Synergistic effects between sphingosine-1-phosphate receptor antagonists and antimicrotubule agents

This invention is based on the discovery that the administration of a sphingosine-1-phosphate receptor antagonist (S1P) and at least one chemotherapeutic agent selected from the the group of antimicrotubule agents provides an unexpectedly superior treatment for cancer. Antimicrobial agents such as the taxane compounds are known in the art, for example, paclitaxel (available as TAXOL® from Bristol-Myers Squibb, Princeton, N.J.), docetaxel (available as TAXOTERE® from Sanofi-aventis, Bridgewater, N.J.) and the like and other compounds that act as antimicrotubule agents, such as Vincristine (ONCOVIN®, VINCASAR PFS®, VCR), Vinblastin (VELBAN®, VELSAR®) and Vinorelbine, and similar compounds. The present invention also provides methods of modulating the growth of selected cell populations, such as cancer cells, by administering a therapeutically effective amount of at least one sphingosine-1-phosphate 1 (S1P1R) receptor antagonists, and at least one antimicrotubule agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This disclosure relates to the S1P receptor antagonists, compositions comprising the S1P receptor antagonists and methods for using and processes for making the S1P receptor antagonists described in a patent application entitled “Sphingosine-1-Phosphate Receptor Antagonists” co-owned by Exelixis, Inc, filed concurrently and whose U.S. Provisional Application No. is 61/196,495, the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention is based on the discovery that the administration of a sphingosine-1-phosphate receptor antagonist (S1P) and at least one chemotherapeutic agent selected from the the group of antimicrotubule agents provides an unexpectedly superior treatment for cancer. Antimicrobial agents such as the taxane compounds are known in the art, for example, paclitaxel (available as TAXOL® from Bristol-Myers Squibb, Princeton, N.J.), docetaxel (available as TAXOTERE® from Sanofi-aventis, Bridgewater, N.J.) and the like. Other compounds that act as antimicrotubule agents, are the vinca alkaloids, such as Vincristine (available as ONCOVIN® from Eli Lilly and Company, Indianapolis, Ind., VINCASAR PFS®, VCR), Vinblastin (available as VELBAN® from Eli Lilly and Company, Indianapolis, Ind., VELSAR®) and Vinorelbine, and similar compounds are also known in the art. The present invention also provides methods of modulating the growth of selected cell populations, such as cancer cells, by administering a therapeutically effective amount of at least one sphingosine-1-phosphate 1 (S1P1R) receptor antagonists, and at least one antimicrotubule agent such as a taxane or a vinca alkaloid.

BACKGROUND OF THE INVENTION

Sphingosine 1-phosphate (S1P) is derived from sphingosine, which provides the backbone to all sphingolipids. Phosphorylation of sphingosine, a metabolite of the pro-apoptotic lipid ceramide, to S1P, is mediated by lipid kinases called sphingosine kinases (SphK). There are two SphK isoenzymes: SphK1 or SphK2. SIP may be reversibly deactivated through dephosphorylation by several phosphatases or irreversibly deactivated by S1P lyase. S1P is produced intracellularly in organelles and the plasma membrane and then secreted. The newly generated S1P is then secreted and is bound extensively by albumin and other plasma proteins. This provides a stable reservoir in extracellular fluids, presumably at higher total concentrations than in tissues, and rapid delivery to cell surface receptors. S1P, via its five cognate G-protein coupled receptors (GPCRs), S1P1-5 Rs, regulates diverse biological functions, including inflammatory responses, cell proliferation, apoptosis, cell migration, lymphocyte trafficking and cell senescence. Thus, coordinated activities of biosynthetic and biodegradative enzymes help maintain and regulate concentrations of S1P in the range required for physiological activities.

S1P has been shown to be an important mediator of angiogenesis and tumorigenesis. One way to modulate S1P levels is to target SphK, and thereby affect biosynthesis of S1P. SphK1 has been shown to stimulate proliferation in vitro, and is tumorigenic in vivo. It also imparts resistance to radiotherapy and chemotherapy and is elevated in some solid tumors. SphK1 inhibitors have been shown to have anti-cancer effects in vivo. These effects have been attributed to the inhibition of formation of S1P. Further, a monoclonal antibody against S1P reduces progression of or eliminates tumors in murine xenograft and allograft models. Thus, lowering levels of S1P by inhibiting SphK or by an S1P-specific antibody has anti-tumorigenic effects.

Since many, if not all effects of S1P are mediated by five GPCRs, an alternative approach to cancer therapy may be inhibition of S1P receptors. Of the five known S1P receptors, S1P1R has been shown to play an important role in vascular permeability and S1P1R knock-out mice have an embryonic lethal phenotype. Furthermore, there is increasing evidence for cross-talk between S1P1R and other growth factor receptors such as PDGFR. Thus, S1P1R receptor antagonists have the potential to offer clinical benefit as anti-cancer therapeutics.

Antimicrotubule drugs such as taxanes, vinca alkaloids and epothilones are a major category of anticancer agents (Rowinsky, E. K., and Tolcher, A. W.,Antimicrotubule agents. In: V. T. Devita, Jr., and S. Hellman, and S. A. Rosenberg (eds.)Cancer Principles and Practice, Ed. 6, pp 431-452. Philadelphia: Lippincott Williams and Wilkins, 2001). Antimicrotubule drugs work by interfering with the function of cellular microtubules, particularly the mitotic spindle. The disruption of normal spindle function leads to apoptotic cell death.

Taxanes are antimicrotubule agents and are part of a class of compounds called diterpenes. Compounds of this type are produced by and originally isolated from plants of the genusTaxus. For example, paclitaxel was originally isolated from the bark of the Pacific yew treeTaxus brevivolia. Recently, taxanes and their intermediates were isolated from other plant species as well, (Ottaggio et al.,J. Nat. Prod.2008, 71:58-60). Presently, most of the drug used for clinical use is produced by a semisynthesis (Holton et al., inTaxol Science and applications; Suffness, M., Ed.; CRC Press: Boca Raton, 1995; pp 97-121), starting from a natural precursor, 10-deadetylbaddatin III, that is more redily available from the needles of yew species as a renewable source (Hook, I. et al.,Phytochemistry1999, 52:1041-1045, van Rocendaal, E. L. M., et al.,Phytochemistry2000, 53:383-389).

The taxanes are a group of drugs that are used in the treatment of cancer. Taxanes have a unique way of preventing the growth of cancer cells, they are anti-mitotic and anti-microtubule agents. Taxanes are microtubule stabilizing agents and interfere with microtubule breakdown which results in cessation of cancer cell growth and division. Taxanes have been used in the treatment of a wide variety of cancers.

Vinca alkaloids are antimicrotubule agents and are part of a class of compounds called plant alkaloids. Vinca compounds of this type are produced and were originally isolated from plants of the genusVincaand specifically fromVinca rosea. Unlike the taxanes which are microtubule stabilizing agents, vinca alkaloids are microtubule destabilizing agents that cause microtubule depolymerization and inhibit mitotic progression and ultimately result in apoptotic cell death (Perez, E. A.,Molecular Cancer Therapeutics2009, 8:2086-2095). Vinca alkaloids have also been used in the treatment of a wide variety of cancers.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the use of at least one S1P1R receptor antagonist and at least one antimicrotubule agent produces unexpectedly superior results in the treatment of cancer.

The present invention describes methods of treating cancer in a patient in need thereof by administering to the patient a therapeutically effective amoung of at least one S1P1R receptor antagonist and at least one antimicrotubule agent, preferably a taxane compound or a vinca alkaloid compound. The S1P1R receptor antagonists of the invention are described in the Patent Application entitled “Sphingosine-1-Phosphate Receptor Antagonists” co-owned by Exelixis, Inc, filed concurrently and whose U.S. Provisional Application No. is 61/196,495. The taxanes of the invention are compounds well known in the art and are part of a class of compounds called diterpenes. Compounds of this type are produced by and originally isolated from plants of the genusTaxus. Compounds of this type interfere with microtubule breakdown which results in cessation of cancer cell growth and division. The vinca alkaloids of the invention are also well known in the art and are part of the class of compounds called plant alkaloids. Compounds of this type are produced by and originally isolated from the plantVinca rosea. Compounds of this type destabilize the microtubule resulting in the cessation of cancer cell growth and division. The S1P1R receptor antagonist and the antimicrotubule agent, preferably a taxane compound or a vinca alkaloid compound can be administered separately or as components of the same composition.

The present invention describes methods of modulating the growth of selected cell populations, such as cancer cells, by administering a therapeutically effective amoung of at least one S1P1R receptor antagonist and at least one antimicrotubule agent, preferably a taxane compound or a vinca alkaloid compound. The S1P1R receptor antagonists of the invention are described in the Patent Application entitled “Sphingosine-1-Phosphate Receptor Antagonists” co-owned by Exelixis, Inc, filed concurrently and whose Provisional Application No. is 61/196,495. The taxanes of the invention are compounds well known in the art and are part of a class of compounds called diterpenes. Compounds of this type are produced by and originally isolated from plants of the genusTaxus. Compounds of this type interfere with microtubule breakdown which results in cessation of cancer cell growth and division. The vinca alkaloids of the invention are also well known in the art and are part of the class of compounds called plant alkaloids. Compounds of this type are produced by and originally isolated from the plantVinca rosea. Compounds of this type destabilize the microtubule resulting in the cessation of cancer cell growth and division. The S1P1R receptor antagonist and the antimicrotubule agent, preferably a taxane compound or a vinca alkaloid compound, can be administered separately or as components of the same composition.

The present invention also describes compositions comprising at least one S1P1R receptor antagonist and at least one antimicrotubule agent, preferably a taxane compound or a vinca alkaloid compound. The S1P1R receptor antagonists of the invention are described in the Patent Application entitled “Sphingosine-1-Phosphate Receptor Antagonists” co-owned by Exelixis, Inc, filed concurrently and whose Provisional Application No. is 61/196,495. The taxanes of the invention are compounds well known in the art and are part of a class of compounds called diterpenes. Compounds of this type are produced by and originally isolated from plants of the genusTaxus. Compounds of this type interfere with microtubule breakdown which results in cessation of cancer cell growth and division. The vinca alkaloids of the invention are also well known in the art and are part of the class of compounds called plant alkaloids. Compounds of this type are produced by and originally isolated from the plantVinca rosea. Compounds of this type destabilize the microtubule resulting in the cessation of cancer cell growth and division. The composition can comprise a pharmaceutically acceptable carrier, excipient or diluent. These and other aspects of the invention are described in detail herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unexpected discovery that the administration of at least on S1P1R receptor antagonist and at least one antimicrotubule agent preferable a taxane compound or a vinca alkaloid compound, produces superior results in the treatment of cancer. Appropriate S1P1R receptor antagonists, taxanes and vinca alkaloids are described herein.

“Administration” and variants thereof (e.g., “administering” a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., surgery, radiation, chemotherapy, and the like), “administration” and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents such as taxanes.

Preferrably, cancer refers to AIDS related Kaposi's Sarcoma, angiosarcoma, breast cancer, carcinoma of the bladder, carcinoma of the esophagus, carcinoma of the fallopian tube, carcinoma of the pancreas, carcinoma of the prostate, cervical cancer, colorectal cancer, gastric cancer, head and neck cancer, Hodgkin's disease, leukemia, malignant glioma, malignant lymphoma, malignant melanoma, malignant neoplasm of endometrium of corpus uteri, malignant neoplasm of liver, malignant tumor of nasopharynx, malignant tumor of peritoneum, multiple myeloma, non-small cell lung carcinoma, oligodendroglioma of the brain, osteosarcoma, ovarian cancer, small cell lung carcinoma, soft tissue sarcoma and testicular cancer.

“Metabolite” refers to the break-down or end product of a compound or its salt produced by metabolism or biotransformation in the animal or human body; for example, biotransformation to a more polar molecule such as by oxidation, reduction, or hydrolysis, or to a conjugate (see goodman and gilman, “The Pharmacological Basis of Therapeutics” 8.sup.th Ed., Pergamon Press, gilman et al. (eds), 1990 for a discussion of biotransformation). As used herein, the metabolite of a compound of the invention or its salt may be the biologically active form of the compound in the body. In one example, a prodrug may be used such that the biologically active form, a metabolite, is released in vivo. In another example, a biologically active metabolite is discovered serendipitously, that is, no prodrug design per se was undertaken. An assay for activity of a metabolite of a compound of the present invention is known to one of skill in the art in light of the present disclosure.

“Patient” for the purposes of the present invention includes humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications. In another embodiment the patient is a mammal, and in another embodiment the patient is human.

A “pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found inRemington's Pharmaceutical Sciences,17thed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference or S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977;66:1-19 both of which are incorporated herein by reference. It is also understood that the compound can have one or more pharmaceutically acceptable salts associated with it.

“Prodrug” refers to compounds that are transformed (typically rapidly) in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. Common examples include, but are not limited to, ester and amide forms of a compound having an active form bearing a carboxylic acid moiety. Examples of pharmaceutically acceptable esters of the compounds of this invention include, but are not limited to, alkyl esters (for example with between about one and about six carbons) the alkyl group is a straight or branched chain. Acceptable esters also include cycloalkyl esters and arylalkyl esters such as, but not limited to benzyl. Examples of pharmaceutically acceptable amides of the compounds of this invention include, but are not limited to, primary amides and secondary and tertiary alkyl amides (for example with between about one and about six carbons). Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.

“Therapeutically effective amount” is an amount of a compound of the invention, that when administered to a patient, effectively treats the disease. The amount of a compound of the invention which constitutes a “therapeutically effective amount” will vary depending upon a sundry of factors including the activity, metabolic stability, rate of excretion and duration of action of the compound, the age, weight, general health, sex, diet and species of the patient, the mode and time of administration of the compound, the concurrent administration of adjuvants or additional therapies and the severity of the disease for which the therapeutic effect is sought. The therapeutically effective amount for a given circumstance can be determined without undue experimentation.

“Treating” or “treatment” of a disease, disorder, or syndrome, as used herein, includes (i) preventing the disease, disorder, or syndrome from occurring in a human, i.e., causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (iii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. As is known in the art, adjustments for systemic versus localized delivery, the age, weight, general health, sex, diet and species of the patient, the mode and time of administration of the compound, the concurrent administration of adjuvants or additional therapeutically active ingredients and the severity of the disease for which the therapeutic effect is sought may be necessary, and will be ascertainable with routine experimentation.

Representative S1P1R Receptor antagonist compounds of the inventions are set forth in the following Table 1.

Taxane compounds prevent the growth of cancer cells by affecting cell structures called microtubules, which play an important role in cell functions. In normal cell growth, microtubules are formed when a cell starts dividing. Once the cell stops dividing, the microtubules are broken down or destroyed. Taxane compounds stop the microtubules from breaking down, such that the cancer cells become clogged with microtubules so that they cannot grow and divide.

Other compounds that act as antimicrotubule agents, are the vinca alkaloids, such as Vincristine (available as ONCOVIN® from Eli Lilly and Company, Indianapolis, Ind., VINCASAR PFS®, VCR), Vinblastin (available as VELBAN® from Eli Lilly and Company, Indianapolis, Ind., VELSAR®) and Vinorelbine, and similar compounds are also known in the art. Compounds of this class act as microtubule destabilizing agents, causing microtubule depolymerization and ultimately cessation of cell growth and division resulting in apoptotic cell death.

Other compounds that can be used in the invention are those that act through a taxane mechanism. Compounds that act through a taxane mechanism include compounds that have the ability to exert microtubule-stabilizing effects and cytotoxic activity against rapidly proliferating cells, such as tumor cells or other hyperproliferative cellular diseases. Such compounds include, for example, epothilone compounds, such as, for example, epothilone A, B, C, D, E and F, and derivatives thereof. Other compounds that act through a taxane mechanism (e.g., epothilone compounds) that become approved by the FDA or foreign counterparts thereof are also preferred for use in the methods and compositions of the present invention. Epothilone compounds and derivatives thereof are known in the art and are described, for example, in U.S. Pat. Nos. 6,121,029, 6,117,659, 6,096,757, 6,043,372, 5,969,145, and 5,886,026; and WO 97/19086, WO 98/08849, WO 98/22461, WO 98/25929, WO 98/38192, WO 99/01124, WO 99/02514, WO 99/03848, WO 99/07692, WO 99/27890, and WO 99/28324, the disclosures of which are incorporated herein by reference in their entirety.

As described herein, the present invention is based on the unexpected discovery that the use of at least one S1P1R Receptor antagonist and at least one antimicrotubule agent, preferably a taxane or a vinca alkaloid, produces superior results in treating cancer. In one embodiment, the present invention provides methods of treating cancer and/or modulating the growth of selected cell populations (e.g., cancer cells) by administering at least one S1P1R Receptor antagonist and at least one antimicrotubule agent, preferably a taxane or avincaalkaloid. In another embodiment, the present invention provides methods of treating cancer and/or modulating the growth of selected cell populations (e.g., cancer cells) by administering at least one S1P1R Receptor antagonist and at least one compound that acts via a taxane mechanism. One skilled in the art will appreciate that the methods described in the present invention encompass administering at least one S1P1R Receptor antagonist with taxane compounds, or compounds that act through a taxane mechanism. In the methods of the present invention, the S1P1R Receptor antagonist and the antimicrotubule agent, preferably a taxane compound or a vinca alkaloid compound, can be administered simultaneously, about the same time, or at different times, or can be components of a single composition.

Compositions and Administration:

A pharmaceutical composition of the invention will contain a therapeutically effective amount of a compound of the invention, or an individual stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, with the remainder of the pharmaceutical composition comprised of one or more pharmaceutically acceptable excipients. Generally, a compound of the invention, or an individual stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof will comprise from 1% to 99% by weight of a pharmaceutically acceptable composition, with the remainder of the composition comprised of one or more pharmaceutically acceptable excipients. Typically, a compound of the invention, or an individual stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof will comprise from 5% to 75% by weight of a pharmaceutically acceptable composition, with the remainder of the composition comprised of one or more pharmaceutically acceptable excipients. Methods for preparing the dosage forms of the invention are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990).

A therapeutically effective amount of a compound of the invention will vary depending upon a sundry of factors including the activity, metabolic stability, rate of excretion and duration of action of the compound, the age, weight, general health, sex, diet and species of the patient, the mode and time of administration of the compound, the presence of adjuvants or additional therapeutically active ingredients in a composition and the severity of the disease for which the therapeutic effect is sought.

The compounds of the invention can be administered to human patients at dosage levels in the range of about 0.1 to about 10,000 mg per day. Thus, a normal human adult having a body weight of about 70 kilograms can be administered a dosage in the range of from about 0.15 μg to about 150 mg per kilogram of body weight per day. Typically, a normal adult human will be administered from about 3 mg to about 100 mg per kilogram of body weight per day. The optimum dose of a compound of the invention for a particular patient can be determined by one of ordinary skill in the art.

The S1P1R antagonist assay used to identify or analyze the compounds in Table 1 and list 1 is a fluorescent membrane potential dye measurement assay, indicative of intracellular cAMP changes due to G protein-coupled receptor activation. HEK293 cells engineered to express human S1P1 receptors and a cyclic nucleotide-gated (CNG) channel are obtained from BD Biosciences, 80300-250. The CNG channels are activated by elevated levels of cAMP, resulting in ion flux and cell membrane depolarization. Membrane depolarization is detected with a membrane potential dye. Stimulation of the cells with 5′-(N-ethylcarboxamido)adenosine (NECA), an A2b receptor agonist (Sigma, E2387), elicits an A2bR-dependent increase in cAMP.

Subsequent exposure of the cells to a S1P1R agonist suppresses the NECA induced increase in cAMP through S1P1R-specific signaling by inhibiting adenylyl cyclase and the formation of cAMP from ATP. The degree to which a test compound overcomes the S1P1R agonist suppression of the NECA induced increase in cAMP is a measure of S1P1R antagonist activity. Antagonist activity is quantified as the IC50(i.e., the concentration needed to elicited one-half of the maxium response of the test compound) and/or as the EC50(i.e., the concentration needed to elicited one-half of the NECA induced stimulation).

Day 2: Membrane potential dye (20 μL, BD Biosciences, 341833) was added to each well and the plates were incubated for 2.5 hours at ambient temperature. Test compounds (20 μL) were added to the wells at various concentrations (≦10 μM, 1 to 3 dilutions) in a NECA base solution and incubated for 90 minutes in the presence of the S1P1R agonist {4-((4-phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzyl}-3-azetidinecarboxylic acid (10 nM). The NECA-base solution contained Dulbecco's phosphate-buffered saline (Invitrogen, 14190-136), 2.5% DMSO (Fluka, 41648), 25 μM Ro 20-1724 (Sigma, B8279), and 500 nM NECA).

A HEK293 cell line that expresses the human CB1 receptor and a CNG channel (BD Biosciences, 80500-211) were used as the counterscreen. CB1R cells were stimulated with 500 nM NECA and with CB1R agonist CP-55940 (10 nM) causing a CB1R-specific decrease of NECA-induced elevated levels of cAMP. Specific S1P1R antagonists will have no effect on CB1R activation.

Assay plates were read with a PerkinElmer EnVision reader (Excitation 350 nm, Emission 590 nm) at time T=0 minutes, before compound addition and at time T=90 minutes. The signal was calculated as the ratio T90/T0. Data was analyzed in ActivityBase XE and graphs showing log of compound concentration (X-axis) vs. % activity (Y-axis) were generated for IC50determinations. Percent activity was calculated with the following formula: (Signal−Agonist Control Signal)/(NECA Control Signal−Agonist Control Signal)×100.

Suitable S1P1R and CB agonists for use in the S1P1R antagonist assay are known in the art. For example, fingolimod or 2-amino-2-(4-nonylphenethyl)propane-1,3-diol, is a known S1P1R agonist. Methods for making and using fingolimod are found in European Patent Appliation EP0627406. 1-{4-((4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzyl}-3-azetidinecarboxylic acid is a known S1P1R agonist and methods for making and using it are found in WO 03/062252. Suitable CB1R agonists are known in the art, for example, WIN55,212-2 and CP-55940 are commerically available (Sigma, W102 and C1112, respectively).

The S1P1R antagonists of this invention were assayed by the methods described in Example 1 and were found to inhibit the S1P1R agonist elicited effects at IC50and/or EC50values ranging from about 1 nM to about 2 ηM concentrations. In contrast, the S1P1R antagonists of this invention were found not to inhibit CB1 R elicited effects. The activities the S1P1R antagonists of this invention are indicated in Table 2, wherein the letters A, B and C denote, respectively, that a compound has an EC50or IC50value of (i) less than or equal to 0.3 μM, (ii) greater than 0.3 μM but less than or equal to 1 μM, and (iii) greater than 1 μM.

Synergistic Effects of an S1P1R Receptor Antagonist and a Taxane Compound in an in Vivo Non-Small Cell Lung Carcinoma Animal Model

In this experiment, a maximum efficacy dose of an S1P1R receptor antagonist was used with an optimal dose of paclitaxel (MP Biomedicals cat #193532). Compound #2, 50, 52 or 75 from Table 1 were used in these experiments and a representative result is shown inFIG. 1. Nude mice (athymic female mice, Taconic Farms, Inc., 10 animals per group) were inoculated subcutaneously with human non-small cell lung carcinoma cells (ATCC #HTB-56) (1×106cells per animal). After tumors were allowed to grow for 9 days, one group of animals was treated with a S1P1R receptor antagonist (100 mg/kg qd po). A second group of animals was treated with paclitaxel at a dose of 15 mg/kg q3d ip. A third group of animals was treated with a S1P1R receptor antagonist and paclitaxel using the same dose and schedule used for the individual agents. A fourth control group was treated with the vehicles used for each agent individually. Tumor dimensions were measured by calipers and then converted to weight using the formula: tumor weight (mg)=[tumor volume=length (mm)×width (mm2)]/2. Animals were also monitored for weight loss as an indicator of signs of toxicity. The S1P1R receptor antagonist was formulated in a compostion containing 10% NMP (n-methyl pyrrolidone)+90% corn oil. The taxol was formulated in a composition containing 12.5% Cremophor/12.5% ethanol/50% water.

Results of an experiment with one of the S1P1R receptor antagonists are shown inFIG. 1. Cells were inoculated subcutaneously on day 0 and allowed to grow for 9 days. At time of first treatment, tumors averaged approximately 100 mgs. Dosing continued until day 31 and tumors from some animals in each of the four groups were measured and averaged. Some animals in the paclitaxel group and the combined treatment group were monitored after treatment stopped for a period of time. At the end of the treatment period, tumors in the control group of animals grew rapidly to an average size of about 1500 mgs. After treatment the tumors in animals dosed with an S1P1R receptor antagonist averaged about 1150 mgs and tumors in animals treated with paclitaxel averaged about 550 mgs. Tumors in animals treated with both agents exhibited a 52% regression after treatment, averaging approximately 40 mgs. There was no evidence of toxicity in these animals. These data indicate that treatment with 51P1R receptor antagonists and paclitaxel had an unexpectedly superior (e.g. synergistic) anti-tumor effect.

Synergistic Effects of an S1P1R Receptor Antagonist and a Taxane Compound in an in Vivo Breast Cancer Animal Model

In this experiment, 4 different doses of an S1P1R receptor antagonist, 0.3 mgs/kg, 3 mgs/kg, 10 mgs/kg or 30 mgs/kg was used with an optimal dose of paclitaxel (MP Biomedicals cat #193532). Compound #2, 50, 52 or 75 were used in these experiments and a representative example is shown inFIG. 2. Nude mice (athymic female mice, Taconic Farms, Inc., 10 animals per group) were inoculated subcutaneously with human MDA-MB-231 cells breast cancer cells (ATCC #HTB-26) (1×106cells per animal). After tumors were allowed to grow for 21 days, one group of animals was treated with paclitaxel at a dose of 7.5 mg/kg q3d ip. Four groups of animals were treated with a specific concentration of S1P1R receptor antagonist and the same concentration of paclitaxel using the same schedule used for the individual paclitaxel group. Finally, a control group was treated with the vehicles used for each agent individually. Tumor dimensions were measured by calipers and then converted to weight using the formula: tumor weight (mg)=[tumor volume=length (mm)×width (mm2)]/2. Animals were also monitored for weight loss as an indicator of signs of toxicity. The S1P1R Receptor antagonist was formulated in a compostion containing 10% NMP (n-methyl pyrrolidone)+90% corn oil. The taxol was formulated in a composition containing 12.5% Cremophor/12.5% ethanol/50% water.

Results of an experiment with one of the S1P1R receptor antagonists are shown inFIG. 2. Cells were inoculated subcutaneously on day 0 and allowed to grow for 21 days. At time of first treatment, tumors averaged 100 mgs. Dosing continued for 19 days and tumors in each of the four groups were measured and averaged. At the end of the 19 day treatment period, tumors in the control group of animals grew rapidly. After 19 days of treatment the tumors in animals dosed with paclitaxel averaged about 325 mgs. Tumors in animals treated with both agents exhibited a decreasing amount of tumor growth corresponding to the increasing amount of S1P1R recepter antagonist used in combination with paclitaxel. Animals treated with the highest dose of an S1P1R receptor antagonist exhibited tumor regression at the end of treatment, tumor weight less than 100 mgs. There was no evidence of toxicity in these animals. These data indicate that treatment with S1P1R receptor antagonists and paclitaxel had an unexpectedly superior (e.g. synergistic) anti-tumor effect.

Synergistic Effects of an 51P1R Receptor Antagonist and a Vinca Compound in an in Vivo Breast Cancer Animal Model

In this experiment, a maximum efficacy dose of an S1P1R receptor antagonist was used with doses of vincristine (Yes Pharma, Ltd., CAS#2068-78-2, Lot#80702). Compound #2, 50, 52, or 75 were used in these experiments. Nude mice (athymic female mice, Taconic Farms, Inc., 10 animals per group) were inoculated subcutaneously with human MDA-MB-231 breast cancer cells (ATCC# HTB-26) (1×106cells per animal). After tumors were allowed to grow for 19 days, two groups of animals were treated with vincristine at doses of 0.1 or 1 mg/kg q3d iv. One group of animals was treated with a specific concentration of S1P1R receptor antagonist. Two groups of animals were treated with a specific dose of S1P1R receptor antagonist and the same dose of vincristine using the same schedule used for the individual vincristine groups. Finally, a control group was treated with the vehicles used for each agent individually. Tumor dimensions were measured by calipers and then converted to weight using the formula: tumor weight (mg)=[tumor volume=length (mm)×width2(mm2)]/2. Animals were also monitored for weight loss as an indicator of signs of toxicity. The S1P1R receptor antagonist was formulated in a composition containing 10%NMP (n-methyl pyrrolidone)+90% corn oil. The vincristine was formulated in normal saline (0.9% NaCl).

Results of an experiment with one of the S1P1R receptor antagonists and vincristine are as follows. Cells were inoculated subcutaneously on day 0 and allowed to grow for 19 days. At time of first treatment, tumors averaged 95 mgs. Dosing continued for 8 cycles (q3d) and tumors in each of the six groups were measured and averaged. After 8 cycles of treatment, the tumors in animals dosed with 0.1 mg/kg and 1 mg/kg vincristine averaged 659 and 26 mg, respectively. Tumors in animals treated with the S1P1R receptor antagonist and 0.1 mg/kg or 1 mg/kg vincristine averaged 260 and 5 mg, respectively, and represents 72% and 95% regression after treatment. There was no evidence of toxicity in these animals. These data indicate that treatment with S1P1R receptor antagonists and vincristine had an unexpectedly superior (eg, synergistic) anti-tumor effect.

Synergistic Effects of an S1P1R Receptor Antagonist and a Taxane Compound in an in Vivo Breast Cancer Animal Model

In this experiment, a maximum efficacy dose of an S1P1R receptor antagonist was used with an optimal dose of docetaxol (Haorui Pharma Chem, Inc; CAS#114977-28-5). Compound #2, 50, 52, or 75 were used in these experiments. Nude mice (athymic female mice, Taconic Farms, Inc., 10 animals per group) were inoculated subcutaneously with human MDA-MB-231 breast cancer cells (ATCC#HTB-26) (1×106cells per animal). After tumors were allowed to grow for 17 days, one group of animals was treated with docetaxel at a dose of 7 mg/kg q3d ip. One group of animals was treated with a specific dose of S1P1R receptor antagonist and the same dose of docetaxel using the same schedule used for the individual docetaxel group. Finally, a control group was treated with the vehicles used for each agent individually. Tumor dimensions were measured by calipers and then converted to weight using the formula: tumor weight (mg)=[tumor volume=length (mm)×width2(mm2)]/2. Animals were also monitored for weight loss as an indicator of signs of toxicity. The S1P1R receptor antagonist was formulated in a composition containing 10% NMP (n-methyl pyrrolidone)+90% corn oil. The docetaxel was formulated in a composition containing 12.5% Cremephor, 12.5% ethanol, and 75% saline.

Results of an experiment with one of the 51P1R receptor antagonists and doxetaxel are as follows. Cells were inoculated subcutaneously on day 0 and allowed to grow for 17 days. At time of first treatment, tumors averaged 96 mgs. Dosing continued for 7 cycles (q3d) and tumors in each of the three groups were measured and averaged. After 7 cycles of treatment, the tumors in animals dosed with docetaxel averaged about 310 mgs. Tumors in animals treated with both agents averaged 186 mgs. There was no evidence of toxicity in these animals. These data indicate that treatment with S1P1R receptor antagonists and docetaxel had an unexpectedly superior (eg, synergistic) anti-tumor effect.