AU2018101071A4 - Methods and compositions for treating multidrug-resistant cancer - Google Patents

Abstract

A method of treating a subject suffering from a multidrug-resistant cancer comprising the step of administering an effective amount of a compound of Formula (1) or its pharmaceutically acceptable salt or solvate to the subject. A method of potentiating the 5 activity of a chemotherapeutic compound in multidrug-resistant cancer cells comprising contacting the multidrug-resistant cancer cells with a compound of Formula (1) or its pharmaceutically acceptable salt or solvate and the chemotherapeutic compound. Also, a pharmaceutical composition comprising a compound of Formula (1) or its pharmaceutically acceptable salt or solvate and the chemotherapeutic compound. Fig. for the Abstract: Fig. 4b TXT(1.4tM) - - + + Rg5(gM) -0 8 8 4 2 Fig. 4a lcow A2780/T tII 753 ,80 m 1 11~i 27 CD ----- a - 3. 2 3 4 5 6 TXT(LZpAM)- + - t + + RG5(izM) - 0 8 8 4 2 Fig. 4b

Description

TECHNICAL FIELD

The present invention relates to a method of treating a multidrug-resistant cancer by administering a saponin to a subject, in particular but not exclusively in combination with a chemotherapeutic compound. The present invention also pertains to a pharmaceutical composition for treating multidrug-resistant cancer.

BACKGROUND OF THE INVENTION

Drug-resistance in cancer is the major impediment to a successful treatment of cancer. Multidrug-resistance (MDR) in cancer cells is a phenotype whereby cells display a reduced sensitivity to chemotherapeutic compounds based on several mechanisms in particular including an increase in drug efflux. Said multidrug-resistance can be a preexisting one and, thus, evident at the onset of therapy (intrinsic) or alternatively be acquired after onset of therapy. Chemotherapy is one of the most effective ways to cure cancer at present, yet failure in chemotherapy is common, which is associated with the occurrence of drug-resistance. For example, docetaxel has been used as first-line chemotherapy drug since 2004, but the occurrence of resistance to docetaxel has been a major obstacle leading to the failure of treatment.

Numerous mechanisms have been described to explain MDR, including transport25 based classical MDR mechanisms and non-classical mechanism related with alterations in the biochemistry of in transporter-overexpressing cells. The transportbased classical MDR mechanisms are related to the ATP-binding cassette (ABC) family of membrane transports which were overexpressed in cancer cells and pump anticancer drugs out of the cell resulting in a lack of effective concentrations of drugs for therapy. The first to third generations of MDR modular were inhibitors of ABC transporters. Even the clinical trials with the new third generation agents are ongoing; however, none of them have found a general clinical use so far.

Although there has been increased research in this regard, there remains a strong need for methods and compositions allowing for an effective therapeutic treatment of cancer, especially of multidrug-resistant cancer and cancer cells with a multidrug-resistant

2018101071 02 Aug 2018 phenotype. Also, efficacious treatment options are urgently required for specifically treating subjects with a disorder associated with an overexpression of ABC transporter proteins.

SUMMARY OF THE INVENTION

The first aspect of the present invention relates to a method of treating a subject suffering from a multidrug-resistant cancer comprising the step of administering an effective amount of a compound of Formula (I) or its pharmaceutically acceptable salt or solvate to the subject,

OH

Formula (I).

Preferably, the multidrug-resistant cancer is a multidrug-resistant ABC-protein15 dependent cancer in particular a multidrug-resistant P-glycoprotein-dependent cancer.

In an embodiment, the compound of Formula (I) is administered in combination with an effective amount of a chemotherapeutic compound selected from paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

The second aspect of the present invention pertains to a method of potentiating the activity of a chemotherapeutic compound in multidrug-resistant cancer cells, the method comprising contacting the multidrug-resistant cancer cells with (i) the compound of Formula (I) or its pharmaceutically acceptable salt or 25 solvate; and (ii) the chemotherapeutic compound in particular selected from paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

2018101071 02 Aug 2018

The third aspect refers to a pharmaceutical composition comprising (i) the compound of Formula (I) or its pharmaceutically acceptable salt or solvate; and (ii) a chemotherapeutic compound selected from the group consisting of paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

Further, the present invention also relates to use of the compound of Formula (I) in the preparation of a medicament for treating a multidrug-resistant cancer. Preferably, the use of the compound of Formula (I) in combination with a chemotherapeutic compound in the preparation of a medicament for treating a multidrug-resistant cancer.

The inventors unexpectedly found that the compound of Formula (I) in particular the compound of Formula (II) is capable of potentiating the activity of a chemotherapeutic compound in multidrug-resistant cancer cells in particular those having elevated expression of ABC-protein such as P-glycoprotein (P-gp). The combination of the compound of Formula (II) and the chemotherapeutic compound effectively inhibits the cell growth, inhibits cell colony formation, induces apoptosis, and increases accumulation of the chemotherapeutic compound in the multidrug-resistant cancer cells The inventors also found that the compound of Formula (II) can reduce Nrf2 expression by down-regulating the PI3K-Akt and ERK pathways and reverse the resistance of the MDR cancer cells. Accordingly, the methods and pharmaceutical compositions as described in the present application are useful in treating a subject suffering from MDR cancer and inhibiting the MDR cell growths so as to provide an advantageous treatment exceptionally suitable to specifically address multidrug-resistant cancer.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all steps and features referred to or indicated in the specification, individually or collectively, and any and all combinations of the steps or features.

Other features and aspects of the invention will become apparent by consideration of the following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

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Fig. 1a to 1d show the expression of ABCB1 transporter in A2780, A2780/T, A549 and A549/T cells. Fig. 1a is a plot of relative mRNA level of P-gp in A2780 and A2780/T cells. Fig. 1b is a western blot showing the expression of P-gp protein level in A2780 and A2780/T cells. Fig. 1c is a plot showing relative mRNA level of P-gp in A549 and

A549/T cells. Fig. 1d is a western blot showing the expression of P-gp protein level in

A549 and A549/T cells.

Fig. 2a to 2d show the cytotoxicity of the compound of Formula (II) (abbreviated as Rg5) and docetaxel (TXT) towards A2780, A2780/T, A549 and A549/T cells as determined by the SRB assay. Fig 2a shows A2780 and A2780/T cell survival percentage against various concentrations of Rg5. Fig 2b shows A2780 and A2780/T cell survival percentage against various concentrations of TXT. Fig 2c shows A549 and A549/T cell survival percentage against various concentrations of Rg5. Fig 2d shows A549 and A549/T cell survival percentage against various concentrations of TXT. The cells were treated with various concentrations of Rg5 or TXT for 48 hours.

Fig. 3a to 3d show the cytotoxicity of TXT alone and in combination with various concentrations of Rg5 toward A2780, A2780/T, A549 and A549/T cells as determined by the SRB assay. Fig 3a is a plot showing A2780/T cell survival percentage against various concentrations of TXT and in combination with Rg5 at a concentration of 2μΜ, 4μΜ or 8μΜ. Fig 3b is a plot showing A2780 cell survival percentage against various concentrations of TXT and in combination with Rg5 at a concentration of 2μΜ, 4μΜ or 8μΜ. Fig. 3c is a table demonstrating the reversal effect achieved by Rg5 in A2780/T cells. Fig 3d is a plot showing A549/T cell survival percentage against various concentrations of TXT and in combination with Rg5 at a concentration of 2μΜ, 4μΜ or 8μΜ. Fig 3e is a plot showing A549 cell survival percentage against various concentrations of TXT and in combination with Rg5 at a concentration of 2μΜ, 4μΜ or 8μΜ. Fig. 3f is a table demonstrating the reversal effect achieved by Rg5 in A549/T cells. The cells were treated with various concentrations of TXT alone or in combination with Rg5 at a concentration of 2μΜ, 4μΜ or 8μΜ for 48 hours.

Figs. 4a and 4b illustrates the colony formation of A2780/T cells treated with TXT alone and in combination with various concentrations of Rg5. Fig. 4a includes a set of microscopic images showing the colony formation of A2780/T cells after treatment with

TXT (1.2μΜ) alone and in combination with Rg5 at indicated concentrations. Fig. 4b is

2018101071 02 Aug 2018 a plot showing the number of colonies of A2780/T cells after treatment with TXT (1.2μΜ) alone and in combination with various concentrations of Rg5. ## or **, R<0.01; ### or ***, P<0.001, vs absence of Rg5.

Figs. 5a to 5d show the effect of TXT in combination with various concentrations of Rg5 on apoptosis and cell cycle of A2780 and A2780/T cells. Fig. 5a is a plot showing the proportion of apoptosis of A2780 and A2780/T cells after treatment with TXT alone and in combination with Rg5 (2, 4, and 8μΜ) for 24, 48 and 72 hours. Fig. 5b shows the flow cytometry patterns of A2780/T cells after treatment with Rg5 (0, 8μΜ) and/or TXT (0, 1.2 and 14.22μΜ) for 48 hours. Fig. 5c is a plot showing the cell cycle distribution percentage of A2780 and A2780/T cells after treatment with TXT alone and in combination with Rg5 (2, 4, and 8μΜ) for 48 hours. Fig. 5d is a set of histograms obtained from flow cytometry analysis of A2780/T cells after treatment with Rg5 (0, 8μΜ) and/or TXT (0, 1.2 and 14.22μΜ) for 48 hours. The data are representative of three different experiments and are shown as mean ± SD (n=3). ## or **, R<0.01. ### or ***, vs the control group.

Figs. 6a to 6d are quantitative diagnostic plots showing the synergistic effect between Rg5 and TXT generated by computer stimulation. Fig. 6a is a plot of fraction affected (Fa) against dose for Rg5, TXT, and the combination thereof. Fig. 6b is a plot of combination index (Cl) against Fa for the combination of Rg5 and TXT. Fig. 6c is a isobologram for different Fa values (0.5, 0.75 and 0.9). Fig. 6d is a plot of dose reduction index (DRI) against Fa for Rg5 and TXT in a constant ratio combination design. Cl<1, synergism; Cl=1, additive; Cl>1, antagonism. DRI>1, Reduced dose and reduced toxicity.

Figs. 7a and 7b are histograms obtained from flow cytometry showing the intracellular accumulation of rodamin123 (Rho123) doxorubicin (DOX). Fig. 7a shows a set of histograms obtained from flow cytometry analysis of A2780 and A2780/T cells after treatment with Rho123 (5μΜ) for 8 hours in the absence or presence of Rg5 (8μΜ) and the positive control quinidine (QND) (20μΜ). Fig. 7b shows a set of histograms obtained from flow cytometry analysis of A2780 and A2780/T cells after treatment with DOX (10μΜ) for 8 hours in the absence or presence of Rg5 (8μΜ) and the positive control quinidine (QND) (20μΜ). The experiment were repeated for at least 3 times, presented are representative images.

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Figs. 8a and 8b are plots showing the transport of Rho123 in Caco2 cells. Fig. 8a is a plot showing the Rho123 Papp values of Caco2 cells in the absence or presence of QND (20μΜ) and/or Rg5 (2, 4 and 8μΜ). Fig. 8b is a plot showing the Rho123 efflux ratio of Caco2 cells in the absence or presence of QND (20μΜ) and/or Rg5 (2, 4 and 8μΜ).

Figs. 9a and 9b are plots of ATP consumed per microgram P-gp per minute against different concentrations of Rg5. Fig. 9a shows the ECso measurements for stimulating the P-gp ATPase activity by Rg5. Fig. 9b shows the IC50 measurements for inhibiting verapamil (200pM)-stimulated P-gp ATPase activity. # or *, P<0.05; ## or **, P<0.01, vs absence of Rg5.

Figs. 10a to 10c show the expression of P-gp, total AKT, phosphorylated AKT and ERK, and NRF2 in A2780/T cells. Fig. 10a is a western blot showing the expression of P-gp in A2780/T cells after treatment with TXT (1.2μΜ) in the absence or presence of Rg5 (2, 4 and 8μΜ). Fig. 10b is a western blot showing the expression of total AKT, phosphorylated AKT and ERK in A2780 and A2780/T cells after treatment with or without TXT (1.2μΜ) in the absence or presence of Rg5 (2, 4 and 8μΜ). Fig. 10c is a western blot showing the expression of NRF2 in A2780 and A2780/T cells after treatment with or without TXT (1.2μΜ) in the absence or presence of Rg5 (2, 4 and 8μΜ).

Figs. 11 a and 11 b are diagrams obtained from docking analysis of Rg5 binding with P25 gp. Fig. 11a shows a diagram obtained from docking analysis of Rg5 binding with Pgp, the binding site is magnified for a better illustration. The putative binding pattern of Rg5 and P-gp is shown in the binding site. Fig. 11b is a schematic diagram showing the two-dimensional interaction mode between Rg5 and P-gp. Hydrogen bonds and ππ/σ-π interactions are shown as lines. Bubbles represent hydrophobic and polar amino acid residues.

Figs. 12a to 12d show the effect of Rg5 in combination with TXT in the A549/T cell xenograft nude mice model. Fig. 12a is a plot of body weight against time after implantation. Fig. 12b is a plot of tumor volume against time after implantation. Fig. 12c is a photograph showing the size of tumors of different treatment groups after the tumor

2018101071 02 Aug 2018 xenografts were excised. Fig. 12d is a bar chart showing the tumor weight of tumor xenografts in different treatment groups on the 27^th day after implantation. The data shown are expressed as the mean ± SD for each group (n=9 or 10), *P<0.05, **P<0.01, ***P<0.001

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention belongs.

As used herein, “comprising” means including the following elements but not excluding others. “Essentially consisting of” means that the material consists of the respective element along with usually and unavoidable impurities such as side products and components usually resulting from the respective preparation or method for obtaining the material such as traces of further components or solvents. The expression that a material is certain element is to be understood for meaning “essentially consists of” said element. As used herein, the forms “a,” “an,” and “the,” are intended to include the singular and plural forms unless the context clearly indicates otherwise.

In the first aspect of the present invention, there is provided a method of treating a subject suffering from a multidrug-resistant cancer, i.e. a cancer with multidrugresistant phenotype. Said method of treating multidrug-resistant cancer comprises the step of administering an effective amount of a saponin to said subject. The saponin can be a synthetic one or obtained from extracts of respective plants. Preferably, the saponin is a ginsenoside. More preferably, the saponin is a compound of Formula (I), including any salt or solvate thereof:

OH

2018101071 02 Aug 2018

Formula (I).

Also contemplated by the present invention are any pharmaceutically acceptable salts, hydrates, solvates, anhydrates as well as enantiomers and their mixtures, stereoisomeric forms, racemates, diastereomers and their mixtures of the compound of the present invention.

As used herein, the term solvate refers to a complex of variable stoichiometry formed by a solute, i.e. the compound of Formula (I), and a solvent. If the solvent is water, the solvate formed is a hydrate. Suitable pharmaceutically acceptable salts are those which are suitable to be administered to subjects, in particular mammals such as humans and can be prepared with sufficient purity and used to prepare a pharmaceutical composition. The terms stereoisomers, diastereomers, enantiomers and racemates are known to the skilled person.

Preferably, the compound of the present invention is of Formula (II):

Formula (II).

including any salt or solvate thereof and including any stereoisomer, diastereomer, enantiomer or racemate thereof.

Said compound of Formula (II) is known as ginsenoside Rg5, commercially available and can be prepared according to methods known to the skilled person or isolated, in particular, from genus Panax in particular from Panax ginseng.

The expression “effective amount” and “effective dose” generally denote an amount sufficient to produce therapeutically desirable results, wherein the exact nature of the

2018101071 02 Aug 2018 result varies depending on the specific disorder which is treated. When the disorder is cancer, the result is usually an inhibition or suppression of the proliferation of the cancer cells, a reduction of cancerous cells or the amelioration of symptoms related to the cancer cells.

The effective amount of the compound of the present invention may depend on the species, body weight, age and individual conditions of the subject and can be determined by standard procedures such as with cell cultures or experimental animals. A concentration of the compound of Formula (I) such as the compound of Formula (II) for treating the subject may, for example, be 1 μΜ, 2 μΜ, 4 pM, 6 pM, 8 pM or at least 8 pM. Alternatively, the dose of the compound of Formula (I) such as the compound of Formula (II) for treating the subject may, for example, be 10mg/kg, 20mg/kg, 30mg/kg, 40mg/kg, or 50mg/kg.

The subject can be a human or animal, in particular the subject is a mammal, preferably a human. The subject is, thus, preferably a human having a cancer with a multidrugresistance. Said subject, thus, includes human subjects having a drug resistance to conventional chemotherapeutic compounds which induce cell death in cancer cells, i.e. which are used to treat cancer.

The terms “cancer” and “cancerous” refer to or describe a physiological condition in subjects in which a population of cells are characterized by unregulated cell growth. The term “tumor” simply refers to a mass being of benign (generally harmless) or malignant (cancerous) growth.

The multidrug-resistant cancer can be a multidrug-resistant cancer of any origin, in particular human origin. In particular, the multidrug-resistant cancer is selected from the group consisting of multidrug-resistant:

• leukemia, · lymphoma, • kidney cancer and renal carcinoma, respectively, • pancreatic cancer, • ovarian cancer, • liver cancer, · myeloma,

2018101071 02 Aug 2018 • sarcoma, • lung cancer, • breast cancer • gastric cancer, and · colon cancer.

Preferably, the cancer is selected from multidrug-resistant:

• lung cancer • breast cancer · ovarian cancer; or • colon cancer.

Still more preferably, the cancer is selected from multidrug-resistant lung cancer or multidrug-resistant ovarian cancer.

The provided method is used and particularly effective in treating subjects whose cancer has become multidrug-resistant. The term multidrug-resistance is generally used for an acquired or natural, i.e. intrinsic, resistance of a cancer or more specifically of a cancer having cancer cells being simultaneously resistant to a range of chemotherapeutic compounds that usually differ structurally and functionally. Multidrugresistant cancer with acquired drug resistance is characterized by a resumption of its growth and/or reappearance after having seemingly gone into remission, despite the administration of increased doses of a chemotherapeutic compound.

Cancers with cancer cells that have developed resistance to or are naturally resistant to two or more chemotherapeutic compounds are said to be multidrug-resistant in the present patent application such as to chemotherapeutic compounds selected from the group consisting of topoisomerase-ll inhibitors, anthracyclines, coordination complexes of platinum, taxanes, protein kinase inhibitors, vinca alkaloids or derivatives thereof, topoisomerase-l inhibitors and nucleotide analogs or precursor analogs. In preferred embodiments of the present invention, the multidrug-resistant cancer is a cancer having multidrug-resistant cancer cells, i.e. cancer cells which have developed resistance to or are naturally resistant to two or more chemotherapeutic compounds, wherein said multidrug-resistant cancer cells are resistant against at least one of paclitaxel,

2018101071 02 Aug 2018 docetaxel, doxorubicin, 5-fluorouracil and daunorubicin, in particular against one of taxol or doxorubicin or both of them, more preferably against docetaxel.

A cancer is multidrug-resistant if it comprises cancer cells which are multidrug-resistant, in particular if more than 30% of cancer cells, more preferably more than 50% of cancer cells in said cancer are multidrug-resistant. Accordingly, the cancer cells with multidrugresistant phenotype will show less sensitive or more tolerant to most common chemotherapeutic agents. In practice, this can be determined by taking a sample of the cancer and determining the percentage of cancer cells with multidrug-resistance.

A multidrug-resistance can be detected in a subject, cancer, tissue, or cell by administering to the subject, tissue, or cell, compounds such as chemotherapeutic compounds and determining the activity of the chemotherapeutic compounds such as the induction of cell death or the inhibition of the proliferation of cancer cells compared to a reference control, namely cells or tissue of the same cell or tissue type, a cancer or a subject that do not have multidrug-resistance or non-cancerous cells.

The multidrug-resistance according to the present invention is in particular at least one of:

· ABC-protein dependent, i.e. mediated by ABC transporter proteins (hereinafter “ABC-proteins”) such as by P-glycoprotein, i.e. is associated with an enhanced expression and/or enhanced functional activity of at least one ABC-protein in the multidrug-resistant cancer cells, in particular of P-glycoprotein, and/or • apoptosis-deficient, i.e. associated with a decreased expression of at least one pro25 apoptotic protein including gene knockout and/or decreased pro-apoptotic activity of at least one pro-apoptotic protein and/or enhanced expression of at least one anti-apoptotic protein and/or enhanced anti-apoptotic activity of at least one antiapoptotic protein in the multidrug-resistant cancer cells; in particular apoptosisdeficient refers to at least one of p53-deficient, Bax-deficient or Bak-deficient.

In one embodiment of the present invention, the multidrug-resistance and hence the multidrug-resistant cancer is ABC-protein dependent, in particular P-glycoprotein dependent.

2018101071 02 Aug 2018

ABC-proteins are transporter proteins that may act to remove chemotherapeutic compounds from cells. The, thus, resulting multidrug-resistant phenotype can be specifically detected in a subject, tissue, cancer or cell by administering to the subject, tissue, or cell, a compound such as a chemotherapeutic compound which is transported by the ABC-proteins, i.e. is a substrate to ABC-proteins such as to P-glycoprotein. The method then encompasses determining the amount of said chemotherapeutic compound in the cells compared with the amount in a reference control, i.e. a subject, a tissue, cancer or a cell of the same cell or tissue type that does not express said multidrug-resistance phenotype, namely with ABC-protein expression as present in non-cancerous cells, in particular cancer cells or tissue without the multidrug-resistance phenotype or non-cancerous cells or tissue.

Said ABC-protein is in particular selected from the “B” subfamily, “C” subfamily or “G” subfamily of ABC-proteins. Most preferably, said ABC-protein is P-glycoprotein, i.e. in embodiments of the present invention, the multidrug-resistant cancer is a Pglycoprotein-dependent multidrug-resistant cancer. Preferred “B” subfamily members include the protein encoded by ABCB1 (MDR1), ABCB4 (MDR2), ABCB5 or ABCB11 in humans or corresponding genes in other mammals. Preferred “C” subfamily members include the protein encoded by ABCC1 (MRP1) in humans or corresponding genes in other mammals. Preferred “G” subfamily members include the protein encoded by ABCG2 (BCRP) in humans or corresponding genes in other mammals. More preferably, the ABC-protein is of the “B” subfamily, in particular the ABC-protein is the protein encoded by ABCB1, ABCB4, ABCB5 or ABCB11 in humans or corresponding genes in other mammals which can transport drugs, in particular ABCB1 and/or ABCB5, most preferably ABCB1 or corresponding genes in other mammals, i.e. most preferably P-glycoprotein.

P-glycoprotein as used herein refers to the protein as encoded by the ABCB1 (MDR1) gene in humans or respective genes including SNPs and naturally occurring mutations to said gene and as encoded by corresponding genes in other mammals, respectively.

An enhanced expression and/or enhanced functional activity of at least one ABCprotein, i.e. ABC-protein-dependent multidrug-resistant cancer, means an expression and/or functional activity exceeding, in particular significantly exceeding, the one in normal cells or tissue, i.e. non-cancerous cells or tissue, or cancer cells without the multidrug-resistant phenotype. The term “enhanced expression” or “enhanced

2018101071 02 Aug 2018 functional activity” of at least one ABC-protein such as P-glycoprotein includes embodiments in which the multidrug-resistant cancer cells express the ABC-protein such as P-glycoprotein, whereas in the reference control, i.e. cancer cells without the multidrug-resistant phenotype or non-cancerous cells of the same cell or tissue type, said ABC-protein such as P-glycoprotein is not expressed, at all. I.e. when said reference control does not express the ABC-protein such as P-glycoprotein, multidrugresistant cancer cells having a detectable expression or functional activity of the ABCprotein such as P-glycoprotein are ABC-protein-dependent such as P-glycoproteindependent by definition.

Whether a multidrug-resistant cancer is an ABC-protein-dependent such as Pglycoprotein-dependent multidrug-resistant cancer can be determined by methods known to the skilled person in particular comprising immunological methods accompanied by the use of MDR-specific antibodies, immunocytochemistry and immunohistochemistry, respectively, by determining respective mRNA levels such as with Northern blots or quantitative RT-PCR, with MDR-specific antibodies in vivo or with an ABC-protein such as P-glycoprotein efflux assay detecting the efflux of a marker.

In particular, an ABC-protein such as P-glycoprotein efflux assay can be used for determining the functional activity of ABC-proteins, i.e. for determining whether multidrug-resistant cancer cells are ABC-protein-dependent. Markers which can be used in said efflux assay include drugs which are a substrate for the respective ABCprotein, a radionuclide or a dye like a fluorescent dye selected from Rhodamine123 (also referenced as “Rho123”, 6-amino-9-(2-methoxycarbonylphenyl) xanthen-325 ylidenejazanium chloride), DiOC2 (3,3’-diethyloxacarbocyanine iodide) or Calcein AM (calcein ο,ο'-diacetate tetrakis(acetoxymethyl)ester). Elimination from or, alternatively, retention of the marker in the multidrug-resistant cells can be determined and compared with a reference control, i.e. cells with ABC-protein expression as present in noncancerous cells such as cancer cells that do not have a multidrug-resistance phenotype or non-cancerous cells of the same cell or tissue type. For example, fluorescence of a fluorescent marker can be determined by flow cytometry.

Preferably, an ABC-protein-dependent such as a P-glycoprotein-dependent multidrugresistant cancer is a cancer comprising multidrug-resistant cancer cells with an expression of ABC-protein or ABC-protein functional activity exceeding the one in the reference control by at least 10%, in particular by at least 20%. For example, the

2018101071 02 Aug 2018 expression or functional activity of P-glycoprotein in P-glycoprotein-dependent multidrug-resistant cancer cells is at least 10% or at least 20% higher than the expression or functional activity of P-glycoprotein in the reference control.

In particular embodiments of the present invention, an ABC-protein efflux assay is carried out to determine whether a multidrug-resistant cancer is ABC-proteindependent. Thereby, the amount of marker, in particular a fluorescent dye, taken up by a multidrug-resistant cancer cell or a sample with such cancer cells is compared with the amount taken up by a reference control, namely cells with ABC-protein expression as present in non-cancerous cells, such as cancer cells that do not have a multidrugresistance phenotype or non-cancerous cells of the same cell or tissue type. The multidrug-resistant cancer cells or the sample of multidrug-resistant cancer cells and, thus, the cancer is preferably considered for being ABC-protein-dependent according to the present invention, if the multidrug-resistant cancer cells have a reduced amount of marker such as dye, in particular an at least 50%, and more preferably at least 60% reduced amount of marker in the cells compared to the amount of marker in the reference control as revealed by the efflux assay or, alternatively, if the sample of multidrug-resistant cancer cells has a reduced percentage of cells with marker, namely an at least 50 percentage points and in particular at least 60 percentage points reduced percentage of cells with marker after carrying out the efflux assay compared to the reference control. In particular, a sample of multidrug-resistant cancer cells and, thus, a cancer having those cells, is preferably considered for being P-glycoproteindependent, if it comprises less cells with marker such as dye like Rho123 as revealed by the P-glycoprotein efflux assay compared to the reference control which is a cell sample with P-glycoprotein expression as present in cancer cells that do not have a multidrug-resistance phenotype or non-cancerous cells ofthe same cell or tissue type. Namely, the percentage of cells with marker is preferably at least 50 percentage points, more preferably at least 60 percentage points and in particular at least 70 percentage points lower than the percentage of cells with marker in the reference control as revealed by the P-glycoprotein efflux assay.

The multidrug-resistant cancer is an embodiment of the present invention a cancer comprising multidrug-resistant P-glycoprotein-dependent cancer cells, i.e. multidrugresistant cancer cells having an enhanced expression of P-glycoprotein and/or an enhanced functional activity of P-glycoprotein, in particular comprising more than 30% of said cancer cells, more preferably more than 50% of said cancer cells.

2018101071 02 Aug 2018

The term “apoptosis-deficient” used herein refers to a cancer having at least one of (i) decreased expression including gene knockout and/or decreased pro-apoptotic activity of at least one pro-apoptotic protein or (ii) enhanced expression and/or enhanced anti5 apoptotic activity of at least one anti-apoptotic protein or both of them, i.e. (i) and (ii). Pro- and/or anti-apoptotic proteins in particular include p53, mitogen-activated protein kinase (MAPK)-family members and B cell lymphoma 2 (Bcl-2) family members.

The compound of the present invention is in preferred embodiments of the present invention administered in combination with an effective amount of at least one chemotherapeutic compound. As used herein, the term chemotherapeutic compound includes drugs which are advantageously and commonly administered to cancer or cancer cells without the multidrug-resistance phenotype, i.e. which have been known to affect cancer cells.

In an embodiment, the chemotherapeutic compound is preferably selected from the group consisting of a topoisomerase-ll inhibitor, an anthracycline, a coordination complex of platinum, a taxane, a protein kinase inhibitor, a vinca alkaloid or derivative thereof, a topoisomerase-l inhibitor and a nucleotide analog or precursor analog. Such chemotherapeutic compounds include etoposide, doxorubicin, daunorubicin, cisplatin, paclitaxel (taxol), docetaxel, staurosporine, vinblastine, vincristine, topotecan and methotrexate. Preferably, the chemotherapeutic compound is selected from paclitaxel, docetaxel, doxorubicin, or daunorubicin. Further chemotherapeutic compounds which are non-substrates for the P-glycoprotein efflux can also be used in combination with the compound of the present invention such as 5-fluorouracil. In a particular embodiment, the compound of Formula (I) preferably a compound of Formula (II) is used in combination with a chemotherapeutic compound selected from paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

The chemotherapeutic compound can be administered before, after or simultaneously with the compound of Formula (I), in particular simultaneously with the compound of Formula (I).

According to the invention is also the compound of Formula (I) of the present invention, in particular of Formula (I), for use as a medicament for the treatment of multidrugresistant cancer, in particular P-glycoprotein dependent multidrug-resistant cancer. The

2018101071 02 Aug 2018 compound of the present invention, in particular of Formula (I) or preferably (II) can be used in an effective amount for treating an animal or a human, in particular a mammal, preferably a human. Another aspect of the invention refers to the use of the compound of the present invention, in particular of Formula (I) or preferably (II) for preparing a medicament for treating a multidrug-resistant cancer, in particular P-glycoproteindependent cancer. The compound of the present invention, in particular of Formula (I) or preferably (II) is used in combination with at least a further chemotherapeutic compound, preferably chemotherapeutic compounds which are used for treating cancer.

The compound of the present invention is effective for potentiating the activity of the chemotherapeutic compound, i.e. for increasing the effectiveness of the chemotherapeutic compound to inhibit proliferation of the multidrug-resistant cancer cells, inducing cell death of the multidrug-resistant cancer cells, and/or indirectly inhibiting development of the multidrug-resistant cancer cells. In particular, the activity of the chemotherapeutic compound to inhibit proliferation or inducing cell death, i.e. apoptosis, is increased. “Potentiating the activity” as used herein means any measurable increase such as of at least 5%, preferably of at least 10% and more preferably of at least 20%.

For example, potentiating the activity of a chemotherapeutic compound can be an increase with regard to cell death, in particular the percentage of total cell deaths after contacting the multidrug-resistant cancer cells with the chemotherapeutic compound and the compound of the present invention preferably for at least 12 h compared to the percentage of cell deaths in multidrug-resistant cancer cells which have been contacted with the chemotherapeutic compound, but not with the compound. The percentage of total cell deaths and cell viability is preferably measured with a MTT assay and annexin V flow cytometry analysis.

In an embodiment of the present invention, potentiating the activity of a chemotherapeutic compound refers to a decrease in IC50 of the chemotherapeutic compound towards the multidrug-resistant cells in the presence of the compound of the present invention compared to the IC50 of the chemotherapeutic compound towards the multidrug-resistant cells in the absence of the compound.

2018101071 02 Aug 2018

The step of contacting the cells with the compound of the present invention in particular of Formula (I) preferably Formula (II) and the chemotherapeutic compound may be carried out by applying at least one incubation solution comprising the compound and/or the chemotherapeutic compound to said cells which incubation solution may further comprise suitable excipients such as solvents, buffers or a suitable growth medium.

The multidrug-resistant cancer cells are contacted with the chemotherapeutic compound before, after or simultaneously with the compound of the present invention, in particular simultaneously with the compound of the present invention. The chemotherapeutic compound is preferably selected from paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

Preferably, the cancer cells are selected from multidrug-resistant:

· lung cancer cells • breast cancer cells • ovarian cancer cells; or • colon cancer cells.

Most preferably, the multidrug-resistant cancer cells are multidrug-resistant lung cancer cells or multidrug-resistant ovarian cancer cells.

Preferably, the multidrug-resistant cancer cells are contacted with the compound of

Formula (II) or its pharmaceutically acceptable salt of solvate.

2018101071 02 Aug 2018

Further in accordance with the present invention is a pharmaceutical composition comprising an effective dose of (i) a compound of Formula (I) or its pharmaceutically acceptable salt or solvate as described above

oh

Formula (I); and (ii) at least one chemotherapeutic compound as described above, in particular selected from paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

The pharmaceutical composition may comprise excipients, in particular pharmaceutically acceptable excipients, such as a carrier, salt, buffer, water, or a combination thereof. The skilled person is able to select suitable excipients.

The compound according to the invention can be present in solid, semisolid or liquid form to be administered by an oral, rectal, topical, parenteral or transdermal or inhalative route to a subject, preferably a human.

Preferably, the compound is of Formula (II)

Formula (II).

2018101071 02 Aug 2018

EXAMPLES

Chemicals and materials

The compound of Formula (II) was isolated and purified. The structure and purity were confirmed with LC-MS. Paclitaxel (abbreviated as PTX), docetaxel (abbreviated as TXT), doxorubicin (abbreviated as DOX), quinidine (abbreviated as QND), verapamil (abbreviated as Ver), 5-fluorouracil (abbreviated as 5-FU), daunorubicin (abbreviated as DON), dimethylsulfoxide (DMSO), RNase A, leupeptin, aprotinin, phenyl methyl sulfonyl fluoride, Triton X-100, propidium iodide (PI) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The RPMI 1640 medium, fetal bovine serum, penicillin and streptomycin were obtained from Life Technologies Inc., Grand Island, NY. ERK 1/2 and actin antibodies were purchased from Santa Cruz Biotechnology, USA; P-gp antibodies were purchased from Calbiochem; Nrf2 antibodies were purchased from Abeam, Hong Kong; other antibodies such as AKT, P15 AKT, and P-ERK1/2 were purchased from Cell Signaling Technology, Inc.

Cell culture

Human ovarian cancer cells A2780 cells and paclitaxel-resistant ovarian cancer cells A2780/T cells, human non-small cell lung cancer (NSCLC) A549 cells and paclitaxelresistant NSCLC cancer cells A549/T cells were purchased from KeyGen Biotech Co.,

Ltd. (Nanjing, China). Cells were grown as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum (GIBCO, Paisley, Scotland) at 37°C in a humidified 5% CO2 atmosphere. Paclitaxel at a concentration of 0.94μΜ was added to the culture medium for A2780/T cells and a concentration of 0.24μΜ paclitaxel was added to the culture medium for A549/T cells. The expression of P-gp in A549/T and

A2780/T cancer cells at mRNA and protein level has been determined. As shown in

Fig. 1a and 1 b, A549/T and A2780/T cancer cells have an elevated expression of P-gp. Also, human colon carcinoma cells Caco-2 cells were purchased from the ATCC, and cells at passage numbers 25-35 were used for the assays.

Statistical analysis

All experiments were repeated at least three times and the data were presented as the mean ± SD unless noted otherwise. Statistical analysis was carried out using Student’s t-test or one-way analysis of variance with Microsoft Excel 2010, and the level of significance was set at a P value of <0.05(*), <0.01 (**) or <0.001 (***).

2018101071 02 Aug 2018

EXAMPLE 1

Cytotoxic effect of the compound of Formula (II) in multidrug-resistant cancer cells

Cell cytotoxicity assay

Sulphorhodamine B (SRB) assays were used to measure the drug-induced cytotoxicity and cell proliferation. In brief, cells were harvested, counted and seeded into 96-well flat-bottomed plates at an initial density of 7.5* 10³ per well before treatment. Cells were exposed to various concentrations of chemotherapeutic compounds in the absence or presence of the compound of Formula (II) which is also denoted as Rg5 herein at a concentration of 2, 4, or 8μΜ. 50μΙ 50% (W/V) TCA (with final concentration of 10% TCA) was add to each well and incubated at 4°C for 1 h. After five washings with water, the cells were stained with 0.4% SRB dissolved in 1% v/v acetic acid (ΙΟΟμΙ/well) for 10 min, and quickly washed with 1% acetic acid before dissolution with 200μΙ of 10 mM Tris base solution (pH=10.5). The bound protein stain was measured at wavelengths

515nm using a plate reader (Spectramax® paradigm®Multi-mode Detection platform,

Molecular Devices, California, United States). The degree of resistance was estimated by comparing the IC50 (concentration of 50% inhibition) for the multidrug resistant (MDR) cells to that of parent sensitive cells which are have no obvious resistance against the chemotherapeutic compounds. The degree of reversal of MDR was calculated by dividing the IC50 of cells incubated with the chemotherapeutic compounds in the absence of Rg5 by that obtained in the presence of Rg5.

With reference to Fig. 2a, the cytotoxicity of Rg5 indicated with IC50 values were 64.59 and 54.36μΜ in MDR A2780/T cells and sensitive A2780 cells, respectively. This compound showed antitumor effects against both resistant and sensitive human ovarian and lung cancer cell lines in the absence of 0.94μΜ PTX in the culture medium. The cytotoxicity of Rg5 is lower than that of PTX, TXT, DOX, or the like as shown in Fig. 2b. Moreover, Rg5 does not inhibit the growth of MDR cell lines at concentration of 8μΜ. Therefore, the maximum concentration of Rg5 used by the inventors in the reversal assays was 8μΜ.

The inventors then tested whether Rg5 could reverse the resistance of A2780/T cells.

With reference to Fig. 3b, A2780/T cells treated with Rg5 have significant reduction in the IC50 of TXT in a concentration-dependent manner, as shown with the left shift of the cytotoxicity curves in Fig. 3b. Specifically, the results in Fig. 3c show that A2780/T cells

2018101071 02 Aug 2018 treated with 2, 4, and 8μΜ Rg5 have a reduced IC50 of TXT by 1.95, 4.55, and 17.38 fold, respectively. However, Rg5, at tested concentrations, had no effect on the IC50 of TXT in the parental sensitive A2780 cells as shown in Fig. 3a. Moreover, at the concentration of 8μΜ, Rg5 also reduced the IC50 values of PTX, DOX and DON with reversal fold of 6.68, 6.38 and 5.31, respectively, whereas it also decreased the IC50 values of 5-fluorouracil (non-substrate of ABCB1) with a reversal fold of 6.67 as shown in Table 1.

Table 1 Cytotoxicity effect of Rg5 on MDR cells in the presence or absence of chemotherapeutic compounds such as paclitaxel, docetaxel, 5-fluorouracil, daunorubicin and doxorubicin.

A2780/T	A549/T
Drug	ICsoiSD (μΜ)	fold reversal	IC5o±SD (μΜ)	fold reversal
Paclitaxel	3.55±0.88	1.00	3.35±0.38	1.00
+2μΜ Rg5	1.78±0.69	1.20	1.89±0.27	1.78
+4μΜ Rg5	0.94±0.45*	3.76	1.12±0.35*	2.99
+8μΜ Rg5	0.53±0.36**	6.68	0.60±0.26**	5.62
Docetaxel	14.22±1.31	1.00	8.41±1.17	1.00
+2μΜ Rg5	7.28±0.75*	1.95	5.31 ±0.84	1.58
+4μΜ Rg5	3.13±0.66**	4.55	1.88±0.76**	4.47
+8μΜ Rg5	0.82±0.27***	17.38	0.75±0.28***	11.22
Doxorubicin	6.76±0.79	1.00	3.58±0.85	1.00
+2μΜ Rg5	5.62±0.92	1.20	3.25±1.04	1.13
+4μΜ Rg5	1.87±0.66*	3.59	2.25±0.56	1.58
+8μΜ Rg5	1.08±0.59**	6.38	1.42±0.23*	2.52
Daunorubicin	8.91 ±0.97	1.00	6.38±0.89	1.00
+2μΜ Rg5	6.68±1.26	1.33	4.16±0.59	1.35
+4μΜ Rg5	3.34±0.83*	2.66	2.32±0.72*	2.43
+8μΜ Rg5	1.68±0.78**	5.31	0.82±0.26**	6.90
5-Fluorouracil	149.87±12.19	1.00	119.05±9.68	1.00
+2μΜ Rg5	95.50±7.93	1.48	94.57±7.69	1.27
+4μΜ Rg5	42.24±3.44*	3.34	59.67±4.86*	1.99

2018101071 02 Aug 2018 +8μΜ Rg5 21.17±1.73** 6.67 22.39±2.43** 5.01

Cell growth was determined using the SRB assay as described above. The data are representative of three different experiments and are shown as mean ± SD (n=3). *, P<0.05, **, P<0.01, ***, P<0.001, means significantly different from the control group in the absence of Rg5.

As shown in Table 1, Rg5 also achieves a similar reversal effect in ABCB1overexpressing non-small cell human lung cancer cell line A549/T which is resistant against PTX and the corresponding parental and sensitive cells A549 cells. The reversal effects of Rg5 to TXT and other chemotherapeutic agents are similar to that in

A2780/T cells. With reference to Figs. 3d to 3f, the addition of Rg5 at 2, 4, and 8μΜ significantly decreased the IC50 of TXT with reversal fold of 1.58, 4.47, and 11.22, respectively. At the same concentrations, Rg5 had no effect on the IC50 of TXT in the parental sensitive A549 cells. All these results demonstrate that Rg5 can potentiate the activity of a chemotherapeutic compound in multidrug-resistant cancer cells, i.e. is capable of sensitizing multidrug resistance cancer cells to chemotherapeutic compounds.

Colony formation assay

Colony formation assay was conducted to measure the effect of Rg5 and the combination of Rg5 and a chemotherapeutic compound in cancer cells. This assay is particularly useful in determination of long term reversal effect of Rg5 on multidrugresistant ABC-protein-dependent cancer cells in particular ABCB1 mediated MDR cancer cells. A2780/T cells (1200 cells/well) in 6-well plates were treated with Rg5 at different concentrations or culture medium containing 1.2μΜ of TXT. Triplicate wells were set up for each condition. After 8 days, the cells were stained with crystal violet (0.2% in buffered formalin) at room temperature for 20 min. The plates were rinsed with phosphate buffered saline (PBS) for 3 times, and the colony numbers were counted using the software of Quantity one-Colony counting (BIO-RAD, California, USA).

Referring to the results in Figs. 4a and 4b, complete inhibition of colony formation was achieved by the combination of 1.2μΜ TXT and different concentrations of Rg5, whereas no inhibition was observed at either 8μΜ Rg5 or 1.2μΜ TXT alone. Taken together, these results indicate that combination of Rg5 with TXT elicited significantly

2018101071 02 Aug 2018 higher cytotoxic response in multidrug-resistant ABC-protein-dependent cancer cells in particular ABCB1 mediated MDR cancer cells.

EXAMPLE 2

Effect of the compound of Formula (II) on apoptosis in multidrug-resistant cancer cells

Cell cycle and apoptosis analysis

Flow cytometry was used to determine the effect of the compound of Formula (II) in cell cycle and apoptosis of multidrug-resistant ABC-protein-dependent cancer cells. In cell cycle analysis, A2780/T cells were harvested after 24 hours, 48 hours or 72 hours treatment and washed twice with ice-cold PBS. The cells were fixed and permeabilized with 70% ice-cold ethanol overnight at 4°C or 2 h at -20°C. After that, the cells were washed with PBS once, stained with a staining solution containing PI (50pg/ml) and RNase A (200pg/ml) for 30 min at room temperature. Then they were pelleted, washed and re-suspended in PBS to a final concentration of 1x10⁶/ml and analyzed by flow cytometry BD FACS Aria (San Jose, CA).

In apoptosis analysis, 1*10⁶ cells were collected after treatment, washed and suspended in 100μΙ of binding buffer (10mM of N-2-hydroxyethylpiperazine-N’-220 ethanesulfonic acid/NaOH, 140mM of NaCl, 2.5mM of CaCb, pH 7.4). Apoptotic cells were identified by double supravital staining with 5μΙ of recombinant FITC (fluorescein isothiocyanate)-conjugated Annexin-V and 5μΙ of PI (50μg/ml). The cells were stained for 15 min at room temperature in the dark, and analyzed by fluorescence-activated cell sorting eater-plus flow cytometry. Data acquisition and analysis were performed in BD

FACS Aria with FlowJo software.

Referring to Fig. 5a, A2780/T cells treated with 2, 4, and 8μΜ Rg5 show a significantly increased apoptosis induced by 1.2μΜ TXT in a concentration-dependent manner. Treatment with 2μΜ Rg5 can boost the apoptosis induced by 1.2μΜ TXT to a similar degree as that of 14.22μΜ TXT (ICso of A2780/T). While single treatment of 8μΜ Rg5 or 1.2μΜ TXT did not show remarkable apoptosis induction.

The inventors then determined whether the induction is related to synergistic effect caused by the combination of Rg5 and TXT. Asynchronously growing A2780/T cells and its sensitive parental cell line A2780 both treated with TXT in the absence or

2018101071 02 Aug 2018 presence of Rg5 were examined for their cell cycle progressions by flow cytometry. In the untreated control group, the percentage of A2780/T cells in G0/G1-, S- and G2/Mphases were 79.3%, 7.31% and 12.6%, respectively. Single exposure for 48 hours to TXT (14.22μΜ) resulted in G2-M arrest in A2780/T cells, see Fig. 5b. In the absence of

Rg5 treatment, there were 71.9% of cells at G1 phase and 17.1% of cells at G2 phase after incubated with 1.2μΜ TXT, whereas these distributions were significantly shifted to 37.9% of G1 and 41.8% of G2 phase cells after treatment of Rg5 at 8μΜ in combination with 1.2μΜ TXT. This pattern was evident after 24 h and persisted over the 72 h of treatment. Thus, while A2780/T cells were remarkably resistant to 1.2μΜ

TXT, the combination of Rg5 with TXT was found to greatly increase the proportion of G2/M arrested cells to >41%. Rg5 (8μΜ) alone had no obvious effect on cell cycle distribution of A2780/T.

EXAMPLE 3

Synergistic effect of the compound of Formula (II) and chemotherapeutic compounds

The Chou-Talalay Methods as described by T-C Chou and P. Talalay (1997: Cambridge (UK): Biosoft.) were further used to evaluate the synergistic therapeutic effect for the combination of Rg5 and TXT. “Combination index” (Cl) was calculated by this method to quantitatively depict synergism (Cl<1), additive (Cl=1), or antagonism (Cl>1) effect. In brief, multidrug-resistant A2780/T cells were exposed to a serially diluted mixture of Rg5 and TXT for 48 hours. The 2-fold serial dilution with several concentration points above and below its IC50 value was used for evaluating cytotoxicity of combination by SRB method as described above. With the use of CalcuSyn software

v. 2.1 (Bio-soft), synergy is further refined as synergism (Cl = 0.3-0.7), strong synergism (CI=0.1-0.3), and very strong synergism (Cl <0.1).

The combination index (Cl) values calculated at 50% (ED₅o) and 90% (ED90) of cell killing were 0.23 and 0.05 as shown in Table 2, demonstrating strong synergistic cytotoxic effect (CI<0.3) of the Rg5-TXT combination in the ABCB1-overexpressing A2780/T cells. With CalcuSyn simulation, an ED50 is produced by 69.56μΜ of Rg5 or 16.02μΜ of TXT in A2780/T cells, but the ED50 of TXT is 1.79 μΜ in combined with 8.12μΜ Rg5 which is a 8.95-fold decrease for the ED50 value. The quantitative diagnostic graphics for this synergistic effect between Rg5 and TXT are shown in Figs.

6a to 6d. The Cl values were plotted as a function of the particular inhibitory effect. Cl

2018101071 02 Aug 2018 values <1 represent a synergistic combination, Cl values equal to 1 are additive and Cl values >1 represent antagonistic combinations.

Table 2 The values of Cl and the synergism dose of Rg5 and TXT at Fa 0.5 (ED₅o) and 5 Fa 0.9 (ED₉₀)

Data for Fa = 0.5	Cl value	Dose Rg5(gM)	Dose ΤΧΤ(μΜ)
Rg5	/	69.56	/
TXT	/	/	16.02
Rg5+TXT	0.23	8.12	1.79
Data for Fa = 0.9	Cl value	Dose Rg5(gM)	Dose ΤΧΤ(μΜ)
Rg5	/	6.29	/
TXT	/	/	1.8
Rg5+TXT	0.05	0.18	0.04

The above results proved that Rg5 have a significant effect on reversing multidrugresistance. The inventors then determined the effect of Rg5 on intracellular accumulation of doxorubicin and Rhodamin123 which is a reference P-gp substrate probe in multidrug-resistant cancer cells. In particular, Rhodamin123 (Rho123, 5μΜ) and DOX (1 ΟμΜ) were added to A2780 or A2780/T cells and incubated in the presence of absence of Rg5 (8μΜ) for 8 h. Cells were detached, re-suspended in 500μΙ of PBS after washed twice with cold PBS, and analyzed by flow cytometry (BD FACS Aria, BD Biosciences, San Jose, CA). Excitation and emission wavelengths (nm) used for DOX and Rho123 were as follows: 480 to 585, and 496 to 524. Quinidine (QND, 20μΜ), a known ABCB1 inhibitor, was used as a positive control.

Referring to Figs. 7a and 7b, the intracellular accumulation of DOX and Rho123 were significantly higher in A2780 than that in A2780/T. When the drug-resistant cells were treated with 8μΜ Rg5 or 20μΜ QND (a reference P-gp inhibitor), the intracellular accumulation of Rho123, and DOX were higher than that in the untreated A2780/T. In contrast, Rg5 had no obvious effect on DOX and Rho123 levels in the parental A2780 cells. Taken together, these results showed that Rg5 significantly increased the intracellular accumulation of chemotherapeutic compounds in ABCB1-overexpressing cells, thus increasing the cytotoxicity in these MDR cells.

2018101071 02 Aug 2018

EXAMPLE 5

Effect of the compound of Formula (II) on the transport of ABC-protein

Human colorectal carcinoma Caco-2 cells are widely used as an in vitro model to determine human drug absorption and efflux activity of transporters. To further confirm the effect of Rg5 on P-gp function, the efflux ratio of the P-gp substrate Rho 123 in the presence or absence of Rg5 was determined.

Caco-2 cells were seeded on 6-well Corning transwell insert products (Corning Incorporated Life Sciences, MA, USA) and they formed a confluent monolayer over 21 days culture prior to the experiment. The integrity of the cell monolayers was checked by measuring the transepithelial electrical resistance (TEER) before and after the transport experiments using a WPI EVOM2 volt-ohmmeter (Word Precision

Instruments, Inc. Sarasota, FL USA) fitted with STX2 chopstick electrodes (World Precision Instruments, Sarasota, FL, USA). On day 21, the transport assay included apical-to-basolateral (A—>B) and basolateral-to-apical (B—>A) transport rate determinations for Rho123 (5μΜ) and DOX (10μΜ) in Caco-2 cell line was carried out over a time period of 2 hours. Briefly, samples (100μΙ) were collected from apical I basolateral side of Caco-2 cell monolayer at predetermined times of 30, 60, 90 and 120 min, and immediately detected for the fluorescence intensity in 96 well black plate using a microplate reader (infinite M200 PRO, TECAN, Switzerland). Bidirectional transport of target compound was conducted in Caco2 cell monolayer with Rg5 added in both apical and basolateral chambers. Quinidine was used as a potent control inhibitor of P25 gp.

The apparent permeability coefficients (Papp) were calculated as:

p - ^d(\ ¹ ^^app_ At C₀A where dQ/dt (mM/sec) is the rate of permeation of compound across the cells, Co (mM) is the donor compartment concentration at time zero and A (cm²) is the area of the cell monolayer. The decrease in Efflux Ratio (ER= Papp (B to A) /Papp (A to B)) in the presence of Rg5 and putative inhibitor QND was determined to assess their relative inhibitory potency to transporter P-gp.

2018101071 02 Aug 2018

With reference to Fig. 8a, two hours after administration, the values of Papp (A-B) of Rho 123 increased dose-dependently in the presence of Rg5. Moreover, the efflux ratio of Rho123 was significant decreased (>50%) in a concentration-dependent manner as shown in Fig. 8b . These results prove that Rg5 increased Rho 123 accumulations in the MDR cells by inhibiting ABCB1 transporter.

The efflux function of ABCB1 has a close relationship with ATP hydrolysis. Therefore, the inventors further measured ABCB1-mediated ATP hydrolysis with different concentrations of Rg5. The impact of Rg5 on P-gp ATPase activity was estimated by

Pgp-Glo™ assay systems (Promega, USA). The inhibitory effects of Rg5 were examined against a verapamil-stimulated ABCB1 ATPase activity. Sodium orthovanadate (Na3VO4) was used as an ABCB1 ATPase inhibitor. Following manufacture’s instruction, 0.25mM of Na3VO4, 0.5mM of verapamil, or Rg5 in various concentrations were incubated with assay buffer, 25pg of recombinant human ABCB1 membranes and 5mM of MgATP at 37°C for 40 min. Then, 200μΜ verapamil was added with Rg5 together. Luminescence was initiated by ATP detection buffer. The plate (white opaque 96-well, corning, USA) was further incubated at room temperature for 20 min to develop luminescent signal, and was read with luminometer (infinite M200 PRO.TECAN, Switzerland). The changes of relative light units (ARLU) were determined by comparing Na3VO4-treated samples with Rg5 alone or Rg5 and verapamil combination-treated samples, and hence, the ATP consumed was calculated by comparing to a standard curve.

As shown in Fig. 9a, Rg5 stimulated the ATPase activity of ABCB1 in a dose-dependent manner, with ECso of 9.75μΜ and a maximal stimulation of 3-fold of the basal activity, suggesting that Rg5 is capable of affecting the ATPase activity of ABCB1 and might interact at the drug-substrate-binding site as a substrate of ABCB1.

The inventors also examined the effects of Rg5 on verapamil stimulated ABCB1

ATPase activity. Verapamil is sometimes referred as an ABCB1 inhibitor because, as a substrate for transport, it inhibits ABCB1 activity with other substrates by interfering with their transport in a competitive mode. Fig. 9b shows a reduction of 200μΜ verapamil-stimulated ATPase activity by Rg5 with an ICso value of 9.16μΜ, indicating Rg5 is an ABCB1 ATPase inhibitor.

2018101071 02 Aug 2018

Next, the reversal of multidrug-resistant ABC-protein-dependent cancer can be achieved either by reducing ABC-protein expression or by inhibiting the function of ABC-protein transporter. The inventors investigated the effect of Rg5 on the expression of ABCB1 at protein level.

Referring to Fig. 10a, Rg5 did not significantly alter the protein level of ABCB1 in A2780/T cells at the selected concentrations in the reversal assays. These findings revealed that the reversal effect of Rg5 was not caused by the inhibition of ABCB1 expression. Therefore, Rg5 may be involved in the inhibition of ABCB1 transporter which results in an increase in intracellular accumulation of chemotherapeutic compounds.

The inventors then examined the effect of Rg5 on the expression of total and phosphorylated AKT and ERK in A2780/T cells. After treatment with TXT and Rg5 for

48 h, there was an inhibition on phosphorylated AKT (8μΜ Rg5 in combination with

1.2μΜ TXT group or 24μΜ Rg5 group), but not on total AKT, ERK and phosphorylated ERK as shown in Fig. 10b, indicating the inhibition of PI3K/AKT pathways by the combination treatment. Moreover, there was a significant decrease in the phosphorylated AKT level after treatment with 24μΜ of Rg5. These results indicated that enhanced cytotoxic response by co-treatment with Rg5 and TXT in ABCB1 overexpression MDR cancer cells is associated with inhibition of PI3K/AKT pathways.

Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that up-regulates expression of a number of genes to combat oxidative and electrophilic stress. Referring to Fig. 10c, the results show a remarkably higher level of Nrf2 in A2780/T cells as compared to A2780 cells. Rg5 in combination with TXT reduced the protein level of Nrf2 in a dose-dependent manner. These results clearly demonstrated that the reversal effect of Rg5 to TXT is associated with the inhibition of Nrf2, i.e. Rg5 is capable of reducing the protein expression level of Nrf2 in the presence of a chemotherapeutic compound.

EXAMPLE 7

Effect of the compound of Formula (II) in an animal model

2018101071 02 Aug 2018

The inventors then determined whether Rg5 is capable of overcoming docetaxel resistance in A549/T xenograft model with nude mice. All animal studies were approved by Animal Care and Use Committee at Guangzhou University of Chinese Medicine (No # ZYYL20150807). A549/T cells (1 x10⁶) in solution of 100 μΙ of RPMI 1640 medium and 50 μΙ of matrigel were injected into subcutaneously in the right flank of 6-8-week old nude mice. When the tumors reached a volume of approximately 100-200 mm³, mice were randomly divided into six groups (9-10 for each group). Saline, TXT (10mg/kg), Rg5 (50mg/kg), mixture of TXT (10mg/kg) plus low dosage Rg5 (10mg/kg), mixture of TXT (10mg/kg) plus middle dosage Rg5 (30 mg/kg), and mixture of TXT (10mg/kg) plus high dosage Rg5 (50mg/kg), were intra-peritoneal injection (IP) every 2 days to a total of nine injections and tumor volume was measured every 2 days until it reached 2,000 mm³. The animals were sacrificed on the 27th day and tumors were detached and weighed. The tumor volume was calculated using the following equation: volume = (width² x length)/2.

The total cellular samples were harvested and rinsed twice with ice-cold PBS buffer. Cell extracts were lysed in RIPA buffer (50mM of Tris (pH 7.4), 150mM of NaCI, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride and EDTA) containing protease inhibitor cocktails (Roche Life Science, USA).

Protein concentration was determined using the BCA protein assay kit. Equal amounts of cell lysates were resolved by SDS-PAGE and subsequently electrophoretically transferred onto PVDF membranes (Millipore, Darmstadt, Germany). After blocking in tris-buffered saline containing 0.1% Tween20 (TBST) with 5% (w/v) skim milk (Nestle Carnation, New Zealand) for 2 h at room temperature, the membranes were incubated with primary and secondary antibodies and subsequently visualized with an enhanced chemiluminescence detection kit (Thermo Scientific™ SuperSignal™ West Pico Chemiluminescent Substrate, USA). β-Actin was used as the loading control for the experimental data analysis.

The dose of TXT was 10mg/kg for the docetaxel, and the combination of docetaxel and Rg5 groups, which is within the safety range and tolerated in this study with increased body weight. According to Fig. 12a and 12b, TXT alone slightly inhibited tumor progression with little difference in tumor volume compared to that in the saline group, confirming that TXT alone did not work very well at that particular dose. With reference to Figs. 12 to 12d, the tumor growth in all groups treated with both TXT and Rg5 was

2018101071 02 Aug 2018 significantly inhibited in a dose dependent manner, indicating considerable therapeutic efficacy. As expected, the efficacy of high dose of Rg5 (50mg/kg) with TXT was greatly diminished against the taxol-resistant xenograft, accomplishing suppression of tumor growth by 48%. At the end of the experiment, all tumors were removed and weighed.

The high dose of Rg5 with TXT group had the smallest tumor weight, which was 2.3fold smaller than that in the saline group and nearly twofold smaller than that in the TXT and Rg5 groups. Moreover, no weight loss was observed in the combination treatment group indicating without a significant increase in docetaxel toxicity. These results demonstrate that Rg5 with TXT overcomes the resistance both in vitro and in vivo.

EXAMPLE 8

Determination of the crystal structure of P-gp

To understand the binding mechanism between Rg5 and P-gp, Surflex-dock embedded in Tripos Sybyl X 2.0 was performed using the crystal structure of QZ59-RRR bound to P-gp (PDB ID: 4M2S). The crystal structure of P-gp with a bound inhibitor QZ59-RRR (PDB ID 4M2S) was used for molecular docking. All the residues in P-gp were protonated at pH 7.0. Partial charges of the atoms were assigned by the Sybyl force field. The protomol which represents a set of molecular fragments to characterize the active site was generated by a ligand-based approach, and the bound ligand was utilized for protomol generation. The proto_thresh and proto_bloat parameters represent how much the protomol can be buried in the protein and how far the protomol extends outside the cavity, respectively, which were assigned by the default value 0.5 and 0. For the reliability of the molecular docking method, the bound inhibitor QZ5925 RRR was re-docked back to the protein with the average RMSD less than 1.5A for the docking poses compared with the original one.

The selected pose of Rg5 fitted the binding site pocket of P-gp well with the docking score (Total_Score) of 5.95, which is close to that (6.01) of the reference compound quinidine. Observed from the docking result, Rg5 formed several kinds of specific interactions with P-gp as shown in Figs. 11 a and 11 b, Rg5 formed four hydrogen bonds with Tyr306, Gln721, and Tyr949. The π-σ interaction was also observed between Rg5 and P-gp. Besides these interactions, Rg5 also formed Van der Waals interactions and hydrophobic interactions with such residues as Met68, Phe331, Phe332, Leu335,

Ile336, Phe339, Gln343, Phe724, and Phe979 in the binding pocket. All these interactions contributed to the putative binding pattern of Rg5 in P-gp.

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CONCLUSION

The compound of Formula (II) (Rg5) at non-cytotoxic concentrations is capable of potentiating the activity of a chemotherapeutic compound in multidrug-resistant cancer cells, in particular multidrug-resistant ABC-protein-dependent cancer preferably multidrug-resistant P-glycoprotein-dependent cancer. The results discussed above clearly demonstrate that the compound of Formula (II) can potentiate the effect of DOX, PTX, TXT and DON in at least MDR ovarian cancer cells, and non-small lung cell cancer cells. The application of the compound of Formula (II) also does not product significant therapeutic effect on parental cells which are not resistant against the chemotherapeutic compounds. The concentrations of the compound of Formula (II) are lower than the maximal plasma concentration (22.5μΜ) obtained in in vivo pharmacokinetic study. Moreover, the compound of Formula (II) significantly enhanced the in vivo antitumor activity of TXT in the A549/T xenograft model without a significant increase in TXT toxicity which is comparable with the effect of third-generation P-gp inhibitors such as OC144-093 and LY335979. The compound of Formula (II) can inhibit the efflux activity of ABCB1 transporter in Caco-2 monolayer cell model. Docking study clearly illustrates that the ligand of the compound of Formula (II) is well-fitted into an active cavity of ABCB1 with similar affinity as quinidine.

The activity of ATPase was stimulated by the compound of Formula (II) in a concentration dependent manner and therefore it is potentially a substrate of ABCB1. Moreover, verapamil-stimulated ATPase activity was reduced by Rg5. Therefore, it may competitively bound to the substrate-binding site of ABCB1, leaving little place for other agents to bind to the transporter, which resulted in decreased activity of ABCB1 transporter.

Down-regulating the AKT/ERK and Nrf2 signaling pathways can enhance MDR cancer cells sensitivity to drugs such as paclitaxel, doxorubicin, 5-fluorouracil, etc. As the compound of Formula (II) has been demonstrated with inhibitory effect on the phosphorylation of AKT and ERK, therefore the effect of the compound of Formula (II) on AKT/ERK phosphorylation in A2780/T cells has been confirmed using Western Blot analysis. Moreover, co-treatment of the compound of Formula (II) and TXT significantly suppressed Nrf2 expression in A2780/T cells which have a significantly higher level of

2018101071 02 Aug 2018

Nrf2 than that of A2780 cells. These help to explain the reversal effect of the compound of Formula (II) to 5-fluorouracil which is not a P-gp substrate. It was reported that the inhibition of Nrf2 expression could be carried out through PI3K/AKT and ERK signaling pathway. Thus, mechanistically, the compound of Formula (II) sensitizes the MDR cancer cells to chemotherapeutic compounds through significantly reducing Nrf2 expression by down-regulating the PI3K-Akt and ERK pathways. In addition, the MDRreversal properties of the compound of Formula (II) to TXT and other chemotherapeutic compounds observed in this study would be the results of activation of multiple proteins and signaling pathways related to the MDR.

2018101071 02 Aug 2018

Claims (16)

1. A method of treating a subject suffering from a multidrug-resistant cancer comprising the step of administering an effective amount of a compound of Formula (I)

5 or its pharmaceutically acceptable salt or solvate to the subject,

OH

Formula (I).

2. The method of claim 1, wherein the multidrug-resistant cancer is a multidrug10 resistant ABC-protein-dependent cancer.

3. The method of claim 1, wherein the multidrug-resistant cancer is a multidrugresistant P-glycoprotein-dependent cancer.

15 4. The method of claim 1, wherein the multidrug-resistant cancer is resistant against a chemotherapeutic compound selected from paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

5. The method of claim 1, wherein the compound of Formula (I) is administered in

20 combination with an effective amount of a chemotherapeutic compound selected from paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

6. The method of claim 5, wherein the compound of Formula (I) is administered simultaneously with the chemotherapeutic compound.

7. The method of claim 1, wherein the multidrug-resistant cancer is multidrugresistant lung cancer, multidrug-resistant ovarian cancer, multidrug-resistant colon cancer or multidrug-resistant breast cancer.

8. The method of claim 1, wherein the compound is of Formula (II)

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Formula (II).

9. A method of potentiating the activity of a chemotherapeutic compound in multidrug-resistant cancer cells, the method comprising contacting the multidrugresistant cancer cells with (i) a compound of Formula (I) or its pharmaceutically acceptable salt or

10 solvate, oh

Formula (I); and (ii) the chemotherapeutic compound.

10. The method of claim 9, wherein the multidrug-resistant cancer cells are 15 multidrug-resistant ABC-protein-dependent cancer cells.

11. The method of claim 9, wherein the multidrug-resistant cancer cells are multidrug-resistant P-glycoprotein-dependent cancer.

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12. The method of claim 9, wherein the multidrug-resistant cancer cells are resistant against the chemotherapeutic compound selected from paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

5 13. The method of claim 9, wherein the multidrug-resistant cancer cells are contacted with the chemotherapeutic compound simultaneously with the compound of Formula (I).

14. The method of claim 9, wherein the multidrug-resistant cancer cells are

10 multidrug-resistant lung cancer cells, multidrug-resistant ovarian cancer cells, multidrug-resistant colon cancer cells or multidrug-resistant breast cancer cells.

15. The method of claim 9, wherein the compound is of Formula (II)

15 Formula (II).

16. A pharmaceutical composition comprising (i) a compound of Formula (I) or its pharmaceutically acceptable salt or solvate,

20 oh

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Formula (I); and (ii) a chemotherapeutic compound selected from paclitaxel, docetaxel, doxorubicin, 5-fluorouracil or daunorubicin.

5 17. The pharmaceutical composition of claim 16, wherein the compound is of

Formula (II)

Formula (II).

1 /16

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Fig. 1a

A2780 A2780/T

Fig. 1b

MDR1 £0. 15

0. 1 •^0.05

A549/T

A549

A549/T

ΡβΡ β-actin

Fig. 1d

A549

Fig. 1c

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Cells IC^ of Rg5 (μΜ) _Ι^μΜ)_ _Fo|_d

A2780 54.36 ±2.65 ^PfM« ^A278° ^A2780/T

A27B0/T 64.59±2.11 TXT 0.11±0.09 14.22±1.31 129

Fig. 2a Fig. 2b

RgS^M)

Cells

0.00001 0.001 0.1 10 ΤΧΤ(μΜ)

IC^of Rg5{pM)

A549

A549/T

55X1 ±3.53 69.21 ±2.25

Fold

Drug A549 A549/T reversal

TXT 0.08 ±0.02 8.41 ±1.17 105

Fig. 2c

Fig. 2d

3/16

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Fig. 3b

O.OOO1 0.01 1 100

ΤΧΤ(μΜ)

Fig. 3a

Drug IC^o οίΤΧΤ(μΜ) A2780/T Fold reversal TXT 14.22 ±1.31 1.00 +2μΜ Rg5 7.28 ±0.75* 1.95 +4μΜ Rg5 3.13±0.66·· 4.55 +8μΜ Rg5 0.82 ±0.27*·· 17.38 Fig. 3c

~ A549

E100 L \ u «*»

O * ^⁵⁰ >

I « 0

-β— 8μΜ R¢5+TXT 4μΜ R<5+TXT — 2μΜ Rg5+TXT · - TXT

......

0.00001

0.001

ΤΧΤ(μΜ)

Fig. 3d

0.1

ΤΧΤ(μΜ)

Fig. 3e

Drug ICm οίΤΧΤ(μΜ) Fold reversal A549/T TXT 8.41 ±1.17 1.00 +2μΜ Rg5 5.31 ±0.84 1.58 +4μΜ Rg5 1.88 ±0.76·* 4.47 +8μΜ Rg5 0.75 ±0.28··· 11.22

Fig. 3f

4/16

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ΤΧΤ(1.2μΜ)

Rg5^M) + - +

0 8 8

Fig. 4a

8 8 4 2

ΤΧΤ(1.2μΜ)RG5(pM)2 +

Fig. 4b

5/16

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Fig. 5a

Annexin V-FITC

Fig. 5b oo

6/16

Ο (Μ

OO (Μ

Ο ο

ο οο ο

(Μ

Α278Ο ΐ*ΐί··Μΐ

4£h □GO/GH*! ·5(%} QG2/M(%)

1,2 14.22

Fig. 5c

Fig. 5d

7/16

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Fig. 6a

Fig. 6b

Dose B

Fig. 6c

Fig. 6d

8/16

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Fig. 7a

9/16

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Fig. 7b

10/16

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Papp:A-B S nPapp:B'A

QND(20pM}:- +

Fig. 8a

Rg5(pM):- 8 4 2

QND(20pM):- + . . .

Fig. 8b

11 /16

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Fig. 9b

12/16

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ΤΧΤ(1.2μΜ):

RgS(pM):

P'gp β-actin

Fig. 10a

A2780

ΤΧΤ(1.2μΜ): +++ +

RgS^M): - 2 4 8 - 24 AKT P-AKT — — — — — ERK1/2 — Ί--- • — P-ERK1/2 p-dCIiii

Fig. 10b

A2780 ΤΧΤ(1.2μΜ): +++ + - RgS^M): - 2 4 8 - NRF2 lv>* β-actin —

Fig. 10c

13/16

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z z

z z

z z

Fig. 11a

14/16

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Fig. 11b

15/16

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Fig. 12b

16/16

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CTR

SOmg/kg RgS _t j lOmg/kg TXT lOmg/kg RgS ♦lOmg/kg TXT

30mg/kg RgS ♦lOmg/kg TXT

SOmg/kg Rg5 ♦lOmg/kg TXT

Fig. 12c

Fig. 12d