Technical field

The present invention relates to a kind of pharmaceutical composition and the application in the medicine of preparation treatment cancer thereof, be specifically related to contain the pharmaceutical composition and the application in the medicine of preparation treatment pulmonary carcinoma, cancer of pancreas, colon cancer, hepatocarcinoma, carcinoma of prostate, renal carcinoma, gastric cancer, cerebroma, sarcoma, ovarian cancer or breast carcinoma thereof of Sorafenib and arteannuin and artemisinin derivatives.

Background technology

World Health Organization's investigation report shows that global cancer condition is serious day by day, and 20 years from now on new patients' number will be increased to 1,500 ten thousand by present every year 1000 ten thousand, because of the number that cancer is dead also will be by increasing to 1,000 ten thousand 6,000,000 of every year.Wherein pulmonary carcinoma is one of common malignancy, comes from bronchiolar epitheliums at different levels, is divided into cell lung cancer and nonsmall-cell lung cancer; The cancer of pancreas pilosity is born in head of pancreas portion, and 90% derives from the ductus pancreaticus epithelial cell, and all the other are the digestive system common malignancy from pancreatic acini, and sickness rate is ascendant trend year by year.Because onset concealment lacks effective method of early diagnosis, often reach an advanced stage when making a definite diagnosis or shift, the patients with terminal median survival interval is no more than six months; The morbidity of colon cancer and environment, living habit, especially diet style is relevant.It is generally acknowledged that high fat diet and cellulose deficiency are main pathogenic factors.Along with growth in the living standard, the change of dietary structure, the sickness rate of colon cancer is ascendant trend year by year; Primary hepatocarcinoma is one of human modal malignant tumor for occurring in the epithelial canceration of hepatocyte and stones in intrahepatic bile duct; Carcinoma of prostate is most important a kind of in the male genitourinary system tumor, is human distinctive disease.Carcinoma of prostate is a senile disease, mostly at 50 years old with sequela.Along with the prolongation of human average life, the raising of diagnostic techniques, the change of life style, the sickness rate of carcinoma of prostate is in continuous rising, and it is extremely urgent therefore to study the treatment of prostate cancer medicine.

The antitumor drug that has gone on the market at present is more, and as alkylating agent medicine, antimetabolite, antitumor antibiotics, immunomodulator etc., but medicine is because toxicity is bigger mostly, and patient does not tolerate.The Study on Molecular Mechanism that develops along with the generation to tumor is more and more clearer, and the multiple malignant tumor of molecular targeted treatment has been subjected to paying close attention to widely and paying much attention to.Molecular targeted agents selectivity height, wide spectrum are effective, and its safety is better than the cytotoxicity chemotherapeutics, are the new directions of present oncotherapy field development.

Arteannuin is the sesquiterpene lactone that contains peroxide bridge, and its derivant has artesunate, Artemether and dihydroarteannuin etc.Arteannuin and derivant thereof are that a class is efficient, the antimalarial agent of low toxicity, along with going deep into of research, it is found that arteannuin and artemisine compounds also have other a lot of important pharmacologically actives, as schistosomicide, immunomodulating, arrhythmia, antitumor etc., especially its antitumor action more and more causes the attention of researcheres.A lot of experimentatioies show, arteannuin and artemisine compounds have significant inhibitory effect to the growth of kinds of tumor cells, and it is very low to normal histiocytic toxicity, mechanism of action may with the generation and the oxidative stress of free radical in the tumor cell, postpone cell cycle, cell death inducing generates relevant with antineoplastic vascular.Dihydroarteannuin is an active stronger derivant in the artemisinin-based drug, artesunate is one of most important derivant of arteannuin, has good water solubility, artesunate antitumor mechanism has direct killing effect or relevant with cell death inducing with it to tumor cell line, also may be relevant with its inhibition tumor tissues angiogenesis etc.Arteannuin and artemisinin-based drug can the selective killing tumor cells, and do not have crossing drug resistant with traditional chemotherapeutic, multidrug resistance phenomenon that can the reversing tumor cell.

Sorafenib is a kind of many target spots inhibitors of kinases by Bayer and the common development of ONYX, targeting is serine/threonine kinase receptor and tyrosine kinase receptor in tumor cell and tumor vessel, suppress tumor growth by suppressing the Raf/MEK/ERK signal transduction path, simultaneously also by suppressing to generate the activity of relevant tyrosine kinase receptor with new vessels, block tumor neovasculature generation, suppress the growth of tumor cell indirectly.Sorafenib is 800mg/ days at the clinical dosage that uses for the adult at present.Yet Sorafenib has more untoward reaction, as, " toxic and side effects of Sorafenib and processing thereof ", what " cancer progression magazine " July in 2007, the 5th volume the 4th phase 370-373 page or leaf was put down in writing, untoward reaction comprises brothers' syndrome, tired, diarrhoea, dermal toxicity and gastrointestinal reaction etc., and often causes the interruption of drug administration or reduce drug dose.Therefore seek the focus of cancer therapy drug that therapeutic effect is better, side effect is low for studying at present.

Along with the progress of oncomolecularbiology, the molecular targeted treatment of tumor has become the focus of tumor research, has brought into play important effect in the treatment of kinds of tumors.Yet, the biological behaviour of most of tumor is arranged by single signal transduction pathway, but a plurality of signal transduction pathway concur, therefore drug combination carries out targeted therapy at many target spots and will not only be intended to reduce or delay chemical sproof appearance, reduce toxicity, and by multiple medicine the synergism that cancerous cell kills and wounds is obtained better therapeutic.

Summary of the invention

At above technological deficiency, the invention provides a kind of pharmaceutical composition and the application in the medicine of preparation treatment pulmonary carcinoma, cancer of pancreas, colon cancer, hepatocarcinoma, carcinoma of prostate, renal carcinoma, gastric cancer, cerebroma, sarcoma, ovarian cancer or breast carcinoma thereof, be specially the application of pharmaceutical composition in the medicine of preparation treatment pulmonary carcinoma, cancer of pancreas, colon cancer, hepatocarcinoma, carcinoma of prostate, renal carcinoma, gastric cancer, cerebroma, sarcoma, ovarian cancer or breast carcinoma that contains Sorafenib and arteannuin and artemisinin derivatives.

The present invention contains in the pharmaceutical composition of Sorafenib and arteannuin and artemisinin derivatives, and described arteannuin and artemisinin derivatives can be arteannuin, artesunate, dihydroarteannuin, Artemether or arteether, or their corresponding derivative.

Arteannuin and artemisinin derivatives in the pharmaceutical composition of the present invention are preferably: artesunate, dihydroarteannuin or Artemether, its corresponding structure formula is respectively formula I, formula II and formula III.

In the pharmaceutical composition of the present invention, described component is not limited to artesunate, dihydroarteannuin and Artemether medicine itself, can also be its pharmaceutically useful salt, hydrate or derivant etc.

Among the present invention, described Sorafenib (English name: Sorafenib) be 4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-uride] phenoxy group }-the pyridine-2-carboxylic acids methylamine, its structural formula is formula IV.

Among the present invention, Sorafenib is not limited to this medicine itself, can also be its pharmaceutically useful salt, the analog of the derivant of Sorafenib or the various Sorafenibs disclosed in the WO2000041698 patent application; During using, the present invention simultaneously Sorafenib can also be replaced with the other medicines of many target spots inhibitors of kinases.

The present invention contains in the pharmaceutical composition of Sorafenib and arteannuin and artemisinin derivatives, and the mol ratio of Sorafenib and arteannuin and artemisinin derivatives is 1.0-15.0: 1.0-25.0; Further the mol ratio of preferred described Sorafenib and arteannuin and artemisinin derivatives is 2.0-7.5: 2.5-12.5.

The pharmaceutical composition that the present invention contains Sorafenib and arteannuin and artemisinin derivatives can be used for the treatment of various tumors, and described tumor includes but not limited to pulmonary carcinoma, cancer of pancreas, colon cancer, hepatocarcinoma, carcinoma of prostate, renal carcinoma, gastric cancer, cerebroma, sarcoma, ovarian cancer or breast carcinoma.

The pharmaceutical composition of the preferred Sorafenib of the present invention and arteannuin and artemisinin derivatives is used for preparing the application of the medicine for the treatment of pulmonary carcinoma, cancer of pancreas, colon cancer, hepatocarcinoma and carcinoma of prostate.

Be used for preparing in the application for the treatment of lung cancer drugs at pharmaceutical composition of the present invention, the mol ratio of described Sorafenib and arteannuin and artemisinin derivatives is 2.0-4.0: 3.0-10.0; The mol ratio that is preferably Sorafenib and arteannuin and artemisinin derivatives is 3.0-4.0: 5.0-10.0; The mol ratio that further is preferably Sorafenib and arteannuin and artemisinin derivatives is 4.0: 10.0.

Be used for preparing in the application of the medicine for the treatment of cancer of pancreas at pharmaceutical composition of the present invention, the mol ratio of described Sorafenib and arteannuin and artemisinin derivatives is 2.5-7.5: 2.5-10.0; The mol ratio that is preferably Sorafenib and arteannuin and artemisinin derivatives is 5.0-7.5: 5.0-10.0; The mol ratio that further is preferably Sorafenib and arteannuin and artemisinin derivatives is 7.5: 10.0.

Be used for preparing in the application of the medicine for the treatment of colon cancer at pharmaceutical composition of the present invention, the mol ratio of described Sorafenib and arteannuin and artemisinin derivatives is 2.5-6.0: 2.5-7.5; The mol ratio that is preferably Sorafenib and arteannuin and artemisinin derivatives is 4.0-6.0: 5.0-7.5; The mol ratio that further is preferably Sorafenib and arteannuin and artemisinin derivatives is 6.0: 7.5.

Be used for preparing in the application of the medicine for the treatment of hepatocarcinoma at pharmaceutical composition of the present invention, the mol ratio of described Sorafenib and arteannuin and artemisinin derivatives is 2.0-4.0: 3.0-10.0; The mol ratio that is preferably Sorafenib and arteannuin and artemisinin derivatives is 3.0-4.0: 6.0-10.0; The mol ratio that further is preferably Sorafenib and arteannuin and artemisinin derivatives is 4.0: 10.0.

Be used for preparing in the application of the medicine for the treatment of carcinoma of prostate at pharmaceutical composition of the present invention, the mol ratio of described Sorafenib and arteannuin and artemisinin derivatives is 2.5-5.0: 5.0-12.5; The mol ratio that is preferably Sorafenib and arteannuin and artemisinin derivatives is 3.5-5.0: 7.5-12.5; The mol ratio that further is preferably Sorafenib and arteannuin and artemisinin derivatives is 5.0: 12.5.

The pharmaceutical composition that contains Sorafenib and arteannuin and artemisinin derivatives is in preparation treatment pulmonary carcinoma, cancer of pancreas, colon cancer, hepatocarcinoma, carcinoma of prostate, renal carcinoma, gastric cancer, cerebroma, sarcoma, in the application of the medicine of ovarian cancer or breast carcinoma, in the scheme of the medicament of the present composition being made administration simultaneously, arteannuin and artemisinin derivatives and Sorafenib can be contained in in a kind of pharmaceutical preparation such as tablet or the capsule, also arteannuin and artemisinin derivatives and Sorafenib can be made preparation respectively, as making tablet or capsule respectively, and the mode that adopts this area routine is with their packings or combine, and the patient takes simultaneously according to the indication of package insert then; In the scheme of the medicament of the present composition being made administration successively, arteannuin and artemisinin derivatives can be made different preparations respectively with Sorafenib, and the mode that adopts this area routine is with their packings or combine, the patient takes according to the sequencing of package insert indication then, or two kinds of compositions in the above-mentioned composition are made a kind of preparation of controlled release, a kind of composition in elder generation's release composition and then the another kind of composition in the release composition, the patient only need take this controlled release composition preparation; In the scheme of the medicament that the present composition is prepared into the intersection administration, arteannuin and artemisinin derivatives can be made different preparations respectively with Sorafenib, and the mode that adopts this area routine is with their packings or combine, the patient takes according to the chi sequence of package insert indication then, the controlled release preparation that perhaps this preparation of pharmaceutical compositions is become arteannuin and artemisinin derivatives and Sorafenib intersection to discharge.

In the application of the pharmaceutical composition of Sorafenib and arteannuin and artemisinin derivatives in the medicine of preparation treatment pulmonary carcinoma, cancer of pancreas, colon cancer, hepatocarcinoma, carcinoma of prostate, renal carcinoma, gastric cancer, cerebroma, sarcoma, ovarian cancer or breast carcinoma, arteannuin in the described compositions and artemisinin derivatives and Sorafenib can use or simultaneously with the using in order of any priority, as arteannuin and artemisinin derivatives and Sorafenib being taken to the patient simultaneously; Also can earlier Sorafenib be taken, be taken then to arteannuin and artemisinin derivatives medicine to the patient, or take Sorafenib earlier, take arteannuin and artemisinin derivatives medicine then, the interval of taking for both does not have special requirement, but the interval of preferably taking two kinds of medicines is no more than one day; Perhaps two kinds of medicines replace administration.

Among the present invention, the method of arteannuin of the present invention and artemisinin derivatives and Sorafenib employing this area routine can be prepared into the pharmaceutical preparation that is suitable for gastrointestinal administration or parenteral administration, the pharmaceutical preparation that the present invention preferably makes gastrointestinal administration with arteannuin and artemisinin derivatives and Sorafenib, its dosage form can be conventional tablet or capsule or controlled release, slow releasing preparation.In the pharmaceutical preparation of arteannuin of the present invention and artemisinin derivatives and Sorafenib compositions, according to different dosage forms and preparation specification, the content of described compositions in preparation can be counted 1-99% for quality, is preferably 10%-90%; The adjuvant that preparation uses can adopt the adjuvant of this area routine, reacts or the curative effect that do not influence medicine of the present invention is a prerequisite with the discord present composition; The preparation method of described preparation can adopt the preparation method of this area routine to be prepared.

Among the present invention, the preparation of compositions method does not have any restriction, arteannuin and artemisinin derivatives and Sorafenib can directly mix makes preparation then, or respectively and/or corresponding auxiliary material mix and to make preparation respectively, and then be packaging together, or mix and then mix and make preparation with corresponding auxiliary material respectively according to the mode of this area routine.

The dosage of the pharmaceutical composition among the present invention can carry out suitable variation according to the dosage form difference of administration object, route of administration or medicine, but is prerequisite to guarantee that this pharmaceutical composition can reach effective blood drug level in mammalian body.

The present invention has carried out the test that H460 (lung cancer cell line), Panc1 (pancreas cancer cell strain), HCT-116 (colon cancer cell line), HepG2 (hepatoma cell strain) and 22RV1 (prostate gland cancer cell strain) are killed in the combination of Sorafenib and arteannuin and artemisinin derivatives respectively, results suggest, Sorafenib of the present invention and arteannuin and artemisinin derivatives combined therapy pulmonary carcinoma, cancer of pancreas, colon cancer, hepatocarcinoma and carcinoma of prostate have significant cooperative effect, improved the curative effect of medicine, reduce dosage, reduced the generation of side effect.

The specific embodiment

The invention will be further elaborated with the following Examples, but the present invention is not limited to this.

Embodiment

Reagent and method:

Cell: H460 (lung cancer cell line), Panc 1 (pancreas cancer cell strain), HCT-116 (colon cancer cell line), HepG2 (hepatoma cell strain) and 22RV1 (prostate gland cancer cell strain), all available from American Type Culture Collection (ATCC), Rockville, MD, USA.Medicine: institute's pharmaceutical composition is all by following method 1 or 2 described preparations of method in following examples; Artemisinin derivatives artesunate, dihydroarteannuin and Artemether are all available from Sigma; Sorafenib referenced patent US2003207872 is synthesized into.

Method 1: each component of accurate weighing corresponding pharmaceutical compositions, dissolve respectively with dimethyl sulfoxide, be made into the stock solution of 10mM separately, preserve down at-20 ℃, be diluted to suitable concentration with fresh culture medium during use, the solution of each component of 1 microlitre of respectively asking for then mixes standby.In all tests, the ultimate density of dimethyl sulfoxide is answered≤5g/L, so that do not influence cell activity.

With all cells in RPMI 1640 culture medium that contain 10% calf serum, 100kU/L penicillin, 100mg/L streptomycin, 37 ℃, 5%CO ₂Damp condition cultivate down, in the previous day of dosing, on six orifice plates, carry out cell inoculation 2 * 10 ⁵/ hole adds the pharmaceutical composition solution of preparation as stated above then in cell, make each component reach its working concentration, 1-15 in specifically seeing Table.

After the drug treating, measure cell death by trypan blue (Trypan Blue), cell turns into 10 minutes by carrying out pancreatin at 37 ℃ with trypsin sodium/EDTA.Because dead cell comes off from incubator enter the culture medium,, and then, mix with the trypan blue dyestuff with culture medium suspended sediment again by all cells of centrifugal collection under 1200 rev/mins.After the dyeing, count with optical microscope and hematimeter.Dyed the blue dead cell of counting by dyestuff.500 cells of picked at random are counted, and dead cell is recently expressed with the percentage that accounts for the grand total cell.

Method 2: each component of accurate weighing corresponding pharmaceutical compositions, dissolve respectively with dimethyl sulfoxide, be made into the stock solution of 10mM separately, preserve down at-20 ℃.Be diluted to suitable concentration with fresh culture medium during use, the solution for standby of each component of 1 microlitre of respectively asking for then.In all tests, the ultimate density of dimethyl sulfoxide is answered≤5g/L, so that do not influence cell activity.

With all cells in RPMI 1640 culture medium that contain 10% calf serum, 100kU/L penicillin, 100mg/L streptomycin, 37 ℃, 5%CO ₂Damp condition cultivate down, in the previous day of dosing, on six orifice plates, carry out cell inoculation 2 * 10 ⁵/ hole adds each component solution of the pharmaceutical composition of preparation as stated above with any order then in cell, make each component reach its working concentration, 16-27 in specifically seeing Table.

After the drug treating, measure cell death by trypan blue (Trypan Blue), cell turns into 10 minutes by carrying out pancreatin at 37 ℃ with trypsin sodium/EDTA.Because dead cell comes off from incubator enter the culture medium,, and then, mix with the trypan blue dyestuff with culture medium suspended sediment again by all cells of centrifugal collection under 1200 rev/mins.After the dyeing, count with optical microscope and hematimeter.Dyed the blue dead cell of counting by dyestuff.500 cells of picked at random are counted, and dead cell is recently expressed with the percentage that accounts for the grand total cell.

Tabulate down in the drug regimen shown in 1, the combination of 1-15 prepares by method 2 by the combination of method 1, the 16-27.

Table 1

The artesunate of embodiment 1 different proportion and the combination Synergistic of Sorafenib promote the test of H460 cell death, see Table 2.

Table 2

Cause in the test of lung cancer cell line H460 cell death investigating related compound, find when use separately 10.0 μ M artesunate only have an appointment 20% cell death, use 4.0 μ M Sorafenibs, 15% cell death of only having an appointment separately; (5.0 μ M artesunate+3.0 μ M Sorafenibs) then produce the obvious synergistic effect when both share under low concentration, cause about 80% cancer cell death; When both share with the ratio of 10.0 μ M artesunate+4.0 μ M Sorafenibs, then produce significant more synergism, cause 99% cancer cell death.

The dihydroarteannuin of embodiment 2 different proportions and the combination Synergistic of Sorafenib promote the test of H460 cell death, see Table 3.

Table 3

Cause in the test of lung cancer cell line H460 cell death investigating related compound, find when use separately 10.0 μ M dihydroarteannuins only have an appointment 20% cell death, use 4.0 μ M Sorafenibs, 15% cell death of only having an appointment separately; (5.0 μ M dihydroarteannuins+3.0 μ M Sorafenibs) then produce the obvious synergistic effect when both share under low concentration, cause about 65% cancer cell death; When both share with the ratio of 10.0 μ M dihydroarteannuins+4.0 μ M Sorafenibs, then produce significant more synergism, cause 99% cancer cell death.

The Artemether of embodiment 3 different proportions and the combination Synergistic of Sorafenib promote the test of H460 cell death, see Table 4.

Table 4

Cause in the test of lung cancer cell line H460 cell death investigating related compound, find when using 10.0 μ M Artemether, 4.0 μ M Sorafenibs 15% cell death of only having an appointment separately; (5.0 μ M Artemether+3.0 μ M Sorafenibs) then produce the obvious synergistic effect when both share under low concentration, cause about 50% cancer cell death; When both share with the ratio of 10.0 μ M Artemether+4.0 μ M Sorafenibs, then produce significant more synergism, cause about 88% cancer cell death.

The artesunate of embodiment 4 different proportions and the combination Synergistic of Sorafenib promote the test of Panc1 cell death, see Table 5.

Table 5

Cause in the test of pancreas cancer cell strain Panc1 cell death at the investigation related compound, find when use 10.0 μ M artesunate or lower concentration separately, to have only very a spot of cell death, even use 7.5 μ M Sorafenibs, 20% cell death of also only having an appointment separately; (5.0 μ M artesunate+5.0 μ M Sorafenibs) then produce the obvious synergistic effect when both share under low concentration, cause 68% cancer cell death; When both share with the ratio of 10.0 μ M artesunate+7.5 μ M Sorafenibs, then produce significant more synergism, cause 92% cancer cell death.

The dihydroarteannuin of embodiment 5 different proportions and the combination Synergistic of Sorafenib promote the test of Panc1 cell death, see Table 6.

Table 6

Cause in the test of pancreas cancer cell strain Panc1 cell death at the investigation related compound, find when use 10.0 μ M dihydroarteannuins or lower concentration separately, to have only very a spot of cell death, even use 7.5 μ M Sorafenibs, 20% cell death of also only having an appointment separately; (5.0 μ M dihydroarteannuins+5.0 μ M Sorafenibs) then produce the obvious synergistic effect when both share under low concentration, cause 51% cancer cell death; When both share with the ratio of 10.0 μ M dihydroarteannuins+7.5 μ M Sorafenibs, then produce significant more synergism, cause 81% cancer cell death.

The artesunate of embodiment 6 different proportions and the combination Synergistic of Sorafenib promote the test of HCT116 cell death, see Table 7.

Table 7

Cause in the test of colon cancer cell line HCT116 cell death at the investigation related compound, find 20% cell death of when using 7.5 μ M artesunate or 6.0 μ M Sorafenibs separately, only having an appointment; (5.0 μ M artesunate+4.0 μ M Sorafenibs) then produce the obvious synergistic effect when both share under low concentration, cause 76% cancer cell death; When both share with the ratio of 7.5 μ M artesunate+6.0 μ M Sorafenibs, then produce significant more synergism, cause about 95% cancer cell death.

The dihydroarteannuin of embodiment 7 different proportions and the combination Synergistic of Sorafenib promote the test of HCT116 cell death, see Table 8.

Table 8

Cause in the test of colon cancer cell line HCT116 cell death at the investigation related compound, find 20% cell death of when using 7.5 μ M dihydroarteannuins or 6.0 μ M Sorafenibs separately, only having an appointment; (5.0 μ M dihydroarteannuins+4.0 μ M Sorafenibs) then produce the obvious synergistic effect when both share under low concentration, cause 67% cancer cell death; When both share with the ratio of 7.5 μ M dihydroarteannuins+6.0 μ M Sorafenibs, then produce significant more synergism, cause about 90% cancer cell death.

The artesunate of embodiment 8 different proportions and the combination Synergistic of Sorafenib promote the test of HepG2 cell death, see Table 9.

Table 9

Cause in the test of hepatoma cell strain HepG2 cell death at the investigation related compound, find 15% cell death of when using 10.0 μ M artesunate or 4.0 μ M Sorafenibs separately, only having an appointment; (6.0 μ M artesunate+3.0 μ M Sorafenibs) then produce the obvious synergistic effect when both share under low concentration, cause 42% cancer cell death; When both share with the ratio of 10.0 μ M artesunate+4.0 μ M Sorafenibs, then produce significant more synergism, cause about 91% cancer cell death.

The dihydroarteannuin of embodiment 9 different proportions and the combination Synergistic of Sorafenib promote the test of 22RV1 cell death, see Table 10.

Table 10

Cause in the test of prostate gland cancer cell strain 22RV1 cell death at the investigation related compound, find 10% cell death of when using 7.5 μ M dihydroarteannuins, 3.5 μ M Sorafenibs or lower concentration separately, only having an appointment; Even also only have an appointment 20% cell death when increasing concentration to 12.5 μ M dihydroarteannuin, the 5.0 μ M Sorafenibs of single medicine; (7.5 μ M dihydroarteannuins+3.5 μ M Sorafenibs) then produce the obvious synergistic effect when both share under low concentration, cause 44% cancer cell death; When both share with the ratio of 12.5 μ M dihydroarteannuins+5.0 μ M Sorafenibs, then produce significant more synergism, cause 81% cancer cell death.