CN112656784A - Novel indications and administration methods for paclitaxel - Google Patents

Abstract

The invention discloses a new clinical application of paclitaxel in enhancing alcohol ablation treatment of liver cancer under image guidance. Paclitaxel is a first-line chemotherapeutic drug widely used for gynecological tumors and lung cancer. The research result of the invention shows that the paclitaxel dissolved in alcohol can obviously improve the liver cancer treatment effect of alcohol ablation. The invention provides a new strategy of combined and synergistic treatment of liver cancer based on paclitaxel drug therapy and alcohol ablation therapy for the first time, which is beneficial to improving the interventional therapy level of liver cancer and increasing the application range of paclitaxel in tumor therapy.

Description

Novel indications and administration methods for paclitaxel

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a new indication and a drug delivery method of paclitaxel.

Background

The absolute alcohol injection is also called chemical ablation, and the chemical ablation agent such as alcohol is directly injected into the center of a tumor under the guidance of B ultrasonic or CT, so that the tumor cells and nearby vascular endothelial cells are quickly dehydrated, and protein is denatured and solidified, thereby causing the necrosis/ischemia of the tumor cells. At present, absolute alcohol injection is mainly used for treating liver cancer. Because the spatial diffusion limitation of alcohol may have residual focus on the large heterogeneous liver cancer, the traditional alcohol ablation is mostly limited to the treatment of the primary or recurrent liver cancer with lesion diameter less than 3cm and lesion number less than 3. Because PEI treatment lacks standardization in clinic, the curative effect of PEI often depends on clinical experience, and the reports of the PEI on the curative effect are different among different families. Currently, alcohol ablation is rarely used clinically, and other thermal ablation technologies such as radio frequency ablation are mostly used. However, thermal ablation has contraindications, such as high surgical risk for liver cancer near large blood vessels, near the gall bladder, on the surface of the liver, or under the hepatic capsule. At present, no report is available on improving ablation therapeutic effect by adding chemical anticancer drugs into alcohol.

Paclitaxel, also called taxol, taxol and Temin, is the most excellent natural anticancer drug, and has been widely used for treating breast cancer, ovarian cancer, partial head and neck cancer and lung cancer clinically. Paclitaxel is taken as diterpene alkaloid compound with anticancer activity, has novel and complex chemical structure, wide and remarkable biological activity, completely new and unique action mechanism and scarce natural resources, is greatly favored by phytologists, chemists, pharmacologists and molecular biologists, and becomes an anticancer star which draws attention in the next half of the 20 th century and research focus. The molecular formula of the taxol is C47H51NO14, and the relative molecular mass is 853.91. Paclitaxel is white crystal powder, odorless, tasteless, insoluble in water, and soluble in organic solvents such as methanol, acetonitrile, chloroform, and acetone. In 1979, the unique anti-tumor mechanism of action of paclitaxel was elucidated by the molecular pharmacologist Horwitz, the american academy of einstein medicine: paclitaxel can make tubulin and tubulin dimer composing microtubule lose dynamic balance, induce and promote tubulin polymerization, microtubule assembly, prevent depolymerization, stabilize microtubule and inhibit cancer cell mitosis and trigger apoptosis, thereby effectively preventing cancer cell proliferation and playing a role in resisting cancer. In fact, tubulins which are closely related to mitosis of cells are ubiquitous in almost all eukaryotic cells and can reversibly polymerize into microtubules, which are required for chromosome isolation. Following mitosis, these microtubules are again depolymerised to tubulin. The transient collapse of the spindle-like microtubules preferentially kills abnormally dividing cells, and some important anticancer drugs such as colchicine (colchicin), vinblastine (vinblastine), vincristine (vincristine) and the like play an antitumor role by preventing tubulin from repolymerizing. In contrast to antimitotic antineoplastic drugs, paclitaxel was the first drug to be found to interact with tubulin polymers, i.e., by binding tightly to microtubules, stabilizing them and acting, and was also found to exhibit good effects on a variety of solid tumor cells. Because paclitaxel is insoluble in water, the addition of castor oil, a diluent solvent, is required to increase the solubility of paclitaxel in glucose injections, which increases the complexity of the drug configuration, i.e., the injections must not contact PVC devices, catheters, or devices. Because castor oil dissolves hepatotoxic excipients from PVC. It is therefore preferred to use glass containers for this purpose and the most common and fatal side effect is that 15-20% of patients experience acute anaphylactic shock, mainly associated with castor oil, but not with paclitaxel itself.

The chemotherapy of liver Cancer by paclitaxel has been studied clinically, and both D.Stremberg in Germany (European Journal of Cancer, 1998, 34, 1290-1292,) and S-D Lee in Taiwan in China (Brtish Journal of Cancer, 1998, 78, 34-39) have conducted clinical trials of paclitaxel in the nineties, respectively, and it was consistently concluded that: paclitaxel chemotherapy did not significantly benefit liver cancer, and paclitaxel was administered intravenously as an injection in both reports.

Disclosure of Invention

Aiming at the defects and problems in the prior art, the invention aims to provide a new indication and a drug delivery method of paclitaxel.

The invention is realized by the following technical scheme:

the invention provides a medical application of paclitaxel, which treats liver cancer by using paclitaxel to assist alcohol ablation.

The invention provides a method for treating liver cancer by paclitaxel, which comprises the step of mixing paclitaxel and alcohol and injecting the mixture into a liver cancer focus by percutaneous puncture under the guidance of images.

Further, paclitaxel is solid powder raw material drug, and before administration, paclitaxel is dissolved in absolute alcohol to form paclitaxel alcohol injection.

Further, the concentration of paclitaxel in absolute alcohol is 0-40 mg/ml.

The specific action mechanism of the paclitaxel for enhancing alcohol ablation treatment of liver cancer in the invention is as follows:

high concentrations of alcohol are able to penetrate into tumor tissue and rapidly cause cell dehydration, protein denaturation and platelet aggregation, leading to tissue necrosis, small vessel embolization and fibrous tissue formation;

the limited spatial diffusion of alcohol results in distant tumors remaining viable under the stimulation of alcohol if any, as viable tumor tissue is found to remain in the margins or septal separated parts of the tumor, causing intrahepatic and extrahepatic metastases, ultimately leading to low long-term survival; in addition, the side effect of using alcohol in a large amount is also an important problem in clinic;

it has been found that paclitaxel can inhibit cell proliferation and tissue growth by inhibiting microtubule depolymerization in tumor cells, so that paclitaxel dissolved in alcohol can act on tumor cells which are already weakly stimulated by alcohol to produce enhanced therapeutic effect through synergistic effect; the dehydration function of alcohol and the cytotoxicity of paclitaxel are utilized to kill tumor cells to the maximum extent and reduce relapse and metastasis.

Compared with the prior art, the invention discloses a new application of paclitaxel as a medicinal active ingredient for treating liver cancer by liver cancer ablation treatment, and when the medicament is matched with alcohol for use, the defects of the existing alcohol ablation can be overcome.

The invention provides a novel therapeutic strategy for treating liver cancer by ablation and chemical drug combination, which is expected to delay the progress of liver cancer and improve the life quality of patients.

Drawings

FIG. 1 is a schematic representation of the present invention, wherein FIG. 1a is a schematic representation of a method of administration according to the present invention; FIG. 1b is a schematic of tumor location and drug injection.

FIG. 2 is a table of experimental groupings according to the present invention.

Fig. 3 is a comparison of necrosis rates between groups of ultrasound imaging and pathological evaluation for each group of experiments, wherein fig. 3a is the measure of radio necrosis of the tumor by ultrasound and fig. 3b is the percentage of tumor necrosis rate by H & E histology.

FIG. 4 is an ultrasound image of the calculated iconographic necrosis ratio (RNR) for each set of experiments.

Figure 5 is an image of the calculated Histological Necrosis Rate (HNR) in each set of experiments.

FIG. 6 is a graph showing the relationship between paclitaxel dose and tumor necrosis, wherein FIG. 6a shows the relationship between paclitaxel dose and tumor necrosis imaged; FIG. 6b is a graph of paclitaxel dose vs. pathological tumor necrosis rate.

FIG. 7 is a photograph of the cross section of the tumor after formalin immersion in formalin for each experiment.

Detailed Description

Technical solutions in the embodiments of the present invention are clearly and completely described, and the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that do not have the inventive result, which can be implemented on this basis, fall within the scope of protection of the present invention.

The liver cancer animal model is used as an experimental object, paclitaxel and alcohol are mixed and injected into a liver cancer focus to carry out an experiment for synergistically treating the liver cancer, and meanwhile, the experiment is compared with a conventional comparative example.

1. Experimental methods

1.1 subjects and procedures

a) Establishing a liver cancer animal model: establishing a liver tumor model of a rabbit VX2, namely inoculating a frozen rabbit VX2 tumor cell homogenate to a right hindlimb lateral muscle group of a healthy rabbit after recovery to prepare a tumor-bearing seed rabbit; taking out the tumor completely after 2 weeks, cutting into pieces, and placing into a 16G lumbar puncture needle; taking a healthy rabbit, placing a puncture needle with a small tumor block at the lower edge of the left inner lobe of the liver under the guidance of ultrasound, slowly pushing the small tumor block out by using a matched needle core, then pulling out the needle, and suturing layer by layer after the needle channel is subjected to hemostasis by compression; after the rabbit is fed, the rabbit is put back into a cage for feeding, ultrasonic examination is carried out every day after the rabbit is normally fed for 10 days, the diameter of the tumor is about 0.8-1.5 cm after 2 weeks, and the rabbit is immediately put into the group when the diameter is more than 1.5 cm.

b) Administration and surgery: the paclitaxel administration method in this embodiment is direct administration by percutaneous puncture to the tumor site, and is consistent with the clinical ultrasound-guided chemical ablation, and the difference is the difference of the injection, and the specific schematic diagram is shown in fig. 1 a. Performing intravenous injection on the ear margin by using 3% sodium isoamyl barbital (1ml/Kg), fixing the rabbit in a supine position on an animal operating table after the rabbit is in a coma, preparing skin, disinfecting, paving a sterile hole towel, observing the size, the shape and the internal echo of the tumor by conventional ultrasound, judging whether a sac deterioration dead zone exists or not, evaluating the blood supply condition (Alder grading) of the tumor by CDFI, performing routine ultrasonic contrast examination before operation, and observing the blood perfusion condition of the tumor to obtain the enhanced peak intensity; after observing the tumor condition by ultrasonic, selecting a maximum tumor section, dividing the maximum section into three equal parts, selecting two middle points (1/3 and 2/3, as shown in figure 1 b), selecting cytological fine needle puncture (23G) which is a tail end hole, wherein tumor necrosis is related to the total amount of alcohol injection and is unrelated to the tail end hole or a side hole of the puncture needle, theoretically, the design of the side hole is beneficial to the alcohol, and the design of a dispersion puncture needle does not influence the tumor necrosis degree), and under the guidance of real-time ultrasonic monitoring, the puncture needle is punctured from the side edge of the probe, and the needle inserting direction is kept parallel to the long axis of the probe, so that a needle sheath and a needle point can be clearly displayed; in order to avoid the influence on the needle tip display after the injection of the medicine (the ultrasonic display of the tumor area is a high echo area, which influences the needle tip display), the distal end is injected first, the needle core is pulled out after the needle tip reaches the distal end 2/3, the medicine (absolute alcohol or absolute alcohol-paclitaxel mixed liquid) of 1/2 is injected, and the needle is slowly withdrawn after the injection until the tumor 1/3 is injected with the residual medicine of 1/2.

1.2 Experimental groups

The VX2 tumor rabbit model 36 was selected and randomly divided into 6 groups, and the grouping is shown in FIG. 2:

group 1 was saline injection; group 2 and group 3 were both alcohol ablated with the difference that the injection of alcohol in group 2 was 2 ml, in contrast to the three subsequent combination treatment groups; and group 3 was an alcohol ablation group, with alcohol usage calculated as tumor volume (V ═ 4/3 pi (r +0.5)³) Where r is the tumor radius (in cm), for example, a tumor radius of 0.8 cm, 9.2 ml of alcohol needs to be injected, i.e., the injection amount per time depends on the tumor size measured by the ultrasound image. This is also the standard dose for clinical use with alcohol ablation.

The three groups 4 to 6 are combined treatment groups, the alcohol content used is 2 ml, and the paclitaxel content is 12.5, 25 and 37.5 mg respectively, and the injection is completed once.

1.3 Observation indicators and methods

A week of routine ultrasound and ultrasound contrast examination was performed after the above 6 sets of VX2 tumor rabbit models: evaluating the residual activity and necrosis of the tumor; conventional ultrasound observation including tumor size, morphology, boundary, presence or absence of necrotic areas (range, observation of residual tumor active area, and delineating tumor area and enhanced area by using "small parts" mode, and calculating the imagewise necrosis ratio (abbreviated as RNR).

Killing tumor-bearing rabbits, taking tumor tissue sections for pathology and immunohistochemistry, wherein the pathological sections are obtained by immersing the taken tumor in formalin and embedding the tumor in paraffin blocks, taking a sample with the maximum diameter of 5 mm, then cutting the sample into two adjacent five-micron slices by a slicer, staining one slice with hematoxylin-eosin (H & E), further examining the stained slice with a microscope, calculating the histological tumor necrosis rate (HNR) by using image pro plus6.0 software, staining the other slice with KI-67 antibody according to the kit specification, further examining the stained slice with the microscope, and calculating the corrected proliferation index (m-PI).

The m-PI calculation method is as follows: the entire live cells in the H & E sections were observed from a field (e.g., 10 live cells were observed at 40 x magnification), then Ki-67 positive cells were identified from those sections in adjacent Ki-67 sections from the same field (e.g., only 8 cells were positive out of the 10 live cells, and therefore the proliferation index was calculated as 80%), and for each section we randomly selected three regions and averaged to give a corrected proliferation index for each tumor.

1.4 statistical methods

All data are expressed as mean ± SD and statistically analyzed using SPSS 22.0 and Graphpad Prism 6.01. The relationship between paclitaxel dose and radiology/histology necrosis or proliferation index was evaluated by using least squares linear regression and calculation of a determination coefficient (R2).

The difference in pathological necrosis rates of the modules was analyzed using Welch variance based on the characteristics of the data obtained, the imagewise necrosis rate was analyzed using the Kruskal-Wallis H test, proliferation indices were compared using one-way ANOVA, P values less than 0.05 were considered to indicate statistically significant differences, and P values less than 0.100 indicated statistical trends.

2. Results

2.1 comparison of necrosis Rate between groups for ultrasound imaging and pathological evaluation

Radionecrosis of tumors as shown in fig. 3a, different treatment strategies lead to various outcomes.

Analysis showed that the RNR was significantly elevated (P < 0.001) for the conventional ablation group (group 3) compared to the saline group (group 1), but there was no significant difference in necrosis between group 3 and the 2 ml alcohol ablation group (group 2) (P ═ 0.048) and the low paclitaxel dose group (group 4) (P ═ 0.307). The results indicate that large doses of alcohol are relatively beneficial for the benefit of ablation therapy because the RNR values are unsatisfactory at the clinically used dose, only 52.75 ± 8.92%.

At the same time, the RNR increased by almost 50% (77.97 ± 8.17%, P ═ 0.020) in the dose group (group 5) in the co-treatment with respect to group 3; for the high dose group (group 6), the differences were statistically large (95.00 ± 3.58%, P < 0.001), since only very weak dynamically enhanced signals were detected for all tumors.

Histological necrosis results are shown in figure 3b, and the HNR values were lower than the RNR for each group, but their statistical differences or trends were the same. The rate of necrosis was significantly higher in group 3 than in group 1(P ═ 0.003), comparable to group 2(P ═ 0.380), comparable to group 4(P ═ 0.989) and group 5(P ═ 0.291), but significantly lower than in the high dose group (group 6) (P < 0.001).

The ultrasound images of groups 1 to 6, in which the ratio of the imaging necrosis (RNR) was calculated, are shown in FIG. 4, and each tumor is a black-and-white image of the entire tumor area obtained by B-mode ultrasound, and the subsequent pseudo-color image is an arterial phase enhancement image, in which the active tumor area can be calculated, and each tumor image is marked with the percentage necrosis. The average percentage and standard deviation of the entire group are on the left.

Images of calculated Histological Necrosis Rate (HNR) as shown in fig. 5, the software calculated the percentage of necrotic area by color, the percentage of necrosis is labeled in each tumor image, and the left value is the average percentage and standard deviation of the entire group.

2.2 relationship between paclitaxel dose and tumor necrosis

Linear regression showed a strong positive correlation between paclitaxel dose and RNR (R)²0.946; as shown in fig. 6 a).

The drug dose has statistical significance for necrosis rate (F (1, 16) ═ 154.389, P<0.001) of the drug, accounting for 90.6% of the change in necrosis rate, with a similar trend for HNR ((R)²0.843; as shown in FIG. 6 b), P<0.001)。

Correlation analysis shows that the paclitaxel dose and tumor necrosis have obvious correlation, which indicates that the paclitaxel has obvious cytotoxicity to VX2 cells.

2.3 photograph of tumor tissue

The photographs of the cross sections of the tumors of groups 1 to 6 after immersion in formalin are shown in fig. 7, in which the active tumor tissues are visible to the naked eye, the active tissues in the control group occupy most of them, and the ablation of alcohol can cause partial necrosis, in contrast to the case where the mixture of alcohol and paclitaxel causes more severe necrosis, and in the case of the high dose group, we sometimes even do not observe the tumor residues.

3. Conclusion

In the research, the results of the imaging and pathological results of the tumors of each group after operation are compared to find that the effect of the ablation-chemotherapy synergistic treatment is better than that of the single ablation group, and the synergistic treatment has obvious taxol dosage correlation, so that the taxol greatly improves the ablation treatment effect.

This combination therapy demonstrated synergy between ablation and chemotherapy, suggesting that transdermal alcohol-paclitaxel combination therapy may be a treatment strategy for intervention in liver cancer, with similar or preferential clinical outcomes to other ablation techniques. And because the paclitaxel is injected in tumor and the injection amount of alcohol can be greatly reduced, no obvious toxic or side effect is caused.

The foregoing merely represents preferred embodiments of the invention, which are described in some detail and detail, and therefore should not be construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, various changes, modifications and substitutions can be made without departing from the spirit of the present invention, and these are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (4)

1. Medical use of paclitaxel characterized by: the paclitaxel and absolute alcohol are mixed and injected into the liver cancer focus for the synergistic treatment of liver cancer.

2. A method of administering paclitaxel for the synergistic treatment of liver cancer, comprising: paclitaxel and absolute alcohol are mixed and injected into the liver cancer focus by percutaneous puncture under the guidance of images.

3. The method of administering paclitaxel according to claim 2, wherein: the paclitaxel adopts solid powder state raw material medicine, and before administration, the paclitaxel is dissolved in absolute alcohol to form paclitaxel alcohol injection.

4. The method of administering paclitaxel according to claim 3, wherein: the concentration of the paclitaxel in the absolute alcohol is 0-40 mg/ml.

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