WO2009126211A2 - Controlled release formulation of water insoluble drug and methods of preparation and treatment - Google Patents

Abstract

An implant for use in method of treatment is disclosed whereby cancer cells are brought into contact with a formulation comprising an inhibitor of tyrosine kinase receptors. The formulation may be comprised of an injectable carrier and two or more tyrosine kinase receptor inhibitors which may be nordihydrogluaiaretic acid (NDGA) and doxyrubicine.

Description

CONTROLLED RELEASE FORMULATION OF WATER INSOLUBLE DRUG AND METHODS OF PREPARATION AND TREATMENT

BACKGROUND OF THE INVENTION

[0001] Many tumor cells depend on the activity of tyrosine kinases, which act, among other functions, to depress apoptosis in the cell. The tyrosine kinases are usually overproduced in malignant cells, which contributes to the cell's ability to resist apoptosis. Modulating the activity of these proteins provides an effective means of treating cancer while not unduly damaging normal tissues. For example, about 25% of breast tumors express unusually high levels of the Her2 protein, a tyrosine kinase receptor that normally plays a part in the development of the mammary epithelium. Herceptin® (Trastuzumab) is a humanized antibody that is currently used to treat breast cancer by targeting and blocking the function of the Her2 protein. Other treatments focus on interfering with the receptors to overexpressed tyrosine kinase proteins. Receptors include HER2/neu and IGF-IR. See, Meric et al. (Apr. 2002) J. Am. Coll. Surg. 194(4):488-501.

[0002] The major lignin in chaparral, known as nordihydroguaiaretic acid (NDGA) is a potent antioxidant and was originally used in commercial food products as a preservative. See, U.S. Pat. No. 2,644,822. Later, it was discovered that NDGA is useful in the treatment of diabetes. Hsu et al. (2001) Cell Transplant. 10(3):255- 262. More recently, NDGA was investigated as a treatment for cancer because it inhibits the platelet derived growth factor receptor and the protein kinase C intracellular signalling family, which both play an important role in proliferation and survival of cancers. Moreover, NDGA induces apoptosis in tumor xenografts. Although it is likely to have several targets of action, NDGA is well tolerated in animals. However, high concentrations of NDGA are required for efficacy and it has been suggested that more potent analogs may be required. See, McDonald et al. (2001) Anticancer Drug Des. 16(6):261-270.

[0003] Other cancer drugs include doxorubicin hydrochloride (DOX), which is used alone or in combination with other drugs for treatment of malignant lymphomas and leukemias. DOX is believed to bind DNA and inhibit nucleic acid synthesis. Examples of tumors amenable to treatment with DOX are acute lymphoblastic leukemia, acute myeloblasts leukemia, Wilm's tumor, soft tissue and bone sarcomas, breast carcinoma and ovarian carcinoma. The dosage needs to be closely monitored because it can cause irreversible cardiac damage. A typical dose for adults, when given intravenously is 60-75 mg/m2 once in 21 days, or 30 mg/m2 daily for 3 days every four weeks, where the total cumulative dose should not exceed 550 mg/m2 without monitoring for cardiac function.

[0004] It is well established that breast cancer is regulated by receptors for the female sex steroids, estrogen and progesterone. It is now appreciated that receptor tyrosine kinases (RTKs) are also very important for breast cancer growth (Arteaga CL, Moulder SL, Yakes FM: HER (erbB) tyrosine kinase inhibitors in the treatment of breast cancer. Semin Oncol 29:4-10, 2002; Averbuch S, Kcenler M, Morris C, Wakeling A: Therapeutic potential of tyrosine kinase inhibitors in breast cancer. Cancer Invest 21 :782-791, 2003; Baserga R: The IGF-I receptor in cancer research. Exp Cell Res 253:1-6, 1999; Dickson RB, Lippman ME: Growth factors in breast cancer. Endocr Rev 16:559-589, 1995; Gross JM, Yee D: The type-1 insulin-like growth factor receptor tyrosine kinase and breast cancer: biology and therapeutic relevance. Cancer Metastasis Rev 22:327-336, 2003; and Nahta R, Hortobagyi GN, Esteva FJ: Growth factor receptors in breast cancer: potential for therapeutic intervention. Oncologist 8:5-17, 2003).

[0005] Accordingly, RTKs are targets for anti-tumor therapy. RTKs are transmembrane proteins that typically contain an extracellular ligand binding domain, activated by peptide hormones, and an intracellular tyrosine kinase domain. Two RTKs of demonstrated importance in breast and other cancers are the insulin-like growth factor receptor (IGF-IR) (Heinemann V: Present and future treatment of pancreatic cancer. Semin Oncol 29:23-31, 2002) and c- erbB2/HER2/neu (HER2/neu) (Morin MJ: From oncogene to drug: development of small molecule tyrosine kinase inhibitors as anti-tumor and anti-angiogenic agents. Oncogene 19:6574-6583, 2000). Based on their major role in regulating cancer cell growth and survival, inhibitors of these RTKs are undergoing drug development (Morin MJ: From oncogene to drug: development of small molecule tyrosine kinase inhibitors as anti-tumor and anti-angiogenic agents. Oncogene 19:6574-6583, 2000; Bruns CJ, Solorzano CC, Harbison MT, Ozawa S, Tsan R, Fan D, Abbruzzese J, Traxler P, Buchdunger E, Radinsky R, Fidler IJ: Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma. Cancer Res 60:2926- 2935, 2000; Bruns CJ, Harbison MT, Davis DW, Portera CA, Tsan R, McConkey DJ, Evans DB, Abbruzzese JL, Hicklin DJ, Radinsky R: Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms. Clin Cancer Res 6:1936-1948, 2000; Blum G, Gazit A, Levitzki A: Substrate competitive inhibitors of IGF-I receptor kinase. Biochemistry 39: 15705- 15712, 2000).

[0006] Signaling via the IGF-IR is important for normal cell growth and differentiation. In addition, the IGF-IR stimulates mitogenesis and suppresses apoptosis of cancer cells (Lowe WL: Biological actions of the insulin-like growth factors. In LeRoith D (ed): Insulin-like growth factors: molecular and cellular aspects. Boca Raton, CRC Press, 1991). Following binding of the ligand to the IGF- IR, a conformational change induces trans-autophosphorylation of the β-subunits on select tyrosine residues, and subsequent activation of tyrosine kinase activity (Lowe WL: Biological actions of the insulin-like growth factors. In LeRoith D (ed): Insulin-like growth factors: molecular and cellular aspects. Boca Raton, CRC Press, 1991). Phosphorylation of several target substrates activates divergent signaling cascades, though the anti-apoptotic effects of the IGF-IR are primarily mediated via the Akt/PKB pathway (Kulik G, Klippel A, Weber MJ: Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. MoI Cell Biol 17:1595-1606, 1997).

[0007] Tyrosine phosphorylation of the insulin receptor substrate (IRS) family of proteins by the IGF-IR allows binding of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K) to the IRS proteins via SH2 domains. Activated PI3K serine phosphorylates and activates the serine kinase Akt (Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA: Mechanism of activation of protein kinase B by insulin and IGF-I . EMBO J 15:6541-6551, 1996). Akt can phosphorylate the protein BAD, which prevents BAD from forming a pro-apoptotic complex with Bcl-2 proteins (Virdee K, Parone PA, Tolkovsky AM: Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival. Curr Biol 10:1151-1 154, 2000).

[0008] Interruption of the IGF- 1 R signaling system, either by reducing effective

IGF-I levels or targeting the receptor, can block growth and proliferation of cancer cells (Kahan Z, Varga JL, Schally AV, Rekasi Z, Armatis P, Chatzistamou L, Czompoly T, Halmos G: Antagonists of growth hormone-releasing hormone arrest the growth of MD A-MB-468 estrogen-independent human breast cancers in nude mice. Breast Cancer Res Treat 60:71-79, 2000; Neuenschwander S, Roberts CT, Jr., LeRoith D: Growth inhibition of MCF-7 breast cancer cells by stable expression of an insulin-like growth factor I receptor antisense ribonucleic acid. Endocrinology 136:4298-4303, 1995; Prager D, Li HL, Asa S, Melmed S: Dominant negative inhibition of tumorigenesis in vivo by human insulin-like growth factor I receptor mutant. Proc Natl Acad Sci U S A 91 :2181-2185, 1994; Weckbecker G, Tolcsvai L, Liu R, Bruns C: Preclinical studies on the anticancer activity of the somatostatin analogue octreotide (SMS 201-995). Metabolism 41:99-103, 1992; and Yee D, Jackson JG, Kozelsky TW, Figueroa JA: Insulin-like growth factor binding protein 1 expression inhibits insulin-like growth factor I action in MCF-7 breast cancer cells. Cell Growth Differ 5:73-77, 1994). While overexpression of the IGF-IR can drive transformation and mitogenesis, it is the requirement for its constitutive presence in cancer cells (Rubin R, Baserga R: Insulin-like growth factor-I receptor. Its role in cell proliferation, apoptosis, and tumorigenicity. Lab Invest 73:31 1-331, 1995) that makes this RTK an attractive target for anti-tumor therapies. The HER2/neu (c-erbB-2) protooncogene encodes a 1,255 amino acid, 185 kDa member of the class I RTK family. HER2/neu is overexpressed in 20-30% of breast cancers, most commonly via gene amplification, and overexpression is associated with poor prognosis in these patients (Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, .: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707-712, 1989; Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177-182, 1987). Evidence from transgenic animal studies indicates that HER2/neu overexpression directly contributes to transformation and tumor progression (BoI D, Kiguchi K, Beltran L, Rupp T, Moats S, Gimenez-Conti I, Jorcano J, DiGiovanni J: Severe follicular hyperplasia and spontaneous papilloma formation in transgenic mice expressing the neu oncogene under the control of the bovine keratin 5 promoter. MoI Carcinog 21 :2-12, 1998; Bouchard L, Lamarre L, Tremblay PJ, Jolicoeur P: Stochastic appearance of mammary tumors in transgenic mice carrying the MMTV/c-neu oncogene. Cell 57:931-936, 1989; and Lucchini F, Sacco MG, Hu N, Villa A, Brown J, Cesano L, Mangiarini L, Rindi G, Kindl S, Sessa F, .: Early and multifocal tumors in breast, salivary, harderian and epididymal tissues developed in MMTY-Neu transgenic mice. Cancer Lett 64:203-209, 1992), and suggests that its prognostic significance arises from the particularly aggressive phenotype it confers (Hynes NE, Stern DF: The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta 1 198:165-184, 1994). The efficacy of targeting HER2/neu in anti-cancer therapy has been demonstrated by the clinical use of an antibody to HER2/neu to treat certain patients with breast cancer (Albanell J, Baselga J: Trastuzumab, a humanized anti-HER2 monoclonal antibody, for the treatment of breast cancer. Drugs Today (Bare ) 35:931-946, 1999). Nordihydroguaiaretic acid (NDGA) is a phenolic compound that was identified as a major component of a tea made from resinous extracts of the creosote bush Larrea divaricatta. It has been used for centuries by Native North Americans as a remedy for diverse illnesses, including tumors (Duisberg PC: Desert Plant Utilization. Texas J Sci 4:269, 1952; Hawthorn P: Medicinal uses of plants of Nevada used by Indians. Contr Flora Nevada 45: 1-139, 1957). NDGA has been reported to inhibit the growth of various tumors both in vitro and in animals (Wilson DE, DiGianfilippo A, Ondrey FG, Anderson KM, Harris JE: Effect of nordihydroguaiaretic acid on cultured rat and human glioma cell proliferation. J Neurosurg 71 :551-557, 1989; Avis IM, Jett M, Boyle T, Vos MD, Moody T, Treston AM, Martinez A, Mulshine JL: Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J Clin Invest 97:806-813, 1996; Rose DP, Connolly JM: Effects of fatty acids and inhibitors of eicosanoid synthesis on the growth of a human breast cancer cell line in culture. Cancer Res 50:7139-7144, 1990; and Shimakura S, Boland CR: Eicosanoid production by the human gastric cancer cell line AGS and its relation to cell growth. Cancer Res 52:1744-1749, 1992). NDGA also has been reported to induce apoptosis in a variety of cell lines (Ding XZ, Kuszynski CA, El Metwally TH, Adrian TE: Lipoxygenase inhibition induced apoptosis, morphological changes, and carbonic anhydrase expression in human pancreatic cancer cells. Biochem Biophys Res Commun 266:392-399, 1999; La E, Kern JC, Atarod EB, Kehrer JP: Fatty acid release and oxidation are factors in lipoxygenase inhibitor-induced apoptosis. Toxicol Lett 138:193-203, 2003; Seufferlein T, Seckl MJ, Schwarz E, Beil M, Wichert G, Baust H, Luhrs H, Schmid RM, Adler G: Mechanisms of nordihydroguaiaretic acid-induced growth inhibition and apoptosis in human cancer cells. Br J Cancer 86: 1188-1 196, 2002; Tong WG, Ding XZ, Witt RC, Adrian TE: Lipoxygenase inhibitors attenuate growth of human pancreatic cancer xenografts and induce apoptosis through the mitochondrial pathway. MoI Cancer Ther 1 :929- 935, 2002; and Tong WG, Ding XZ, Adrian TE: The mechanisms of lipoxygenase inhibitor-induced apoptosis in human breast cancer cells. Biochem Biophys Res Commun 296:942-948, 2002). Still, the mechanism of this anti-cancer effect of NDGA is not well understood. It has been reported that NDGA inhibits the tyrosine kinase activity of the platelet-derived growth factor receptor (PDGFR), but not the epidermal growth factor receptor (EGFR), in cells and in vitro (Domin J, Higgins T, Rozengurt E: Preferential inhibition of platelet-derived growth factor-stimulated DNA synthesis and protein tyrosine phosphorylation by nordihydroguaiaretic acid. J Biol Chem 269:8260-8267, 1994). While one report suggests that NDGA is inactive against the IGF-IR (Seufferlein T, Seckl MJ, Schwarz E, Beil M, Wichert G, Baust H, Luhrs H, Schmid RM, Adler G: Mechanisms of nordihydroguaiaretic acid-induced growth inhibition and apoptosis in human cancer cells. Br J Cancer 86:1 188-1196, 2002), a compound with a very high degree of structural homology to NDGA has been described as a potent inhibitor of this receptor(Blum G, Gazit A, Levitzki A: Substrate competitive inhibitors of IGF-I receptor kinase. Biochemistry 39:15705-15712, 2000; Blum G, Gazit A, Levitzki A: Development of new insulin- like growth factor- 1 receptor kinase inhibitors using catechol mimics. J Biol Chem 278:40442-40454, 2003). The effects of NDGA on the HER2/neu receptor, which also plays a critical role in breast cancer, have not been explored. We have now found that NDGA antagonizes the activation of both the IGF-I and HER2/neu receptors, inhibits the cellular anti-apoptotic signaling pathway of the IGF-IR, and inhibits the growth of breast cancer cells both in vitro and in vivo.

[0011] Many drugs such as NDGA and DAU are substantially insoluble in water.

This makes it difficult to create an aqueous injectable formulation containing such drugs. The present invention includes a formulation which is comprised of drugs which are substantially water insoluble in a formulation which is injectable and provides for controlled release of the included drug.

SUMMARY OF THE INVENTION

[0012] An injectable formulation of a water insoluble drug dispersed in a biocompatible polymer is disclosed. The injectable formulation is administered intramuscularly or subcutaneously as a liquid and subsequently solidifies in situ and thereafter slowly dissolves providing for controlled release of the water insoluble drug.

[0013] In order to prepare the formulation the biocompatible polymer is dissolved within a water-miscible biocompatible solvent. The polymer is dissolved in the solvent using physical systems such as mixing, stirring and/or grinding and may include heating the polymer and the solvent together. At this point the drug may not be present. If heat is applied the polymer solution can then be cooled under ambient conditions and the drug or drugs dispersed into the polymer solution such as by homogenization. Alternatively the drug may be dissolved in a second biocompatible solvent which is miscible with the polymer solvent and water.

[0014] The resulting formulation comprised of the biocompatible polymer, one or more solvents, and one or more drugs, has a viscous consistency. However, the resulting formulation is sufficiently flowable so that it can be injected, i.e. the formulation is syringable and can be injected into a patient intramuscularly or subcutaneously using a convention syringe and needle.

[0015] When the formulation is injected into the patient it comes into contact with the surrounding conditions which include an aqueous environment and various dissolved components which are normally present. When the formulation comes into contact with the in vivo conditions present in the body of the patient the formulation solidifies and forms a gel matrix which entraps the drug. Over time the polymer solvent dissipates and diffuses out of the system and water in the surrounding environment diffuses into the polymer matrix. Due to the water- insoluble nature of the polymer, the polymer precipitates or coagulates to form a solid implant in situ. Once the matrix or implant is formed in situ it dissolves slowly.

[0016] The formulation can be produced with any type of drug. However, there are certain advantages when producing the formulation when using the water insoluble drug because it is difficult to produce implantable formulations which have water insoluble drug dispersed therein. In addition to being a water insoluble drug the formulation preferably includes a drug which has more beneficial effects when it is administered very slowly over a long period of time. Some drugs of this type are described below. [0017] Nordihydroguaiaretic acid (NDGA) is a phenolic compound isolated from the creosote bush Larrea divaricatta that has anti-cancer activities both in vitro and in vivo. These anti-cancer properties in breast cancer cells are created by the ability of NDGA to directly inhibit the function of two receptor tyrosine kinases (RTKs), the insulin-like growth factor receptor (IGF-IR) and the c-erbB2/HER2/neu (HER2/neu) receptor. In MCF-7 human breast cancer cells, low micromolar concentrations of NDGA inhibited activation of the IGF-IR, and downstream phosphorylation of both the Akt/PKB serine kinase and the pro-apoptotic protein BAD.

[0018] In mouse MCNeuA cells, NDGA also inhibited ligand independent phosphorylation of HER2/neu. This inhibitory effect in cells is due to a direct action on these receptors. The IGF-I -stimulated tyrosine kinase activity of isolated IGF-IR is inhibited by NDGA at 10 μM or less. A composition of NDGA is also effective at inhibiting autophosphorylation of isolated HER2/neu receptor at similar concentrations. In addition, NDGA inhibits IGF-I specific growth of cultured breast cancer cells with an IC50 of approximately 30 μM. Treatment with NDGA (intraperitoneal injection 3 times per week) also decreases the activity of the IGF- 1 R and the HER2/neu receptor in MCNeuA cells implanted into mice. Inhibition of RTK activity is associated with decreased growth rates of MCNeuA cells in vivo. Accordingly, the anti-breast cancer properties of NDGA are related to the inhibition of two important RTKs and as such formulations of RTK inhibitors provide a means of treating breast cancer.

[0019] An aspect of the invention is a controlled release pharmaceutical composition which is comprised of an insoluble drug such as a tyrosine kinase inhibitor and a biodegradable polymer which polymer may be dissolved at least in part in a solvent which is a biocompatible solvent.

[0020] A more specific aspect of the invention is such a formulation wherein the biodegradable polymer is poly(lactide-co-glycolide) wherein the lactide to glycolide ratio is in a range of lactide to glycolide of 75 ± 10:25 ± 10.

[0021] Another aspect of the invention is a method of preparing a formulation where a biocompatible polymer is dissolved in a biocompatible solvent and a water insoluble drug is dispersed therein.

[0022] Another aspect of the invention is a method of treatment wherein a biocompatible polymer is dissolved in a biocompatible solvent and an insoluble drug dissolved therein to create a flowable formulation which is injectable from a syringe and after injection the formulation solidifies into an implant and slowly dissolves thereby releasing the drug to the surrounding tissue. [0023] These and other aspects of the invention will become apparent to those persons skilled in the art upon reading the details of the formulations and methods as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows the chemical structure of nordihydroguaiaretic acid

(NDGA).

[0025] Figure 2 shows the chemical structure of diarylurea 21834 (DAU).

[0026] Figure 3 is a graph showing the percent of total drug released versus time for NDGA and DAU in vitro.

[0027] Figure 4 is a graph of relative tumor volume plotted versus days after treatment showing the in vivo anti-tumor activity of an injected formulation of the invention comprised of NDGA and poly(lactide-co-glycolide).

[0028] Figure 5 is a graph of relative tumor volume plotted against days after treatment showing the activity for the control where no treatment occurs versus three different treatments wherein the first includes poly(lactide-co-glycolide) by itself without drug, the second includes the drug DAU by itself and the third includes a formulation of the invention wherein the DAU is dispersed within poly(lactide-co-glycolide).

DETAILED DESCRIPTION OF THE INVENTION

[0029] Before the present compositions for and methods of treating cancer are described, it is to be understood that this invention is not limited to particular compositions and methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0030] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0032] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cancer cell" includes a plurality of such cancer cells and reference to "the methods of administration" includes reference to one or more methods and equivalents thereof known to those skilled in the art, and so forth.

[0033] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DEFINITIONS

[0034] The term "nordihydroguaiaretic acid" is also referred to as "NDGA" and is the compound shown within the structure of Figure 1 and see U.S. Patent 2,644,822 incorporated here to disclosed NDGA as well as related compounds and their method of manufacture. It is pointed out that pharmaceutically acceptable salts and amines of the acid may be formed during use and are considered to be encompassed by the term unless specifically indicated otherwise.

[0035] The term tyrosine kinase receptor blocker and inhibitor of tyrosine kinase are used interchangeably to describe compounds which selectively and specifically bind to tyrosine kinase receptors. The binding preferably has an antagonist effect. Such compounds include compounds such as Her2 inhibitors, doxorubicine and Herceptin™.

[0036] The terms "treatment," "treating," and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. In general, methods of the invention involve treating diseases referred to as cancer and may be applied to a variety of different types of cancer by utilizing combinations of compounds such as tyrosine kinase receptor inhibitors which are known to bind to the receptor site. "Treatment" as used herein covers any treatment of such a disease in a mammal, particularly a human, and includes:

[0037] (a) preventing and/or diagnosing the disease in a subject which may be predisposed to the disease which has not yet been diagnosed as having it;

[0038] (b) inhibiting the disease, i.e. arresting its development; and/or

[0039] (c) relieving the disease, i.e. causing regression of the disease.

[0040] The invention is directed towards treating patients with cancer and is particular directed towards treating particular types of cancer which are not generally treatable with normal surgical methods. More specifically, "treatment" is intended, in preferred circumstances, to mean providing a therapeutically detectable and beneficial effect on a patient suffering from cancer.

FORMULATIONS AND METHODS

[0041] Although a range of different polymers could be used to produce a formulation of the invention the polymers should be biodegradable so that when the polymer is injected it can degrade over time and allow the drug dispersed therein to contact the target tissue such as cancerous tissue and have the desired effect. The biodegradable polymer may be a government approved polymer such as an FDA approved polymer which includes poly(lactide-co-glycolide) (PLGA). The polymer PLGA is useful because it undergoes hydrolysis in the body and produces lactic acid and glycolic acid. Under normal physiological conditions the two acids produced which are lactic acid and glycolic acid are the natural bi-products of various metabolic pathways in the body. A normal mammalian body such as a human body effectively deals with these two monomers resulting in minimal systemic toxicity when PLGA is used for delivery. There are commercially available PLGA products which are FDA approved for use in treatment of humans.

[0042] There are other formulations of PLGA which are comprised of microparticles of PLGA and a drug. However, the microparticles need to be reconstituted in an aqueous media before they can be injected into the body. Further, other formulations may require the use of organic solvents such as methylene chloride for the solubilization of the PLGA polymer. These formulations and others include a residual organic solvent in the microsphere product which can have side effects.

[0043] Implants have also been produced which are comprised of PLGA polymer which has a drug dispersed therein. Although these products can be useful they are difficult to administer in that they require at least minor surgery or a special type of pellet injector such as a trocar which results in an inconvenience to the patient.

[0044] The present invention does not require surgery to be implanted because the formulation is in an injectable or syringable and remains in a flowable state until it is injected inside of the patient. It can be injected intramuscularly, subcutaneously or intratumorally and then allowed to solidify in situ.

[0045] In order to produce the formulation of the invention the polymer such as

PLGA polymer is dissolved such as by heating in a water-miscible, biocompatible solvent. Different polymers and solvents can be used and other techniques such as mixing, stirring, chopping and grinding can be utilized in order to dissolve the polymer. If the polymer has been heated it is then cooled under ambient conditions and a drug such as a water soluble drug is dispersed into a polymer/solvent combination such as by using homogenization. Other methods can also be utilized. For example, the drug can be dissolved in the biocompatible solvent and the solvent drug combination can be miscible with the polymer/solvent combination with water. The basic concept is to produce a polymer/solvent/drug combination that has a viscous consistency that is sufficiently syringable that it can be injected intramuscularly, subcutaneously or intratumorally using a conventional syringe and needle. Two or more polymers and two or more solvents can be used and water can be added to provide the resulting flowable formulation.

[0046] Once the desired viscosity is obtained the formulation can be injected into the patient. At this point the formulation comes into contact with the various physiological fluids present in the body. For example, the body has a substantially aqueous environment with various salts dissolved therein which come into contact with the polymer. After the contact has occurred the polymer solidifies and forms a gel matrix which entraps the drug. This is desirable in that the drugs administered using the formulation of the invention are generally drugs which need to be delivered in small amounts over a long period of time in order to have the best effect.

[0047] When the polymer solidifies the solvent used in preparing the formulation dissipates and diffuses out of the matrix. In order to solidify the polymer and form the implant the solvent diffuses out of the polymer into the surrounding water or aqueous solution in the body and the surrounding solution diffuses into the polymer matrix. Due to the water- insoluble nature of the polymer the polymer precipitates and coagulates thereby forming a solid implant in situ. The solid implant is allowed to remain in place and degrade over a long period of time such as from one day to five days or one day to ten days or one day to fifteen days or longer.

[0048] Various types of drugs can be used in the formulation of the invention.

However, the invention has some particular advantages when the drug is a water insoluble drug and in particular when the drug is a water insoluble drug which is efficacious when administered in small amounts over a very long period of time. Examples of such drugs include NDGA as shown within Figure 1 and DAU 21834 show within Figure 2.

[0049] In order to carry out examples of the invention NDGA or DAU were dissolved in DMSO or glycofurol and mixed with PLGA. The PLGA may, itself, be dissolved in DMSO or glycofurol. The resulting formulation is injectable and can be injected into an aqueous solution in vitro in order to study the results. Those results were found to be the precipitation and coagulation of the formulation into a solid pellet. The drugs such as NDGA or DAU were released with an initial burst followed by a slower controlled release as shown within Figure 3 which results are injection into an aqueous in vitro environment. [0050] When the formulation was injected directly into the tumor which was established in a mammal such as a mouse, solid implants were formed in situ. The injection directly into the tumor of NDGA dispersed in PLGA or DAU dispersed in PLGA resulted in complete or partial regression of tumors whereas injection with PLGA alone had little effect on tumor growth as shown in Figures 4 and 5.

TREATING NEUROBLASTOMA

[0051] Neuroblastoma is a common pediatric malignancy that metastasizes to the liver, bone, and other organs, and is often resistant to available treatments. Insulin- like growth factors (IGFs) stimulate neuroblastoma growth, survival, and motility, and are expressed by neuroblastoma cells and the tissues they invade. Administration of formulations of the invention disrupt the effects of IGFs on neuroblastoma tumorigenesis and thereby slow disease progression. Nordihydroguaiaretic acid (NDGA), a phenolic compound isolated from the creosote bush (Larrea divaricatά), has anti-tumor properties against a number of malignancies. NDGA inhibits the phosphorylation and activation of the Her2/neu and IGF-I receptors (IGF-IR). The present invention shows that NDGA inhibits IGF-I-mediated activation of the IGF-IR in human neuroblastoma cell lines. NDGA inhibits neuroblastoma growth and disrupts activation of ERK and Akt signaling pathways induced by IGF-I. NDGA induces apoptosis at higher doses, causing IGF-I-resistant activation of caspase-3 and a large increase in the fraction of sub-Go cells. NDGA inhibits the growth of xenografted human neuroblastoma tumors in nude mice by 50%. The results provided show that small molecules that prevent activation of the IGF-IR, such as NDGA, are useful in the treatment of neuroblastoma.

[0052] Neuroblastoma affects an estimated 1 in 7000 children under age 15

(Carlsen NL. Neuroblastoma: epidemiology and pattern of regression. Problems in interpreting results of mass screening. Am J Pediatr Hematol Oncol 1992; 14: 103- 1 10), making it the second most common solid tumor in children. Neuroblastoma tumors are believed to arise from neural crest cells in the adrenal gland and spinal ganglia. Neuroblastoma often regresses spontaneously in children under 1 year of age, but neuroblastoma in older children is difficult to treat with conventional radiation and chemical therapies (Philip T. Overview of current treatment of neuroblastoma. Am J Pediatr Hematol Oncol 1992;14:97-102). Metastasis to bone, meninges, the liver, and other organs contributes to the difficulty in eliminating the disease.

[0053] The development of effective treatments for neuroblastoma is hampered by an incomplete understanding of the factors that lead to neuroblastoma tumorigenesis, although several key abnormalities are associated with a significant subset of aggressive tumors. Although several chromosomal abnormalities have been described, amplification of MYCN is still the best understood genetic abnormality and is associated with advanced disease.

[0054] While a primary defect in growth factor signaling has not been observed in neuroblastoma, growth factor responsiveness is believed to support tumor growth, survival, and invasiveness. Thus, therapeutic approaches that disrupt growth factor signaling may have an impact on disease progression. Recently, nordihydroguaiaretic acid (NDGA), a naturally occuring compound isolated from creosote {Larrea divaricata), was found to inhibit the activation of partially purified insulin-like growth factor I (IGF-I) and her2/neu receptor tyrosine kinases.

[0055] NDGA has been extensively studied as an inhibitor of arachidonic acid metablolism, where it blocks lipoxygenase activity, but its ability to inhibit receptor tyrosine kinase activation was previously unappreciated. In breast cancer cells, NDGA inhibited ligand activation of the IGF-I and her2/neu receptors and subsequent activation of signaling intermediates downstream of these receptors. Both the in vitro and in vivo growth of breast cancer cells is inhibited by NDGA, potentially via its ability to suppress responsiveness to growth factors.

[0056] IGF-I and II are peptide growth factors that regulate cell mitogenesis and survival. IGFs bind to the tyrosine kinase IGF-I receptor (IGF-IR), causing receptor autophosphorylation that initiates the mitogen activated protein kinase (MAPK) and phosphatidylinositol 3 -kinase (PI-3K) signaling pathways. MAPK regulates mitogenesis (De Meyts P, Wallach B, Christoffersen CT, et al. The insulin-like growth factor-I receptor. Structure, ligand-binding mechanism and signal transduction. Horm Res 1994;42:152-169), while PI-3K activates targets that impact apoptosis, such as Akt (Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta and Gonzalez-Baron M. PI3K/Akt signaling pathway and cancer. Cancer Treat Rev 2004;30: 193-204).

[0057] IGFs promote neuroblastoma tumorigencity by stimulating proliferation, inhibiting apoptosis, and stimulating motility. IGFs are expressed in all neuroblastoma tumor stages and in neuroblastoma tumor lines (Martin DM, Yee D, Carlson RO and Feldman EL. Gene expression of the insulin-like growth factors and their receptors in human neuroblastoma cell lines. Brain Res MoI Brain Res 1992; 15:241-246), and can act as either autocrine or paracrine mitogens (Martin DM and Feldman EL. Regulation of insulin-like growth factor-II expression and its role in autocrine growth of human neuroblastoma cells. J Cell Physiol 1993;155:290-300). IGF-I and IGF-IR expression prevent neuroblastoma cells from undergoing apoptosis (Singleton JR, Dixit VM and Feldman EL. Type I insulin- like growth factor receptor activation regulates apoptotic proteins. J Biol Chem 1996;271 :31791-31794) by regulating the activity of caspases and BcI proteins. IGFs also regulate the metastatic capabilities of neuroblastoma cells by stimulating actin polymerization, lamellipodium extension, and motility.

[0058] The present invention is based, in part, on an understanding of the ability of

NDGA to inhibit growth and IGF-IR-related signaling events in breast cancer. The present invention shows the anti -tumor effects of NDGA in three human neuroblastoma cell lines. Results provided here quantify IGF-I- and serum- dependent growth of neuroblastoma cells treated with NDGA, and characterize IGF-I-dependent phosphorylation of IGF-IR, extracellular regulated kinases (ERKs), and Akt in the presence of NDGA. Results provided here show that IGF- IR blockade mediated by NDGA resulted in decreased proliferation, increased apoptosis, decreased motility, and decreased tumor growth in xenograft models. Further, the results provided here show that NDGA is a potent inhibitor of neuroblastoma growth and survival, and of IGF-I-stimulated signaling events associated with tumorigenesis in neuroblastoma.

[0059] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMSThat which is claimed is:

1. A controlled release pharmaceutical composition, comprising : a tyrosine kinase inhibitor; and a biodegradable polymer.

2. The composition according to claim 1, further comprising: a pharmaceutically acceptable solvent of the biodegradable polymer, wherein said biodegradable polymer comprises poly(lactide-co-glycolide); and wherein the solvent is selected from the group consisting of glycofurol and DMSO.

3. The composition according to claim 2, wherein the polymer is comprised of lactide and glycolide and has a ratio of lactide to glycolide of 75 ±10:25±10 and wherein the tyrosine kinase inhibitor is comprised of two aromatic groups.

4. An implant solidified in situ in a mammal, the implant comprising: a water insoluble, pharmaceutically active drug; and a biodegradable polymer wherein aqueous solution in the mammal has replaced solvent in the polymer.

5. The implant of claim 4, manufactured for use in treating cancerous tissue.

6. The implant according to claim 4, wherein the implant is manufactured for injection into the cancerous tissue in liquid form: after which the formulation will solidify to form an implant in situ.

7. The implant according to claim 5, wherein the tyrosine kinase inhibitor is selected from the group consisting of nordihydroguaiaretic acid (NDGA), l-(2-methoxy-5-methyl- phenyl)-3 -(2 -methyl quinolin-4-yl)-urea (DAU), pharmaceutically acceptable salts of NDGA, pharmaceutically acceptable salts of DAU, and mixtures thereof.

8. The implant according to claim 7, wherein said biodegradable polymer comprises poly(lactide-co-glycolide) in a solvent.

9. A method of making an injectable pharmaceutical composition as claimed in claim 6, comprising: combining a biocompatible polymer with a solvent for the polymer; heating the polymer and solvent; cooling the polymer and solvent; adding a tyrosine kinase inhibitor to the polymer and solvent; and mixing to obtain homogenization of the tyrosine kinase inhibitor with the polymer and solvent.

10. The method according to claim 9, wherein the solvent is a biocompatible solvent is miscible with both water and the polymer solvent.

1 1. The method according to claim 10, wherein the solvent is selected from the group consisting of glycofurol and DMSO and wherein said biodegradable polymer comprises poly(lactide-co-glycolide).

12. The method according to claim 1 1, wherein the tyrosine kinase inhibitor is selected from the group consisting of nordihydroguaiaretic acid (NDGA), l-(2-methoxy-5-methyl- phenyl)-3-(2-methyl quinolin-4-yl)-urea (DAU 21834), pharmaceutically acceptable salts of NDGA, pharmaceutically acceptable salts of DAU 21834, and mixtures thereof.

13. A controlled release pharmaceutical composition, comprising : a tyrosine kinase inhibitor selected from the group consisting of nordihydroguaiaretic acid (NDGA), l-(2-methoxy-5 -methyl -phenyl)-3-(2-methyl quinolin-

4-yl)-urea (DAU), pharmaceutically acceptable salts of NDGA, pharmaceutically acceptable salts of DAU, and mixtures thereof; and a biodegradable polymer comprising poly(lactide-co-glycolide), wherein the composition is characterized by an ability to dissolve in situ over a period of eight days or longer.

14. A method of treatment, comprising the steps of: injecting cancerous tissue with a liquid controlled release composition comprising a tyrosine kinase inhibitor and a biodegradable polymer; allowing the liquid composition to solidify in situ; permitting the solidified composition to dissolve in situ over a period of eight days or longer.

15. A method of treatment, comprising the steps of: contacting cancerous tissue with a controlled release composition comprising a tyrosine kinase inhibitor and a biodegradable polymer.

16. The method according to claim 15, wherein the composition is injected into the cancerous tissue in liquid form, the method further comprising: allowing the formulation to solidify to form an implant in situ after injection.

17. The method according to claim 16, further comprising: allowing the implant to release the tyrosine kinase inhibitor continuously over an extended period of time.

18. The method according to claim 17, wherein said extended period is five days or longer.

19. The method according to Claim 18, wherein the cancerous tissue is chosen from cancerous pancreatic tissue and cancerous breast tissue.

20. The method according to Claim 15, wherein the cancerous tissue is mammalian tissue and wherein the tyrosine kinase inhibitor is selected from the group consisting of nordihydroguaiaretic acid (NDGA), l-(2-methoxy-5-methyl-phenyl)-3-(2-methyl quinolin- 4-yl)-urea (DAU), pharmaceutically acceptable salts of NDGA, pharmaceutically acceptable salts of DAU, and mixtures thereof.

21. The method according to claim 20, wherein said biodegradable polymer comprises poly(lactide-co-glycolide) in a solvent.

22. The method according to claim 16, further comprising: dissolving the polymer in a biodegradable solvent; and heating the polymer and solvent.

23. A controlled release formulation, for use in: injecting cancerous tissue with a liquid controlled release composition comprising a tyrosine kinase inhibitor and a biodegradable polymer; allowing the liquid composition to solidify in situ; permitting the solidified composition to dissolve in situ over a period of eight days or longer.