阅读说明：本技术 供用于治疗乳腺癌的2-羟丙基-β-环糊精（HPβCD） (2-hydroxypropyl-beta-cyclodextrin (HP beta CD) for use in the treatment of breast cancer ) 是由 曼迪普·考尔 苏拉尼·塔鲁·萨哈 于 2020-01-28 设计创作，主要内容包括：本发明涉及供用于治疗乳腺癌的2-羟丙基-β-环糊精(HPβCD)。特别地,本发明涉及供用于治疗三阴性乳腺癌的HPβCD,其中HPβCD用于给药于有此需要的患者。本发明扩展到制备包含HPβCD的药物组合物的方法,并且另外扩展到通过将所述组合物给药于有此需要的患者来治疗乳腺癌,通常为三阴性乳腺癌的方法。(The present invention relates to 2-hydroxypropyl-beta-cyclodextrin (HP β CD) for use in the treatment of breast cancer. In particular, the present invention relates to HP β CD for use in the treatment of triple negative breast cancer, wherein the HP β CD is for administration to a patient in need thereof. The present invention extends to a method of preparing a pharmaceutical composition comprising HP β CD, and additionally to a method of treating breast cancer, typically triple negative breast cancer, by administering said composition to a patient in need thereof.)

1. 2-hydroxypropyl-beta-cyclodextrin (HP β CD) for use in the treatment of breast cancer in a human or animal body.

2. 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to claim 1, wherein the breast cancer is triple negative breast cancer.

3. The 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to claim 1 or claim 2, wherein the 2-hydroxypropyl- β -cyclodextrin (HP β CD) is formulated as a pharmaceutical composition.

4. 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to claim 3, wherein the pharmaceutical composition is for administration via parenteral and/or non-parenteral routes.

5. 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to claim 4, wherein the parenteral administration is at least one selected from the group of: intravenously, intramuscularly or by implantation into the human or animal body.

6. 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to claim 4, wherein the non-parenteral administration is at least one selected from the group of: delivering said pharmaceutical composition into said human or animal body orally-, rectally-, vaginally-, sublingually-, buccally-and intranasally.

7. 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to any one of claims 3 to 6, wherein the pharmaceutical composition further comprises an excipient and/or an additional active pharmaceutical ingredient.

8. A method of inducing and/or favoring apoptosis of a breast cancer cell, said method comprising the step of contacting said breast cancer cell with 2-hydroxypropyl- β -cyclodextrin (HP β CD) and/or a pharmaceutical composition comprising 2-hydroxypropyl- β -cyclodextrin (HP β CD) in a human or animal body.

9. The method of claim 8, wherein the breast cancer cells are triple negative breast cancer cells.

10. The method according to claim 8 or claim 9, wherein said step of contacting said breast cancer cells with 2-hydroxypropyl- β -cyclodextrin (HP β CD) and/or said pharmaceutical composition comprising 2-hydroxypropyl- β -cyclodextrin (HP β CD) comprises said administration of 2-hydroxypropyl- β -cyclodextrin (HP β CD) and/or said administration of said pharmaceutical composition comprising 2-hydroxypropyl- β -cyclodextrin (HP β CD) to said human or animal body by parenteral and/or non-parenteral means.

Technical Field

The present invention relates to 2-hydroxypropyl-beta-cyclodextrin (HP β CD) for use in the treatment of breast cancer. In particular, the present invention relates to HP β CD for use in the treatment of triple negative breast cancer, wherein the HP β CD is for administration to a patient in need thereof. The present invention extends to a method of preparing a pharmaceutical composition comprising HP β CD, and additionally to a method of treating breast cancer, typically triple negative breast cancer, by administering said composition to a patient in need thereof.

Background

Cancer is one of the leading causes of death worldwide. According to WHO, the number of new cancer cases worldwide is expected to increase by about 70% in the next two decades. Breast cancer is one of the most common malignant tumors in women worldwide, and nearly 170 ten thousand new cases occur in 2012. In 2012, more than 8000 new cases of breast cancer diagnosed in south africa accounted for 21.79% of the total number of National cancer diagnoses (National Health Laboratory Service, 2012).

Currently available treatments for breast cancer include the use of Selective Estrogen Receptor Modulator (SERM) drugs, which include tamoxifen (a gold standard drug) which is known to have several side effects. These side effects include the development of uterine cancer, cataracts, blood clots, and heart disease. In addition, over time, patients develop resistance to the drug. Therefore, there is a need to develop alternative therapies to successfully treat cancer.

Compared to north america and europe, africa and asia have the least number of breast cancer survivors. Without being limited by theory, such abnormalities are believed to include genetic components. It has been seen that breast cancer patients do not receive typical hormone-based treatments in the african and asian populations. In contrast to other populations, african and asian populations most often develop so-called triple negative breast cancer. Triple negative breast cancer means that the three most common receptor types (estrogen, progesterone and HER-2/neu genes) known to stimulate the growth of most breast cancers are not present in cancer tumors. Therefore, common pharmaceutical active ingredients (APIs) designed to target such receptors are not effective. Triple negative breast cancer has no known effective treatment regimen and therefore patients have little or no chance of recovery.

This is particularly disadvantageous in african and asian populations where the opportunity to obtain pharmaceuticals is often hindered and where poverty aggravates the lack of opportunities to obtain pharmaceuticals and treatments. Therefore, there is an urgent need to develop effective and cost-effective pharmaceutical compositions and/or treatment regimens for breast cancer, particularly triple negative breast cancer.

The invention described below is directed to improving at least one of the problems described above and/or known in the art.

Disclosure of Invention

Broadly, according to a first aspect of the invention there is provided 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use in the treatment of breast cancer in a human or animal body.

Preferably, the breast cancer is a triple negative breast cancer.

2-hydroxypropyl-beta-cyclodextrin (HP beta CD) can be formulated as a pharmaceutical composition.

The pharmaceutical compositions may be administered via parenteral and/or non-parenteral routes.

Parenteral administration can include, but is not limited to, intravenous, intramuscular, or implantation into the human or animal body.

Non-parenteral administration can include, but is not limited to, oral, rectal, vaginal, sublingual, buccal and intranasal delivery of the pharmaceutical composition to the human or animal body.

The pharmaceutical composition may include an excipient.

The pharmaceutical composition may comprise additional active pharmaceutical ingredients.

The pharmaceutical composition may additionally comprise a cyclodextrin from the cyclodextrin family.

According to a second aspect of the present invention there is provided a pharmaceutical composition comprising 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use in the treatment of breast cancer in the human or animal body.

Preferably, the breast cancer is a triple negative breast cancer.

The pharmaceutical compositions may be formulated for administration via parenteral and/or non-parenteral routes.

Parenteral administration can include, but is not limited to, intravenous, intramuscular, or implantation into the human or animal body.

Non-parenteral administration can include, but is not limited to, oral, rectal, vaginal, sublingual, buccal and intranasal delivery of the pharmaceutical composition to the human or animal body.

The pharmaceutical composition may include an excipient.

The pharmaceutical composition may comprise additional active pharmaceutical ingredients.

The pharmaceutical composition may additionally comprise a cyclodextrin from the cyclodextrin family.

According to a third aspect of the present invention there is provided the use of 2-hydroxypropyl- β -cyclodextrin (HP β CD) in the manufacture of a pharmaceutical composition for the treatment of breast cancer in a human or animal body.

Preferably, the breast cancer is a triple negative breast cancer.

The pharmaceutical compositions may be formulated for administration via parenteral and/or non-parenteral routes.

Parenteral administration can include, but is not limited to, intravenous, intramuscular, or implantation into the human or animal body.

Non-parenteral administration can include, but is not limited to, oral, rectal, vaginal, sublingual, buccal and intranasal delivery of the pharmaceutical composition to the human or animal body.

The pharmaceutical composition may include an excipient.

The pharmaceutical composition may comprise additional active pharmaceutical ingredients.

The pharmaceutical composition may additionally comprise a cyclodextrin from the cyclodextrin family.

According to a fourth aspect of the present invention there is provided a method of treating breast cancer, said method comprising the step of administering 2-hydroxypropyl- β -cyclodextrin (HP β CD) to a human or animal in need thereof.

Preferably, the breast cancer is a triple negative breast cancer.

2-hydroxypropyl-beta-cyclodextrin (HP beta CD) can be formulated as a pharmaceutical composition.

The pharmaceutical compositions may be administered via parenteral and/or non-parenteral routes.

Parenteral administration can include, but is not limited to, intravenous, subcutaneous, intramuscular, or implantation into the human or animal body.

Non-parenteral administration can include, but is not limited to, oral, rectal, vaginal, sublingual, buccal and intranasal delivery of the pharmaceutical composition to the human or animal body.

The pharmaceutical composition may include an excipient.

The pharmaceutical composition may comprise additional active pharmaceutical ingredients.

The pharmaceutical composition may additionally comprise a cyclodextrin from the cyclodextrin family.

According to a fifth aspect of the present invention there is provided a method of inducing and/or favouring apoptosis of a breast cancer cell, the method comprising the step of contacting said breast cancer cell with 2-hydroxypropyl- β -cyclodextrin (HP β CD) and/or a pharmaceutical composition comprising 2-hydroxypropyl- β -cyclodextrin (HP β CD) in a human or animal body.

The breast cancer cells in the method can be triple negative breast cancer cells.

The step of contacting said breast cancer cells with 2-hydroxypropyl- β -cyclodextrin (HP β CD) and/or a pharmaceutical composition comprising 2-hydroxypropyl- β -cyclodextrin (HP β CD) in the method comprises administering to the human or animal body 2-hydroxypropyl- β -cyclodextrin (HP β CD) and/or a pharmaceutical composition comprising 2-hydroxypropyl- β -cyclodextrin (HP β CD) by parenteral and/or non-parenteral means.

There is additionally provided 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to the above-described first aspect of the invention, substantially as herein described, illustrated and/or exemplified with reference to any one of the accompanying drawings and/or examples below.

There is additionally provided a pharmaceutical composition according to the above-described second aspect of the invention, substantially as herein described, illustrated and/or exemplified with reference to any one of the accompanying drawings and/or examples below.

There is further provided the use of 2-hydroxypropyl- β -cyclodextrin (HP β CD) in the manufacture of a pharmaceutical composition according to the above-described third aspect of the invention, substantially as herein described, illustrated and/or exemplified with reference to any one of the accompanying drawings and/or examples below.

There is additionally provided a method of treating breast cancer according to the above fourth aspect of the invention, substantially as herein described, illustrated and/or exemplified with reference to any one of the accompanying drawings and/or examples below.

There is further provided a method of treating breast cancer according to the above-described fifth aspect of the invention, which method induces and/or favours apoptosis of breast cancer cells, substantially as herein described, illustrated and/or exemplified with reference to any of the accompanying drawings and/or examples below.

Drawings

FIG. 1: representative graphs comparing the percent growth inhibition of MCF7 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, and 50 mM. Plumbagin (Plumbagin) (40. mu.M) was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 2: representative graphs comparing percent inhibition of growth of MDA-MB-231 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, and 50 mM. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 3: representative graphs comparing the percent growth inhibition of MRC-5 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, and 50 mM. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 4: representative graphs comparing percent growth inhibition of HEK-293 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM and 50 mM. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 5: representative graphs comparing the percent apoptosis of MCF7 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 6: light microscopy images showing the amount of apoptosis of MCF7 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control.

FIG. 7: representative graphs comparing the percent apoptosis of MDA-MB-231 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 8: light microscopy images showing the amount of apoptosis of MDA-MB-231 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control.

FIG. 9: representative graphs comparing the percent apoptosis of MRC-5 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 10: light microscopy images showing the amount of apoptosis of MRC-5 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control.

FIG. 11: representative graphs comparing the percent apoptosis of HEK-293 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 12: light microscopy images showing the amount of apoptosis of HEK-293 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control.

Fig. 13A to C: MOMP map of MCF7 cells. (A) The method comprises the following steps In untreated cellsLoss of mitochondrial membrane potential, (B): positive sample (10mM H₂O₂) Loss of mitochondrial membrane potential and (C): loss of mitochondrial membrane potential under 10mM HP β CD.

Fig. 14A to C: MOMP map of MDA-MB-231 cells. (A) The method comprises the following steps Loss of mitochondrial membrane potential in untreated cells, (B): positive sample (10mM H₂O₂) Loss of mitochondrial membrane potential and (C): loss of mitochondrial membrane potential under 10mM HP β CD.

Fig. 15A to C: MOMP map of MRC-5 cells. (A) The method comprises the following steps Loss of mitochondrial membrane potential in untreated cells, (B): positive sample (10mM H₂O₂) Loss of mitochondrial membrane potential and (C): loss of mitochondrial membrane potential under 10mM HP β CD.

Fig. 16A to C: ROS production in MCF7 cells. (A) The method comprises the following steps ROS generation in untreated cells (7.6%) (B): positive sample (10mM H₂O₂) ROS generation in (88.5%) and (C): ROS generation at 10mM HP β CD (17.9%).

Fig. 17A to C: ROS production in MDA-MB-231 cells (A): ROS production in untreated cells (3.7%), (B): positive sample (10mM H₂O₂) ROS generation (73%) and (C): ROS production at 10mM HP β CD (13.7%).

Fig. 18A to C: ROS production in MRC-5 cells. (A) The method comprises the following steps ROS production in untreated cells (2.8%), (B): positive sample (10mM H₂O₂) ROS generation (64.1%) and (C): ROS generation at 10mM HP β CD (10.8%).

Fig. 19A to C: caspase 3/7 profile in MCF cells. (A) The method comprises the following steps Total apoptosis/viable cell production of untreated cells, total apoptosis-6.4%, (B): total apoptosis/surviving cell production in positive samples (40 μ M plumbagin), total apoptosis-23.55% and (C): total apoptosis/surviving cells in treated (10mM HP β CD) cells, total apoptosis-12.5%.

Fig. 20A to C: caspase 3/7 profile in MDA-MB-231 cells. (A) The method comprises the following steps Total apoptosis/surviving cytogenesis of untreated cells (total apoptosis-19.6%), (B): total apoptosis/surviving cell production (total apoptosis-80.45%) in positive samples (40 μ M plumbagin) and (C): total apoptotic/surviving cells in treated (10mM HP β CD) cells (total apoptosis-48.75%).

Fig. 21A to C: caspase 3/7 profile in HEK-293 cells. (A) The method comprises the following steps Total apoptosis/viable cell production of untreated cells (total viable cells-71.90%), (B): total apoptotic/surviving cell production (total apoptosis-72.40%) in positive samples (40 μ M plumbagin) and (C): total apoptotic/viable cells in treated (10mM HP β CD) cells (total viable cells-66.75%).

FIG. 22: representative graphs comparing total, free and esterified cholesterol levels of MCF7 cells at selected concentrations of HP β CD at 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 23: representative graphs comparing total, free and esterified cholesterol levels of MDA-MB-231 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

FIG. 24: representative graphs comparing total, free and esterified cholesterol levels of MRC-5 cells at selected concentrations of HP β CD of 1mM, 5mM, 10mM, 20mM, 50mM and negative samples. Plumbagin was used as a positive control. Data are mean ± standard deviation s.d. (n ═ 3) from the raw data, where p <0.05, p <0.01, and p <0.001 were significantly different from the untreated control.

Fig. 25A to C: fluorescence microscope after staining for non-lippin (Filipin) ((Cell imaging station) that compares the overall cholesterol levels in MCF7 cells. Protruding cells are marked with arrows. (A) Fluorescence in untreated cells is shown, (B):fluorescence in positive samples (5mM MBCD) and (C) fluorescence in 10mM HP β CD treated cells.

Fig. 26A to C: fluorescence microscopy after staining for nonlevel: (Cell imaging station) that compares the overall cholesterol levels in MDA-MB-231 cells. Protruding cells are marked with arrows. (A) The method comprises the following steps Fluorescence in untreated cells, (B): fluorescence in positive sample (5mM MBCD), (C): fluorescence in 10mM HP β CD treated cells.

Fig. 27A and B: western blot analysis to detect SREBP-1 protein expression in MCF7 cells. (A) The method comprises the following steps Protein levels in untreated cells, (B): protein levels in HP β CD treated cells (10mM HP β CD). Beta-tubulin was used as a loading control.

Fig. 28A and B: western blot analysis to detect SREBP-1 protein expression in MDA-MB-231 cells. (A) The method comprises the following steps Protein levels in untreated cells, (B): protein levels in HP β CD treated cells (10mM HP β CD). Beta-tubulin was used as a loading control.

Fig. 29A and B: western blot analysis to detect SREBP-1 protein expression in MRC-5 cells. (A) The method comprises the following steps Protein levels in untreated cells, (B): protein levels in HP β CD treated cells (10mM HP β CD). Beta-tubulin was used as a loading control.

Fig. 30A to C: post euthanized mouse images showing tumor size in the advanced stages of the untreated group injected with MDA-MB-231 cells. (A) The method comprises the following steps Euthanized mice, (B): harvested tumors and (C): tumor size.

Fig. 31A to C: post-euthanized mouse images showing tumor size in advanced stages of treated groups injected with MDA-MB-231 cells and treated with HP β CD (3000mg/kg b.w.). (A) The method comprises the following steps Euthanized mice, (B): harvested tumors and (C): tumor size. The results showed a reduction of 73.9% in tumors compared to the untreated group.

Fig. 32A to C: post euthanasia mouse images showing tumor size in the treated group at mid-stage, injected with MDA-MB-231 cells and treated with HP β CD (3000mg/kg b.w.) for 10 doses. (A) The method comprises the following steps Euthanized mice, (B): harvested tumors and (C): tumor size. The results showed a 94% reduction in tumor compared to the untreated group.

Fig. 33A and B: mouse images showing tumor size in the early stage of untreated group injected with MDA-MB-231 cells (a): initiation of tumor and (B): size of tumor

Fig. 34A and B: mouse images showing tumor size in early stages of treated groups injected with MDA-MB-231 cells and treated with HP β CD (3000mg/kg b.w.) 10 agents (a): tumor termination and (B): the tumor is completely cured.

FIG. 35: all three cured mice (nos. 2, 4 and 7) were tested for recurrence of ER-tumors over a 4-week period. The results showed no recurrence at all.

Fig. 36A to F: the MDA-MB-231 mice are shown for hematoxylin and eosin staining and examination in untreated, intermediate and advanced stages. (A) Untreated cells are shown, where dissecting tumor cells between skeletal muscle fibers (arrows) can be seen in the micrograph (magnification x 100). (B) Non-treatment is shown, in which necrotic regions (arrows) were identified (magnification x 100). (C) Metaphase is shown, where many apoptotic cells (arrows) were identified (magnification x 200). (D) Metaphase is shown, where the micrograph has many mitotic images (magnification x 200). (E) The late phase is shown, where many apoptotic cells (arrows) were identified (magnification x 200). (F) Late stage is shown, where large areas of local necrosis (asterisk) are present together with scattered necrotic foci (arrows) (magnification x 100).

FIG. 37: graphical representation of ALT/AST levels of all 14 mice throughout the study. The allowable range for mice, ALT 28-132U/L and AST 59-247U/L. Data are mean ± standard deviation s.d. (n-3 or n-4) from raw data, where p <0.05 and "ns" corresponds to no significant difference from untreated controls.

FIG. 38: list of genes up-regulated (red) and down-regulated (green) in human lipocalin signaling. Results in particular in MDA-MB-231 showed upregulation of the ABCA1 gene (highlighted), indicating an increased efflux of cholesterol and phospholipids to lipid-poor lipocalins (apoA1 and apoE), which then formed nascent High Density Lipoproteins (HDL) after treatment with HP β CD. In black and white plots, asterisks indicate up-regulation, and double asterisks indicate down-regulation.

FIG. 39: list of genes up-and down-regulated in human breast cancer, where 'red' represents up-regulation and 'green' represents down-regulated genes. In black and white plots, asterisks indicate up-regulation, and double asterisks indicate down-regulation.

FIG. 40: cytoscape assay of up-and down-regulated genes in human lipocalin signaling (MDA-MB-231). Green to orange represent low to high expression levels, respectively. In the black and white figures, orange is indicated by an asterisk, and green is indicated by a double asterisk.

FIG. 41: cytoscape analysis of genes up-and down-regulated in human breast cancer (MDA-MB-231). Green to orange represent low to high expression levels, respectively. In the black and white figures, orange is indicated by an asterisk, and green is indicated by a double asterisk.

FIG. 42: cytoscape analysis of up-and down-regulated genes in human lipocalin signaling (MCF 7). Green to orange represent low to high expression levels. In the black and white figures, orange is indicated by an asterisk, and green is indicated by a double asterisk.

FIG. 43: cytoscape analysis of up-and down-regulated genes in human breast cancer (MCF 7). Green to orange represent low to high expression levels, respectively. In the black and white figures, orange is indicated by an asterisk, and green is indicated by a double asterisk.

FIG. 44: HP β CD was shown to be able to bind to human proteins, which shows a representative SPR sensorgram representing the direct interaction of HP β CD with: (A) ABCA 1; (B) ADAM 3; (C) AKT 1; (D) GATA 3; (E) cathepsin and (F) SFRP 1. Sensorgrams were analyzed to generate the kinetics in table 3.

Fig. 45(a) to (D): (A) to (C) shows non-uniform staining of mouse sections (for cholesterol). (A)4 from untreated, (B)3 from mid-stage and (C)4 from late stage. The results show a reduction in cholesterol in mid and late stage tumors compared to the untreated group as seen in (D).

Fig. 46(a) to (D): (A) to (C) shows nonlevel staining of MDA-MB-231 cells for cholesterol. The results show that cholesterol is reduced in treated cells compared to untreated cells. (A) Showing untreated cells, (B) showing positive cells, (C) showing treated cells, and (D) showing a graphical representation showing cholesterol reduction in treated cells compared to untreated cells.

Fig. 47(a) to (D): (A) to (C) shows BODIPY staining of MDA-MB-231 cells against lipid droplets. Green shows lipid staining, while blue is nuclear staining. The results show a reduction in lipid droplets in treated cells compared to untreated cells. Lipid droplets store cholesterol, while HP β CD can reduce the accumulation and storage of cholesterol in cancer cells. (A) Showing untreated cells, (B) showing positive cells, (C) showing treated cells, and (D) showing a graphical representation showing cholesterol reduction in treated cells compared to untreated cells.

Fig. 48(a) to (D): (A) to (C) shows ALEXA FLUOR staining of MDA-MB-231 cells against lipid raft dissociation. The results show a reduction in lipid rafts in treated cells compared to untreated cells, as shown graphically (D).

Fig. 49(a) to (D): (A) to (D) shows photographs showing the development of tumors in 4 mice (ER +). (A) Mouse No. 29 tumor 173.21mm is shown³(B) mice No. 30 tumor 163.46mm³(C) mouse No. 31 tumor 188.01mm³And (D) shows 103.74mm mouse tumor No. 32³. Mean tumor size 157.1mm³。

Fig. 50(a) to (D): (A) to (D) show photographs of ER + mice treated with HP β CD for 5 weeks. (A) Show mouse No. 29, where the tumor was completely cured, (B) show mouse No. 32, where the tumor was completely cured, (C) show mouse No. 31, where the tumor was reduced to 21.28mm³And (D) shows mouse No. 30 in which the tumor was completely cured. Mean tumor size 5.3mm³. The tumor was reduced by 96.6%.

Fig. 51(a) to (D): (A) to (C) shows nonlevel staining of MCF7 cells against cholesterol, where (a) was untreated, (B) was positive, and (C) was treated. (D) Graphically, the reduction of cholesterol in treated cells compared to untreated cells is shown.

Fig. 52(a) to (D): (A) to (C) shows BODIPY staining of MCF7 cells against lipid droplets. Green (darker areas in black and white) shows lipid staining, while blue (lighter defined points in black and white) is nuclear staining. (A) Untreated, (B) positive, and (C) treated. (D) The results are shown graphically, where lipid droplets are reduced in treated cells compared to untreated cells. Lipid droplets store cholesterol, and HP β CD can reduce the accumulation and storage of cholesterol in cancer cells.

Fig. 53(a) to (D): (A) to (C) shows ALEXA FLUOR staining of MCF7 cells against lipid raft dissociation, where (a) was untreated, (B) was positive, and (C) was treated. (D) The results are shown graphically, where lipid rafts are reduced in treated cells compared to untreated cells.

Detailed Description

The summary of the invention (including all first to further aspects) is repeated below by reference only to avoid repetition. Specific but non-limiting embodiments of the invention will now be described.

Generally, according to a first aspect of the present invention there is provided 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use in the treatment of breast cancer in the human or animal body. Preferably, the breast cancer is a triple negative breast cancer. Applicants have surprisingly found that HP β CD can treat, ameliorate and/or prevent solid tumor cancers, particularly breast cancer, more particularly triple negative breast cancer. This is particularly surprising and unexpected because cyclodextrins are generally the only excipients in pharmaceutical compositions and are generally inactive and/or inert. This surprising and unexpected effect is additionally advantageous because cyclodextrins, and in particular HP β CD, are safe for human and animal use. Furthermore, the cost problem of developing a medicament comprising HP β CD for the treatment of triple negative breast cancer will be relatively low. Without being limited by theory, applicants believe that HP β CD extracts additional cholesterol from breast cancer cells, thereby depriving the breast cancer cells of essential fuel for cell division and proliferation, resulting in cell death, as supported by experimental data herein.

Additional experimental protocols for developing dosage regimens will also be performed in time. Generally, the amount of HP β CD administered to a human or animal in need thereof is related to, but not limited to, body weight, tumor size, tumor weight, and/or whether the cancer has metastasized.

Typically, 2-hydroxypropyl- β -cyclodextrin (HP β CD) is formulated as a pharmaceutical composition when used to treat breast cancer.

The pharmaceutical compositions may be administered via parenteral and/or non-parenteral routes.

Parenteral administration can include, but is not limited to, intravenous, subcutaneous, intramuscular, or implantation into the human or animal body.

Non-parenteral administration can include, but is not limited to, oral, rectal, vaginal, sublingual, buccal and intranasal delivery of the pharmaceutical composition to the human or animal body.

The pharmaceutical composition may include an excipient. It is to be understood that other ingredients may also be included in the composition, such as fillers, taste-masking agents, colorants, vitamins, minerals, other Active Pharmaceutical Ingredients (APIs), and the like.

The pharmaceutical composition may additionally comprise a cyclodextrin from the cyclodextrin family.

The invention extends to the second, third, fourth and fifth aspects of the invention as described herein.

Examples

The following examples are not intended to limit the scope of the invention in any way, and are merely provided as examples and/or illustrative of certain embodiments of the invention.

HP β CD was tested for cytotoxic activity in four different cell lines constituting in vitro studies. These four cell lines are: estrogen-positive (ER +) MCF7 breast cancer cells, triple-negative MDA-MB-231 breast cancer cells, MRC-5 (normal human lung fibroblasts), and HEK-293 (normal human embryonic kidney cells). Assays based on cytotoxicity, apoptosis and cholesterol were performed in vitro. The results are shown below. In vivo studies (mouse models) are also presented, and additional studies of ER + and triple negative breast cancer cells are envisioned. Based on preliminary studies (in vitro and in vivo), the mechanism of combined action will be elucidated.

Cells were supplemented with 10% FCS, penicillin (100U/ml) and streptomycin (100. mu.l)g/ml) (Gibco) in a suitable medium at 37 ℃ in an incubator with 5% CO₂And (5) culturing.

In vitro studies:

growth inhibition assay-MTT assay

Four different cell lines MCF7(ER +), MDA-MB-231(ER-), MRC-5 (lung fibroblasts) and HEK-293 (embryonic kidney) were used. MRC-5 and/or HEK-293 were used as control cell lines depending on availability.

To determine whether HP β CD has the ability to induce cancer cell death, a primary screen using the MTT assay was performed. Three different time points of treatment at 24 hours, 48 hours and 72 hours were selected in order to establish the efficacy of HP β CD. The results are shown in fig. 1 to 4.

Table 1: IC showing MCF7, MDA-MB-231, MRC-5, and HEK-293 cells₅₀The value is obtained.

The results obtained show a growth inhibition of about 45% at a concentration of 10mM HP β CD. Subsequently, the percent growth inhibition increased with increasing HP β CD concentration. The results apply for all three time points, thus indicating its potential ability to disrupt mitochondrial processes (without being limited by theory) and its possible slow effects (fig. 1). 40 μ M Plumbagin (PL) was used as a positive control and showed almost 80%, 90% and 100% growth inhibition at 24 hours, 48 hours and 72 hours, respectively. Plumbagin is a well-known compound that has been shown to inhibit the growth of cancer cells.

As with MCF7, three different time points were selected for MDA-MB-231 cells, 24 hours, 48 hours, and 72 hours. The results obtained show that the growth inhibition is about 45% at a concentration of 10mM HP β CD, and the percentage of growth inhibition increases gradually with increasing HP β CD concentration. The results were similar for all three time points, thus indicating that HP β CD is quite effective on different breast cancer cells (fig. 2). 40 μ M Plumbagin (PL) was used as a positive control and showed almost 85%, 92% and 95% growth inhibition at 24 hours, 48 hours and 72 hours, respectively.

Similar time points (24 hours, 48 hours, 72 hours) were selected for control cell lines MRC-5 and HEK-293 to maintain consistency. The results obtained show that the growth inhibition is absolutely zero before the HP β CD concentration of 10 mM. The results apply for both time points (24 hours and 48 hours). Interestingly, at 72 hours (black box in fig. 3 and 4), even 20mM was not toxic to the cells, thus indicating that HP β CD was actually helping the cells survive, and all unhealthy cells were reprogramming and appeared normal. 50mM was considered to be very cytotoxic (FIGS. 3 and 4) because the dose was too high. Each cell has a threshold for a specific drug concentration beyond which it becomes toxic and each drug follows a dose response protocol. IC at the concentration calculated to cause 50% growth inhibition₅₀After the values, it can be seen that at 24 hours (table 1), the concentration required to cause 50% growth inhibition in both cell lines was almost twice the HP β CD concentration, while at 72 hours the concentration rose almost five-fold compared to cancer cells, thus indicating that HP β CD is specifically toxic to cancer cells.

Cell-apoercentage apoptosis assay

The apotercentage assay gives a measure of total apoptosis in cells. Apoptosis is an early event, and therefore only one time point (24 hours) was selected and tested on four cell lines, MCF7, MDA-MB-231, MRC-5 and HEK-293 cells. 10 μ M plumbagin was used as a positive control, rather than 40 μ M from MTT, because higher concentrations of plumbagin were too cytotoxic and required the optimal dose to capture early activity.

To confirm that the growth inhibition obtained from the MTT assay was actually due to apoptosis, the Cell-apoercentage apoptosis assay was performed according to a known method.

The results of MCF7 gave us approximately 45% cell death at 10mM HP β CD concentration. The percentage of cell death increased with increasing HP β CD concentration in all treatments (figure 5). 10 μ M plumbagin was used as a positive control and produced almost 90% apoptosis. Our graphical data were validated in photographs taken under light microscopy (fig. 6) after staining with apoercentage dye, where it can be clearly seen that as the concentration of HP β CD increases, the pink intensity of the dye (in the colour version of the figure) increases, thus indicating increased apoptosis. In black and white images, the number of defined dark gray spots relative to background increased with increasing HP β CD concentration, indicating increased apoptosis.

For MDA-MB-231 cells, at a 10mM concentration of HP β CD, the results obtained gave us about 40% cell death (less than MCF7) (FIG. 7). As with MCF7, the percent cell death continued to increase at all higher concentrations of HP β CD and the maximum cell death achieved at 50mM HP β CD was approximately 70%. 10 μ M plumbagin was used as a positive control and the percentage of apoptosis observed was almost 90%. Images taken under light microscopy after staining with APO percent dye validate our graphical data (fig. 8), where as with MCF7, we can clearly see that as the HP β CD concentration increases, the pink intensity of the dye (in the color version of the figure) increases, indicating a gradual progression of apoptosis. In black and white images, the number of defined dark grey spots relative to background increases with increasing HP β CD concentration, indicating the progression of apoptosis.

Finally, for normal cells (MRC-5 and HEK-293), results were obtained that were nearly zero percent cell death before the HP β CD concentration was 10mM (FIG. 9). For both cell lines, 20mM and 50mM brought about 15% and 18% cell death, respectively, to us. The photographs taken under the light microscope after staining with APO percent dye validate our graphical data (fig. 10) and it is clear that the cells did not take up dye until the HP β CD concentration was 10 mM. At higher concentrations we do see little pink intensity (in the color version of the figure), which is confirmed by our graphical data, which thus confirms the fact that HP β CD is indeed toxicologically benign to normal cells. In a black and white image, the number of spots defined with respect to the background does not increase too much. See FIGS. 11 and 12 for the response of the HEK-293 cell line to HP β CD, showing similarities to MRC-5.

Mitochondrial outer Membrane potential measurement (MOMP)

One of the key hallmarks of apoptosis is the loss of mitochondrial membrane potential. To investigate this aspect, we performed MOMP assays, which validated our Cell-apoercentage assay. The assay was performed on three cell lines, MCF7, MDA-MB-231, and MRC-5. The upper right and lower quadrants of each figure represent healthy (FL2-A) and apoptotic (FL1-A) cells, respectively (see FIGS. 13 to 15).

According to slave MTT (IC)₅₀Values) and Cell-aporpercentage assay, it is clear that 10mM HP β CD causes Cell growth inhibition by apoptosis of cancer cells, while having little effect on normal cells. Therefore, we selected only 10mM HP β CD as the only working concentration for HP β CD treatment. The 24 hour time point (HP. beta. CD and 10mM H) was selected₂O₂) Since mitochondrial membrane potential loss is an early cellular event.

The results obtained for MCF7 (fig. 13) showed an apoptotic population of 92.1% at 10mM concentration of HP β CD (treated), while the healthy population in untreated cells was 90.0%. Positive sample (10mM H₂O₂) The percentage of apoptotic cells in was 92.1%. Like PL, H₂O₂Are well known agents that cause apoptosis.

MDA-MB-231 cells produced 78.1% cell death at 10mM concentration of HP β CD (FIG. 14), while the cell death rate of untreated healthy population was still quite high, 94.2%. As expected, a positive sample (10mM H)₂O₂) Very high 92.4% apoptotic cells were produced.

For normal cells (MRC-5), the results obtained brought us 98.2% of non-apoptotic/healthy population and 12.8% of apoptotic cells at 10mM HP β CD concentration (fig. 15). Thus, it was confirmed that HP β CD is indeed non-toxic to normal cells, while to cancer cells significantly apoptotic via the intrinsic apoptotic pathway and leading to loss of mitochondrial membrane potential. This is the most surprising and unexpected result.

ROS assay (reactive oxygen free radical)

To additionally validate the occurrence of apoptosis and study the exact mechanism of apoptosis, ROS assays were performed to capture ROS production. This is because ROS generation has also been shown to be characteristic of apoptosis. To support this fact, ROS assays were performed in three cell lines: MCF7, MDA-MB-231, and MRC-5. HP β CD and Positive (10mM H) as before₂O₂) Was treated for 24 hours. The right-hand section in each figure represents ROS production.

Cancer cells generally have enhanced metabolism because they divide rapidly compared to normal cells, resulting in the production of ROS in large quantities.

As can be seen from fig. 16C, the percentage of ROS production was 17.9% in 10mM HP β CD treated MCF7 cells, compared to 7.6% in the untreated case (fig. 16A). Although this was approximately 2.5 fold higher compared to the untreated samples, it was not as significant, particularly if we looked at the level of ROS production in the positive samples with very high 88.5% ROS production (fig. 16B) and in normal cells (MRC-5) showing 10.8% ROS activity under 10mM HP β CD treatment (fig. 18C).

MDA-MB-231 cells produced similar results to MCF7, and the percentage of ROS production was 13.7% for 10mM HP β CD treatment (FIG. 17C), versus 3.7% without treatment. Again, this was approximately 4-fold higher compared to untreated cells, but this was not too surprising when we looked at positive samples showing 73% ROS production (fig. 17B) and normal cells that seen 10.8% ROS production under 10mM HP β CD treatment (fig. 18C).

MRC-5 cells generally showed 2.8% ROS activity in untreated cells (FIG. 18A), and ROS production rose almost 4-fold under 10mM HP β CD treatment (10.8%) as MCF and MDA-MB-231 cells. Thus, it was concluded that HP β CD does cause apoptosis, but not via the massive production of ROS. Down-regulation of ROS in apoptosis is often observed and may occur given that ROS have an important role in physiological processes. It is highly likely that detoxification enzymes such as catalase and glutathione reductase responsible for maintaining free radicals are overexpressed. However, additional research is required to elucidate the exact mechanism.

Caspase 3/7 assay

The final step in apoptosis following the intrinsic pathway is activation of the executioner caspases (executioner caspases), such as caspase 3 and caspase 7, which causes the release of cytochrome c. Therefore, to confirm that cell death is due to a mitochondria-mediated apoptotic pathway, caspase 3/7 assay was performed.

Caspases are activated and break down cells in response to myriad cell death stimuli. Caspase 3 and caspase 7 are involved in most of the proteolytic cleavage that occurs in apoptosis. As a final step in the confirmation and validation of apoptosis via the mitochondrial intrinsic pathway, caspase 3/7 assays were performed on three cell lines: MCF7, MDA-MB-231, and HEK-293. HP β CD and positive (40 μ M PL) for 24 hours. The percentage of dead/apoptotic cells is clearly indicated in the specific corners of each quadrant in the upper panel.

For MCF7 cells, the total apoptosis observed in untreated cells was 6.4% (fig. 19A), while in treated cells (10mM HP β CD) apoptosis rose to 12.5% (fig. 19C), which was almost two-fold increased compared to the untreated sample. For positive samples (40 μ M PL), the total apoptotic population was 23.55% (fig. 19B). The overall low caspase 3/7 activity was because MCF7 cells do not generally express caspase 3 activity, and this has been well reported. Since this particular assay gives measurements of caspase 3 and caspase 7, the overall measurement of apoptosis is offset by the absence of caspase 3 activity, even though caspase 7 activity is quite high. Thus, the cell death activity in MCF7 was entirely attributed to caspase 7, and it is likely that caspase 7 activity in MCF7 cells was quite low overall.

On the other hand, MDA-MB-231 cells showed better caspase 3/7 activity. In untreated cells, the total apoptotic population reached 19.6% (fig. 20A), which rose to 48.75% in the 10mM HP β CD treated samples (fig. 20C). This confirms that in MDA-MB-231 cells, HP β CD does act via intrinsic pathways and leads to mitochondrial disruption, thereby activating the performers caspase 3 and caspase 7. In the positive sample (40 μ M PL), the total apoptotic population was 80.45% (fig. 20B).

In normal cells (HEK-293), the overall healthy population in untreated cells was 71.90% (fig. 21A), which was only partially reduced (fig. 21C) and down to 66.75% in the 10mM HP β CD treated samples. In the positive sample (40 μ M PL), the total apoptotic population was 72.4% (fig. 21B). This now absolutely ensures that HP β CD is indeed non-toxic to normal cells. The non-toxicity of HP β CD was additionally verified later in our in vivo assay.

Quantitative determination of cholesterol

To investigate whether the apoptosis observed from all the above assays was due to cholesterol depletion, cholesterol assays were performed on all three cell lines: MCF7, MDA-MB-231, and MRC-5. HP β CD and positive PL (known as cholesterol depleting agents) were treated for 24 hours.

It is well known that cholesterol levels in cancer cells are elevated, as cholesterol is an integral part of the cell membrane and is recruited by cancer cells to undergo rapid division.

HP β CD and positive (40 μ M PL) for 24 hours. This assay gives us estimates of total cholesterol, cholesterol esters and free cholesterol.

The results against MCF7 (fig. 22) clearly show that HP β CD treated cells reduced total cholesterol levels at all concentrations starting from 1mM, 5mM, 10mM, 20mM and 50mM compared to untreated cells. At 10mM (which is the safe use concentration we screened from earlier assays), total cholesterol levels were reduced to over 75% compared to untreated samples.

Compared to MCF7, MDA-MB-231 cells had lower cholesterol levels (FIG. 23). The results clearly show that HP β CD treated cells reduced total cholesterol levels at all HP β CD concentrations starting from 1mM, 5mM, 10mM, 20mM and 50mM compared to untreated cells. At 10mM, total cholesterol levels were reduced to over 50% compared to untreated samples such as MCF7 cells (fig. 23).

Non-cancerous MRC-5 cells have lower cholesterol levels compared to MCF7 and MDA-MB-231 cells because they are less aggressive in dividing and therefore require less cholesterol. The results clearly show that HP β CD treated cells reduced total cholesterol levels at all concentrations starting from 1mM, 5mM, 10mM, 20mM and 50mM compared to untreated cells (figure 24). At 10mM the total cholesterol level was reduced to over 50% compared to untreated samples similar to MCF7 and MDA-MB-231 cells, but this depletion was sufficient to actually kill the cells, as observed from our earlier assay. Upon quantification of total cholesterol content, it can be seen that MCF7 and MDA-MB-231 cells have approximately 7.7 and 2.7 times more cholesterol than normal MRC-5 cells.

Cholesterol staining

To additionally verify the cholesterol depletion observed from the cholesterol assay, a cholesterol staining based on felodipine was performed. MCF7, MDA-MB-231 cells were used. HEK-293 cells could not be used because they were not available. HP β CD and positive (5mM MBCD) for 24 hours. Like PL, MBCD is also a well-known cholesterol depleting agent.

Fluorescence microscopy has become a powerful tool for studying intracellular transport of proteins. While not ruled in binding specifically to cholesterol and can bring about an overall quantification, it has recently been clinically used for the diagnosis of Niemann Pick Type C (Type C Niemann-Pick disease).

For MCF7 cells after staining with non-ruled flat, the untreated sample (fig. 25A) and the 10mM HP β CD treated sample (fig. 25C) showed significant differences in fluorescence intensity measured using ImageJ software of NIH. The fluorescence of the treated cells was reduced to a quarter compared to untreated cells. This is consistent with our cholesterol quantification, where 10mM HP β CD treatment resulted in almost 75% cholesterol depletion.

The fluorescence of the positive sample (5mM MBCD) was reduced to almost one third compared to the untreated sample after quantification (fig. 25B).

Similarly, there was a significant difference in fluorescence intensity between untreated cells (FIG. 26A) and 10mM HP β CD treated cells (FIG. 26C) for MDA-MB-231 cells.

After quantification with ImageJ, the fluorescence of the treated cells was reduced to one sixth compared to untreated cells. This was again correlated with our cholesterol quantitation data, where almost 50% of total cholesterol was seen to be depleted at 10mM HP β CD. The fluorescence of the positive sample (5mM MBCD) was reduced to almost one eighth compared to the untreated sample after quantification (FIG. 25B). Thus, it was confirmed that apoptosis by my HP β CD was indeed due to global depletion of cholesterol. Although normal cells cannot be studied, we have fully appreciated that their cholesterol levels are indeed lower and therefore show significantly less fluorescence than the aforementioned cancer cells.

Western blotting method

To investigate in depth the more detailed mechanism of cholesterol depletion, western blot analysis was performed on SREBP-1 protein, a transcription factor known to be involved in cholesterol homeostasis. Lysates from MCF7, MDA-MB-231, and MRC-5 cells were used. 10mM HP β CD for 24 hours. SREBP is a key element responsible for gene expression of important enzymes involved in fatty acid synthesis, and includes SREBP 1 and SREBP 2. Down-regulation of SREBP may be associated with degeneration of cell growth and migration, and has also been shown to cause apoptosis in several cancers. To investigate this characteristic of SREBP, western blot analysis was performed to see if HP β CD treatment did alter SREBP levels.

For MCF7, after quantification using Image LabTM software, it can be seen that the SREBP-1 activity of the treated (10mM HP. beta. CD) protein sample (FIG. 27A) was reduced by about one fifth compared to the untreated lysate (FIG. 27B) after equivalent loading of the corresponding sample. Thus, SREBP-1 was indicated to be significantly downregulated after HP β CD treatment.

Similar observations were also made in MDA-MB-231. After quantification using Image LabTM software, it can be seen that the SREBP-1 activity of the treated (10mM HP. beta. CD) protein sample (FIG. 28A) was reduced to about two fifths compared to the untreated lysate after equal loading of the corresponding sample (FIG. 28B).

Interestingly, in normal MRC-5 cells, after quantification, SREBP-1 activity was observed to remain equal and at basal levels in both untreated (FIG. 29A) and treated lysates (FIG. 29B). This western blot analysis is a confirmation and demonstrates that HP β CD treatment significantly reduces SREBP-1 activity in cancer cells, while it remains unchanged in normal cells and does not affect cholesterol biosynthesis

In vivo studies (mouse xenografts)

Since the efficacy of HP β CD on breast cancer was determined in our in vitro assay, a thorough study was performed in a mouse model (nude mice, MF-1 strain). MCF-7 and MDA-MB-231 cells were injected into 32 mice (16 per cell type) and, after tumor development, treated with HP β CD (3000mg/kg b.w.). The dose treatment schedule was followed three times a week throughout the study.

To examine whether HP β CD can be used as a future anti-breast cancer drug, mouse xenografts were prepared. MCF7 cells (500 ten thousand) were injected into 16 mice and weekly supplemented with β -estradiol. Studies have been completed for triple negative induced breast cancer (MDA-MB-231). Up to 400 ten thousand cells were injected in 16 mice. This is subdivided into three groups, namely: advanced, intermediate and early estrogen-negative cancers. The 4 mice were divided into untreated (not receiving HP β CD), treated (late) 4, treated (mid) 3 and treated (early) 3 groups.

Approximately 5 weeks after cell injection, tumors were observed in the untreated group (n-4) and the mean size rose to 13178.12mm after two weeks³(FIG. 30A, B, C). This is because MDA-MB-231 cells are inherently very aggressive, which is why any appropriate treatment is possible in addition to the side effects that are likely to cause chemotherapy. These 4 mice had to be euthanized at this time due to the humane endpoint. In the advanced stage treatment group, when the tumor size is 900mm³To 1350mm³In between, the process is started (fig. 30A, B, C). After following a strict 10 dose regimen in which HP β CD was administered intraperitoneally 10 times (once daily) for 2-3 weeks, we seen a significant reduction in tumor progression and a significant reduction in the mean size of the tumors to 73.9% compared to the untreated group (fig. 30C).

In the middle stage group (n ═ 3), when the tumor size reached-250 mm³The process is started. Also, after 10 doses of HP β CD treatment, we seen a reduction in the mean tumor size to 784.18mm³This was a dramatic 94% reduction compared to the untreated group (fig. 31B, C). This is very surprising given the invasiveness of MDA-MB-231 cells and their ability to be acclimated by HP β CDs. Finally, in the early group (n-3), when the tumor size was-20 mm³The process is started (fig. 33A, B). HP β CD was administered 10 doses and approximately one and a half weeks later, the tumor was completely cured (fig. 34A, B). These 3 mice were then kept for 4 weeks to check whether the tumors had relapsed, since estrogen-negative tumors are highly prone to relapse. After 4 weeks of relapse trial (fig. 35), no relapse of the tumor was clearly observed. These mice cannot be retained for long periods due to financial and technical limitations.

As shown in fig. 36, hematoxylin and eosin staining of mice was performed in untreated, mid-stage and late-stage MDA-MB-231 mice.

Conclusion (in vivo studies)

The applicant believes that 2-hydroxypropyl-beta-cyclodextrin (HP β CD) for use in the treatment and/or prevention of breast cancer, in particular triple negative breast cancer, where triple negative breast cancer is generally known to remain unresponsive to chemotherapeutic treatment regimens, at least ameliorates the disadvantages known in the prior art. Furthermore, the use of HP β CD does not include the serious side effects of typical chemotherapeutic treatment regimens, wherein patient compliance and quality of life of a human or animal receiving breast cancer treatment is improved.

Hematoxylin and eosin staining of mice:

microscopic examination of the tumor (see table 2 and fig. 36) showed solid growth patterns, with some sections showing dissection of tumor cells between skeletal muscles. The untreated, mid-and late-stage hematoxylin and eosin examinations in MDA-MB-231 mice are shown in FIG. 36. The single cell has obvious polymorphism and the nuclear-to-cytoplasmic ratio is improved. The tumor has irregular nuclear boundary, and the cell is vesicular and has one or more protruding nucleoli. Active mitotic activity is evident. However, areas of peri-neural and lymphatic vascular infiltration were not recorded.

Tumor sections in the untreated group showed focal necrotic areas at the periphery of 1-5% of the examined tissue. One case (UT3) showed extensive muscle infiltration. Apoptotic cells were identified. One case from this group of tumors (UT10) showed the greatest number of mitoses (77 per 10 high power fields). There are many atypical mitoses. Sections of the metaphase group of tumors showed scattered necrotic foci throughout the tumor, accounting for approximately 6-10% of the total tumors examined. Active mitotic activity was noted, similar to that seen in the untreated group. Atypical mitotic images were observed. The number of apoptotic cells in this group of tumors was comparable to that in the untreated group.

Sections from the advanced group showed necrotic pockets throughout the tumor, some of which showed necrosis in large localized areas. The necrotic areas are much more visible than the areas visible in the other two groups. One case (late stage 12) showed necrosis to be 20% of the examined tissue, thus illustrating an increase in necrosis of approximately 4-fold compared to that observed in the untreated group. A higher number of apoptotic cells was noted in this group of tumors, with the late group showing an increase in apoptotic cell number of approximately 1.7-fold compared to the untreated group and 2.9-fold compared to the metaphase group. Mitotic activity in the late group was approximately 1.1-fold higher than that indicated in the other two groups of tumors.

TABLE 2 case study of MDA-MB-231 hematoxylin and eosin stained (H & E) sections

Thus, from the conclusions of the mouse study, it can be concluded that, possibly in humans, HP β CD treatment can slow the progression of cancer if the tumor is diagnosed in late or intermediate stages, thereby improving the life expectancy of humans and if diagnosed in early stages. It is also hypothesized that breast cancer, usually triple negative breast cancer, can be cured completely without any of the serious complications and side effects of chemotherapy.

HP beta CD does not induce hepatotoxicity

AST/ALT assay

After euthanasia, mouse sera were analyzed for AST/ALT (alanine aminotransferase/aspartate aminotransferase) levels to see if HP β CD treatment had any toxic effect on the liver of treated mice (fig. 37). The AST/ALT ratio gives an estimate of hepatotoxicity or damage. Experiments showed that HP β CD had no toxic effect on mice. All early and mid-term groups were not different from the untreated group in AST/ALT ratios below the recommended ratio of 2, and were below the reference range (40U/L) except for the late-term group AST levels. In the latter groups, the AST/ALT ratio was 2.2, slightly above the recommended limit, but no statistical difference was found from the untreated group. These results show that HP β CD does not cause toxicity when administered to mice. In this regard, please see fig. 37.

Additional work was performed on MDA-MB-231 cells to identify the mechanism of action in cancer cell models (using RT-PCR [ reverse transcription polymerase chain reaction ] array and staining), and to validate the mechanism of action and confirm drug-protein interactions as described above.

Identification of HP beta CD mechanism of action

PCR (polymerase chain reaction) arrays were run to identify changes in gene expression in cancer cells after treatment with HP β CD. Differentially expressed genes and their significance for cancer, some genes were selected for additional drug-protein interaction studies.

The results of RT2 ProfilerTMPCR Array human lipoprotein signaling and cholesterol metabolism (MCF7 and MDA-MB-231) are shown in FIG. 38.

The results of RT2 ProfilerTMPCR Array human breast cancer (MCF and MDA-MB-231) are shown in FIG. 39.

After gene expression analysis, five genes were selected from this array. In MDA-MB-231 cells, upregulation of Akt1 was observed, suggesting that breast cancer cell migration was reduced, for example, by modulating TSC2, palladin, and EMT proteins. SFRP1, an important inhibitor of the Wnt pathway, is a known tumor suppressor gene, whose epigenetic silencing is also observed to be upregulated in a variety of tumors. Interestingly, GATA3, a useful marker for luminal tumors, particularly breast cancer, was down-regulated following treatment with HPCD, indicating the efficacy of this compound as a possible treatment. Previous studies highlighted that CTSD gene transcription was increased by estrogen and growth factors in estrogen receptor positive breast cancer cells and by unknown mechanisms in estrogen receptor negative cells, thus making it an interesting study candidate. From the above gene expression studies, it was observed that CTSD was up-regulated in MDA-MB-231 cells and significantly down-regulated in MCF7, which could indicate that HP β CD treatment is likely to be a causative agent. Last but not least, ADAM23 was selected that caused breast cancer progression and mesenchymal circulating tumor cell spread. In our case, ADAM23 was upregulated in MDA-MB-231 cells, many-fold downregulated in MCF7, which makes it an interesting research target, and the efficacy of HP β CD as a potential drug could be validated in all respects, and the potential mechanism of action of HP β CD could also be studied.

The graph in figure 40 shows that most of the genes involved in the cholesterol-associated pathway are down-regulated following treatment with HP β CD. This information was first generated in cancer cells. This observation strongly suggests that HP β CD acts by disrupting cholesterol homeostasis in cancer cells, and that cholesterol depletion may be used as a therapeutic strategy for treating cancer.

These results show that HP β CD blocks several signaling pathways in cancer cells, in particular DNA damage and repair, AKT signaling, Hedgehog signaling and EMT (epithelial to mesenchymal transition) which is the cause of metastasis of tumors. No significant change was observed in steroid receptor mediated signaling, thus suggesting that HP β CD is independent of hormone receptors to induce its effects (see figure 41).

The graph in figure 42 shows that most of the genes involved in cholesterol-related pathways are down-regulated after treatment with HP β CD, similar to MDA-MB-231 cells. This observation strongly suggests that HP β CD acts by disrupting cholesterol homeostasis in cancer cells, and that cholesterol depletion may be used as a therapeutic strategy for treating cancer.

The above results show similar trends as those obtained for MDA-MB-231 cells, and HP β CD was observed to block signaling pathways such as DNA damage and repair, AKT signaling, Hedgehog signaling, and EMT (epithelial to mesenchymal transition) as a cause of metastasis of tumors. Also, no significant change was observed in steroid receptor mediated signaling, thus suggesting that HP β CD is independent of hormone receptors to induce its effects (see fig. 43).

Confirmation of mechanism of action

Identification of drug-protein interactions between HP β CD and selected proteins based on RT-PCR array data

Six proteins were selected from the array based on gene expression analysis of the following proteins in both MCF7 and MDA-MB 231: akt1, ADAM23, Cathepsin D (CTSD), SFRP1, GATA3 and ABCA1, which were of major interest for MDA-MB-231 since they were used to complete the mouse study.

The binding affinity of HP β CD to Akt1, ADAM23, Cathepsin D (CTSD), SFRP1, GATA3 and ABCA1 was determined.

Binding affinity assays for HP β CD for six human proteins were performed using BioNavis tm420A ILVES MP-SPR (BioNavis, tanpelere, Finland) at 25 ℃. As running buffer, degassed PBS Tween 20 was used. The recombinant protein was immobilized as a ligand at 0.5ug/ml on a functionalized 3D carboxymethyl dextran sensor (CMD 3D 500L; BioNavis, Tanperel, Finland). Immobilization of the ligand is achieved by: coupling of the amine after activation of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) [ Sigma Aldrich in Germany (Germany) ] and N-hydroxysuccinimide (NHS) [ Sigma Aldrich in Germany ] was achieved according to the protocol provided by the manufacturer (BioNavis, finland) to reach <200 RU. The reference channel without immobilized protein served as control for nonspecific binding and refractive index change. As analytes, HP β CD was prepared as 0, 1.25, 2.5, 5 and 10nM aliquots and injected 3 times into each flow cell at a flow rate of 50 μ l/min. Injections with buffer only were used as controls. Binding between ligand and analyte was allowed for 3 minutes and dissociation was monitored for a total of 10 minutes. The kinetic steady state equilibrium constant data were processed after double referencing the sensorgrams using global fitting using TraceDrawer software version 1.8 (Ridgeview Instruments, sweden).

As a result:

HP β CD interacts with six human proteins (fig. 44). Kinetic analysis of HP β CD with human proteins showed the highest affinity for SFRP1 in the nanomolar range, while ADAM3 protein had the lowest binding affinity in the micromolar range (table 3).

TABLE 3 kinetic data of HPCD interaction with human recombinant proteins

The kinetics of direct interaction of HP β CD with various human proteins is represented by the association rate (Ka), dissociation rate (Kd) and steady state affinity (Kd) determined by SPR analysis. The ligand represents the corresponding immobilized protein on the CMD 3D 500L chip surface, and the analyte (HP. beta. CD) was injected onto the chip surface at least 3 times at a flow rate of 50. mu.l/min. Data were analyzed after double referencing as baseline (no change in refractive index of the chip surface with protein immobilized and no buffer with analyte injected). Data are presented as mean plus/minus measurement standard error. The chi-square (χ 2) values determined show the 1:1langmuir curve fit residuals.

Conclusion

The affinity of HP β CD binding to protein is, in order, high: SPRF1> ABCA1> AKT1> GATA3> Cathepsin > ADAM3, with low affinity.

2. Binding rate, rapid binding: SFRP1> AKT1> ABCA1> GATA3> Cathepsin > ADAM3 bound slowest.

3. Off rate, fast off: SRFP1> ADAM > AKT > ABCA > GATA3> Cathepsin dissociates slowly.

Cholesterol staining of mouse tumor tissue with felodipine

Figure 45 shows non-unilamellar staining of mouse sections (against cholesterol). Untreated 4 cases, medium 3 cases, late 4 cases. The results show a reduction in cholesterol in mid and late stage tumors compared to untreated.

The results show that with the identification of a significant decrease in cholesterol, HP β CD reaches and extracts cholesterol from the tumor. This confirms that HP β CD reduces tumor size by extracting cholesterol, thereby preventing proliferation of cancer cells.

Cholesterol staining of cancer cells using felodipine

FIG. 46 shows nonlevel staining of MDA-MB-231 cells for cholesterol. The results show a decrease in cholesterol in treated cells compared to untreated cells.

Lipid staining of cancer cells using BODIPY

FIG. 47 shows BODIPY staining of MDA-MB-231 cells against lipid droplets. Green represents lipid staining, while blue is nuclear staining. The results show a reduction in lipid droplets in treated cells compared to untreated cells. Lipid droplets store cholesterol, while HP β CD can reduce the accumulation and storage of cholesterol in cancer cells.

Using Alexa Staining of lipid rafts in cancer cells by flours

FIG. 48 shows ALEXA FLUOR staining of MDA-MB-231 cells for lipid raft dissociation. The results show a reduction in lipid rafts in treated cells compared to untreated cells.

Conclusion

The different activities performed enable us to determine:

anti-tumor potential of HP β CD in cell and mouse models,

2. identifying mechanisms of action in cancer cell models, and

3. the mechanism of action was verified and drug-protein interactions were confirmed.

We have confirmed that HP β CD affects several molecular mechanisms in cancer cells, particularly cholesterol-related pathways (as confirmed by mouse tumor samples and RT-PCR data), confirming evidence for the anti-cancer potential of HP β CD. We also identified several protein targets in the cells that have been confirmed to bind to HP β CD. One of the key proteins is SFRP1, which is known to regulate cancer-associated signaling pathways, SFRP1 shows the strongest binding to HP β CD in the nanomolar range. This suggests a role for HPCD stabilizing proteins in cells. The down-regulation of SFRP1 is found in many cancers, including breast cancer, and therefore, prolonged expression of this protein will help prevent cancer progression. In our study, we found that HP β CD up-regulates the expression of mRNA of SFRP1, and that it also binds to proteins.

In summary, we have confirmed that HP β CD inhibits tumor growth and progression by depleting cholesterol in cancer cells as a result of changes in the molecular levels of several cholesterol-associated pathways. These effects are amplified by modulating several cancer-associated signaling pathways, ultimately inhibiting the growth of cancer cells. The main advantage of HP β CD is safety, since it is non-toxic to humans (as confirmed by previous studies), thus providing a completely new approach to cancer treatment.

We have shown the efficacy of HP β CD on breast cancer tumors, particularly Triple Negative Breast Cancer (TNBC). We also elucidated the mechanism of action of its molecules. HPCD can be a very cost-effective treatment for patients with TNBC, whereas to date there is no other treatment for HPCD except surgery and chemotherapy, both of which have serious side effects and a survival rate of less than 5 years.

Additional work was also performed on a mouse model of MCF-7 cell-induced ER + breast cancer.

HP beta CD significantly reduces estrogen positive breast cancer tumor size

HP β CD was also tested for its anti-cancer effect on tumors induced by estrogen positive MCF7 cells in a xenograft mouse model. This study was not completed because only 4 out of 16 mice developed tumor development. However, mice that developed tumors were treated with HP β CD and a 96.6% tumor reduction was observed (fig. 49 and 50). Fig. 49 shows a photograph showing tumor development in 4 mice (ER +). (A)Mouse No. 29 tumor 173.21mm is shown³(B) mice No. 30 tumor 163.46mm³(C) mouse No. 31 tumor 188.01mm³And (D) shows 103.74mm mouse tumor No. 32³。

Fig. 50(a) to (D) show photographs of ER + mice treated with HP β CD for 5 weeks. (A) Mouse No. 29, where the tumor was completely cured, (B) mouse No. 32, where the tumor was completely cured, (C) mouse No. 31, where the tumor was reduced to 21.28mm³And (D) shows mouse No. 30, in which the tumor was completely cured. Mean tumor size 5.3mm³. The tumor was reduced by 96.6%.

Cholesterol staining of cancer cells using felodipine

The results show a reduction in cholesterol in treated cells compared to untreated cells (see figure 51). Fig. 51(a) to (C) show nonlipine staining of MCF7 cells for cholesterol, where (a) was untreated, (B) was positive, and (C) was treated. (D) Graphically, cholesterol was reduced in treated cells compared to untreated cells.

Lipid staining of cancer cells using BODIPY

The results show a reduction in lipid droplets in treated cells compared to untreated cells. Lipid droplets store cholesterol, and HP β CD can reduce the accumulation and storage of cholesterol in cancer cells (see fig. 52). Fig. 52(a) to (C) show BODIPY staining of MCF7 cells against lipid droplets. Green (darker areas in black and white) shows lipid staining, while blue (lighter defined areas in black and white) is nuclear staining. (A) Untreated, (B) positive, and (C) treated. (D) The results are shown graphically, where lipid droplets are reduced in treated cells compared to untreated cells. Lipid droplets store cholesterol, and HP β CD can reduce the accumulation and storage of cholesterol in cancer cells.

Using Alexa Fluor staining of lipid rafts in cancer cells

The results show a reduction in lipid rafts in treated cells compared to untreated cells (see figure 53). Fig. 53(a) to (C) show ALEXA FLUOR staining of MCF7 cells against lipid raft dissociation, where (a) was untreated, (B) was positive, and (C) was treated. (D) The results are shown graphically, where lipid rafts are reduced in treated cells compared to untreated cells.

HP β CD is safe for use in humans and animals, and provides a viable solution, providing a cost-effective and efficient treatment regimen for triple negative breast cancer where solid tumors often metastasize and lead to death. Furthermore, since HP β CD is known to be safe in human clinical trials, and thus the drug registration procedure can be performed quickly. Applicants have surprisingly found that HP β CD does not interact with healthy cells, but has a significant effect on breast cancer cells.

While the invention has been described in detail with respect to specific embodiments and/or examples thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing, may readily conceive of alterations to, variations of, and equivalents to these embodiments. The scope of the invention should, therefore, be assessed as that of the appended claims and any equivalents thereto.

The claims (modification according to treaty clause 19)

1. 2-hydroxypropyl-beta-cyclodextrin (HP β CD) for use in the treatment of triple negative breast cancer in a human or animal.

2. The 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to claim 1, wherein the 2-hydroxypropyl- β -cyclodextrin (HP β CD) is formulated as a pharmaceutical composition.

3. 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to claim 2, wherein the pharmaceutical composition is for administration via parenteral and/or non-parenteral routes.

4. 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to claim 3, wherein the parenteral administration is at least one selected from the group of: intravenously, intramuscularly or by implantation into the human or animal body.

5. 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to claim 3, wherein the non-parenteral administration is at least one selected from the group of: delivering said pharmaceutical composition into said human or animal body orally-, rectally-, vaginally-, sublingually-, buccally-and intranasally.

6. 2-hydroxypropyl- β -cyclodextrin (HP β CD) for use according to any one of claims 2 to 5, wherein the pharmaceutical composition further comprises an excipient and/or an additional active pharmaceutical ingredient.