TECHNICAL FIELD

[01] The present disclosure relates to pharmaceutical compositions for the treatment of cancer, and more particularly for the treatment of cancer that overexpress HER2.

BACKGROUND INFORMATION

[02] Despite decades of basic and clinical research and trials of promising new therapies, cancer remains the second leading cause of death worldwide with a high morbidity and mortality rate, accounting for nearly 10 million deaths in 2020. The term “cancer” is mainly defined as a collection of related diseases that are characterized by the development and growth of abnormal cells which can divide uncontrollably, thus invading and destroying other parts of the body.

[03] The key role of the human epidermal growth factor receptor 2 (“HER2”) in tumorigenesis has been widely acknowledged in cancer research. HER2, a transmembrane tyrosine kinase receptor protein, functions as a mediator of cell growth, differentiation and survival. However, the amplification of HER2 gene and receptor overexpression can lead to the development of various types of human cancer including breast, colon, endometrial, cervical, urothelial, lung, ovarian, gastric, gastroesophageal junction, head and neck, biliary tract, prostate, and pancreatic cancer. Significantly, HER2 is highly expressed in about 5-20% of breast cancer patients and acts as an important marker for poor prognosis.

[04] The relationship between abnormal HER2 expression and cancer is important in cancer therapeutics. Therefore, HER2 has been determined as an ideal biological target for treatment of cancer, particularly breast cancer. This is because the formation and spread of cancer cells may be restricted by inhibiting HER2. As a result, many therapeutic strategies were directed at the complex mechanisms which regulate the function of HER2 in order to selectively inhibit the growth of HER2-overexpressing cancer cells. Moreover, many studies have focused on conducting ligand-based and structure-based studies to assess the potency of existing therapeutic agents or compounds as potential HER2 inhibitors. For instance, a study titled “Design, synthesis, and antitumor activity of new bis-aminomethylnapthalenes” published by Mariela Bollini et al in 2008 discloses the preparation of a series of bis-aminomethylnaphthalenes through a simple synthetic strategy using reductive amination. The synthesized compounds were evaluated for their anticancer activity and ability to act as DNA-binding ligands. None of the prior art solutions disclosed the use of these synthesized derivatives, particularly (6-Methoxy- naphthalen-2-ylmethyl)-[2-(2-{2-[(6-methoxy-naphthalen-2-ylm ethyl)-amino]-ethoxy}- ethoxy)-ethyl] -amine of general formula C30H37CIN2O4 as a potential HER2 inhibitor for treatment of cancer.

SUMMARY

[05] Therefore, it is an object of the present disclosure to provide a pharmaceutical composition including a compound of the general formula (I), or a pharmaceutically acceptable salt therefore, and at least one pharmaceutically acceptable carrier for treating cancer:

Formula (I)

[06] In aspects of the present disclosure, the pharmaceutical composition may be formulated as a solid, liquid, or semi-solid dosage form. [07] In some aspects, the pharmaceutical composition may be administered via different routes such as oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, ocular, vaginal, rectal, or intraventricular routes.

[08] In some aspects, the pharmaceutical composition may further include one or more therapeutic agents as combination therapy.

[09] In aspects of the present disclosure, the pharmaceutical composition may be used for treating different types of HER2-amplified cancer.

[010] In some aspects, the different types of HER-2 amplified cancer may be breast, colon, endometrial, cervical, urothelial, lung, ovarian, gastric, gastroesophageal junction, head and neck, biliary tract, prostate, and pancreatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[Oil] The present disclosure will now be described with reference to the accompanying drawings, which illustrate embodiments of the present disclosure, without however limiting the scope of protection thereto, and in which:

[012] FIG. 1 illustrates a line chart of tumor growth versus time in mice treated with different doses of the pharmaceutical composition prepared in accordance with embodiments of the present disclosure.

[013] FIG. 2 illustrates a bar chart of tumor weight harvested from mice after a four-week treatment with different doses of the pharmaceutical composition prepared in accordance with embodiments of the present disclosure.

[014] FIG. 3 illustrates a bar chart of percentage inhibition of tumor growth in mice after a four-week treatment with different doses of the pharmaceutical composition in accordance with embodiments of the present disclosure.

DETAILED DESCRIPTION

[015] Embodiments of the present disclosure provide a pharmaceutical composition comprising a compound of the general formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may further include one or more therapeutic agents as combination therapy.

Formula (I)

[016] In embodiments of the present disclosure, the composition may be formulated as a solid, liquid, or semi-solid dosage form.

[017] In some embodiments of the present disclosure, the composition may be administered via different routes such as oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, ocular, vaginal, rectal, or intraventricular routes.

[018] Embodiments of the present disclosure provide a use of the pharmaceutical composition for treating different types of HER2-amplified cancer. In some embodiments of the present disclosure, the pharmaceutical composition may act as a selective HER2 inhibitor.

[019] In some embodiments of the present disclosure, the different types of HER2- amplified cancer may be breast, colon, endometrial, cervical, urothelial, lung, ovarian, gastric, gastroesophageal junction, head and neck, biliary tract, prostate, and pancreatic cancer.

[020] The disclosure is now further illustrated on the basis of examples and a detailed description from which further features and advantages may be taken. It is to be noted that the following explanations are presented for the purpose of illustrating and description only; they are not intended to be exhaustive or to limit the disclosure to the precise form disclosed.

Example 1

Preparation of the pharmaceutical composition

[021] The pharmaceutical composition of the present disclosure comprising the compound of general formula (I) was synthesized with reference to a previously published method with few modifications.

[022] The synthesis of the compound was initiated by formation of bis-imines, followed by subsequent reduction of these imines with NaBFE in methanol to give the desired compound (6-Methoxy-naphthalen-2-ylmethyl)-[2-(2-{2-[(6-methoxy-napht halen-2- ylmethyl)-amino] -ethoxy} -ethoxy)-ethyl] -amine of general formula C30H37CIN2O4 with a good yield. The compound was then precipitated by HC1. All the needed characterization (NMR and 2D NMR and Mass spectroscopy) were also done to accurately determine and ensure the structure of the needed compound and its purity.

[023] The compound was then identified as a potential HER2 inhibitor through molecular modeling and in silico screening on national cancer institute (“NCI”) database.

Example 2

In vitro assay of pharmaceutical composition

[024] Anti-proliferative experiments were conducted in order to evaluate the effect of the compound on different normal and breast cancer cell lines with distinguished expression level of HER2. The compound was tested in vitro against three different breast cancer cell lines namely, SKBR3 cells (over expressed HER2 receptors), MDA 231 (not expressing HER2 cells), and MCF7 (not expressing HER2 cells). The normal noncancerous cells were the normal skin fibroblast, the normal breast cells MCF10 and cardiac cells. In comparison to normal noncancerous cells, the compound showed the best activity and selectivity against cells with HER2 overexpression (SKBR3). Example 3

Mechanism of action of pharmaceutical composition

[025] The use of the prepared compound for treatment of cancer, particularly cancer cells with HER2 overexpression lies in its mechanism of action. The mechanism of action of the compound was studied using western blotting technique. Results of this technique clearly demonstrated the ability of the compound to target HER2 receptors by binding to the ATP-binding pocket of their intracellular kinase domain, thus inhibiting the phosphorylation of HER2. As phosphorylation of HER2 is blocked, its activation along with downstream signaling pathways which promote cancer cell proliferation were also inhibited.

[026] The half maximal inhibitory concentration (IC50) of compound was found to be 450, 2500, and 2000 nM on SKBR3, MCflO and fibroblast cells, respectively.

Example 4

Determination of median lethal dose (LDso) of pharmaceutical composition

[027] Acute toxicity of a drug can be determined by the calculation of its median lethal dose (LD50) . A repeated dose toxicity study was conducted via intraperitoneal administration of compound of present disclosure in 36 male albino mice, followed by a recovery period. The intraperitoneal route was selected as it is a most probable route of human exposure in the environment. The study was based on the internationally recognized guidelines and dose levels for the toxicity study selected were based on set preliminaries in vitro cytotoxicity study.

[028] An approximate LD50 was initially determined as a pilot study by a so called "staircase method" using a small number of mice (2 each dose) and increasing the doses of the drug. Five doses for the determination of LD50 (250, 200, 150, 100, 50 mg/kg body weight) were chosen and administered intraperitoneally to 6 groups of mice, 2 in each group. The mice were observed for first 2 hours and then at the 6th and 24th hour for any toxic symptoms. After 24 hours, the number of deceased mice was counted in each group. Table 1 below shows the mortality at various doses of the tested compounds:

Table (1)

The percentage mortality at each dose level was calculated at transformed to probit as under:

0 25

For 0% mortality: 100 *

For 100% mortality: 100 * ⁿ °' ²⁵

Where n is the number of mice in each group. The probit values were then plotted against log-doses. The dose corresponding to probit 5, i.e. 50% mortality, was found out. As a result, LD50 was determined as 125 mg/Kg body weight.

Example 5

In vivo assay of pharmaceutical composition

[029] The anti-tumor activity of compound was investigated against adenocarcinoma tumor (SKBR3) implanted orthotopically in mammary pad of about 40 athymic NCR nu/nu nude mice aged 4-6 weeks with a weight of about 23 ± 2 g. After two weeks of tumour initiation, the mice were divided randomly into five groups comprising of 6 animals each. The pharmaceutical composition was administered orally by oral gavages (wt/wt) once a day for a period of 4 weeks with doses of 60 mg/kg body weight (group III), 40 mg/kg body weight (group IV), 20 mg/kg (group V). The negative control group of mice (group I) received only 0.2 ml distilled water whereas the positive control group of mice (group II) were injected intraperitoneally with 15 mg/kg Herceptin twice a week. The tumour size and body weight were recorded before starting the treatment and at weekly basis. The tumour size was calculated by applying the formula below:

, ,

Tumour volume (mm

Where L is the length, W is the width and D is the depth of the tumour measured by a calliper.

At the end of experiment, the mice were euthanized by CO2 followed by cervical dislocation. Their xenograft tumors were harvested and cut into two parts. The first tumor half was preserved in 4% paraformaldehyde for histopathology work, vascularization, tissue morphology, and apoptosis. The other tumor half was stored at -80°C until further for assessment of cell cycle parameters using flow cytometry.

[030] Reference in this example is made to FIG. 1-3. FIG. 1 illustrates a line chart showing the tumour growth profile in all the tested mice groups after treatment with different dosages of the compound over a time period of 6 weeks. In comparison to the negative control group (group I), tumour growth was supressed, showing little changes over time with increased dosage of the compound. FIG. 2 demonstrates a bar chart showing weight of harvested tumour from each tested mice group after a 4- week treatment with different dosages of the compound. Untreated mice of group I showed the largest tumour size, followed by mice treated with 20 mg/kg body weight (group III), 40 mg/kg body weight (group IV), 15 mg/Kg Herceptin (group II), and 60 mg/kg body weight (group V), respectively. As a result, it can be noted that the administration of the compound at the dose of 60 mg/kg body weight has shown more pronounced tumour suppressing effects than compared to other doses tested. FIG. 3 illustrates a bar chart of the percentage inhibition of breast tumour after a 4- week treatment of all tested mice groups with different dosages of the compound. The concentration of the compound at 20, 40 and 60 mg/kg exhibited a dose-dependent suppression of SKBR3 tumor growth with 46.3, 62.9 and 75.9% inhibition, respectively. The positive control group II treated with 15 mg/kg Herceptin showed 63.8% reduction of the breast tumour in comparison to the negative control group (group I). [031] Results of this in vivo analysis showed clear evidence that the compound possesses potent anti -tumor activity against the breast adenocarcinoma (SKBR3), thus electing the drug as an effective and potential anti-HER2 agent. Additionally, the compound showed superior activity and selectivity against the breast cancer model than the clinically used drug Herceptin which is very expensive and has many side effects.

[032] While embodiments of the present disclosure have been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various additions, omissions, and modifications can be made without departing from the spirit and scope thereof.

[033] In describing and claiming the present invention, the following terminology was used.

[034] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[035] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a defacto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[036] As used herein, the term “about”, when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[037] As used herein, the terms "treat," "treating," or "treatment," refer to reducing the severity of HER2-amplifying cancer, delayed onset of HER2-amplifying cancer, alleviated growth of HER2-amplified cancer, alleviated cell metastasis ofHER2- amplified cancer, shortened duration of HER2-amplified cancer, hindered development of HER2-amplified cancer. It includes causing cancer regression, alleviating the condition caused by HER2-amplified cancer, or stopping the symptoms caused by HER2-amplified cancer. The terms "treat," "treat," or "treatment" include, but are not limited to, prophylactic and / or therapeutic treatment