SYNERGISTIC EFFECT OF MEMANTINE AND NICOTINAMIDE ON HUMAN GLIOBLASTOMA MULTIFORME CELL LINE

Abstract

The present invention provides a synergistic combination of Memantine and Nicotinamide is provided that has a cytotoxic effect on glioblastoma (U87 cell lines) cancer cell line. Memantine which is a N-methyl-D-aspartate (NMDA) antagonists basically used for treatment of Alzheimer’s disease demonstrates an additional effect by exhibiting cytotoxicity on a glioblastoma cancer cell line. When combined with a specific phytocompound (Nicotinamide), it augments its cytotoxic impact while avoiding any adverse effects on healthy normal cells. The compounds were mixed in different concentration ratios aligned with their respective IC50 values, and an MTT assay was conducted. The synergistic impact between Memantine and Nicotinamide was observed at a ratio of 1:1, for a CI value of 74.95%.

Specification

Description:FIELD OF THE INVENTION

The present invention relates to cancer therapeutics and more particularly, relates to the synergistic cytotoxic effect of Memantine (MEM) and Nicotinamide (NAM) on the cytotoxic effect of the drug on glioblastoma (U87) cancer cell line by in vitro analysis.

BACKGROUND OF THE INVENTION

Glioblastoma, previously known as glioblastoma multiforme (GBM), is the most aggressive and prevalent type of brain cancer, characterized by a very poor prognosis for survival. It accounts for the majority of primary malignant brain tumors, making up roughly 16% of all central nervous system neoplasms. While GBM typically originates within the brain, it can also develop in other regions such as the brain stem, cerebellum, and spinal cord.
The distribution of GBM across the brain is significant, with the majority of primary gliomas, approximately 61%, found in the brain's four lobes. Specifically, about 25% occur in the frontal lobe, 20% in the temporal lobe, 13% in the parietal lobe, and 3% in the occipital lobe. This wide distribution underscores the complexity of the disease, affecting critical areas responsible for vital functions.
The typical timeline for developing a new drug and bringing it to market spans 10 to 15 years. A pioneering approach in drug development entails repurposing existing medications for new applications, a practice commonly referred to as "drug repurposing."
Drug repurposing offers a cost-effective approach to cancer treatment by using existing medications with known safety profiles, reducing the time and expense of drug development. Many repurposed drugs have shown promising anticancer activity, providing new therapeutic options that can overcome drug resistance and improve patient outcomes with fewer side effects.
Memantine (MEM) is FDA approved drug the pharmacokinetic, pharmacodynamics, and toxicity profiles of drugs have been already established in the preclinical and clinical studies which significantly reduces the cost development process. It is widely known for its use in treating dementia related to Alzheimer's disease, functions by targeting the N-Methyl-D-Aspartate (NMDA) receptor subtype within the glutamate receptor system. As an NMDA receptor antagonist, Memantine works to slow down neurotoxic processes that are believed to contribute to neurodegenerative disorders, making it a crucial therapeutic option for patients with Alzheimer's disease.
In recent years, Memantine's potential has extended beyond neurodegenerative diseases, showing promise in cancer treatment. Albayrak et al. (2018) repurposed Memantine for lung cancer therapy, demonstrating its ability to induce apoptosis, or programmed cell death, in A549 lung cancer cell lines. This discovery opened the door to further exploration of Memantine's anti-cancer properties.
Cacciatore et al. (2017) conducted a comprehensive investigation into the effects of Memantine on human U87MG glioblastoma cells, a model often used to study aggressive brain tumors. Their findings revealed that Memantine exhibited a highly efficient cytotoxic effect on glioblastoma cells, suggesting its potential as a therapeutic agent for treating glioblastoma. This study not only highlighted Memantine's ability to inhibit cancer cell growth but also emphasized its relevance in targeting cancers that are typically resistant to conventional treatments.
Further research by Albayrak et al. (2021) explored the anti-cancer potential of Memantine in a Mouse 4T1 breast tumor model. Their findings confirmed Memantine's anti-cancer properties, showcasing its versatility in targeting different cancer types. These studies collectively underscore Memantine's emerging role in oncology, providing a foundation for future research into its use as a potential anti-cancer therapy.
Nicotinamide (NAM), a soluble form of Vitamin B3 (niacin), is widely recognized for its non-toxic nature, exhibiting minimal adverse effects on normal cell lines while demonstrating cytotoxic properties against breast cancer cell lines. It is found in both animal and plant-based foods and is also available as an affordable dietary supplement. The anti-cancer potential of Nicotinamide has been the subject of various studies. Sartini et al. (2013) investigated its efficacy against non-small cell lung cancer cells, while more recent research by Yousef et al. (2022) examined its effects on two human cancer cell lines: HCT-116 (colorectal cancer) and HepG-2 (hepatocellular carcinoma). These studies suggest Nicotinamide's promising role in cancer treatment, as it appears to selectively target cancer cells while sparing healthy ones.
The innovative approach of combining the natural compound with MEM augments improves the cytotoxic effect and sensitivity of the drug. This synergistic interaction between MEM and NAM has been established in the context of Glioblastoma (U87) cells.
Presently available treatments for glioblastoma include radiation therapy, targeted therapy, hormonal therapy, and chemotherapy. However, these traditional approaches face substantial challenges. Cancer cells often develop resistance to conventional therapies, which not only reduces their effectiveness but also leads to toxic adverse effects on normal, healthy cells. These therapies, while aimed at controlling tumor growth, can cause significant collateral damage, resulting in severe side effects and long-term health complications.
Moreover, aggressive or resistant forms of glioblastoma further complicate treatment efforts. The limitations of presently available therapies, such as chemotherapy, radiation, and surgery, extend beyond their immediate effectiveness. These interventions frequently lead to compromised quality of life due to their harsh side effects, and their impact on healthy tissues can cause enduring health issues. Additionally, the high financial cost of prolonged treatments adds another layer of burden, further underscoring the urgent need for more targeted, less toxic therapies that can offer better outcomes for patients while minimizing long-term consequences.
Therefore there is a need for one or more compounds that forms a combination to effectively inhibit the growth of cancer cells. Since MEM is an FDA-approved drug, it's pharmacokinetic, pharmacodynamics, and toxicity profiles have already been established. The synergy observed between MEM and NAM demonstrates a potent cytotoxic effect on breast cancer cell lines. Furthermore, the inherently non-toxic nature of NAM results in minimal adverse reactions on normal cell lines, while exerting a pronounced cytotoxic effect on glioblastoma (U87) cancer cell line.
OBJECTIVES OF THE INVENTION
The primary objective of the present invention is to provide a synergistic effect of Memantine and Nicotinamide on Glioblastoma (U87) cells.

Another objective of the present invention is to provide a synergistic combination of Memantine and Nicotinamide at a ratio of 1:1.

Yet another objective of the present invention is to provide a synergistic combination that exhibits less adverse reaction on normal cell lines and cytotoxic effect on glioblastoma cancer cell lines.
Other objects, features, and advantages will become apparent from the detailed description and appended claims to those skilled in the art.

SUMMARY OF THE INVENTION
An aspect of the present invention is to provide a synergistic combination of Memantine and Nicotinamide is provided that has a cytotoxic effect on glioblastoma cancer cell line (U87 cell line). Memantine, an antagonist demonstrates an additional effect by exhibiting cytotoxicity on a glioblastoma cancer cell line. When combined with a specific phytocompound, it augments its cytotoxic impact while avoiding any adverse effects on healthy normal cells. The compounds were mixed in different concentration ratios aligned with their respective IC50 values, and an MTT assay was conducted. The synergistic impact between Memantine and Nicotinamide was observed at a ratio of 1:1, displaying a CI value of 0.005 for U87 cells.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be described in more detail herein with the detailed description that relates well with the preferred embodiments of the invention as explained with reference to the following accompanying schematic drawings:
Figure 1: illustrates Figure 1(a) the dose effect curve and Figure 1(b) table demonstrating the fraction of affected cells upon treatment with the drug and natural compounds, according to an embodiment of the present invention;
Figure 2: illustrates the wound healing assay for cells treated with Memantine and Nicotinamide in combination and alone Fig. 2, according to an embodiment of the present invention;
Figure 3: illustrates the Annexin V and cell death assay analyzed by Muze cell analyzer cell apoptosis analysis on U87 cells, (a) control (b) Memantine (c) Nicotinamide and (d) MEM + NAM, according to an embodiment of the present invention;
Figure 4: illustrates the cell cycle arrest analyzed apoptosis analysis on U87 cells, (a) control (b) Memantine (c) Nicotinamide and (d) MEM + NAM, according to an embodiment of the present invention;
It will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure, that the examples/results shown in the figures are not necessarily drawn to scale.
DETAILED DESCRIPTION OF THE INVENTION
The preferred embodiments are provided so that the disclosure will be thorough and will fully convey the scope to those who are skilled in the art. Various specific details are set as specific components to provide an overall understanding of the preferred embodiments of the present disclosure. It will be apparent to those skilled in the art that the specific details need not be employed and the embodiments may be embodied in many different forms and the steps followed do not limit the scope of the disclosure.
In any embodiment described herein, the open-ended terms "comprising," "comprises," and the like (which are synonymous with "including," "having," and "characterized by") may be replaced by the respective partially closed phrases "consisting essentially of," consists essentially of," and the like or the respective closed phrases "consisting of," "consists of", and the like.
As used herein, the singular forms "a", "an" and "the" designate both the singular and the plural, unless expressly stated to designate the singular only.
An aspect of the present invention relates to the synergistic effect of Memantine and Nicotinamide on glioblastoma (U87) cancer cell line by in vitro analysis. The components are listed in the below table:

Table 1: List of drug and natural compound

S. No. Drug Nature of Drug
1. Memantine An antagonist of the N-methyl-D aspartate receptor (NMDAR) subtype of glutamate receptor
2. Nicotinamide It is a form of vitamin B3 found in food and used as a dietary supplement and medication

The synergistic effect of Memantine (MEM) and Nicotinamide (NAM) showed a cytotoxic effect on Glioblastoma cancer cell line (U87). (3-(4, 5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT assay) was carried out to assess the cell metabolic activity. The 50% inhibitory concentration, IC50 value was calculated by plotting the dose response curve. The values are tabulated as below:
Table 2: IC50 value of Memantine and Nicotinamide
S. No Drug IC50 Value on U87 cells
1. Memantine 43.97 ± 1.46 µMol
2. Nicotinamide 1297.90 ± 2.46 µMol
3. Memantine + Nicotinamide (1:1) Combination index value was 0.005* (According to Chou Talay theorem, the combinational index (CI) value of the drugs can be interpreted as synergistic (CI 1)
Combination index analysis
The combination index (CI) effect: The CI is calculated for each combination of drugs. The CI quantifies the nature of the drug interaction as follows:
• CI < 1: Synergistic effect (greater than additive)
• CI = 1: Additive effect
• CI > 1: Antagonistic effect (less than additive)
The compounds were combined in various concentration ratios based on their respective IC50 values, followed by the execution of the MTT assay. As mentioned in Figure 1 (a, b) the observed synergistic effect between MEM and NAM was identified at a ratio 1:1, demonstrating a CI value 0.005 for U87 cells.
A wound healing assay was performed to study the migration activity of the cells at IC50 value. Findings indicate that both Memantine and Nicotinamide inhibit the migration of the cells. However, the combination of Memantine and Nicotinamide notably enhanced the inhibition of cell migration as illustrated in Table 3 and Figure 2.
Table 3: % wound healing closure area of U87 cells
Memantine Nicotinamide Memantine+ Nicotinamide
U87 cells 43.51% 55.63 % 31.35%

With reference to Figure 4, the Annexin V and cell death assay, commonly referred to as the cell apoptosis assay, is demonstrated as a method to evaluate the presence of apoptotic cells within a given sample. This assay was conducted to quantitatively assess cell viability by distinguishing between live cells, early apoptotic cells, late apoptotic cells, and necrotic or dead cells. Annexin V, a protein widely used in apoptosis detection, plays a crucial role in this assay. During apoptosis, a key event is the externalization of phosphatidylserine (PS), which is normally located on the inner leaflet of the plasma membrane. As cells undergo apoptosis, PS is translocated to the outer leaflet, where it becomes accessible to Annexin V. By binding specifically to PS, Annexin V enables the identification of apoptotic cells.
To enhance detection, Annexin V is conjugated to a fluorescent dye, allowing for the precise labeling of apoptotic cells. Techniques such as flow cytometry are then employed to analyze and quantify the fluorescence signal, enabling the clear distinction between different cell populations based on their apoptotic or non-apoptotic states. This approach provides an accurate and efficient method for assessing the extent of apoptosis and cell death in a sample.
It was observed the enhanced apoptosis in synergistic combination of Memantine and Nicotinamide 74.95% which was very high as compared to individual drug treatment which was 44.05% and 13.40% in case of Nicotinamide as illustrated in Table 4 & Figure 3.
Table 4: Cell death observation when cell treated alone and in combination of Memantine + Nicotinamide on U87 cells
U87 Cells Memantine Nicotinamide Memantine + Nicotinamide
Live Cells 45.05% 85.90% 25.00%
Early apoptosis 6.65% 1.95% 40.00%
Late Apoptosis/Death 37.40% 11.45% 34.95%
Total apoptosis 44.05% 13.40% 74.95%

With reference to Figure 4, there is shown a Cell Cycle arrest analysis by Guava® and Muse® cell analyzer. In the cell cycle arrest cell temporarily halts its progression through the cell cycle. The cell cycle is the series of events that a cell goes through to prepare for and complete cell division, and it consists of phases such as G1 (Gap 1), S (Synthesis), G2 (Gap 2), and M (Mitosis). Cell cycle arrest can occur at various checkpoints, depending on cellular signals, damage, or specific regulatory proteins.
At various checkpoints, such as the G1/S checkpoint, the G2/M checkpoint, and the spindle checkpoint during mitosis, cells can undergo cell cycle arrest. Before progressing to the subsequent stage of the cell cycle, the cell evaluates the integrity of the DNA, monitors cell growth, and verifies appropriate chromosome alignment at these critical checkpoints.
Numerous internal and external factors, including DNA damage, replication stress, activation of tumour suppressor pathways, reduction in growth factors, exposure to specific medications, or particular environmental circumstances, can trigger cell cycle arrest. This arrest allows cells the opportunity to repair DNA damage, address cellular stress, or, in case of irreparable damage, initiate programmed cell death.
The Muse® Cell Cycle Kit was used for facile and rapid quantitative measurements of the percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle on the Guava® Muse® Cell Analyzer. It was observed that the cell cycle arrest percentage at S, G2/M was significantly higher when treated in combination as compared to the cells treated alone with MEM and NAM as shown in Table 5 & Figure 4.
Table 5: Cell cycle arrest in breast cancer cells when treated in synergistic combination and alone on U87 cells
U87 cells Memantine Nicotinamide Memantine + Nicotinamide
G0/G1 60% 46.2% 29.1%
S 8.1% 31.6% 2.5%
G2/M 19% 13.6 % 0.8%

The drug, originally developed and primarily used for treatment of Alzheimer's disease, has demonstrated promising secondary applications, particularly in its cytotoxic effect on brain cancer cell lines. When combined with specific phytocompounds, the drug's cytotoxic properties are significantly enhanced. Remarkably, this combination exhibits a targeted action, showing no adverse effects on normal, healthy cells, making it a potential candidate for more focused cancer therapies with minimal side effects.
Although a preferred embodiment of the invention has been illustrated and described, it will at once be apparent to those skilled in the art that the invention includes advantages and features over and beyond the specific illustrated construction. Accordingly, it is indented that the scope of the invention be limited solely by the scope of the hereinafter appended claims, and not by the forgoing specification, when interpreted in light of the relevant prior art. , Claims:1. A synergistic pharmaceutical composition comprising an antialzheimer agent and phytocompound wherein, an antialzheimer agent is Memantine and phytocompound is Nicotinamide in a ratio of 1:1.

2. The synergistic pharmaceutical combination as claimed in claim 1, wherein the said combination exhibits a synergistic cytotoxic effect on Glioblastoma U87 cell line.

3. The synergistic pharmaceutical combination as claimed in claim 1, wherein the Combination Index (CI) of Memantine and Nicotinamide is 0.005 for Glioblastoma U87 cells.

4. The synergistic pharmaceutical combination as claimed in claim 1, wherein the combination of Memantine and Nicotinamide shows enhanced apoptosis of 74.95%.

5. The synergistic pharmaceutical combination as claimed in claim 1, wherein the synergistic effect of the Memantine and Nicotinamide investigated by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Documents