NZ623550B2 - Therapeutic combination for the treatment of cancer - Google Patents

Abstract

The disclosure relates to therapeutic combination comprising an anti-neoplastic agent comprising platinum and an extract from a species of the genus Nerium. The anti-neoplastic agent comprising platinum may be cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, or phosphaplatins and the extract from a species of the genus Nerium may be Nerium indicum, Nerium oleander or Nerium odorum.The disclose alos relotes to the use of these combination for the use of treating prostate cancer, melanoma, pancreatic cancer, lung cancer, breast cancer, or colorectal cancer. osphaplatins and the extract from a species of the genus Nerium may be Nerium indicum, Nerium oleander or Nerium odorum.The disclose alos relotes to the use of these combination for the use of treating prostate cancer, melanoma, pancreatic cancer, lung cancer, breast cancer, or colorectal cancer.

Description

TITLE OF THE INVENTION 'I'herapeutic Combination for the Treatment of Cancer . FIELD OF THE INVENTION This invention relates to eutic combinations sing a platinum—based anti— neoplastic agent and an extract from a species of the genus Nerz'um, as well as s of using such combinations to treat patients suffering from certain types of cancer.

BACKGROUND OF THE INVENTION Because of the continuing need for more effective ways to treat cancer, great effort continues to be exerted to develop new drugs that are less toxic to the patient being treated. However, another approach is to try to lower the toxicity of existing anti-cancer drugs by combining them with other drugs.

Platinum-based anti-cancer drugs, such as cisplatin, find use in chemotherapy of various types of cancer, but their toxicity remains a serious problem. rmore, the emergence of cisplatin resistance in some tumors vitiates its utility at the toxicity-limited dosage. To mitigate these limitations, cisplatin is commonly used in combination with other drugs, such as 5-fluorouracil, that exert their toxic effects on different organs than those affected by tin.

Extracts of Nerz'um er comprise various polysaccharides and ns as well as two toxic cardiac glycosides, oleandrin and oleandrigenin, that are known to exhibit anti-cancer activity.

EMBODIMENTS OF THE INVENTION An ment of the invention is a therapeutic combination comprising (I) an anti- neoplastic agent comprising platinum and (II) an t from a species of the genus Nerium.

Another embodiment ofthe invention is the above therapeutic combination, wherein (I) is present in an amount that, in the absence of (II), is effective to produce an increase in anti- stic activity.

Another embodiment of the invention is the above therapeutic combination, wherein (I) is t in an amount that, in the absence of (II), is ineffective to produce an increase in anti— neoplastic activity.

Another embodiment of the invention is the above therapeutic combination, wherein (I) is selected from the group consisting of cisplatin, latin, oxaliplatin, satraplatin, picoplatin, nedaplatin, tin, and phosphapiatins.

Another embodiment ofthe invention is the above therapeutic combination, wherein (I) is cispiatin.

Another embodiment ofthe invention is the above eutic combination, wherein (II) is an extract from a species selected from the group consisting ofNerium m, Nerz'um Oleander, and Nerium odorum.

Another embodiment of the invention is the above therapeutic combination, wherein (II) is an extract from the species Nerium Oleander.

Another embodiment ofthe ion is the above therapeutic combination, wherein (II) is a based extract.

Another embodiment ofthe invention is the above therapeutic combination, wherein (II) is an ased extract.

Another embodiment ofthe invention is the above therapeutic ation, wherein (I) is cisplatin and (II) is an extract from the species Nerium Oleander.

Another embodiment of the invention is the above therapeutic combination, wherein (I) and (II) are present as separate formulations.

Yet another embodiment of the invention is a method for treating cancer comprising administering to a patient in need thereof a therapeutic combination comprising (I) an anti- neoplastic agent comprising platinum and (II) an t from a species of the genus Nerium.

Another embodiment of the invention is the above method, wherein (I) is present in an amount that, in the e of (II), is effective to produce an increase in anti—neoplastic activity.

Another embodiment ofthe invention is the above method, wherein (I) is present in an amount that, in the absence of (II), is ineffective to produce an increase in anti—neoplastic activity.

Another embodiment of the invention is the above method, wherein (I) is selected from the group ting of cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and phosphaplatins.

Another embodiment of the invention is the above method, n (I) is cisplatin.

Another embodiment of the invention is the above method, wherein (II) is an extract from a species ed from the group consisting of Nerz'um indicum, Nerz'um Oleander, and Nerz'um odnrum.

Another embodiment of the invention is the above , n (II) is an extract from the species Nerz'um Oleander. r ment of the invention is the above method, wherein (II) is a water-based extract.

Another embodiment of the invention is the above method, wherein (II) is an aloe—based extract.

Another embodiment ofthe invention is the above method, wherein (I) is cisplatin and (II) is an extract from the species Nerium Oleander.

Another embodiment of the invention is the above method, wherein (I) and (II) of said therapeutic combination are administered to the patient simultaneously, separately, or sequentially. r embodiment of the invention is the above method, n (II) is administered to the patient intramuscularly, sublingually, or intramuscularly and gually.

Another embodiment of the invention is the above , wherein (II) is administered to the patient sublingually in two or more doses.

Another embodiment of the invention is the above method, wherein the cancer treated is te cancer, melanoma, pancreatic cancer, lung cancer, breast , or colorectal cancer.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 s a graph of the relative reduction in cells of the LNCaP prostate cancer cell line in the ce of AnvirzelTM, tin, and a combination of the two.

Figure 2 depicts a graph of the relative reduction in cells of the A375 melanoma cancer cell line in the presence of AnvirzelTM, cisplatin, and a combination of the two.

Figure 3 s a graph of the relative reduction in cells of the PANC-l pancreatic cancer cell line in the presence of AnvirzelTM, cisplatin, and a combination of the two.

Figure 4 depicts a graph of the relative reduction in cells of the 9N lung cancer cell line in the presence of AnvirzelTM, cisplatin, and a combination of the two.

DETAILED DESCRIPTION OF THE ION It has singly been found that combinations of an anti-neoplastic agent comprising platinum and an extract from a species of the genus Nerz'um synergistically exhibit increased anti—neoplastic activity. As a , otherwise ineffective but well-tolerated doses of such platinum-based anti-neoplastic agents, when combined with dosages of the extract that lack efficacy themselves, exert clinically useful anti-neoplastic efficacy.

The therapeutic combinations of this invention se an anti-neoplastic agent comprising platinum and an extract from a species of the genus Nerium.

The anti-neoplastic agent used in the therapeutic combinations of this invention can be any known anti-cancer or anti-tumor agent or drug that contains platinum. Non-limiting es include cisplatin, latin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and phosphaplatins, such as those disclosed in US. Patent No. 8,034,964 and which are incorporated herein by reference. These compounds can be prepared by methods known in the art. Preferably, the anti-neoplastic agent is cisplatin. Cisplatin is a divalent inorganic ‘ water-soluble, platinum containing complex widely used to treat testicular, bladder, and ovarian cancers.

The platinum—based anti—neoplastic agents used in the therapeutic combinations of this invention can be combined with any pharmaceutically acceptable excipient, carrier, adjunct, or t known in the art.

The extract used in the therapeutic combinations of this invention can be derived from any species of the genus Nerz'um. Non-limiting examples include extracts from the species Nerium indicum, Nerium Oleander, and Nerium odorum. ably, the extract is derived from Nerium Oleander.

Extracts ofNerium Oleander can be prepared by methods known in the art. Such methods include extraction with hot water (US. Patent Nos. 6,565,897 and 5,135,745), room temperature water and aqueous alcohol, ularly aqueous methanol and ethanol (US.

Patent App. Pub. No. 2007/0154573 A1), supercritical carbon dioxide (U.8. Patent No. 7,402,325), and aloe (US. Patent App. Pub. No. 2010/00925 85 Al). These aforementioned extraction methods disclosed in US. Patent Nos. 6,565,897, 5,135 ,745, and 325 and U.8. Patent App. Pub. Nos. 154573 A1 and 2010/00925 85 A1 are incorporated herein by reference. AnvirzelTM is a hot water extract ofNerium Oleander that contains oleandrin and oleandrigenin as its main constituents.

The extracts used in the combinations of the invention can contain any ceutically acceptable excipient, carrier, t, or diluent known in the art. Examples include mannitol, ascorbic acid, sodium ascorbate, methyl n, propyl paraben, and mixtures thereof. ably, the therapeutic combination of the invention comprises cisplatin and an extract from the species Nerium Oleander.

The anti-neoplastic agent comprising platinum and the extract from a species of the genus Nerz'um can be present in the therapeutic combinations of the invention as either separate or combined formulations.

The method of this invention for treating cancer comprises administering to a patient in need thereof a therapeutic combination comprising an eoplastic agent comprising platinum and an extract from a species of the genus Nerz‘um. As used herein, the term "patient" refers to a mammal afflicted with cancer. The red patient is a human.

The anti-neoplastic agent comprising platinum can be present in the therapeutic combinations of the invention and used in the method of this invention in an amount that, in the absence of the extract from a species of the genus Nerz‘um, is either effective or ineffective to produce an increase in eoplastic activity. An amount or dose that is "effective” is that which produces a particular cancer cell growth— or proliferation-inhibiting effect, tumor growth inhibiting effect, tumor volume increase—inhibiting , or cancer treatment effect in a cancer cell or tumor.

The anti-neoplastic agent comprising platinum can be stered to the patient via any mode known in the art, such as intravenously in the case of cisplatin, carboplatin, and oxalplatin, and orally in the ease of satraplatin. A typical dosage of cisplatin for an adult is 70-100 mg/m2 a day for three days, followed by two weeks off, constituting one cycle.

Patients can receive a maximum of six cycles of therapy, but owing to toxicity problems most patients can only endure four cycles of such therapy. A typical adult (170 cm tall and weighing 70 kg), has 1.83 m2 (The Journal of Pediatrics 1978 93:1:62-66; Gehan EA, George SL), corresponding to a dosage of about 125—185 mg.

The extract used in this method can be administered intramuscularly, sublingually, or intramuscularly and sublingually, either in a single dose or in le doses. The normal dosage for AnvirzelTM is 0.5 to 1.0 mL intramuscularly, 1.0 to 2.0 mL gually, or a ation of intramuscular and gual administration so that the total amount in a 24 hour period is no greater that 2 mL. AnvirzelTM administered sublingually only is divided into a series of smaller sub-doses, typically two to four doses of 0.5 mL or less per 24 hours.

As is common with pharmaceutical agents, the respective therapeutic doses of the components ofthe therapeutic combinations of the invention may vary with the condition of the patient and the route by which the drug is administered. The dose, and perhaps the dose ncy, will also vary according to the age, body weight, and response of the individual t. It may be necessary to use dosages outside these ranges in some cases, as will be apparent to those skilled in the art. Further, it is noted that the ian or ng physician will know how and when to interrupt, adjust, or ate therapy in conjunction with individual patient response. The terms "therapeutic amount" and "therapeutically effective amount" are encompassed by the above-described dosage amounts and dose frequency schedules.

The therapeutic combinations of the invention can be administered to the patient simultaneously, separately, or sequentially.

The therapeutic combinations of the invention can be used according to this method to treat any type of cancer. es include prostate cancer, ma, pancreatic cancer, lung cancer, breast cancer, and colorectal .

The following non-limiting examples demonstrate the synergistic cytostatic effect of co- administration of a Nerium Oleander extract with cisplatin.

While these examples show certain specific embodiments of the invention, it will be manifest to those skilled in the art that s modifications and ngements of the parts may be made without departing from the spirit and scope of the underlying inventive t and that the same is not limited to the particular forms herein shown and described.

EXAMPLES Viability Assays Three different viability assays were chosen to complement each other and yielded results dependent on concentration and cell line: the Methyl Tetrazolium dye assay, the Sulforhodarnine B fluorescent dye assay, and the Crystal Violet dye assay.

The -Tetrazolium dye assay measures the activity of enzymes in mitochondria. The dye precursor is taken up by cells, where it undergoes reduction to the purple an. It measures only the mitochondrial activity and, thus, it is not always a reliable tion of whether the cells themselves are alive or dead, since even a recently dead cell has lower enzyme activity in mitochondria. ellular glucose concentration, pH, and time before assay all affect the ements.

The Sulforhodamine B and Crystal Violet assays provide information about protein and were used to complement the Methyl—Tetrazolium dye assay. The Sulforhodamine B assay is more sensitive for the detection of small number of cells and shows a linear relationship between cell number and its staining intensity. It is used for the quantification of cellular proteins of cultured cells. The assay using Crystal Violet, a dye that binds electrostatically to proteins and stains DNA, provides a reliable, simple assay for measuring viability of cells.

The Methyl olium, Sulforhodamine B, and Crystal Violet assays were used to assess cell ities. The incubation times were 24 hours, 48 hours, and 72 hours. The Nerz'um Oleander extract AnvirzelTM (a hot water extract of Oleander obtained from Salud Integral, Honduras) was tested in concentrations ranging from 0.01 ng/mL to 10 ng/mL. Cisplatin (Sigma, P4394) was tested in concentrations ranging from 0.1 ug/mL to 100 ug/mL.

Cells were detached by trypsinization (Trypsin-0.25% EDTA, Invitrogen, 25200-072) during the logarithmic phase of culture growth and plated in 96-well plates (18.000 cells/well) (Corning, Costar 359) in a final volume of 200ul of medium per well. After 70 to 80% confluence of the culture, the medium was d and the AnvirzelTM diluted in water and tin diluted in N,N—dimethylformamide (Fluka, 40255) were added to the cells in graduated densities. The absorbance was measured after 24 hours, 48 hours, and 72 hours of ' incubation.

For the hodamine B assay, 96-well plates were fixed by 10% trichloroacetic acid (Fluka, 91228) and were incubated at 4 °C for lhour. ards, plates were rinsed with water and cells were stained with 0.4% Sulforhodamine B (Sigma, 34173 8), dissolved in 1% acetic acid (Carlo Ebra, 401422) for 15 minutes at room temperature (RT). The unbound stain was washed twice with 1% acetic acid. Finally, 10 mM Tris Buffer pH 10.5 , T6791) was added to dissolve the dye.

For the Crystal Violet assay, the medium was d from the 96—well plates, the plates rinsed with PBS (Sigma, P3813) and then the cells were rinsed by the addition of 10% formalin (MERCK, 2500) .for 20 minutes at RT. Formalin was removed and 0.25% aqueous crystal Violet (Sigma, HT901), ved in Water, was added for 10 minutes at RT.

Unbound Crystal Violet was rinsed by washing with water and finally 33% acetic acid was added to dissolve the dye.

For the Methyl-Tetrazolium assay, the dye (Sigma, M2128; 5 mg/mL, diluted in phosphate- buffered saline) was added to each well and plates were incubated for 3 hours at 37°C. After the end of the incubation period, the medium was discarded and the cells were rinsed with phosphate-buffered saline. Finally, the formazan crystals were dissolved in dimethylsulphoxide (Sigma, D4540).

The optical density of each plate was measured on a uQuant spectrophotometer and the data were analyzed with Gen5 software (uQuant Biomolecular Spectrophotometer MQXZOO and GenSTM Microplatc Data Collection & Analysis software, ® Instruments.1nc, April 2008). Absorbance was measured at 570 nm for all assays and a second wavelength was measured in order to subtract noise. For the -Tetrazolium assay this second wavelength was 630 nm and for the Sulforhodamine B and Crystal Violet assays it was 690 Cell Lines Human carcinoma cell lines were ed by the European Collection of Cell Cultures from Health Protective Agency, UK, and included lines derived from human te cancer (PC3, LNCaP and 22RV1), human breast cancer (MDA—MB 231, T47D, MCF-7), non-small cell lung carcinoma (CALU-l, COLO699N, COR-L 105), colorectal cancer (HGT—116, HT55, HGT-15), ma (A375), and pancreatic cancer (PANCJ).

Cells were cultured in 75 cm2 flasks (Orange Scientific, 5520200, Belgium) in the medium indicated for each line with the appropriate amount of heat inactivated Fetal Bovine Serum (FBS, Invitrogen, 10106-169, California) and 2 mM L-Glutamine (Sigma, G5792, Germany) for each cell line and incubated at 37°C in a 5% C02 atmosphere.

Statistical Analysis All treatments for each cell line were performed in cate. The tical significance of all effects was ted by “difference of the means” test (p < 0.05). elTM concentrations ranged from 0.01 ng/mL to 10 ng/mL, while those for cisplatin ranged from 0.1 ug/mL to 100 ug/mL. Surprisingly, lower concentrations of both the AnvirzelTM (0.01 to 0.1 ng/mL) and the cisplatin (0.1 ug/mL) gave better results than higher concentrations, which yielded unreliable and irreproducible results. The results were also time-dependent, as was observed after 48 and 72 hours of incubation.

Example 1: LNCaP cell line Table 1 below shows results for LNCaP cells, a prostatic cancer-derived cell line, with cell population densities estimated from optical absorbance data.

By Crystal Violet assay, after 48 hours of incubation, unstimulated cells showed an apparent absorbance of 1.307 (a dimensionless quantity), with essentially the same value (1249) ed in the presence of 0.01 ng/mL of AnvirzelTM. The presence of 0.1ug/mL of cisplatin reduced the apparent absorbance to 0.408, but the presence of 0.01 ofng/mL AnvirzelTM and 0.1 ug/niL of cisplatin further d the value to 0.178 (p=0.00008<0.05). A similar diminution in nt absorbance was found after 72 hours incubation as well.

By Sulforhodamine B assay, after 48 hours of incubation, unstimulated cells showed an apparent absorbance of 2.616, with essentially the same value (2.667) ed in the presence of 0.01 ng/mL of AnvirzelTM. The presence of 0.1 ug/mL of cisplatin reduced the apparent absorbance to 1.681, but the presence of 0.01 ng/mL of AnvirzelTM and 0.1 ug/mL of cisplatin further reduced the value to 1.033 (p=0.007<0.05). A similar diminution in apparent absorbance was found after 72 hours incubation as well.

By Methyl—Tetrazolium assay, after 48 hours of tion, unstimulated cells showed an apparent absorbance of 0.429, with a somewhat lower value (0.292) obtained in the presence of 0.01 ng/mL of AnvirzelTM. The presence ofO.1 ug/mL ofcisplatin reduced the apparent absorbance to 0.161, but the presence of 0.01 ng/mL of AnvirzelTM and 0.1 ug/mL of cisplatin further reduced the value to 0.033 (p=0.005<0.05). A similar diminution in nt absorbance was found after 72 hours incubation as well.

Table 1: tical evaluation of absorbance in LNCaP prostate cancer cell line.

Unstimulated cells . 1.307 0.01 ng/mL el 1 249 2.416 0.1 g/mL cisplatin 0408 0.448 0.01 ng/mL e1+ 0.178 0.187 0.1itg/mL cisplatin (p=0.00008<0.05 20.002<0.05 Unst1mulated cells 0.01 ng/mL Anvirzel 001 ng/mL Anvirzel + 0.1 - mLcis-latin 1m1rnLO. cislatin 0.161 0.17 0.1ug/mL cisplatin (p=0.005<0.05) (p=0.0001<0.05) These results in Table l are graphically depicted in Figure 1.

Example 2: A375 Cell Line Table 2 below shows results for A375 cells, a ma—derived cell line, with cell population ies estimated from optical absorbance data.

By Crystal Violet assay, after 48 hours of incubation, unstimulated cells showed an apparent absorbance of 2.791, with essentially the same value (2.71) ed in the presence of 0.01 ng/mL of AnvirzelTM. The presence of 0.1 ug/mL of cisplatin reduced the apparent absorbance to 1.304, but the presence of 0.01 ng/mL of AnvirzelTM and 0.1 ug/mL of cisplatin reduced the value to 0.178 0008<0.05). A similar diminution in apparent absorbance was found after 72 hours incubation as well.

By Sulforhodamine B assay, after 48 hours of incubation, unstimulated cells showed an apparent absorbance of 2.836, with essentially the same value (2.854) obtained in the presence of 0.01 ng/mL of AnvirzelTM. The presence of 0.1 ug/mL of cisplatin reduced the apparent absorbance to 2.421, but the ce of 0.01 ng/mL of AnvirzelTM of and 0.1 ug/mL of cisplatin further reduced the value to 1.89 (p=0.01<0.05). A similar diminution in apparent absorbance was found after 72 hours incubation as well.

By Methyl—Tetrazolium assay, after 48 hours of incubation, ulated cells showed an apparent absorbance of 0.45, with essentially the same value (0.49) obtained in the presence of 0.01 ng/mL of elTM. The presence of 0.1 ug/mL of cisplatin reduced the apparent absorbance to 0.332, but the presence of 0.01 ng/mL of AnvirzelTM and 0.1 ug/mL of cisplatin reduced the value to 0.072 02<0.05). A similar diminution in apparent absorbance was found after 72 hours incubation as well.

Table 2: Statistical evaluation of absorbance in A375 melanoma cancer cell line.

Unstimulated cells 2.791 2.767 0.01 ng/mL Anvirzel 2.71 2.846 0.1ug/mL cisplatin 1.3 04 0.663 0.01 ng/mL Anvirzel + 0.306 0.123 O lug/mL cisplatin (p=0.002<0.05) n=0.0009<0.05) Unstimulated cells 0.01 ng/mL Anvirzel 2.854 rnL cisplatin 2.421 2.487 0.01 ng/mL Anvirzel + . 0.1 - mL cislatin inhstihiuiated cells A H 0.922 0.01 ng/mL Anvirzel 0.999 0.1ug/mL cisplatin 0.332 0.479 0.01 ng/mL Anv1rzel + 0.072 0.053 0 lug/mL ClS latln (p=0.002<0.05) (p=0.005<0.05) These results in Table 2 are graphically depicted in Figure 2.

Example 3: PANC-l Cell Line Table 3 below shows results for PANC—l cells, a pancreatic cancer-derived cell line, with cell population densities estimated from optical absorbance data.

By Crystal Violet assay, after 48 hours of incubation, unstimulated cells showed an apparent absorbance of 0.964, with ially the same value (1.04) obtained in the presence of 0.01 ng/mL ofAnvirzelTM. The ce of 0.1 ug/mL of cisplatin reduced the apparent absorbance to 0.753, but the presence of 0.01 ng/mL of elTM and 0.1 pg/mL of cisplatin reduced the value to 0.283 (p=0.01<0.05). A similar diminution in apparent absorbance was found after 72 hours incubation as well.

By Sulforhodamine B assay, after 48 hours of tion, unstimulated cells showed an apparent absorbance of 2.332, with essentially the same value (2.328) obtained in the presence of 0.01 ng/mL of AnvirzelTM. The presence of 0.1 ng/mL of tin reduced the apparent absorbance to 1.861, but the ce of 0.01 ng/mL of AnvirzelTM and 0.1 ng/mL of cisplatin further reduced the value to 1.25 (p=0.002<0.05). A similar diminution in apparent absorbance was found after 72 hours incubation as well.

By Methyl—Tetrazolium assay, after 48 hours of tion, unstimulated cells showed an apparent absorbance of 0.546, with a similar value (0.518) obtained in the presence of 0.01 ng/mL of AnvirzelTM. The presence of 0.1 ug/mL of cisplatin reduced the apparent absorbance to 0.286, but the presence of 0.01 ng/mL of AnvirzelTM and 0.1 ug/mL of cisplatin reduced the value to 0.143 (p=0.0007<0.05). A similar diminution in apparent ance was found after 72 hours incubation as well.

Table 3: Statistical evaluation of absorbance in PANC-l pancreatic cancer cell line.

Unstimulated cells 0 964 2.862 0 01 ng/mL Anvnzel 2.711 0 lug/mL Cisplatln 0.753 0.872 0.01 ng/mL Anvirzel + 0.283 0.336 0.1ug/rnL latin -=0.0l<0.05 (p=0.001<0.05) Unstimulated cells 2.332 2.924 0.01 ng/mL Anvirzel 2.328 2.882 mL cisplatin 1.861 2.536 0 01 ng/mL Anvnzel + 1.25 1 837 0 lug/mL ClS latin <0.05 =0.004<0.05) I i V r VUnstimulated cells . . 0.01 ng/mL Anvrrzel 0.518 0.954 0.1ug/mL Cisplatln 0.286 0.344 0.01 ng/mL Anvirzel + 0.143 0.115 0.1 1 mL cis-latin =0.0007<0.05) (p=0.002<0.05) These results in Table 3 are cally depicted in Figure 3.

Example 4: COLO699N Cell Line Table 4 below shows results for COLO699N cells, a lung cancer—derived cell line, with cell population densities estimated from optical absorbance data.

By Crystal Violet assay, after 48 hours of incubation, unstimulated cells showed an apparent absorbance of 0.918, with a similar value ) obtained in the presence of 0.01 ng/mL of AnvirzelTM. The presence of 0.1 ug/mL of cisplatin reduced the apparent absorbance slightly to 0.801, but the presence of 0.01 ng/mL of AnvirzelTM and 0.1 ug/mL of cisplatin d the value to 0.361 (p=0.01<0.05). A similar diminution in apparent absorbance was found after 72 hours incubation as well.

By Sulforhodamine B assay, afier 48 hours of incubation, unstimulated cells showed an apparent absorbance of 2.636, with essentially the same value (2.633) obtained in the ce of 0.01 ng/mL of AnvirzelTM. The presence of 0.1 ug/mL of cisplatin reduced the apparent ance to 1.975, but the presence of 0.01 ng/mL of AnvirzelTM and 0.1 ug/mL of cisplatin further reduced the value to 1.046 (p=0.00001<0.05). A r diminution in apparent absorbance was found after 72 hours tion as well.

By Methyl—Tetrazolium assay, after 48 hours of incubation, unstimulated cells showed an apparent absorbance of 0.82, with a somewhat higher value (0.953) obtained in the presence of 0.01 ng/mL of AnvirzelTM. The presence of 0.1 ug/mL of cisplatin reduced the apparent absorbance to 0.636, but the presence of 0.01 ng/mL of AnvirzelTM and 0.1 ug/mL of cisplatin reduced the value to 0.058 (p=0.001<0.05). A r diminution in apparent absorbance was found after 72 hours incubation as well.

Table 4: Statistical. evaluation of absorbance in COLO699N lung cancer cell line.

U11st1mulatedcells 0.918 1.659 0.01 ng/mL Anvirzel 0.826 1.654 0 lug/mL cisplatin 0-801 0.01 ng/mL Anvirzel + . 0.361 0.138 (-.=001<0.05) (p=0.005<0.05) Unstimulated cells 2.636 2.758 0.01 ng/mL Anvirzel 2.633 2.655 0 lug/mL cisplatin 1.975 . 0.01 ng/mL Anvirzel + 1.046 0.111g/mL cisplatin =0.00001<0.05) .

Unstimulated cells 0.01 ng/mL Anvirzel 0.953 1.184 0.1ug/mL cisplatin 0.636 0.01 ng/mL Anvirzel + 0.058 0.047 mL cisplatin . =0.001<0.05) (p=0.001<0.05) These results in Table 4 are cally depicted in Figure 4.

Claims (25)

1. A therapeutic combination sing (I) an anti-neoplastic agent comprising platinum and (II) a hot extract from a species of the genus Nerium.

2. The therapeutic combination of claim 1, wherein (I) is present in an amount that, in the absence of (II), is effective to produce an increase in anti-neoplastic activity.

3. The therapeutic combination of claim 1, wherein (I) is present in an amount that, in the absence of (II), is ineffective to produce an increase in anti-neoplastic activity.

4. The therapeutic combination of claim 1, wherein (I) is selected from the group consisting of cisplatin, carboplatin, latin, satraplatin, picoplatin, nedaplatin, triplatin, and phosphaplatins.

5. The therapeutic combination of claim 1, wherein (I) is cisplatin.

6. The therapeutic combination of claim 1, wherein (II) is an extract from a species ed from the group consisting of Nerium indicum, Nerium oleander, and Nerium odorum.

7. The therapeutic combination of claim 1, n (II) is an extract from the species Nerium er.

8. The therapeutic combination of claim 1, wherein (II) is a water-based extract.

9. The therapeutic combination of claim 1, wherein (II) is an ased extract.

10. The therapeutic combination of claim 1, wherein (I) is cisplatin and (II) is an extract from the species Nerium oleander.

11. The eutic ation of claim 1, wherein (I) and (II) are t as separate formulations, which are suitable for administration to a patient simultaneously, separately, or sequentially. 2736383v1

12. Use of a eutic combination comprising (I) an anti-neoplastic agent comprising platinum and (II) a hot extract from a species of the genus Nerium for the manufacture of a medicament for the treatment of .

13. The use of claim 12, wherein (I) is present in an amount that, in the absence of (II), is effective to produce an increase in anti-neoplastic activity.

14. The use of claim 12, wherein (I) is present in an amount that, in the absence of (II), is ineffective to produce an increase in anti-neoplastic activity.

15. The use of claim 12, wherein (I) is selected from the group ting of cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and phosphaplatins.

16. The use of claim 12, wherein (I) is cisplatin.

17. The use of claim 12, wherein (II) is an extract from a species selected from the group consisting of Nerium indicum, Nerium oleander, and Nerium odorum.

18. The use of claim 12, wherein (II) is an extract from the species Nerium oleander.

19. The use of claim 12, wherein (II) is a based extract.

20. The use of claim 12, wherein (II) is an aloe-based t.

21. The use of claim 12, n (I) is cisplatin and (II) is an extract from the s Nerium oleander.

22. The use of claim 12, wherein (I) and (II) of said therapeutic combination are suitable for administration to a patient simultaneously, separately, or sequentially.

23. The use of claim 12, n (II) is suitable for administration to the patient intramuscularly, sublingually, or intramuscularly and sublingually.

24. The use of claim 12, wherein (II) is suitable for administration to the patient sublingually in two or more doses. 2736383v1

25. The use of claim 12, wherein the cancer treated is prostate cancer, melanoma, pancreatic cancer, lung , breast cancer, or colorectal cancer. 2736383v1