WO2018106988A1

WO2018106988A1 - Biologically active compositions containing two different classes of chemical compounds for treating solid tumors

Info

Publication number: WO2018106988A1
Application number: PCT/US2017/065241
Authority: WO
Inventors: Tao FU; Zuoxiang XIAO
Original assignee: Smet Pharmaceutical Inc.
Priority date: 2016-12-09
Filing date: 2017-12-08
Publication date: 2018-06-14

Abstract

A pharmaceutical composition for treating a solid tumor includes using a combination of two different classes of chemicals: tricyclic thienotriazolodiazepin, a stain, and a pharmaceutical formulation. Using JQl as a representative of tricyclic thienotriazolodiazepin, and Lovastatin as a representative of statin, the combination has brought forth the achievement of successful treatment of solid tumors, especially with respect to liver tumor and lung tumor.

Description

PATENT COORPERATION TREATY APPLICATION

BIOLOGICALLY ACTIVE COMPOSITIONS CONTAINING TWO DIFFERENT CLASSES OF CHEMICAL COMPOUNDS FOR TREATING SOLID TUMORS

CROSS REFERFENCE

This Patent Cooperation Treaty Application is based on, and claims the benefit of U.S. Provisional Patent Application No. 62/432,224, filed on December 9, 2016.

FIELD

Embodiments of the invention relate to biologically active compositions using a combination of two different classes of compounds and a pharmaceutical formulation for treating solid tumors. The first class of chemical compounds are tricyclic thienotriazolodiazepins that can be represented by JQl. The second class of chemical compounds are naturally occurring and synthetic statins known for lowering cholesterol that can be represented by lovastatin.

BACKGROUND

A solid tumor is a collection of abnormal tissues that can be benign or malignant, and is usually named after the type of tissue that the solid tumor originates, such as liver, lung, colon or breast. In contrast, a hematological tumor is a cancer affecting the blood, bone marrow or lymph node. According to the Centers for Disease Control and Prevention in Atlanta, Georgia, U.S.A., the ten top cancer death rates in 2008 to 2012 per 100,000 people of all races are: #1: lung and bronchus (47.2), #2: female breast (21.9), #3: prostate (21.4), #4: colon and rectum (15.5), #5: pancreas (10.9), #6: ovary (7.7), #7: leukemia (7.0), #8: non-Hodgkin lymphoma (6.2), #9: liver and intrahepatic bile duct (6.0), and #10: urinary bladder (4.4) (hLlps^/nccdxdc.gov/uscA/iopvencancerft.aftpx) (FIG. 1). It should be noted that nine out of ten top cancer rates are related to solid tumors.

The treatment of solid tumors encounters several challenging issues.

According to J.M. Brown and A.J. Giaccia (Cancer Research, 1998, 58, 1408-1416), blood vessels in solid tumors are often abnormal, distended capillaries with leaky walls and sluggish flow. Sold tumors also continuously require the growth of new capillaries, a phenomenon known as angiogenesis. Hypoxic conditions due to lack of oxygen in solid tumor may often result in inefficiency in treatment, such as resistance to chemotherapy and radiation therapy.

Paradoxically, these challenging issues related to solid tumors can be exploited as opportunities of treatment. As a result, four different of approaches to treat solid tumors are available, including using: (1) hypoxia-selective cytotoxins to take advantage of the low oxygen level, (2) liposomes that carry anticancer drugs to enter leaky tumor blood vessels, (3) inhibitors of angiogenesis, and (4) gene therapy that may be activated by low oxygen environment or by necrotic regions.

Treating cancer using at least a combination of two different anticancer agents in a synergistic manner has been reported. For example, R.K. Blackburn et al. use a combination of a heat shock protein family 90 (Hsp90) inhibitory compounds and an epidermal growth factor receptor (EGFR) inhibitor (U.S. Patent 9,205,086) ("the '086 Patent"). However, these combinations utilized in the '086 Patent do not exhibit specific activities toward lung or liver tumor. Another example of treating cancer with a combination of two different compounds is reported by I. Eliaz, who used a synergistic combination of honokiol and modified citrus pectin in cancer therapy (U.S. Patent 8,916,541) ("the '541 Patent") for cell lines related to solid and hematological tumors. Nevertheless, the effectiveness of this approach for solid tumors is not indicated in the '541 Patent.

The use of tricyclic thienotriazolodiazepins as anticancer agent has been reported. Miyoshi et al. reported that antitumor agents containing a

thienotriazolodiazepine ring system generally inhibit the binding between acetylated histone and a bromodomain-containing protein (U.S. Patent 8,476,260) ("the '260 Patent"). While the '260 Patent present biological data against about 12 cancer cell lines, however, it does not report whether these compounds can be applied to treating solid tumors.

Compound 1 of U.S. Patent 8,476,260

P. Filippakopoulos et al. subsequently reported a novel molecule "JQ1" that also contains the thienotriazolodiazepine ring system mentioned above. According to Filippakopoulos et al, JQ1 shows competitive binding with the acetyl-lysine recognition motifs (or bromodomains), and thus generates an intense interest of developing chemical probes for "bromodomain and extra terminal" (BET) domain proteins (Nature, 2010, 468, 1067-1073).

(+)- and (-)-JQl (Nature 2010, 468, 1067-1073)

While examples of BET domain proteins include BRD2, BRD3, BRD4 and BRDT, it was recognized among researchers that BRD4 should be the target for potential cancer treatment. Filippakopoulos et al. found that the (+)-enantiomer of JQl: (a) displaces the BRD4 fusion oncoprotein from chromatin; (b) induces immediate and progressive apoptosis in BRD4-dependent human carcinoma cells without leading to growth arrest or cell death in cells lacking the BRD-NUT fusion; and (c) reduces tumor volume in mice with established NMC 797 xenografts.

J.E. Bradner and J. Qi reported using JQl and similar compounds capable of disrupting interaction of BET domain proteins with chromatin (U.S. Patent

8,981,083) ("the Ό83 Patent"). According to the Ό83 Patent, the compounds therein are potentially inhibitory to the growth of neoplastic cells, and thus the compounds are useful in treatment of neoplasia, inflammation, obesity, diabetes, etc. (see '083 Patent, column 30, lines 8-30). However, the '083 Patent indicates that JQl has a half-life of only 1 hour in FIG. 11 therein, and this observation would diminish the use of JQl in further clinical development as a treatment of cancer.

D. H. Lee et al. use a combination of JQl and rapamycin to treat human osteosarcoma (International J. Cancer, 2014, 136, 2055-2064). Lee et al. observed that JQl alone failed to reduce the size of xenografts of the human osteosarcoma cell line MNNG/HOS in immunocompromised mice. In contrast, by combining JQ1 with rapamycin, which is by itself a mammalian target of rapamycin (mTOR) inhibitor, Lee et al. reported that a synergistic effect of the reduction of tumor size was achieved.

However, no tricyclic thienotriazolodiazepin has been found to be effective for the treatment of solid tumors. More research and investigation are needed.

Traditionally, statins, or 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are associated with lowering long-term cardiovascular events. Recently, statins have been recognized to play an interesting role in treating cancer. K. Hindler et al. report that statins exhibit several effects, including the inhibition of tumor cell growth, the inhibition of angiogenesis, the induction of apoptosis, and the repression of tumor metastases (The Oncologist 2006, 11, 306-315).

The use of statins for the treatment of cancer in several human clinical trials have been known, but further studies must be carried out before meaningful results can be achieved. This is because: (a) the tumor types most susceptible to statin treatment have not been determined; (b) the most effective statin for cancer treatment or prevention has not been identified; and (c) the optimal statin regiment has not been defined (see Hindler et al., The Oncologist 2006).

The statin Lovastatin has been shown to exhibit synergistic cytotoxicity in a variety of tumor derived cell lines when being combined with gefitinib, a potent epidermal growth factor receptor (EGFR) inhibitor by T.T. Zhao et al. (PLoS ONE, 2010, 5(9): el2563). According to this study, Lovastatin inhibits internalization and degradation of vascular endothelial growth factor receptor (VEGFR). Thus, the combination of Lovastatin with a tyrosine kinase inhibitor such as KRN633 induced a significant decrease in the phosphorylation status of three proteins in the human mesothelioma cell line NCI-H28. However, no statin has been found to be effective for the treatment of solid tumors. More research and investigation are needed.

Therefore, there is a need for a new approach to effectively treat solid tumors, such as liver tumor and lung tumor using a synergistic combination of two different classes of compounds that are represented by tricyclic

thienotriazolodiazepins and statins.

SUMMARY

In accordance with an embodiment of the invention, a pharmaceutical composition for treating a solid tumor includes a tricyclic thienotriazolodiazepin; a statin; and a pharmaceutical formulation, wherein the composition is useful for treating the solid tumor.

In an embodiment of the invention, the tricyclic thienotriazolodiazepin is a member of the group consisting of JQ1, Ciclotizolam, Brotizolam, and Metizolam.

In an embodiment of the invention, the statin is a member of the group consisting of Lovastatin, Mevastatin, Pravastatin, Fluvastatin, Rosuvastatin,

In an embodiment of the invention, the pharmaceutical formulation is a member of the group consisting of a pill form, a tablet form, a capsule form, a liquid solution form, a liquid suspension form, a powder form, an intravenous solution form, a gel form, and a suppository form.

In an embodiment of the invention, the tricyclic thienotriazolodiazepin is JQ1 and the statin is Lovastatin. In an embodiment of the invention JQl is at a concentration ranging from about 25 mg/kg to about lOOmg/kg.

In an embodiment of the invention, Lovastatin is at a concentration ranging from about 30 mg/kg to about lOOmg/kg.

In an embodiment of the invention, JQl is at a concentration ranging from about 25 mg/kg to about lOOmg/kg, and Lovastatin is at a concentration ranging from about 30 mg/kg to about lOOmg/kg.

In an embodiment of the invention, JQl and Lovastatin are present in a molar ratio ranging from about 1.0:5.0 to about 5.0:1.0.

In an embodiment of the invention, the solid tumor is a member of the group consisting of liver tumor and lung tumor.

In another embodiment of the invention, a method of treating a solid tumor includes the steps of: (a) providing a tricyclic thienotriazolodiazepin; (b) providing a statin; (c) providing a pharmaceutical formulation; (d) combining the tricyclic thienotriazolodiazepin, the statin and the pharmaceutical formulation to form a therapeutically effective dosage, and (e) administering the dosage to a subject having a condition of a solid tumor.

In an embodiment of the method, the tricyclic thienotriazolodiazepin is a member of the group consisting of JQl, Ciclotizolam, Brotizolam, and Metizolam.

In an embodiment of the method, the statin is a member of the group consisting of Lovastatin, Mevastatin, Pravastatin, Fluvastatin, and Rosuvastatin. In an embodiment of the method, the pharmaceutical formulation is a member of the group consisting of a pill form, a tablet form, a capsule form, a liquid solution form, a liquid suspension form, a powder form, an intravenous solution form, a gel form, and a suppository form.

In yet another embodiment of the invention, a method to prepare a pharmaceutical composition for treating a solid tumor includes the steps of: (a) providing a tricyclic thienotriazolodiazepin; (b) providing a statin; (c) providing a pharmaceutical formulation; and (d) combining the tricyclic thienotriazolodiazepin, the statin and the pharmaceutical formulation to form a therapeutically effective dosage.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a chart showing the top 10 cancer sites in years 2008-2012.

Fig. 2 is a photograph showing the different effects of treating lung tumor with DMSO, drug A, drug B, and the combination of drugs A and B, as well as a normal lung without tumor.

Fig. 3 is a graph showing the distribution of lung tumors according to sizes after being treated with DMSO, drug A, drug B and the combination of drugs A and B.

Fig. 4 is a graph showing the distribution of lung tumors according to weights after being treated with DMSO, drug A, drug B and the combination of drugs A and B. Fig. 5 is a photograph showing the different effects of treating mouse liver tumor and mouse lung tumor with DMSO, drug A, drug B and the combination of drugs A and B.

Fig. 6 is a photograph showing the different effects of treating Human lung SCC cell line SW900 with DMSO, drug A, drug B and the combination of drugs A and B.

Fig. 7 is a photograph showing the different effects of treating

immunocompetent mice bearing lung tumors with DMSO, drug A, drug B, and the combination of drugs A and B.

Fig. 8 is a chart showing the effects on lung tumor volume after being treated by DMSO, drug A, drug B, and the combination of drugs A and B.

Fig. 9A is a bar graph showing the effects on lung tumor weights after being treated by DMSO, drug A, drug B, and the combination of drugs A and B.

Fig. 9B is a bar graph showing the effects on lung tumor weights after being treated with DMSO or the combination of drugs A and B.

Fig. 10A is a chart showing the effects on lung tumor weights after being treated with DMSO, drug A, drug B, the combination of drugs A and B.

Fig. 10B is a chart showing the effects on lung tumor weight after being treated with DMSO or the combination of drugs A and B. DETAILED DESCRIPTION OF THE EMBODIMENTS

In accordance with an embodiment of the invention, a pharmaceutical composition for treating a solid tumor includes a tricyclic thienotriazolodiazepin; a statin; and a pharmaceutical formulation, wherein the composition is useful for treating solid tumors.

It has been discovered by the inventors of this patent application that using a combination of two different classes of compounds for the treatment of solid tumors offers synergistic effect. Synergy in treating cancer may be defined as the working together of two drugs (compounds) to produce an anti-tumor effect greater than the sum of their individual effects.

It is observed that a pharmaceutical composition including a tricyclic thienotriazolodiazepin; a statin, exhibited powerful anti-lung squamous cell carcinoma (SCC) effects in lung tumor-bearing mouse models.

Tricyclic thienotriazolodiazepins are chemicals having the basic features of: (a) a thiophene ring, (b) a triazole ring, and (c) a diazepine ring all fused together to form a ring system as shown here:

Tricyclic thienotriazolodiazepins have been known for their biological activities as anxiolytic, anticonvulsant, hypnotic, sedative and skeletal muscle relaxant. In particular, some tricyclic thienotriazolodiazepins have recently been found to exhibit the effect of inhibiting bromodomain and extra-terminal (BET) proteins.

With respect to this invention, examples of tricyclic thienotriazolodiazepins include JQl, Ciclotizolam, Brotizolam, Metizolam (or Desmethyletizolam), and the like.

JQl Ciclotizolam

Brotizolam Metizolam (or

Desmethyletizolam) According to the invention, the preferred examples of tricyclic

thienotriazolodiazepins are JQl, Ciclotizolam, and Brotizolam; the more preferred examples of tricyclic thienotriazolodiazepins are JQl and Brotizolam; and the most preferred example of tricyclic thienotriazolodiazepins is JQl.

Statins are inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase). The enzyme HMG CoA reductase is the rate- limiting step in cholesterol synthesis.

HMG CoA

Statins are chemicals having the common features of a dihydroxyheptanoic acid moiety that resembles HMG CoA (see above). Statins have been known for their biological activities in preventing heart disease for patients who have high cholesterol level.

Ring

Core Structure of Statins In particular, statins have recently been found to exhibit a reduced risk a some cancers, including esophageal cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, and possibly prostate cancer.

With respect to this invention, examples of statins include Lovastatin, Mevastatin, Pravastatin, Fluvastatin, Rosuvastatin, and the like.

Lovastatin Mevastatin

Pravastatin Fluvastatin

Rosuvastatin

According to the invention, the preferred examples of statins are Lovastatin, Mevastatin and Pravastatin; the more preferred examples are Lovastatin and Mevastatin; the most preferred example is Lovastatin.

According to the invention, the pharmaceutical formulation may be utilized in conjunction with the two classes of compounds for the treatment of solid tumors.

In an embodiment of the invention, a combination of tricyclic

thienotriazolodiazepin and statin is utilized for the treatment of tumor, such that the tricyclic thienotriazolodiazepin is JQl and the statin is Lovastatin. The use of JQl and lovastatin is unique because they resulted a synergistic effect as an anti-tumor treatment in lung cancer-bearing mouse models.

Furthermore, the reason for the uniqueness of using these two particular examples is because tricyclic thienotriazolodiazepins and the statins can work together to interfere the tumor cell metabolisms and boost host anti-tumor immune responses.

In an embodiment of the invention JQ1 is at a concentration ranging from about 25 mg/kg to about lOOmg/kg.

In an embodiment of the invention, JQ1 is at a concentration ranging from about 25 mg/kg to about lOOmg/kg, and Lovastatin is at a concentration ranging from about 30 mg/kg to about lOOmg/kg.

According to the invention, JQ1 and Lovastatin are present in a molar ratio in a range from about 1.0:5.0, to about 5.0:1.0. Preferably, the molar ratio of JQ1 to Lovastatin may be in the range from about 1.0:2.5, to about 2.5:1.0. More preferably, the molar ratio of JQ1 to Lovastatin may be in the range from 1.00:1.25, to about 1.25:1.00.

By "solid tumors" (as illustrated in Lane et al., U.S. Patent 8,877,771) are meant tumors and/or metastasis (wherever located) other than lymphatic cancer, e.g. brain and other central nervous system tumors (e.g. tumors of the meninges, brain, spinal cord, cranial nerves and other parts of central nervous system, e.g. glioblastomas or medulla blastomas); head and/or neck cancer; breast tumors;

circulatory system tumors (e.g. heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue);

excretory system tumors (e.g. kidney, renal pelvis, ureter, bladder, other and unspecified urinary organs); gastrointestinal tract tumors (e.g. oesophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus and anal canal), tumors involving the liver and intrahepatic bile ducts, gall bladder, other and unspecified parts of biliary tract, pancreas, other and digestive organs); head and neck; oral cavity (lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx,

nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx); reproductive system tumors (e.g. vulva, vagina, Cervix uteri, Corpus uteri, uterus, ovary, and other sites associated with female genital organs, placenta, penis, prostate, testis, and other sites associated with male genital organs); respiratory tract tumors (e.g. nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung, e.g. small cell lung cancer or non-small cell lung cancer); skeletal system tumors (e.g. bone and articular cartilage of limbs, bone articular cartilage and other sites); skin tumors (e.g. malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and tumors involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneum and peritoneum, eye and adnexa, thyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites.

In an embodiment of the invention, the solid tumor is a member of the group consisting of tumors that develop in many parts of the body including the brain, kidneys, liver and bones.

According to the National Cancer Institute (NCI), solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancer), or malignant (cancer). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (cancers of the blood) generally do not form solid tumors. With respect to solid tumors in men and women, it has been known that solid tumors are typically named after the types of cells that compose them. Some examples of solid tumors include: breast cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, and melanoma.

With respect to solid tumors in children, it is also known that solid tumors make up about 30% of all pediatric cancers. The most common types of solid tumors in children include brain tumors, neuroblastoma, rhabdomyosarcoma, Wilms' tumor, and osteosarcoma.

Solid tumors present critical challenge to the general public because, despite significant breakthroughs in the understanding, prevention, and treatment, cancer in general continues to affect millions of people worldwide. Cancer's complexity has slowed the rate of progress being made against cancer.

With respect to the invention, the solid tumors that present the most critical challenge to the general public are liver tumor and lung tumor.

In an embodiment of the invention, the solid tumor to be treated by a combination of a tricyclic thienotriazolodiazepin and statin is liver tumor.

In another embodiment of the invention, the solid tumor to be treated by a combination of a tricyclic thienotriazolodiazepin and statin is lung tumor.

As illustrated in Examples 1 to 9, the use of the combination of JQ1 and Lovastatin have been successful in treating mouse liver tumors and mouse lung tumors:

• In Example 1, the combination resulted in a normal looking lung.

• In Example 2, the combination achieved a much decreased lung tumor size. • In Example 3, the combination also achieved a much decreased lung tumor weight.

• In Example 4, the combination gave rise to normal looking liver and lung.

• In Example 5, the combination appeared to inhibit lung SCC cells by about 50%.

• In Example 6, the combination succeeded in treating lung tumors.

• In Example 7, the combination resulted in much decreased lung tumor sizes.

• In Example 8, the combination achieved much decreased lung tumor weights.

• In Example 9, the combination also achieved much decreased lung tumor weights.

Therefore, the combination of JQ1 and Lovastatin as a treatment of liver tumor and lung tumor has been successful and unexpected. This combination will potentially contribute to a new and promising approach in cancer chemotherapy in the U.S. and other parts of the world.

In another embodiment of the invention, a method of treating a solid tumor includes the steps of: (a) providing a tricyclic thienotriazolodiazepin; (b) providing a statin; (c) providing a pharmaceutical formulation; (d) combining the tricyclic thienotriazolodiazepin, the statin, and the pharmaceutical formulation to form a therapeutically effective dosage; and (e) administering the dosage to a subject having a condition of a solid tumor.

In an embodiment of the invention regarding the method of treating solid tumor, the tricyclic thienotriazolodiazepin for the method of treating a solid tumor is a member of the group consisting of JQ1, Ciclotizolam, Brotizolam, and Metizolam (or Desmethyletizolam). In an embodiment of the invention regarding the method of treating solid tumor, the statin for the method of treating a solid tumor is a member of the group consisting of Lovastatin, Mevastatin, Pravastatin, Fluvastatin, and Rosuvastatin.

In an embodiment of the invention regarding the method to treat solid tumor, the pharmaceutical formulation for the method of treating a solid tumor is a member of the group consisting of a pill form, a tablet form, a capsule form, a liquid solution form, a liquid suspension form, a powder form, an intravenous solution form, a gel form, and a suppository form.

In an embodiment of the invention regarding the method to treat solid tumor, the tricyclic thienotriazolodiazepin for the method of treating a solid tumor is JQ1 and the statin is lovastatin.

In an embodiment of the invention regarding the method to treat solid tumor, JQ1 as part of the method of treating a solid tumor is at a concentration ranging from about 25 mg/kg to about lOOmg/kg.

In an embodiment of the invention regarding the method to treat solid tumor, Lovastatin as part of the method of treating a solid tumor is at a

concentration ranging from about 30 mg/kg to about lOOmg/kg.

In an embodiment of the invention regarding the method to treat solid tumor, as part of the method of treating a solid tumor, JQ1 is at a concentration ranging from about 25 mg/kg to about lOOmg/kg, and Lovastatin is at a

concentration ranging from about 30 mg/kg to about lOOmg/kg.

In an embodiment of the invention regarding the method to treat solid tumor, JQ1 and Lovastatin are present in a molar ration ranging from about 1.0:5.0, to about 5.0:1.0. Preferably, the molar ratio of JQ1 to Lovastatin may be in the range from about 1.0:2.5, to about 2.5:1.0. More preferably, the molar ratio of JQl to Lovastatin may be in the range from 1.00:1.25, to about 1.25:1.00.

In an embodiment of the invention regarding the method to treat solid tumor, the solid tumor is a member of the group consisting of liver tumor and lung tumor.

In an embodiment of the invention regarding the method to treat solid tumor, the solid tumor is liver tumor.

In another embodiment of the invention regarding the method to treat solid tumor, the solid tumor is lung tumor.

In yet another embodiment of the invention, a method to prepare pharmaceutical composition for treating solid tumor includes the steps of: : (a) providing a tricyclic thienotriazolodiazepin; (b) providing a statin; (c) providing a pharmaceutical formulation; and (d) combining the tricyclic thienotriazolodiazepin, the statin, and the pharmaceutical formulation to form a therapeutically effective dosage.

In an embodiment of the invention regarding the method to prepare a pharmaceutical composition for treating solid tumor, the tricyclic

thienotriazolodiazepin is JQl, and the statin is lovastatin.

In an embodiment of the invention regarding the method to prepare a pharmaceutical composition for treating solid tumor, JQl is at a concentration ranging from about 25 mg/kg to about lOOmg/kg and Lovastatin is at a

concentration ranging from about 30 mg/kg to about lOOmg/kg. In an embodiment of the invention regarding the method to prepare a pharmaceutical composition for treating solid tumor, the solid tumor is a member of the group consisting of liver tumor and lung tumor.

The success of the present technology in treating solid tumors is shown in the following Examples and Figures.

EXAMPLES

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention. The examples are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments described below are all or the only experiments performed.

To describe which chemical is being used in the following Examples, as well as in Figs. 2-10 unless otherwise indicated, "Drug A" refers to JQl, "Drug B" refers to Lovastatin, and "Drugs A+B" refers to the combination of JQl and Lovastatin.

Example 1 Pharmaceutical Inhibition of Lung Tumor Formation in Mice (Fig. 2)

Fig. 2 is a photograph showing the different effects of treating lung tumor with DMSO, drug A, drug B, and the combination of drugs A and B, as well as a normal lung without tumor. Lung squamous cell carcinoma (SCC) cells (3xl0⁵ per mouse) were injected into 8-wk-old WT FVB mice via tail veins. 10 days later, the randomly grouped mice received IP injections of DMSO, drug A, drug B, and the combination of drugs A+B individually with every other day for 4 times totally. 20 days later following the initial tumor cell injection, the lungs of the treated mice were harvested and analyzed. Pictures are the representatives of lungs from individual group after treatments of DMSO, drug A, drug B, and the combination of drugs A+B. "Normal" represents regular mice lung from mice that have not been injected with lung SCC cells. After using the combination of drugs A+B, the lung tumor appears to be identical to the lung without any tumor. This experiment implies that using the combination of drugs A+B can control lung tumors.

Example 2 The Comparison of the Sizes of Lung Tumors (Fig. 3)

Fig. 3 is a graph showing the distribution of lung tumors according to sizes after being treated with DMSO, drug A, drug B and the combination of drugs A and B. The graph shows the size of each tumor from the lungs of mice with the indicated treatments after 20 days of initial lung SCC cell injection, as analyzed by Student's t- test (mean ± SD of four mice per group). P value indicates the comparison of the group after being treated with drugs A+B with the other three groups that have been treated with DMSO, drug A, and drug B, respectively. The comparison of the tumor sizes shows that while the average tumor size for the DMSO, the drug A, and the drug B groups are about 900 micrometers, the average tumor size for the drugs A+B group is about 150 micrometers. This experiment implies that lung tumors size can be successfully decreased using the combination of drugs A+B.

Example 3 The Comparison of the Weights of Lung Tumors (Fig. 4)

Fig. 4 is a graph showing the distribution of lung tumors according to weights after being treated with DMSO, drug A, drug B and the combination of drugs A and B. The weights of lungs from mice treated with DMSO, drug A, drug B, drugs A+B and regular wild-type FVB mice, as analyzed by Student's t-test (mean ± SD of four mice per group). P value indicates the comparison of the group after being treated with drugs A+B with the other three groups that have been treated with DMSO, drug A, and drug B, respectively. The comparison of the tumor weights shows that while the average tumor weights for the DMSO, the drug A, and the drug B groups are between 0.6 mg to about 0.65 mg, the average tumor weight for the drugs A+B group is about 0.20 mg. This experiment implies that lung tumor weights can be successfully decreased using the combination of drugs A+B.

Example 4 Pharmaceutical Inhibition of Tumor Formation in Mice Livers and Lungs (Fig. 5)

Fig. 5 is a photograph showing the different effects of treating mouse liver tumor and mouse lung tumor with DMSO, drug A, drug B and the combination of drugs A and B. Pictures are the representatives of livers and lungs from individual groups. The experiments were performed as mentioned above. The pictures show that, after being treated with the drugs A+B, the mouse liver tumor and the mouse lung tumor looked normal because they do not show any sign of tumor growth. This experiment implies that mouse liver tumor and mouse lung tumor may be successfully treated using the combination of drugs A+B.

Example 5 Pharmaceutical Effects of Combined Compounds to the Human Lung SCC Cell Lines (SW900) (Fig. 6)

Fig. 6 is a photograph showing the different effects of treating Human lung SCC cell line SW900 with DMSO, drug A, drug B and the combination of drugs A and B. Human lung SCC cell line SW900 were cultured in vitro in the complete cell culture medium. The cells were treated with the DMSO, drug A, drug B and drugs A+B individually. The photographs as showed are the representative photos from the cells after the 40 hrs. treatment in each group. The photographs show that, 40 hours after treating with the drugs A+B, the Human lung SCC cells appeared be less by more than 50%. This experiment implies that there is a positive pharmaceutical effect toward human lung SCC cell line when the combination of drugs A+B is used.

Example 6 Pharmaceutical Inhibition of Lung Tumor Formation in

Immunocompetent Mice (Fig. 7) Fig. 7 is a photograph showing the different effects of treating

immunocompetent mice bearing lung tumors with DMSO, drug A, drug B, and the combination of drugs A and B. Lewis lung carcinoma cells (LLC, 5 x 10⁵ per mouse) were injected subcutaneously into the flanks of C57BL/6 mice. After 5 days, all the tumors from reach the size over 5mm in diameter, and the mice were randomly separated to 4 groups (5-7 mice for each group) and treated with DMSO, drug A,B and compound by intraperitoneal injections. The mice were euthanized at 20^th day after initial tumor inoculation and the tumor were weighed. Pictures showed here are the tumors from individual group. The photograph shows that, after being treated with the drugs A+B, the immunocompetent mice bearing tumors have decreased in size and looked relatively normal. This experiment implies that tumor formation in immunocompetent mice can be successfully inhibited using the combination of drugs A+B.

Example 7 The Combination of Drugs A+B Showed Significant Anti-Tumor Effect in Imunocompetent Mice Bearing Lung Tumors (Fig. 8)

Fig. 8 is a chart showing the effects on tumor volume after being treated by DMSO, drug A, drug B, and the combination of drugs A and B. The experiments were performed as mentioned above and the tumor sizes were measured regularly. The graph shows the comparison of size of from individual group. The analysis was performed by grouped two-way AVOVA statistical test (mean ± SD), P value indicates the comparison of groups. ***P<0.0001. The chart shows that, after 20 days, tumor sizes was about 2,000 mm³ after DMSO treatment: 1,400 mm³ after drug B treatment, about 800 mm³ after drug A treatment; and about 300 mm³ after drugs A+B treatment. This experiment implies that tumor sizes in immunocompetent mice can be successfully decreased using the combination of drugs A+B. Example 8 The Comparison of Weights of Lung Tumors (Figs. 9A and 9B)

Fig. 9A is a bar graph showing the effects on tumor weights after being treated by DMSO, drug A, drug B, and the combination of drugs A and B. Similarly Fig. 9B is a bar graph showing the effects on tumor weights after being treated with DMSO or the combination of drugs A and B. The graphs show the weights of tumors from the mice with the indicated treatments after 20 days of initial tumor inoculation, as analyzed by Student's t-test (mean ± SD), and P value indicates the comparison of the groups A, B or A+B with the DMSO group. Both Figs. 9A and 9B indicate that the treatment with drugs A+B resulted in significant decrease of tumor weights. The experiment implies that tumor weights can be successfully decreased using the combination of drugs A+B.

Example 9 The Comparison of Weights of Lung Tumors (Figs. 10A, and 10B)

Fig. 10A is a chart showing the effects on tumor weights after being treated with DMSO, drug A, drug B, the combination of drugs A and B. Similarly, Fig. 10B is a chart showing the effects on tumor weight after being treated with DMSO or the combination of drugs A and B. The graphs show the weights of tumors from the mice with the indicated treatments after 20 days of initial tumor inoculation, as analyzed by Student's t-test (mean ± SD), and P value indicates the comparison of the groups A, B or the combination of the drugs A+B with the DMSO group. Both Figs. 10A and 10B indicate that the treatment with drugs A+B resulted in significant decrease of tumor weights. The experiment implies that tumor weights can be successfully decreased using the combination of drugs A+B.

Claims

What is claimed is:

1. A pharmaceutical composition for treating a solid tumor, comprising:

a tricyclic thienotriazolodiazepin;

a statin; and

a pharmaceutical formulation,

wherein the composition is useful for treating the solid tumor.

2. The composition according to claim 1, wherein the tricyclic

thienotriazolodiazepin is a member of the group consisting of JQl, Ciclotizolam, Brotizolam, and Metizolam.

3. The composition according to claim 1, wherein the statin is a member of the group consisting of Lovastatin, Mevastatin, Pravastatin, Fluvastatin, and

Rosuvastatin.

4. The composition according to claim 1, wherein the pharmaceutical formulation is a member of the group consisting of a pill form, a tablet form, a capsule form, a liquid solution form, a liquid suspension form, a powder form, an intravenous solution form, a gel form, and a suppository form.

5. The composition according to claim 1, wherein the tricyclic

thienotriazolodiazepin is JQl and the statin is lovastatin.

6. The composition according to claim 5, wherein JQl is at a concentration ranging from about 25 mg/kg to about lOOmg/kg.

7. The composition according to claim 5, wherein Lovastatin is at a

concentration ranging from about 30 mg/kg to about lOOmg/kg.

8. The composition according to claim 5, wherein JQl is at a concentration ranging from about 25 mg/kg to about lOOmg/kg and Lovastatin is at a

concentration ranging from about 30 mg/kg to about lOOmg/kg.

9. The composition according to claim 5, wherein JQl and Lovastatin are present in a molar ratio ranging from about 1.0:5.0 to about 5.0:1.0.

10. The composition according to claim 1, wherein the solid tumor is a member of the group consisting of liver tumor and lung tumor.

11. A method of treating a solid tumor, comprising the steps of:

(a) providing a tricyclic thienotriazolodiazepin;.

(b) providing a statin; [[a«€l]]

(c) providing a pharmaceutical formulation

(d) combining the tricyclic thienotriazolodiazepin, the statin and the pharmaceutical formulation to form a therapeutically effective dosage; and

(e) administering the dosage to a subject having a condition of a solid tumor.

12. The method according to claim 11, wherein the tricyclic

13. The method according to claim 11, wherein the statin is a member of the group consisting of Lovastatin, Mevastatin, Pravastatin, Fluvastatin, and

Rosuvastatin.

14. The method according to claim 11, wherein the pharmaceutical formulation is a member of the group consisting of a pill form, a tablet form, a capsule form, a liquid solution form, a liquid suspension form, a powder form, an intravenous solution form, a gel form, and a suppository form.

15. The method according to claim 11, wherein the tricyclic

thienotriazolodiazepin is JQ1 and the statin is Lovastatin.

16. The method according to claim 15, wherein JQ1 is at a concentration ranging from about 25 mg/kg to about lOOmg/kg.

17. The method according to claim 15, wherein Lovastatin is at a concentration ranging from about 30 mg/kg to about lOOmg/kg.

18. The method according to claim 15, wherein JQ1 is at a concentration ranging from about 25 mg/kg to about lOOmg/kg, and Lovastatin is at a concentration ranging from about 30 mg/kg to about lOOmg/kg.

19. The method according to claim 15, wherein JQ1 and Lovastatin are present in a molar ratio ranging from about 1.0:5.0 to about 5.0:1.0

20. The method according to claim 11, wherein the solid tumor is a member of the group consisting of liver tumor and lung tumor.

21. A method to prepare a pharmaceutical composition for treating a solid tumor, comprising:

(a) providing a tricyclic thienotriazolodiazepin;

(b) providing a statin;

(c) providing a pharmaceutical formulation; and (d) combining the tricyclic thienotriazolodiazepin, the statin and the pharmaceutical formulation to form a therapeutically effective dosage,

wherein

the tricyclic thienotriazolodiazepin is a member of the group consisting of JQ1, Ciclotizolam, Brotizolam, and Metizolam,

the statin is a member of the group consisting of Lovastatin, Mevastatin, Pravastatin, Fluvastatin, and Rosuvastatin, and

the pharmaceutical formulation is a member of the group consisting of a pill form, a tablet form, a capsule form, a liquid solution form, a liquid suspension form, a powder form, an intravenous solution form, a gel form, and a suppository form.

22. The method according to claim 21, wherein the tricyclic

thienotriazolodiazepin is JQ1, and the statin is lovastatin.

23. The method according to claim 22, wherein JQ1 is at a concentration ranging from about 25 mg/kg to about lOOmg/kg and Lovastatin is at a concentration ranging from about 30 mg/kg to about lOOmg/kg.

24. The method according to claim 21, wherein the solid tumor is a member of the group consisting of liver tumor and lung tumor.