CN1146431C - Serum preparation with function of resisting tumor and preparation process and use thereof - Google Patents
Serum preparation with function of resisting tumor and preparation process and use thereof Download PDFInfo
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- CN1146431C CN1146431C CNB011146508A CN01114650A CN1146431C CN 1146431 C CN1146431 C CN 1146431C CN B011146508 A CNB011146508 A CN B011146508A CN 01114650 A CN01114650 A CN 01114650A CN 1146431 C CN1146431 C CN 1146431C
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Abstract
The present invention provides a method for preparing a serum preparation with an anti-tumor function, which comprises the following steps: firstly, an animal is continuously and orally fed with snake venom of certain dosage or external modified and digestive products of the animal for more than one day; after the animal is fed with the snake venom or the modified and digestive products of the animal for certain time, the serum of the animal is extracted, and then, the serum which is extracted is processed into a preparation. The serum preparation which is prepared by the method of the present invention has the advantages of low toxicity, high selectivity, obvious curative effect and good application prospect. The method of the present invention can be used for producing and preparing medicines for treating or preventing various malignant tumors of human beings, such as liver cancers, lung cancers, nasopharyngeal carcinomas, leukemia, etc.
Description
The invention belongs to the antitumor drug technical field, particularly a kind of preparation method of utilizing snake venom to extract serum preparation with antitumor action.
The chemotherapy of cancer is the important measures that reduce cancer mortality.At home and abroad all carry out with snake venom and the anticancer research of active component thereof, and obtain certain achievement, particularly aspect cobra venom, successively inject or the anticancer report of In vitro culture in useful full snake venom preparation or its active component body, but because complicated component in the cobra venom, most of components directly enter in the body all may produce the serious toxicity reaction to body, directly with full snake venom or its a certain composition (component that direct dissolution is arranged as pair cell) though external anticancerly truly have certain effect, clinical practice is used then very risky, therefore, these achievements in research are difficult in clinical practice and popularization.Existing both at home and abroad research with cobra venom component and monoclonal antibody coupling guidance quality killing tumor cell, but, conjugate causes the inefficacy that comes off of cobra venom component because of being easy to disintegrate, or be difficult for entering solid tumor inside more greatly because of molecule, and the present shortage reasons such as monoclonal antibody strong to human tumor cell's affinity, the method of cobra venom component and monoclonal antibody coupling guidance quality killing tumor cell is to human tumor, particularly the uncertain therapeutic efficacy of solid tumor is cut, and still difficulty is used for clinical at present; The domestic scholar of having reports that the cobra venom by oral route has tangible antitumaous effect in zoopery, and there is not obvious toxic and side effects, its curative effect is confirmed through clinic trial, but directly with snake venom oral have for the patient that the digestive tract breakage is arranged directly enter sanguimotor danger, be unfavorable at clinical application, therefore be necessary its preparation is transformed.
The objective of the invention is to overcome the shortcoming of prior art, provide that a kind of toxicity is low, selectivity is high, the preparation method of the serum preparation with antitumor action of determined curative effect.
Another object of the present invention is to provide a kind of serum preparation that makes by said method with antitumor action.
A further object of the present invention is to provide above-mentioned application with serum preparation of antitumor action.
The present invention realizes by following technical scheme: the preparation method that originally has the serum preparation of antitumor action comprises the following steps: that (1) give the oral snake venom of animal or the snake venom product in external digestion, degeneration, and continuous oral is more than one day; (2) after taking snake venom or its digestion, denatured products, animal extracts the serum of this animal; (3) serum that extracts is processed into preparation.
The dosage range of giving the oral snake venom of animal is for being equivalent to the maximum tolerated dose of rabbit dosage 10mg/kg～this animal by the body surface area conversion to larger animal oral dose every day.
Specifically, can give healthy adult rabbit every day oral 11.2～45mg/kg cobra venom or give other larger animals the oral cobra venom of press body surface area conversion and the corresponding dose,equivalent of rabbit dosage 11.2～45mg/kg, the time of continuous oral is 3～5 days.
The invention still further relates to the application of the serum preparation with antitumor action in the medicine for preparing multiple human malignancies such as treatment or prevention hepatocarcinoma, pulmonary carcinoma, nasopharyngeal carcinoma, leukemia that makes by this method.
The present invention compares with existing method with snake venom treatment tumor has following advantage and effect:
(1) toxicity is low: because complicated component in the cobra venom, contain the multiple toxin that body is had severe toxicity, most of natural constituents are injected directly in the body all may produce the serious toxicity reaction to body, the method risk of direct injection is too big, be difficult in clinical practice, directly certain danger also arranged with cobra venom is oral.The component of biologically active is protein substance in the cobra venom, via this method by behind the animal oral administration, first closing eliminated in the digestion regulating liver-QI of these protein through digestive enzyme in the gastrointestinal tract of animal, finally the form with various micromolecule digestion products enters the body circulation, the innate toxicity of cobra venom will not exist, thereby can not produce the toxic reaction of general by the serum preparation that this method makes, use as safe as a house.
(2) selectivity height, side effect are little: previously in the research; in the body with cobra venom or its component, external anticancer experiment shows; they are the same with traditional chemotherapeutic; when killing tumor cell; also to a certain extent to normal body cell, particularly breeding faster, body cell produces cytotoxicity.And the serum preparation that the present invention makes is producing the obvious Cytotoxic while to people's hepatocarcinoma, pulmonary carcinoma, leukemia and nasopharyngeal carcinoma cell, the normal faster human embryonic lung fibroblast of propagation is not had cytotoxicity fully, thereby side effect is less.
(3) determined curative effect: zoopery in advance and clinical verification have confirmed that oral cobra venom has certain antitumaous effect, and hang down and the high advantage of selectivity owing to having toxicity by the serum preparation that the present invention makes, thereby therapeutic domain is big, dose titration space abundance, can not be subjected to the restriction of toxic action, be easy to guarantee the drug level that reaches enough in the body.Experimental studies results proves that also the present invention has tangible active anticancer to kinds of tumors, both anticancer propagation, more promote its apoptosis.Simultaneously, because the molecule of digesting and assimilating product diminishes, the easier solid tumor that enters is inner and stride film and enter tumor cell, helps giving full play to its pharmacological action.
(4) application prospect is good: because the present invention has These characteristics, thereby more help at clinical application than the cobra venom preparation of previously research; Again since raw material resources of the present invention very abundant, preparation method is easy, so but the utmost point produce in batches easily; The product that is made by this method is the serum lyophilized powder, be convenient to preserve, also be convenient to make as required capsule or injection and use, simultaneously, it also goes out active component for further separation and purification, with biochemical pharmacy means exploitation safety, a national class PTS provides good basis efficiently.
In order to embody effect of the present invention better, will the serum preparation that be made by this method inhibitory action and the low toxicity characteristics to human body tumour cell be described with cultured tumor cells in vitro experiment and animal acute toxicity test result below.Can find in the cultured tumor cells in vitro experiment: the animal serum of oral cobra venom really can produce tangible cytotoxicity to multiple human tumor cells such as HpeG2, the tumor cell survival rate is obviously descended, and with it the blank serum effect of the animal of contrast obviously a little less than; This serum is compared with the blank serum of animal, and human embryonic lung fibroblast does not have cytotoxicity fully to breeding rapidly.This serum has obvious suppression to the di of cancerous cell, shows that it has the effect that suppresses tumor cell proliferation; Flow cytometer inspection and DNA strip analysis result show, it is remarkable inducing apoptosis of tumour cell also.The acute toxicity tests with the oral said preparation of Kunming mouse shows, mice greater than 5g/kg, illustrates that said preparation is low toxin preparation to the oral maximum tolerated dose of high dose calmette's serum preparation.Be the related experiment result below:
1. the preparation of serum preparation: 21 of healthy adult new zealand rabbits, be divided into 7 groups, every group 3, irritate stomach with cobra venom (high dose 45mg/kg, low dosage 22.5mg/kg) or distilled water every day, and each is organized dosage regimen and is respectively: snake venom 45mg/kg * 1 day, snake venom 45mg/kg * 3 day, snake venom 45mg/kg * 5 day, snake venom 22.5mg/kg * 1 day, snake venom 22.5mg/kg * 3 day, snake venom 22.5mg/kg * 5 day, distilled water 1ml/kg * 3 day.After the last administration 4 hours, under the etherization state, collect blood as much as possible through animal carotid artery with sterile catheter, treat blood after external condensing,, extract whole supernatants (serum) with centrifugal 15 minutes of 1500 rev/mins of speed, the serum of interior 3 animals is organized in merging, behind 30 minutes deactivation complements of 60 ℃ of water-baths, lyophilization becomes lyophilized powder, and the room temperature sealing is preserved standby.
2. best administration natural law is selected: with people's hepatocarcinoma HepG-2 cell strain In vitro culture according to a conventional method, after treating that cell begins propagation and reaches some, be inoculated into respectively in the culture medium that contains above-mentioned serum lyophilized powder 1mg/ml, under high power lens, count tumor cell with blood cell counting plate after 48 hours, the result shows: the serum that gavages two kinds of dosage snake venom animals be on the cytotoxicity 1 day group a little less than, the group action intensity was similar in 3 or 5 days.Show successive administration after 3 days, serum Chinese medicine active component content has reached steady-state level, and visible 3 days is best administration natural law, below the experiment all with 3 days serum of administration for being tried serum.
3. to four kinds of tumor cells and Normocellular cytotoxicity: measure respectively according to a conventional method and tried serum people's hepatocarcinoma HepG-2 cell, human leukemia HL-60 cell, human nasopharyngeal carcinoma CNE-2 cell and four kinds of tumor cells of human lung adenocarcinoma cell, and the cytotoxicity of normal human embryonic lung fibroblast.The experiment of four kinds of tumor cells is divided into 13 groups and carries out, be respectively: each 3 kinds of concentration (lyophilized powder 1,2.5,5mg/ml) of normal serum, low dosage calmette's serum and high dose calmette's serum, 3 kinds of concentration of positive control drug amycin (5,10,15 μ g/ml), blank (normal culture medium), every group 10 hole.The existence cell counting is made in dosing after 48 hours, and respectively is subjected to the suppression ratio of reagent pair cell with reference to the cell number mean value computation of blank group, and the result sees Table 1～table 2 respectively.
Table 1. Activity of Naja Atra Venom Serum is to the influence (* 10 of four kinds of tumor cell existence numbers
3, x ± s, n=10)
| Amycin 15 μ g/ml | 54±3.8 | 38±1.8 | 52±2.8 | 51±3.3 |
With with concentration normal serum group relatively: * p＜0.05, * * p＜0.01
Compare with amycin (5 μ g/ml) group: #p＜0.05, ##p＜0.01
The suppression ratio that table 2. Activity of Naja Atra Venom Serum is survived to four kinds of tumor cells (%, x ± s, n=10)
With with concentration normal serum group relatively: * p＜0.05, * * p＜0.01
Compare with amycin (5 μ g/ml) group: #p＜0.05, ##p＜0.01
The above results has highly significant difference (p＜0.01) through two-way analysis of variance between group, no significant difference (p＞0.05) between sample in the group.The t assay sees Table internal labeling between group, with with the concentration normal serum relatively, the cytotoxicity of each group of high and low dose calmette's serum all significantly is better than normal serum (p＜0.01), truly have unique cytotoxicity, intensity low, high dose calmette's serum 5mg/ml is better than amycin 5 μ g/ml (p＜0.05 or p＜0.01) slightly.
Normal cell experiment divides 9 groups, is normal serum, each 4 kinds of concentration of high dose calmette's serum (0.5,1,2,5mg/ml), blank (normal culture medium).The cell number of each sample is measured in every group 10 hole before the administration earlier, and the cell counting of surviving is made in dosing after 72 hours, and respectively is subjected to the rate of increase of reagent pair cell with reference to the cell number mean value computation before the administration, the results are shown in Table 3.
Table 3. Activity of Naja Atra Venom Serum to the influence of human embryonic lung fibroblast existence number (x ± s, n=10)
With with concentration normal serum group relatively: * p＜0.05,
The above results is through two-way analysis of variance, and there were significant differences between group (p＜0.05), no significant difference (p＞0.05) between sample in the group.The t assay sees Table internal labeling between group.To normal person's embryo lung fibroblast, calmette's serum shows as the promotion cell proliferation, its effect slightly is better than normal rabbit serum, remove the effect of rabbit anteserum itself, the snake venom digestion product does not have cytotoxicity fully to normal cell, slightly promote the effect of cell proliferation on the contrary, show high selectivity cytotoxicity tumor cell.
4. anti-tumour cell proliferative experiment:
Measure respectively according to a conventional method and tried serum people's hepatocarcinoma HepG-2 cell, human leukemia HL-60 cell, human nasopharyngeal carcinoma CNE-2 cell and four kinds of exponential influences of tumour cell division of human lung adenocarcinoma cell.The experiment of four kinds of tumor cells is divided into 4 groups to be carried out, and is respectively: normal serum, high dose calmette's serum (concentration is 5mg/ml), positive control drug amycin (15 μ g/ml), blank (normal culture medium).Do 10 every 24 hours each groups and smear sheet,, draw the cell division index of this group, observed continuously 7 days, the results are shown in Table 4～table 7 with HE dyeing back several 100 intact cells under high power lens, the somatoblast number in 1000 intact cells of 10 sheets of accumulative total.
Table 4. limit mirror calmette's serum is to the influence (10 of CNE-2 cell division index
-3)
Table 5. Activity of Naja Atra Venom Serum is to the influence (10 of HepG-2 cell division index
-3)
| 4 | 151 | 85 | 27 | 15 |
| 5 | 147 | 72 | 16 | 18 |
| 6 | 82 | 70 | 13 | 15 |
| 7 | 73 | 51 | 15 | 12 |
Table 6. Activity of Naja Atra Venom Serum is to the influence (10 of HL-60 cell division index
-3)
Table 7. Activity of Naja Atra Venom Serum is to the influence (10 of lung adenocarcinoma cell di
-3)
| 5 | 151 | 67 | 22 | 19 |
| 6 | 147 | 47 | 19 | 19 |
| 7 | 77 | 40 | 17 | 16 |
By The above results as seen, the normal rabbit anteserum of the cell proliferation effect of Activity of Naja Atra Venom Serum be by force, and with concentration be that the amycin of 15 μ g/ml is close.The cytotoxicity that it is described is relevant with the inhibition tumor cell proliferation.
5. promote the apoptosis of tumor cells experiment:
On the basis that a last experimental cell is cultivated, measured with the fluidic cell method and to be subjected to of the influence of reagent thing hepatocarcinoma and apoptosis of leukemia, the results are shown in Table 8.
Table 8. Activity of Naja Atra Venom Serum is to the influence of two kinds of apoptosis of tumor cells rates (%)
The result shows that normal rabbit serum can not obviously increase the apoptosis of tumor cell, and calmette's serum then can make apoptosis rate increase more than 10 times, and its effect is near the amycin of 15 μ g/ml concentration.The DNA strip analysis also shows similar results, and this prompting Activity of Naja Atra Venom Serum is mainly relevant with the promotion apoptosis of tumor cells to the cytotoxicity of tumor cell.
6. the acute toxicity test of high dose Activity of Naja Atra Venom Serum preparation oral:
Get 20 of healthy Kunming mouses, male and female half and half, body weight 20.1 ± 1.5g, fasting was once irritated the high dose calmette's serum lyophilized powder solution that stomach gives 25% concentration by 5g/kg dosage after 12 hours, observed through continuous 14 days, do not find any overt toxicity reaction, mice is none death in the observation period.Experimental result shows the maximum tolerated dose of the oral said preparation of mice greater than 5g/kg, this shows that this serum preparation is a kind of low toxin preparation.
The several embodiment of various details, but content of the present invention is not limited to this fully.
Embodiment 1
The thick malicious dry thing of cobra venom is mixed with 4.5% concentration liquid with distilled water, pressing the 1ml/kg body weight every day irritates stomach for the healthy adult rabbit, make dosage be equivalent to 4 times (press body surface area conversion) of bibliographical information to the oral cobra venom effective dose of tumor-bearing mice (30mg/kg), successive administration 3 days; After 4 hours, under the etherization state, collect blood as much as possible with sterile catheter in the last administration through animal carotid artery; Treat blood after external condensing, with centrifugal 15 minutes of 1500 rev/mins of speed, extract whole supernatants (serum), behind 30 minutes deactivation complements of 60 ℃ of water-baths, lyophilization becomes lyophilized powder, and the room temperature sealing is preserved standby.
Embodiment 2
According to the method for describing among the embodiment 1, the oral liquor strength of rabbit makes dosage be equivalent to the effective dose (press body surface area convert) of bibliographical information to the oral cobra venom of tumor-bearing mice for containing the thick malicious dry thing 1.12% of cobra venom.
Embodiment 3
According to the method for describing among the embodiment 1, the oral liquor strength of rabbit makes dosage be equivalent to 2 times (press body surface area convert) of bibliographical information to the oral cobra venom effective dose of tumor-bearing mice for containing the thick malicious dry thing 2.25% of cobra venom.
Embodiment 4
According to the method for describing among the embodiment 1, but the only administration 1 day of the oral cobra venom medicinal liquid of rabbit.
Embodiment 5
According to the method for describing among the embodiment 1, but the oral cobra venom medicinal liquid of rabbit is successive administration 5 days.
Embodiment 6
According to the method for describing among the embodiment 1, but the oral thick poison of cobra venom of rabbit digests at outer body with pepsin earlier, its method is: 0.1 gram pepsin is dissolved in the dilute hydrochloric acid solution of 10ml 0.4% makes Digestive system, then cobra venom 4.5 grams are added in the Digestive system, after the stirring and dissolving digestion 30 minutes, then with distilled water diluting to 100ml.
Embodiment 7
According to the method for describing among the embodiment 1, but the oral thick poison liquid of cobra venom of rabbit before administration earlier with 100 ℃ of heating in water bath 30 minutes, make protein denaturation wherein.
Claims (4)
1, a kind of preparation method with serum preparation of antitumor action is characterized in that comprising the following steps: that (1) give the oral snake venom of animal or the snake venom product in external digestion, degeneration, continuous oral 2～14 days; (2) after taking snake venom or its digestion, denatured products, animal extracts the serum of this animal; (3) serum that extracts is processed into preparation.
2, the preparation method with serum preparation of antitumor action according to claim 1, it is characterized in that: give healthy adult rabbit every day oral 11.2～45mg/kg cobra venom or give cattle, horse, sheep, pig, dog, the oral cobra venom of press body surface area conversion and the corresponding dose,equivalent of rabbit dosage 11.2～45mg/kg of cat, the time of continuous oral is 3～5 days.
3, a kind of serum preparation with antitumor action with the described method preparation of claim 1.
4, the described application of serum preparation in preparing treatment or prevention hepatocarcinoma, pulmonary carcinoma, nasopharyngeal carcinoma, leukemic medicine of claim 3 with antitumor action.
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