Nano-activated Carbon Formulations Of The Drug Pharmacokinetics And Tumor Effect Observed

ObjectTo develop a kind of active carbon particles which is provided with better lymph target, drug adsorption, release effect and no toxicity. Evaluate the main characters such as carry and release drug effect and the anti-tumor effect to the mice with transplanted tumors, to provide the basis in clinic.Methods(1) With dialysis and orthogonal design experiment, we measure the adsorption and release effect of 100nm active carbon suspension to the 5-FU. With ultraviolet-visible spectrophotometry , we measured the concentration of 5-FU and the wavelength was 265nm. Dialysis method: The dialysis film was fixed in the diffusion pond. The 3ml distilled water and 3ml drug suspension were in the each side of the dialysis film. When the diffusion reached balance, we took 50ul sample from the side of the distilled water and measured the absorbency after reasonable dilution. Basing on the standard curve, we got the freeing concentration of 5-FU. Repeating the experiment, if the two values were equal or approximate, we considered the freeing drug quantity and the adsorptive quantity reached dynamic equilibrium. The quantity of 5-FU was from lower to higher and we can get the adsorptive curve basing on a series of dots.Orthogonal design experiment: prescription: the suspension consists of 5-FU 75mg/3ml(basing on pharmacopeia ), nano-carbon 9% and PVP7%; Drug releasing condition: 37℃, velocity of flow was 3ml/min. Recorded the quantity of liquid in each hour. In the same time, we took and diluted the samples and measured the absorbency with 265nm to get the concentration and accumulative total releasing percent of the drugs. (2) With Dialysis method, we measured the adsorptive and release effect of 60nm carbon suspension to HCPT. With Ultraviolet-Visible Spectrophotometry, we measured the concentration of HCPT. The wavelength is 413nm.Put lmg/ml HCPT nona-carbon suspension 3ml in a 10cm long dialysis bag and suspended in a 100ml 0.01mol/L NaOH dialysis liquid and shocked it with constant velocity during 37±1℃. At the same time, we took sample 3ml on time and filled up equi-quantity medium in it. The absorbency to the sample was measured with 413nm and got freeing drug concentration C and releasing percent of drug Q. At the same time, the equi-quantity HCPT was used in the control group. (3) We also measured drug concentration in blood of rats and mice with the method of HPLC to compared the change between HCPT/HCPT-nc and 5-FU/5-FU-nc in pharmacokinetics after intraperitoneal Administration. Their measuring wavelengths were 382nm and 265nm. The experiment animals were divided into groups at random. Basing on their body weight, the animals were administrated. Then we collected veno-blood at different time and measured the samples. By the ratio of HCPT/standard pulse area and5-FU/standard pulse area, we got the concentrations of HCPT and 5-FU. By the statistic treatment, we got the pharmacokinetic paramete in blood. (4) In this study we measured the anti-tumor effects of merely HCPT,5-FU and carbon-HCPT(50nm), crbon-5-FU(100nm) by the model of nude mice which were transplanted human gastric carcinoma cells. The model consisted with 25 BALB/c-nu mice. They were male, 8¹0weeks old and 22²8g weight. They were divided into five groups: 5-FU, 5-FU-nc, HCPT, HCPT-nc and saline. They all were bred under SPF. Then transplanted the SGC7901 human gastric carcinoma cells(8.1 × 10⁵/ml)1ml into the mice abdominal cavities.After 12days, all mice were administrated. 5-FU/5-FU-nc was 0.15mg/g,HCPT/HCPT-nc was 0.02mg/g. After 47days, all mice were administratedagain with the same concentration. After 40 hours, we killed the all miceand got their tumor nodes. Selected the smaller tumor nodes(diameter<2mm) and the cell proliferation and apoptosis were measured,then dyed with HE and practiced microscopic examinations.In addition, we also investigated the effect of survival time to the mice withtransplanted tumors after 100nm carbon suspension binding 5-FU.(5) Measure of proliferation and apoptosis in all groups:All the fresh tumor nodes were washed with the PBS buffer, cut to pieceand filtered. we collected the cell suspension and diluted the cellsuspension to 1×10⁵/ml and dyed them by PI and PI/AnnexinV-FITC, thenmeasured them on flow cytometer. The number of apoptosis cells, theproliferation index at early, middle, late stages and the S-phase fractionwere calculated to judge the effect of proliferation and apoptosis in allgroups.Results(1) The adsorptive quantity of active carbon to 5-FU was increasing along with the drug. Geting the saturation, the adsorption reached the balance. The saturate adsorptive quantity was 0.4752mg/mg. In this interval, the adsorption of active carbon accorded with freundlich's formula M=KC^P(M: adsorptive quantity, C: drug concentration, K,P: constant). In this interval, there was a exponent relationship between the adsorptive quantity of active carbon to 5-FU and the concentration of drugs. The experience formula in this study is Y=0.0152X^0.6835(r=0.9894, r²=0.9789). (2) The total releasing quantity of HCPT and HCPT-nc reached the highest points in 34 hours. They are 97.6% and 60%, which showed the releasing velocity of HCPT-ncwas slower. Then the releasing rate of HCPT dropped sharply after 14 hours (92%). But the releasing rate of HCPT-nc dropped gently (58.7%). The adsorption rate of 3% nona-carbon (50nm) suspension 3ml to 3.108mg HCPT was 40% approximately. The unit adsorptive quantity was 0.0166mg. (3) After intraperitonealAdministration, HCPT and 5-FU were absorbed into the blood rapidly and the concentrations reached the highest points in 30min and 2h. They were 8.07ug/ml and 26.9ug/ml respectively. Then they got the preclude stages. The preclude rate were 0.024/min and0.79/h and the preclude half life were 28min and 0.88h respectively; HCPT can exist in blood three hours and 5-FU can exist eight hours after administration. After drugs were taken into abdominal cavity, the time of absorptive phase extended. HCPT-nc was 90min; 5-FU-ch was 6 hours. At the highest points, the concentrations of HCPT-nc and 5-FU-ch were 4.36ug/ml and 5.23ug/ml. But the existing time in blood extended obviously. The preclude phase's halflives were 45min and 7.7 hours respectively. (4) Comparison on the factors such as weight, the biggest diameter of tumor nodes: By Mann-Whitney test, the differences between case group and control group, HCPT-nc and HCPT were significant (p<0.05). However, there was no significant difference between 5-FU-nc and 5-FU(p>0.05). Analysis of survival time with Kaplan-Meier method: the time was longer in the 5-FU-ch group. Its median time was 88days, control group was 68days, 5-FU group was only 6days. By log rank test, the differences between 5-FU-ch and control group, 5-FU-ch and 5-FU were significant (p=0.011, 0.0473). There was no significant difference between the 5-FU and control group (p=0.7281). (5) It showed in all groups that the dead cells were more and the apoptosis cells at early stage were few; the number of cells in corresponding quadrant had no statistic significance. We analysed the values of SPF and PI in all groups, which showed the difference under the factor PI, but no under the SPF. By the T-test, significant results were got in HCPT-nc, 5-FU-nc and control groups (p<0.05).