in the lung still need further study.Effects of long-circulating microemulsion and tumor-blood vessel-targeting long-circulating microemulsion to the distribution of doxorubicin in the tumor are related to the accumulation of microemulsion in the tumor, extracellular release of the drug, phagocytosis and destruction of endothelial cells or tumor cells for microemulsion, intracellular release of the drug and so on . Related work is in progress.Conclusion:The results of our test showed that tumor-blood vessel-targeting long circulating microemulsion loaded with doxorubicin can significantly prolong the cycle time of doxorubicin in the blood, while the distribution of doxorubicin in the heart, liver and spleen was significantly lower than free doxorubicin, while the distribution of doxorubicin in the tumor significantly higher than free doxorubicin. Compared with long circulating DXR microemulsions, tumor-blood vessel-targeting long circulating DXR microemulsions lowered the distribution of doxorubicin in the kidney, while there were fewer changes in the biodistribution of DXR in heart,liver, spleen and lung. Further work is needed to study the tumor-blood vessel-targeting effects of the tumor-blood vessel-targeting long circulating DXR microemulsions.