WO2025114815A1

WO2025114815A1 - Drug combination with cellular anti-proliferative and self-renewal inhibiting properties

Info

Publication number: WO2025114815A1
Application number: PCT/IB2024/061599
Authority: WO
Inventors: Surajit GANGULY; Subhendu Seth; Anupam Chawla
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-11-27
Filing date: 2024-11-20
Publication date: 2025-06-05
Anticipated expiration: 2026-05-27

Abstract

An anti-proliferative and cell self-renewal inhibiting drug combination comprising diazepam and acetylsalicylic acid, wherein said diazepam and said acetylsalicylic acid is in a ratio 1:3 to 3:1. Also provided are anti-proliferative and cell self-renewal inhibiting pharmaceutical composition comprising said combination.

Description

DRUG COMBINATION WITH CELLULAR ANTIPROLIFERATIVE AND SELF-RENEWAL INHIBITING PROPERTIES

Background and the prior art

Glioblastoma multiforme (GBM, WHO grade IV astrocytoma) is the most common primary and aggressive brain tumour of the adult central nervous system and is associated with a poor prognosis. The standard medical care involves maximal surgical resection followed by concurrent radiotherapy and temozolomide (TMZ, Temodar®) chemotherapy resulting in a median survival of about 14 months (Stupp R, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005 Mar 10;352(10):987-96.'). Currently, there are no drugs available for direct intervention to GBM. The lethality is contributed primarily due to the persistence of post-surgical chemotherapy and radiation resistant glioma stem-like cells (GSCs) that lead to recurrence of the tumour [Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature 2004; 432: 396-401]. These glioma stem cells (GSCs) exhibit remarkable self-renewing property. Thus, one strategy to contain GBM would be to induce disruption of the metastatic and tumorogenic potential of these aggressive, radiation resistant Glioma stem cells (GSC) or also called cancer stem cells (CSC), by inducing differentiation so that they become terminally differentiated cells blocking uncontrolled proliferation and more importantly, eliminating the recurrence or self-renewing potential.

It has been suggested that many types of cancer contain functionally subsets of CSCs or in case of Glioblastoma, GSCs. CSCs or GSCs specifically, comprise only a small portion of the tumour but are known to be critical for aggressiveness of tumour, recurrence and are extremely resistant to anti-neoplastic (anti-cancer) drugs. Recent evidence has emerged that CSCs are similar to tissue-specific stem cells with respect to self-renewal and multi-lineage differentiation capacity, but they differ in their long-term proliferative (cause for aggressiveness of tumour) potential. This uncontrolled renewal potential of CSCs might be the reason for tumour relapse after conventional cancer therapy. Many efforts are currently underway to find therapies that specifically target these CSCs. One promising strategy is the induction of CSC differentiation, as it has been associated with a reduction in tumour malignancy in animal models by reducing the aggressive proliferative characteristics of particular cells.

Over many years, the expression of CD133 has been detected in various stem/progenitor cells, particularly in cells of the human neural systems. In glioma, CD133 proved to be an independent prognostic marker for adverse progression- free and overall survival, thus strengthening its role in tumour growth. Recent studies have suggested that a GBM subpopulation expresses CD133 and is enriched for CSCs. This subpopulation shows an increased tumorigenic potential by rapid self-renewal than subpopulations that are devoid of CD 133 expression. If eradication of cancer-initiating stem cells is the critical determinant in achieving cure [Cho DY et al (2013) Targeting cancer stem cells for treatment of glioblastoma multiforme. Cell Transplant 22(4):731-739', Seymour T, Nowak A, Kakulas F (2015) Targeting aggressive cancer stem cells in glioblastoma. Front Oncol 5:159 , it must be reasoned that depletion of the CD 133 -positive cell pool through controlled, drug-induced differentiation could have profound therapeutic implications.

Diazepam is a known anxiolytic benzodiazepine. It is a fast-acting, long-lasting benzodiazepine commonly used to treat anxiety disorders and alcohol detoxification, acute recurrent seizures, severe muscle spasms, and spasticity associated with neurologic disorders. Aspirin, also known as acetylsalicylic acid (ASA) and a nonsteroidal anti-inflammatory drug (NSAID), is used to reduce fever and relieve mild to moderate pain from conditions such as muscle aches, toothaches, common cold, and headaches. It may also be used to reduce pain and swelling in conditions such as arthritis. Anti-tumour activity of non-steroidal anti-inflammatory drugs: Cyclooxygenaseindependent targets, Jason L. Liggett et al, Cancer Lett. 2014 May 1; 346(2): 217- 224. doi: 10.1016/j.canlet.2014.01.021, discloses non-steroidal anti-inflammatory drugs (NSAIDs) are used extensively for analgesic and antipyretic treatments. In addition, NSAIDs reduce the risk and mortality to several cancers. Their mechanisms in anti-tumorigenesis are not fully understood, but both cyclooxygenase (COX)-dependent and -independent pathways play a role. The researchers here have elucidated molecular targets of NSAID-induced apoptosis. In this review, among various NSAIDs, sulindac sulfide and tolfenamic acid are emphasized because these two drugs have been well investigated for their anti- tumorigenic activity in many different types of cancer.

US6004927 discloses a method for increasing bioavailability of an orally administered hydrophobic pharmaceutical compound, which comprises orally administering the pharmaceutical compound to a mammal in need of treatment with the compound concurrently with a bioenhancer comprising an inhibitor of a cytochrome P450 3A enzyme or an inhibitor of P-glycoprotein-mediated membrane transport, the bioenhancer being present in sufficient amount to provide bioavailability of the compound in the presence of the bioenhancer greater than the bioavailability of the compound in the absence of the bioenhancer. This prior art discloses drugs such as diazepam and quinidine aspirin. However, there are no teachings about selection of specific drugs for treatment of cancer.

US2006024365 discloses a dosage form comprising of a high dose, high solubility active ingredient as modified release and a low dose active ingredient as immediate release where the weight ratio of immediate release active ingredient and modified release active ingredient is from 1: 10 to 1:15000 and the weight of modified release active ingredient per unit is from 500 mg to 1500 mg; a process for preparing the dosage form. However, there are no teachings about selection of specific drugs for treatment of cancer. US2001036943 discloses pharmaceutical compositions comprised of a therapeutically effective combination of a nicotine receptor partial agonist and an analgesic agent and a pharmaceutically acceptable carrier. The analgesic agent is selected from opioid analgesics, NMDA antagonists, substance P antagonists, COX-1 and COX-2 inhibitors, tricyclic antidepressants (TCA), selective serotonin reuptake inhibitors (SSRI), capsaicin receptor agonists, anesthetic agents, benzodiazepines, skeletal muscle relaxants, migraine therapeutic agents, anticonvulsants, anti-hypertensives, anti-arrythmics, antihistamines, steroids, caffeine, and botulinum toxin.

US2016000715 discloses oral pharmaceutical dosage form containing at least two medicaments, in which form the medicaments on the one hand are brought together in a leakproof and in-vivo water soluble wrapping and on the other hand are separated so that the active principle of the combined medicaments cannot come into contact with one another. At least one of the medicaments can be chosen from the following therapeutic classes: non-steroidal anti-inflammatory drug (NSAID), proton pump inhibitor (PPI), beta-blocker, statin, conversion enzyme inhibitor (CEI), biguanide, myorelaxant, calcium inhibitor, corticoid, antidepressant, benzodiazepine, non-atropine-like intestinal transit retarder, intestinal antibacterial, and the following therapeutic molecules: spironolactone, propranolol, clarithromycin, amoxycillin, low-dose acetylsalicylic acid, potassium, and clopidogrel. However, there is no teaching about any specific drug combination having anti-tumour/anticancer activity.

W02009051840 discloses acetylsalicylic acid (ASA or aspirin), salicylic acid (SA) and related salicylate esters and their pharmaceutically acceptable salts, when co-administered in effective amounts with a drug or other bioactive agent which typically (in the absence of the salicylate compound) produces significant hepatotoxicity as a secondary indication, will substantially reduce or even eliminate such hepatotoxicity. Thus, reducing hepatotoxicity associated with the administration of certain drugs and other bioactive agents and in certain instances of allowing the administration of higher doses of a compound which, without the co-administration, would produce hepatotoxicity which limits or even negates the therapeutic value of the compound. Here the bioactive agent can be diazepam. WO’ 840 discloses aspirin having beneficial effect when combined with diazepam. However, there is no teaching about any specific drug combination having anti- tumour/anticancer activity.

Although many therapeutic agents have been studied, only few anticancer drugs affect cancer cell differentiation, e.g., retinoic acid (RA), bone morphogenetic proteins (BMP), and drugs that target tumour epigenetics such as histone deacetylase inhibitors and hypomethylating agents. However, no effective cure or intervention has been achieved so far for brain cancer treatment.

Object of the invention

It is an object of the present invention to overcome the drawbacks of the prior art. It is another object of the present invention to inhibit cell proliferation and selfrenewal capacity by triggering cellular differentiation of aggressive, tumour causing cancer-stem cells.

It is yet another object of the present invention to provide an effective cure for brain cancer treatment.

Brief description of accompanying figures

Figure 1 illustrates the light microscopic image of spheroid formation in control cells and disruption of LN- 18 spheroid formation due to drug administration.

Figure 2 illustrates the anchorage-independent tumour growth assay using LN- 18 cells to determine the anti-tumour efficacy of E+D drug combination. Figure 3 illustrates the analysis of expression of “sternness” marker CD133 protein in spheroid cultures. Lower CD 133 expression is an indicator of reduced self-renewal of cancer cells.

Figure 4 illustrates the cellular levels of different signalling pathway molecules (A) phospho-ERK (p-ERK) and (B) phosphorylated AKT (p-AKT) in control and drug-treated spheroid cells.

Figure 5 illustrates the immunocytochemistry of adherent LN- 18 cells to demonstrate morphological changes (differentiation) in presence or absence of drug treatment.

Figure 6 illustrates that the combined drug treatments enhanced survival rate as compared to the single drug components.

Figure 7 illustrates the tumour regression activity of the drug combination of the present invention.

Figure 8 illustrates the effect at various dose ratio of Acetylsalisylic acid (E) : Diazepam (D) as observed by immuno-fluorescence microscopic imaging of dual stained LN- 18 cells (Glioblastoma cell line).

Summary of the invention

Accordingly, the present invention provides an anti-proliferative and cell selfrenewal inhibiting drug combination comprising diazepam and acetylsalicylic acid, wherein said diazepam and said acetylsalicylic acid is in a ratio 1:3 to 3: 1.

According to another aspect of the present invention there is provided an antiproliferative and cell self-renewal inhibiting pharmaceutical composition comprising a combination of diazepam and acetylsalicylic acid in a ratio 1:3 to 3: 1 and other pharmaceutically acceptable excipients. Detailed description of the Invention

It has been surprisingly found by the present inventor that the drug combination of diazepam and acetylsalicylic acid prevents or reverse or slows down the tumour size or rate of tumour growth and increase the survivability of the tumour bearing animals with respect to the untreated animal group. It has been found that the said combination is more potent than the individual drugs.

The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of various embodiments of the present disclosure as defined by the claims and their equivalents. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary.

Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the various embodiments described herein can be made without departing from the scope and spirit of the present disclosure. In addition, descriptions of well-known functions and constructions may be omitted for clarity and conciseness.

The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the present disclosure. Accordingly, it should be apparent to those skilled in the art that the following description of various embodiments of the present disclosure is provided for illustration purpose only and not for the purpose of limiting the present disclosure as defined by the appended claims and their equivalents.

It is to be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cancer" includes reference to one or more of cancers. The terminology used herein is for the purpose of describing various embodiments only and is not intended to be limiting of various embodiments. It will be further understood that the terms "comprises" and/or "comprising" used herein specify the presence of stated features, integers, steps, operations, members, components, and/or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, members, components, and/or groups thereof.

Also, expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

The present inventors have found that the drugs Diazepam, and Aspirin (ASA or Acetylsalicylate) when combined together, have anti-tumour or tumour inhibitory properties for glioblastoma (GBM) or brain cancer and can reduce the “sternness” or proliferative characteristics of aggressive glioblastoma cell lines like LN-18, U87 or GL261. The combination was found to be also effective in remission of tumours in syngeneic xenograft animal models. Further the combined drug treatment enhanced survival rate. It was found that 75% of animals survived more than the vehicle or single drug-treated groups.

The drug combination was found to be having synergistic effect at a drug combination, wherein diazepam and acetylsalicylic acid is in a ratio 1:3 to 3: 1.

The drugs can be present at a concentration of 5-15 pg/per milliliter in vitro, wherein an effective dosage (in animal models') form comprises about 0.1 mg/kg per day to about 1 mg/kg per day of diazepam or a pharmaceutically acceptable salt and 0.3 mg/kg to 10 mg/kg per day of aspirin or a pharmaceutically acceptable salt, for example which will be equivalent to dosage about 6 mg to 60 mg of diazepam (or a pharmaceutically acceptable salt) and 18 mg to 180 mg of aspirin (or a pharmaceutically acceptable salt) per day for a patient weighing 60 kgs as determined by allometric scaling.

Typically, the drug active may be prepared in a dosage form of about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg of each drug including all ranges and values in between and all possible combinations of doses of the two drugs thereof.

According to one embodiment of the present invention there is provided an antiproliferative and anti-tumourigenic drug combination comprising diazepam and acetylsalicylic acid, wherein said diazepam and said acetylsalicylic acid is in a ratio 1:3 to 3: 1, preferably 1: 1.

According to a further embodiment of the present invention there is provided a pharmaceutical combination comprising a combination of diazepam and acetylsalicylic acid in a ratio 1:3 to 3: 1 and other pharmaceutically acceptable excipients. The drugs diazepam and acetylsalicylic acid is the said combination may be administered together or sequentially, irrespective of the order, at a time gap of not more than 5 minutes.

According to another embodiment of the present invention there is provided a pharmaceutical composition for preventing metastasis or treating cancer comprising a combination of diazepam and acetylsalicylic acid in a ratio ranging from 1:3 to 3: 1, and pharmaceutically acceptable excipients. Diazepam and aspirin may each be present in a dose 5 to 15 pg/ml and preferably be combined in a ratio 1: 1.

In a preferred embodiment the pharmaceutical composition comprises diazepam in a dose from 5 to 15 pg/ml and aspirin in a dose 15 pg/ml. Cancer according to the present invention can be described as tumour development caused by aggressive cancer-stem cells, including but not limited to breast cancer, pancreatic cancer, hepatocellular carcinoma, and the like, preferably glioblastoma.

The present combination as well as composition was found to be effective for preventing post-operative recurrence of cancer.

The pharmaceutical composition according to one embodiment of the present application may be in the form of capsules, tablets, granules, injections; preferably injections.

The dosage form of the pharmaceutical composition of the present application may be variously prepared by mixing with a pharmaceutically acceptable carrier, and may be administered orally or parenterally.

The pharmaceutically acceptable excipients may be binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration, and buffers, preservatives, analgesics, a solubilizer, an isotonic agent, a stabilizer, etc. may be mixed and used, and in the case of topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used.

For example, preparations for parenteral administration may include non-aqueous solutions, suspensions, emulsions, conjugated or trapped in nano-particle carriers, lyophilized preparations, suppositories, and the like.

In the case of an injection, it may be prepared in a unit dose ampoule or multiple dose form, and intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection method may be selected. The dosage of the pharmaceutical composition of the present application can be varied depending on the condition and body weight of the patient, the severity of the disease, the drug type, the route and duration of administration, but can be appropriately selected by those skilled in the art.

The present invention is now explained by way of non-limiting examples.

Example 1

A typical example of an injection formulation in accordance with the present invention can be prepared as follows: 2 mg of Diazepam (D) was dissolved in 1 ml of solvent mixture containing 80% ethanol and 20% DMSO to prepare a 2 mg/ml stock of D. Separately, 2 mg of Aspirin (E) was dissolved in 1ml of PBS (phosphate-buffered saline pH 7.2) to prepare a stock of 2mg/ml of E. Finally, 15 pl (microlitre) from the pre-prepared stock of D was mixed with 15 pl from stock of E in 70 pl of PBS to make 100 pl of drug combinations (D+E). Thus, each 100 pl (i.e., 0.1 ml) of drug combination, containing 30 pg of D and 30 pg of E, was prepared for one injection. The final 100 pl of vehicle solvent, prepared for injection had 12% Ethanol and 3% DMSO in PBS.

Example 2

In-vitro and In-vivo assays

METHODS

2.1. Tumour sphere assay

Method: Tumoursphere or Spheroid formation assay was used to evaluate the anti-tumour properties of the drugs diazepam (D) and acetylsalicylic acid (E).

The APIs in solid forms, diazepam (D) and aspirin (E), were dissolved separately in a) 20% DMSO/80% ethanol solvent mixture and b) phosphate-buffered Saline (PBS, pH-7.2), respectively at a concentration of 5 mg/ml D and 15 mg/ml E as stock. Then, each drug stocks were diluted to attain a final concentration of 15 g/ml by mixing in appropriate volume of cell-culture media (DMEM) for in vitro experiments using adherent cell culture and tumour spheroid cultures. After dilution, the final concentrations of vehicle solvents used in all in vitro experiments were 0.06% DMSO and 0.24% Ethanol.

Spheroids were formed using human glioblastoma cell line LN- 18 (ATCC catalog number CRL-2610) cells following the published method as described previously [Johnson et al 2013]. Briefly, LN-18 cells (IxlO⁵ cells per well) were cultured in 6-well plates using Serum Free DMEM (Dulbecco’s Modified Eagle Medium) medium in presence of B27 supplements (ThermoFisher catalog number 17504044), lOng/ml Basic Fibroblast Growth Factor (Sigma-Aldrich, catalog number: F0291), and lOng/ml Epidermal Growth Factor (Sigma-Aldrich, catalog number: E5036). Penicillin and Streptomycin antibiotic mixture (100 units/ml) was added to the medium to prevent bacterial contamination (purchased from ThermoFisher Scientific; Catalog number 15140122). Cells aggregated to form Tumourspheres or Spheroids appear in absence of diazepam and acetylsalicylic acid within 48 hours.

Results: The efficacy of the combined drugs in disruption of the tumorigenic potential of LN- 18 cells were analysed on tissue culture dish (in-vitro) using tumour spheroid formation assay and is described in Figure 1. Trans -retinoic acid (RA), a known differentiating agent but toxic to humans was used, at 2 pM concentration as a positive control for comparative purposes. LN-18 is a GBM- derived cell line and has been used to develop tumour spheroids in cultures under non-adherent conditions. After 48 hours of treatment with the drug combination (D+E), cells remain in dispersed states without forming spheroids with visibly differentiated individual cells (elongated) attached to the culture dish surface (Fig.l), reminiscent of the RA-treated cells. Figure 1 illustrates the light microscopic image of spheroid formation in control cells and disruption of LN- 18 spheroid formation due to drug administration. Spheroids of LN- 18 cells are formed as described in the method. In Control (Cont; No drug treated) samples of dispersed floating cells at the start (zero hour) is converted into aggregates of tumour-like spheres (spheroids) in culture in 48 hours. The spheroid forming units are indication of tumour forming potential of LN- 18 cells. Lack of formation of larger spheroids in drug-treated (D+E) and retinoic acid (RA) treated samples are an indication of disruption (inhibition) of tumour forming potential of LN- 18 cells. Further, the presence of the elongated cells in D+E and RA samples, as compared to the spherical shapes of the cells in Cont (Control) samples, are an example of cellular differentiation with inhibited self-renewal capacity.

2.2. Anchorage Independent Tumour formation assay

Method : Anchorage-independent growth of cancer causing cells is the ability of those cells to grow independently of a solid surface. The colony formation or tumour spheroid (or tumoursphere or colonies) formation on agar from a single cell is a well-established technique for measuring the efficacy of an anti-cancer drug in vitro. The anchorage-independent growth of LN- 18 cells in presence or absence of D+E were determined by colony formation efficiency in soft agar as described before with some modifications [Endo H, et al. (2013) Enhanced Expression of Long Non-Coding RNA HOTAIR Is Associated with the Development of Gastric Cancer. PLoS ONE 8(10): e77070. https://doi.org/10.1371/journal.pone.0077070]. Anchorage independent tumour forming assays were performed in 6-well plates. The base layer of each well consisted of 1ml of DMEM media supplemented with 10% heat inactivated FBS, containing vehicle or drug compounds (D+E) as mentioned in the results section mixed with melted 1% low melting-point agarose. Plates were chilled at room temperature until the agar layer solidifies. Next, 5 x 10⁴ cells suspended in 1 ml of DMEM media containing vehicle or drugs at appropriate concentration, and 0.3% low melting agarose, were layered over the solidified agar. Plates were again chilled at room temperature until the growth layer hardens. A further 1 ml of media (without agarose) containing appropriate concentration of drug molecules were added on top of the cell layer to prevent the agar layers from drying. Cells were allowed to grow at 37°C for 21 days and colonies were observed under an inverted microscope (Zeiss, Germany) at lOx magnification and images captured as representative images. The images of colonies in control (drug untreated) wells, considered as 100% in size, were compared with the colonies in the drug-treated wells (the drug combination as recited in Example 2.1. Decrease in the number of visually larger colonies determine the efficacy of drug combination.

Results: Figure 2 illustrates the anchorage-independent tumour growth assay using LN- 18 cells to determine the anti-tumour efficacy of E+D drug combination. Colony formation in soft agar assays has been the gold standard for tumorigenicity of cancer cells and is strongly correlated to tumorigenic potential in animal xenograft experiments. LN- 18 cells were allowed to form colonies on soft agar for 10 days, following which cells were treated with either DMSO (control) or drug combination (D+E) to evaluate the effect of these drugs on tumorsphere formation. In this anchorage-independent tumour growth assay, LN- 18 cells (as described in method), drug combination (E+D) treatment demonstrated an inhibition of Tumour formation as compared to the no drug- treated (Control) cells. As demonstrated in Figure 2, tumoursperes (cell colonies) grow in size in the control samples (as indicated by the red arrow in Fig 2) when the cells were incubated for 21 days with no drug treatment. Whereas, in presence of drug combo (D+E) and RA (described earlier) as positive control, no large tumourspheres (colonies) were visible under microscope. This is also an indicator that the drug combination inhibits self-renewal property of the tumour forming LN- 18 cells.

2.3. Immunoblot Technique (Western Blot) for detection of target protein expression.

Method: Immunoblotting techniques and quantification of target proteins were essentially followed as described by Choudhury et al 2016. In brief, about 40pg of total protein extracted from tissue or cell (lysates) were mixed with appropriate volume of sample buffer (100 mM Tris-Cl pH 6.8, 4% SDS, 20% glycerol, 200 mM P-mercaptoethanol, 0.2% bromophenol blue) and heated for 10 min at 95°C in dry heating bath. Proteins in lysate were resolved on 10-12% Tris-glycine gel in SDS running buffer at 100V. Resolved protein in the gel was transferred to Immobilon-P polyvinylidene fluoride membrane (0.45 um pore size; Millipore, India) in transfer buffer containing 48 mM Tris, 39 mM glycine and 200ml/lit methanol at 100V for 1.5 - 2 hours. After transfer, membrane blots were dried and activated in methanol, then washed for 5 min in phosphate buffered saline plus 0.05% Twin-20 (Merck, USA) (PBST). Membranes were then blocked for 30 min in blocking solution containing 5% non-fat dry milk prepared in PBST. Following washing with PBST twice, blots were probed with primary antibody at 4°C overnight or at room temperature for 1-2 hours (or overnight as described in the data sheet), on a rocker. All antibodies were diluted in PBST. Anti-GAPDH antibody or anti-H3 (histone H3), as appropriate, was used to normalize protein loading. After overnight incubation, membranes were washed three times for 5 min each with PBST and incubated with HRP-conjugated secondary antibody for 30min at room temperature on slow rocker. Membranes were washed 3-4 times for 5 min each with PBST. Signals were detected using SupersSignal West Pico enhanced chemiluminescent substrate (ECL) (Pierce, USA) or SuperKine enhanced/hypersensitive ECL substrates (Abbkine, USA) and visualized by ChemiDoc XRS (Bio-Rad, USA) system. The images of protein bands were quantified by ImageJ software (NIH, Bethesda, USA) and were statistically analysed by commercially available GraphPad Prism 5 software.

The following antibodies: Anti-CD 133; phospho-ERK (Thr 202/ Tyr 204), Total Erk, phospho-Akt (Ser 473), were all purchased from Cell Signalling Technology, USA. Anti-Gapdh or Anti-histone H3 (Cell Signaling Technology) was used as loading control for each blot.

Results : It is well-known that the cells with higher expression of marker protein called CD133 in CSC and GSCs are correlated with tumour metastasis, chemo- or radio-resistance and recurrence of aggressive tumorigenesis [Singh et al 2005; Seymour et al 2015; Schmohl and Vallera 2016]. To assess whether the drug combination (combination as recited in Example 2.1) has the ability to suppress CD 133 protein levels, the tumour spheroids were collected from culture dishes and were subjected to immunoblot analysis. For this analysis, LN-18 cells were grown in tumour spheroid culture as described above. After 48 hours, spheroids were collected after each treatment (drug treated versus untreated) as described in Figure 3. The collected spheroid samples were homogenised, total protein extracted and the extracted protein was subjected to immunoblot analysis as described in the method section. The CD 133 protein band was detected on blot using anti-CD133 antibody and the intensity of the detected bands were analysed as shown in Figure 3. Significant decrease in CD133 protein was observed in LN- 18 spheroids when treated with D+E as compared to the controls (no drug) and single drug forms (figure 3). This shows that the drug combination lowers CD133 protein expression in the spheroids and lead to reduction in tumorigenic, metastatic and self-renewal potential of the cells.

Apart from CD133 protein, activation of other signalling pathways regulated by proteins like PI3K/Akt, and ERK enables the cancer cells to maintain enhanced self-renewal capacity and compromised differentiation, consequently leading to aggressive tumorigenesis. The levels of these key proteins by immunoblotting were evaluated in control and drug-treated spheroid culture cells, as above. As shown in Figures 4 A and 4B, drug combination, decreased the level of p-ERK (phosphorylated -ERK), and p-AKT (phosphorylated AKT) proteins respectively, as compared to control. This indicates that these drug combinations induced the differentiation of glioblastoma stem cells by acting on key signalling pathways responsible for maintenance of cellular sternness and aggressive proliferation.

To demonstrate the differentiation ability of the drug combinations, the present inventors have also performed immunocytochemistry using antibody against cellular actin (Green in Figure 5) using adherent culture of LN-18 cells. As shown in Fig. 5, E+D drug combination (as mentioned in Example 2.1) induces morphological changes of typical astrocytes into differentiated forms (elongated cells with extended processes). Unlike the mainly polygonal morphology of control, the shape of drug-treated LN- 18 cells exhibited smaller round cell bodies and much longer, fine, tapering processes.

2.4. In-vivo tumour development (Syngeneic Xenograft) in Mice

Method : To investigate the effects of the drug combination on the tumour growth progression, tumour was developed in C57/BL/6 mouse strain by subcutaneous injection of tumour forming Glioblastoma cell line GL261 as described before [Szatmari et al 2006; Maes et al 2011]. In brief, 3 x 10⁶ GL261 Cells were cultured in T75 Flask using Dulbecco’s modified Eagle’s Medium (Invitrogen) with 10% (vol/vol) Fetal Bovine Serum (FBS), 1% penicillin/ streptomycin mixture, and 2 mM L- Glutamine. On achieving 70-80% confluency, 10⁶cells/ lOOpl were prepared and injected in the hind-leg subcutaneously into 6-8 weeks old mice (Females) using 1ml Syringe (26G) to develop the tumour. After 15-18 days, on achieving tumour size of about 4mm, treatment with drugs were initiated. Doses in mg/kg of mice = 1.2 mg/kg of Aspirin + 1.2 mg/kg of Diazepam is equivalent to what was used in in-vitro experiments, i.e., 15 microgram/ml each of drug component.

E and D were pre-mixed in 1: 1 ratio and injected s.c. (sub-cutaneous) near the tumour for efficacy studies.

The vehicle control was prepared mixing and diluting 20% DMSO/ 80% ethanol in PBS without drugs such that the final injectable concentration of DMSO is 3% and ethanol is about 12%. For animal experiments, the drugs diazepam and aspirin, were typically mixed and injected either sequentially or simultaneously pre-mixed in the vehicle as mentioned in details in Example 1.

Stock of Drug D (2 mg/ml) solution was prepared in a mixture of 20% DMSO and 80% Ethanol as solvent. The Drug E (2 mg/ml) was dissolved in phosphate - buffered saline (IxPBS, pH 7.2). Animals were separated into two groups - Control and Treatment groups. For the treatment group, 15 pl of Drug D (30 pg) was mixed with 15 pl Drug E (30 pg) and the volume was adjusted to 100 pl (by adding 70 pl PBS) for injection in each animal. For Control group, the final lOOpl of vehicle solvent, prepared for injection had of 12% Ethanol and 3% DMSO in PBS (with no drugs) matching the solvent composition of the treatment group, was injected sub-cutaneously at the base of the tumour every day, for next 21 days or till the animals survived. Size of the tumour and weight of the animals in all groups were recorded every alternate day.

Result: The effects of these drugs were ascertained on the survivability and remission of the in-vivo tumour in syngeneic animal models. The combined drugs were found to be effective in increasing the survival rate of the animals with tumours (Figure 6) and remission of tumours (Figure 7). The treatment started when the tumours are about 4 mm diameter. In 57% of the animals with syngeneic xenograft animal models, the combined drug treatment for 15 days completely causes the tumours to disappear or decrease in size to negligible volume. 2 pairs of animals with remission of tumours with pre-drug treatment (starting from zero day) and post (15 days of complete treatment) are shown in figure 7 A, and B, as representative images.

Conclusion:

The combination drugs have shown anti-tumour properties and have efficacy in inhibiting cell proliferation by triggering cellular differentiation of aggressively proliferative cancer-stem cells. The drug combinations were found to possess synergistic effects in anti-tumour activity.

Example 3

Method: Determination of dosage ratio of drug combination (E+D) by immunofluorescence analysis of adherent EN-18 cells by monitoring the expression of CD133. CD133 protein marker is an indicator of reduction in tumorigenic/metastatic potential of the cells as described before. This method of protein expression analysis has been reported previously in Ganguly S, et al., Neural adrenergic/cyclic AMP regulation of the immunoglobulin E receptor alpha-subunit expression in the mammalian pinealocyte: a neuroendocrine/immune response link? J Biol Chem. 2007 Nov 9;282(45):32758- 64. doi: 10.1074/jbc.M705950200. Briefly, about 0.1 million cells per well of a 2- well chambered slide, were treated with increasing concentration of D (0, 1, 5 and 15 microgram per millilitre) while keeping the concentration of E constant at 15 microgram per millilitre. After 48 hours of incubation with drugs, cells were washed, fixed in 2% paraformaldehyde and permeabilized by ice-cold methanol. Following which, the cells were incubated with antibodies as appropriate and images captured under microscope as described previously.

Conclusion:

Figure 8 illustrates the effect at various dose ratio of Acetylsalisylic acid (E) : Diazepam (D) as observed by immuno-fluorescence microscopic imaging of dual stained LN-18 cells (Glioblastoma cell line). The Green colour represents CD133 expression (using anti-CD133 antibody staining) and the blue colour [DAPI or 4',6-diamidino-2-phenylindole staining of the cell nucleus] represents nucleus of each co- stained cell. Cont is the control set of images where only vehicle was used and the numbers represent the increase in concentration of D from 1 (EDI) microgram per millilitre to 15 microgram per millilitre (ED 15) with concentration of E remaining constant at 15 microgram per millilitre. The cells were co-stained with anti-CD133 staining (green) and the DAPI (blue) of the same cells. The image pair of each sample represents visualization of CD133 expression (green only) and corresponding merging of CD 133 image with DAPI (Green and Blue) nuclear staining.

As evident from the images in Figure 8, the intensity of the green colour is decreased with the increase in concentration of D. Thus, it suggests that the expression of CD 133 is inhibited as the concentration of D was increased from 1 (EDI) to 15 microgram per millilitre (ED15), keeping E constant at 15 microgram per ml. The expression of CD 133 in the control sample was almost similar to the samples (EDI) treated with 1 microgram per millilitre concentration. As CD133 is the marker of self-renewal/metastatic/tumerigenic potential of cancer cells, reduction of its expression is an indication of the efficacy of the drug used. Taking together, it can be concluded that concentration range of D from 5 to 15 microgram per millilitre in combination with 15 microgram/ml of E is the most efficacious dose. Thus, if concentration of D is equal or < 1 microgram/ml in combination with 15 microgram /ml of E, the combination is not effective.

Claims

CLAIMS:

1. An anti-proliferative and cell self-renewal inhibiting drug combination comprising diazepam and acetylsalicylic acid, wherein said diazepam and said acetylsalicylic acid is in a ratio ranging from 1:3 to 3: 1.

2. An anti-proliferative and cell self-renewal inhibiting pharmaceutical composition comprising a combination of diazepam and acetylsalicylic acid in a ratio ranging from 1:3 to 3: 1 and pharmaceutically acceptable excipients.

3. The pharmaceutical composition as claimed in claim 2, wherein said diazepam is present in a dose from 5 to 15pgm/ml.

4. The pharmaceutical composition as claimed in claim 2, wherein said aspirin is present in a dose 5 to 15pgm/ml.

5. The pharmaceutical composition as claimed in claim 2, wherein said diazepam is present in a dose from 5 to 15pgm/ml and aspirin in a dose 15pgm/ml.

6. The pharmaceutical composition as claimed in claim 2, wherein said pharmaceutical composition is for treating cancer.

7. The pharmaceutical composition as claimed in claim 2, wherein said pharmaceutical composition is for preventing post-operative recurrence of cancer.

8. The pharmaceutical composition as claimed in claim 2, wherein said cancer is selected on the basis of the presence of cancer stem-cells within the tumour, example but not limited to breast cancer, pancreatic cancer, hepatocellular carcinoma, preferably glioblastoma.

9. The pharmaceutical composition as claimed in claim 2, wherein said pharmaceutical composition resulted in 75% survival rate.