WO2017189849A1 - Nanoparticle compositions comprising adenosine receptor antagonists and methods of use - Google Patents

Abstract

A nanoparticle composition includes an adenosine receptor antagonist, a TGFβ inhibitor in a poly(lactic-co-glycolic acid) (PLGA) copolymer matrix for the nanoparticle composition along with a permeation enhancer. The nanoparticle composition is used in methods to treat cancer and diseases or disorders characterized by immunosuppression.

Description

Nanoparticle Compositions Comprising Adenosine Receptor Antagonists and Methods of Use

Cross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application Serial No.

62/328,311, filed April 27, 2016, which application is incorporated herein by reference in its entirety.

Introduction

[0002] The present disclosure relates to nanoparticulate compositions and their use in inhibiting immunosuppressive pathways. In particular, the present disclosure relates to nanoparticulate compositions comprising adenosine pathway inhibitors in combination with inhibitors of the TGF signaling pathway and their use in ameliorating tumor-mediated immunosuppressive activity.

[0003] Tumor growth has been indicated to be dependent on immunosuppression activity within the tumor microenvironment including two major immunosuppressive pathways: 1) the purinergic pathway involving adenosine signaling particularly through the A2A and A2B receptors and 2) TGF signaling. Tumors typically have high levels of both adenosine and TGF , both of which suppress multiple immune cell types that are part of the anti-tumor immune responses. Adenosine is a natural response signal that keeps potential runaway inflammatory responses in check. Likewise TGFP is a multifunctional cytokine that acts to suppress inflammation and undue cell proliferation. In addition to immunity and

inflammation, TGFP has been indicated to be involved in tissue differentiation, development, hematopoiesis, and tissue remodeling and repair. TGF-pi is a central component of wound healing, stimulates matrix molecule deposition and angiogenesis, and has been indicated as a mediator of pathologic scarring in fibrotic disorders. TGFP plays also significant role in cancers. Massague J. Cell. 134(2):215-30 (2008).

Description of the Drawings

[0004] Figures 1 shows a depiction of an adenosine signaling pathway. [0005] Figure 2A shows a graph of tumor volume versus days post injection in injected tumors with a blank (PBS), a nanoparticle with no drug (NP), and nanoparticle with adenosine antagonist SCH58621 (NP-SCH).

[0006] Figure 2B shows a graph of tumor volume versus days post injection in non- injected tumors on the contralateral side of the injected tumor side shown in Figure 2 A.

[0007] Figure 3 shows a graph of particle concentration versus particle size for an exemplary nanoparticle composition.

[0008] Figure 4 shows a graph of counts versus zeta potential for an exemplary nanoparticle composition.

[0009] Figure 5A shows a graph overlay of absorption versus wavelength of an A2A antagonist (SCH58261) and a TGF inhibitor (SB431542).

[0010] Figure 5B shows a graph of absorption versus wavelength plot for a nanoparticle composition comprising a selective A2A antagonist (SCH58261) and a selective TGF inhibitor (SB431542).

[0011] Figure 6A shows an HPLC trace of SB431542.

[0012] Figure 6B shows an HPLC trace of SCH58261.

[0013] Figure 7 shows an HPLC trace of an extract from nanoparticles embedded with both SCH58261 (peak retention time of 15.884 minutes) and SB431542 (peak retention time of 9.288 minutes).

[0014] Figure 8 A shows a graph of tumor volume versus days post tumor graft in treated (injected) tumors with blank nanoparticles not containing drug (ΤΙ-blank), nanoparticles containing SB431542, a TGF inhibitor (TI-07B), nanoparticles containing SCH58261, an adenosine A2A receptor antagonist, (TI-07H) and, nanoparticles containing both SB431542 and SCH58261, i.e., the combination of a TGF-beta inhibitor and an A2A receptor antagonist, (TI-07).

[0015] Figure 8B shows a graph of tumor volume versus days post tumor graft in untreated tumors on the contralateral side of the injected tumor side shown in Figure 8A.

[0016] Figure 9 shows a bar graph of tumor volumes for injected and uninjected tumors at Day 18 following tumor cell engraftment. Summary

[0017] In some aspects, embodiments herein relate to nanoparticle compositions comprising an adenosine receptor antagonist, a TGF inhibitor a permeation enhancer, and a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.

[0018] In some aspects, embodiments herein relate to methods of treating a disease or disorder characterized by immunosuppression comprising administering to a mammalian subject a nanoparticle composition comprising an adenosine receptor antagonist, a TGF inhibitor, a permeation enhancer, and a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.

[0019] In some aspects, embodiments herein relate to methods of treating cancer comprising administering to a mammalian subject a nanoparticle composition comprising an adenosine receptor antagonist, a TGF inhibitor, a permeation enhancer, and a poly(lactic-co- glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.

[0020] In some aspects, embodiments herein relate to methods of treating cancer or a disease or disorder characterized by immunosuppression comprising administering to a mammalian subject a first nanoparticle composition comprising an adenosine receptor antagonist and co- administering a second nanoparticle composition comprising a TGFP inhibitor, wherein the first and second nanoparticle compositions each comprise a permeation enhancer and a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix.

Detailed Description

[0021] Embodiments herein provide nanoparticle compositions that may inhibit one or both of the adenosine and TGFP pathways implicated in immunosuppression. When delivered into tumors, these compositions may provide an approach to cancer treatment. Without being bound by theory, it is believed that blocking the activity of either the purinergic pathway involving adenosine signaling and/or TGF signaling using small molecule inhibitors inhibits the immunosuppressive signals of these pathways and results in the slowing of tumor growth through stimulation of anti-tumor immune responses. The exemplary nanoparticles disclosed herein are tailored to enhance their uptake into myeloid derived cells including macrophages and dendritic cells that are inactivated in the presence of TGFP and adenosine. In some embodiments, the use of small molecule inhibitors in combination to block both adenosine and TGFP may have distinct advantages over each as monotherapies due to their synergistic activity. The use of the compositions disclosed herein to counter immunosuppressive effects in tumors is expected to be particularly useful in combination with other anti-cancer therapies.

[0022] Figure 1 shows how adenosine has been indicated to interact with four G-coupled protein receptors Ai, A2A, A2B and A3. Hasko G. et al. Nat Rev Drug Discov. 7(9):759-770 (2008), "Hasko 2008." The adenosine receptors (AiAR, A_2AR, A_2BAR and A₃AR) are coupled to adenylate cyclase (AC) through G proteins (G_x). The A₂A and A₂B ARS are coupled through Gs while Ai and A3 are coupled through Gi to either activate or inhibit the production of cyclic AMP (cAMP), respectively. The production of cAMP stimulates Protein Kinase A (PKA) and guanine exchange protein (Epac) leading to calcium (Ca²⁺) influx. A₂B and A3 are coupled to Phospholipase C (PLC) through Gq where activation results in the production of Diacylglycerol (DAG) which activates PKC and Inositol 1,4,5-trisphosphate (IP₃) which mobilizes calcium from intracellular stores. The A^R is coupled to the potassium (K⁺) efflux through Go- Both PKA and Epac-1 have been found in macrophages, a specific target cell for the nanoparticle formulations discussed herein (Ballinger M. et al. PLoS One. 5(11): el3962 (2010), Cheng X. et al. Acta Biochim Biophys Sin (Shanghai). 40(7): 651-662 (2008)). The increase in intracellular cAMP is correlated with immune suppression of macrophages. The formulations described herein are to prevent such immune suppressive effects of adenosine.

[0023] Again, without being bound by theory, it is has been indicated that adenosine attenuates inflammatory responses. Extracellular adenosine concentrations from normal cells are approximately 300 nM; however, in response to cellular damage (e.g. in inflammatory or ischemic tissue), these concentrations are quickly elevated (600-1,200 nM). Thus, in regard to stress or injury, the function of adenosine is primarily that of cytoprotection preventing tissue damage during instances of hypoxia, ischemia, and seizure activity. Activation of A_2A receptors produces a constellation of responses that in general can be classified as antiinflammatory. Hasko 2008; Hasko G. et al. Trends Immunol. 25(l):33-9 (2004).

[0024] Adenosine activity contributes to immunosuppression in tumors through enhanced upregulation of Ecto Enzyme activity CD39 and CD73 which converts AMP to adenosine. Table 1 lists multiple adenosine function that when translated to the tumor microenvironment are pro-tumorigenic. Table 1. Pro-tumor Effects of Adenosine Signaling

• Attenuation of PMNs: Oxidative burst and superoxide production

• Attenuation of α4β1 integrin, VLA-4, P-selectin, ICAM-1

• Attenuation of pro-inflammatory cytokines and chemokines: TNF-a

IFN-γ, RANTES, IL-12P₇₀, IL-2, IL-2 receptor a chain (CD25), IL-Ιβ

• Increase in pro-tumor cytokines and matrix elements: TGF and Collagen- 1

• Increase in T cell-negative costimulatory molecules: PD-1 and CTLA-4 expressed on T cells

• Attenuation of CD4+ T cell and natural killer T cell infiltration and activation (Hellstrom Paradox)

• Attenuation of TNF-a receptors on vascular endothelium and tumor cells.

[0025] These effects have been demonstrated to augment tumor growth and attenuation of these effects has anti-tumor potential. Ohta A. et al. Proc Natl Acad Sci U S A. 103 : 13132- 7 (2006); Ohta A. et al. Front. Immunol. 3: 190 (2012).

[0026] In embodiments, compositions herein comprise an adenosine receptor antagonist; in some instances the adenosine receptor is selected from the group of caffeine, theophylline, 8-phenyl theophylline, SCH58261, istradefylline, pyrazolo[4,3-e]-l ,2,4-triazolo[l,5-c] pyrimidines or substituted derivatives thereof (e.g., methoxy biaryl or quinoline

substitutions), SCH412348, SCH420814, fused heterocyclic pyrazolo[4,3-e]-l ,2,4- triazolo[l,5-c] pyrimidines or substituted derivatives thereof (e.g., tetrahydyroisoquinoline or azaisoquinoline derivatives), aryl piperazine substituted 3H-[l,2,4]-triazolo[5,l-i] purin-5- amines, arylindenopyrimidines, arylindenopyrimidines or substituted derivatives thereof, pyrazolo[ 4,3-e]-l,2,4-trizolo[4,3-c] pyrimidon-3-one and thiazolotriazolopyrimidines, 1,2,4- triazolo[l,5-c] pyrimidines or substituted derivatives thereof, purinones or substituted derivatives thereof, thieno[3,2-d] pyrimidines, pyrazolo[3,4-d] pyrimidines, and 6- arylpurines, benzyl substituted triazolo[4,5-d] pyrimidines, triazolo-9H-purines, aminomethyl substituted thieno[2,3-d]pyrimidines, 2-Aminoimidazopyridines, 4-morpholino- benzothiazoles or substituted derivatives thereof, 4-Aryl and 4-morpholino substituted benzofurans, pyridine substituted pyrazines, heterocyclic substituted 2-amino-thiazoles, trisubstituted pyrimidines, piperazine substituted pyrimidine acetamides,

acylaminopyrimidines, pyrimidine, pyridine, or triazine carboxamides, FSPTP, [3,4- dihydropyrimidin-2(lH)-one chemotype e.g. imidazole, 1,2,4-triazole, or benzimidazole rings fused to the 2,3-positions of the parent diazinone; [(3,7-dimethyl-l-propargylxanthine, 1- alkyl- 8 -(piperazine- 1 - sulf onyl) phenylxanthines : 1 - alkyl- 8- (piperazine- 1 - sulfonyl)phenylxanthinesl-ethyl-8-(4-(4-(4-trifluoromethylbenzyl)piperazine- l- sulfonyl)phenyl)xanthine (PSB-09120), 8-(4-(4-(4-chlorobenzyl)piperazine-l- sulfonyl)phenyl)-l-propylxanthine ( PSB-0788), PSB- 1115, 8-(4-(4-(4- chlorophenyl)piperazine- l-sulfonyl)phenyl)-l-propylxanthine (PSB-603), GS 6201, ISAM 140, MRS 1706, MRS 1754 Compound 38, ATL-801 , CVT-6883, OSIP-339, OSIP-391, mixtures or combinations thereof, or and pharmaceutically acceptable salts thereof.

[0027] In some embodiments, the adenosine receptor antagonist comprises an A2A-type antagonist and/or an A2B-type antagonist. In some such embodiments, the A2A-type antagonist and/or an A2B-type antagonist is selected from the group consisting of [3,4- dihydropyrimidin-2(lH)-one chemotype e.g. imidazole, 1 ,2,4-triazole, or benzimidazole rings fused to the 2,3-positions of the parent diazinone; [(3,7-dimethyl-l-propargylxanthine, 1- alkyl-8-(piperazine- l-sulfonyl) phenylxanthines: l-alkyl-8-(piperazine-l- sulfonyl)phenylxanthinesl-ethyl-8-(4-(4-(4-trifluoromethylbenzyl)piperazine- l- sulfonyl)phenyl)xanthine (PSB-09120), 8-(4-(4-(4-chlorobenzyl)piperazine-l- sulfonyl)phenyl)-l-propylxanthine (PSB-0788), PSB- 1115, 8-(4-(4-(4- chlorophenyl)piperazine- l-sulfonyl)phenyl)-l-propylxanthine (PSB-603)], GS 6201 , ISAM 140, MRS 1706, MRS 1754 Compound 38, ATL-801 , CVT-6883, OSIP-339, OSIP-391, mixtures or combinations thereof, or and pharmaceutically acceptable salts thereof.

[0028] In some embodiments, the adenosine receptor antagonist is an A2A-type antagonist, in some such embodiments, the A2A-type antagonist is selected from the group consisting of caffeine, theophylline, 8-phenyl theophylline, SCH58261, istradefylline, pyrazolo[4,3-e]- l ,2,4-triazolo[l,5-c ] pyrimidines orsubstituted derivatives thereof (e.g., methoxy biaryl or quinoline substitutions), SCH412348, SCH420814, fused heterocyclic pyrazolo[4,3-e]- l ,2,4-triazolo[l,5-c ] pyrimidines or substituted derivatives thereof (e.g., tetrahydyroisoquinoline or azaisoquinoline derivatives), aryl piperazine substituted 3H- [l,2,4]-triazolo[5,li] purin- 5 -amines, arylindenopyrimidines, arylindenopyrimidines or substituted derivatives thereof, pyrazolo[ 4,3-e ]- l,2,4-trizolo[ 4,3-c ]pyrimidon-3-one and thiazolotriazolopyrimidines, l,2,4-triazolo[l,5-c]pyrimidines or substituted derivatives thereof, purinones or substituted derivatives thereof, thieno[3,2-d]pyrimidines, pyrazolo[3,4- d]pyrimidines, and 6-arylpurines, benzyl substituted triazolo[4,5-d]pyrimidines, triazolo-9H- purines, aminomethyl substituted thieno[2,3-d]pyrimidines, 2-Aminoimidazopyridines, 4- morpholino-benzothiazoles or substituted derivatives thereof, 4- Aryl and 4-morpholino substituted benzofurans, pyridine substituted pyrazines, heterocyclic substituted 2-amino- thiazoles, trisubstituted pyrimidines, piperazine substituted pyrimidine acetamides, acylaminopyrimidines, pyrimidine, pyridine, or triazine carboxamides, FSPTP or mixtures or combinations thereof, or and pharmaceutically acceptable salts thereof.

[0029] In particular, selective A2A antagonists include, without limitation, ZM241385 (4-

{2-[7-amino-2-(2-furyl) (1, 2, 4)triazolo(2,3-a) (1, 3, 5)triazin-5-yl-amino]ethyl} -phenol) and

SCH 58621, 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-(s)]-l,2,4-triazolo(l,5-c)- pyrimidine.

[0030] Transforming Growth Factor Beta (1, 2, and 3) are highly pleiotropic cytokines that virtually all cell types secrete. TGF molecules are proposed to act as cellular switches that regulate processes such as immune function, proliferation, and epithelial-mesenchymal transition. Targeted deletions of these genes in mice show that each isoform has some non- redundant functions: 1) is involved in hematopoiesis and endothelial differentiation; 2) affects development of cardiac, lung, craniofacial, limb, eye, ear, and urogenital systems; and 3) influences palatogenesis and pulmonary development. However, TGF 1, 2, and 3 have been found to be largely interchangeable in an inhibitory bioassay. TGF knockout mice die around 4 weeks of age due to extensive inflammatory cell infiltrates.

[0031] TGF ligands are initially synthesized as precursor proteins that undergo proteolytic cleavage. The bioactive cytokine molecule is a dimer composed of a polypeptide chain that is cleaved from a precursor by enzymes such as furins and other convertases. The mature segments form active ligand dimers via a disulfide -rich core consisting of the characteristic 'cysteine knot'. Signaling begins with binding to a complex of the accessory receptor betaglycan (also known as RIII) and a type II serine/threonine kinase receptor termed RII. The active TGF dimer signals by bringing together two pairs of receptor serine/threonine kinases known as the type I and type II receptors, respectively. On binding TGF , the type II receptors phosphorylate and activate the type I receptors (either ALK-1 or RI (also called ALK-5)) that then propagate the signal by phosphorylating Smad transcription factors. Receptors of the TGF branch of the cytokine family phosphorylate Smads 2 and 3, whereas those of the other branch such as BMP receptors phosphorylate Smads 1, 5, and 8. Use of other signaling pathways that are Smad-independent allows for distinct actions observed in response to in different contexts. Once activated, the receptor substrate Smads (RSmads) shuttle to the nucleus and form a complex with Smad4, a binding partner common to all Rsmads. Shi Y. et al. Cell. 113:685-700 (2003). Through combinatorial interaction with different transcription factors, a common TGF stimulus can activate or repress hundreds of target genes at once. Variant signaling branches and Smad-independent pathways coexist with the canonical Smad pathway in the response to TGF . Seven type I receptors and five type II receptors paired in different combinations provide the receptor system for the entire TGF family. Alterations and frame shifts are found at the level of the TGF receptors in cancer.

[0032] As an immunosuppressive cytokine, TGFP inhibits the development, proliferation, and function of both the innate and the adaptive arms of the immune system. Targets of TGFP include CD4⁺ effector T cells (Thl and Th2), CD8⁺ cytotoxic T cells (CTLs), dendritic cells, NK cells, and macrophages. Additionally, TGFP stimulates the generation of regulatory T cells (Treg), which inhibit effector T cell functions, and IL 17 -producing Thl7 cells, which regulate NK cells and macrophages. The formulations that have been developed here are to inhibit the effects of TGFP in dendritic cells and macrophages after they engulf the particles from which the TGFP inhibitor released to block the TGFP receptor from the effects of TGFp. These cells are thus protected from the immunosuppressive effects of TGFP and retain the ability to stimulate anti-cancer immune responses.

[0033] TGFP is a potent inducer of Epithelial-mesenchymal transition (EMT). Cells undergoing EMT lose expression of E-cadherin and other components of epithelial cell junctions. Instead, they produce a mesenchymal cell cytoskeleton and acquire motility and invasive properties. It has been have shown that TGFP in the tumor environment primes cells for metastasis through the angiopoietin-like 4 (ANGPTL4) expression pathway. Padua D. et al. Cell 133:66-77 (2008). TGFP can also enhance cell motility by cooperating with HER2 signals, as observed in breast cancer cells overexpressing HER2. TGFP stimulates the generation of myofibroblasts from mesenchymal precursors. De Wever O. et al. J. Pathol. 200:429-47 (2003). Myofibroblasts have features of fibroblasts and smooth muscle cells and are highly motile. Their presence in tumor stroma, partly as what are called "cancer- associated fibroblasts," facilitates tumor development. Glioma cell cultures proliferate in response to TGFP through the induction of platelet-derived growth factor B (PDGF-B) through epigenetic processes.

[0034] Mutational inactivation of core pathway components occurs in large subsets of colorectal, pancreatic, ovarian, gastric, and head and neck carcinomas. Breast cancers, prostate cancers, gliomas, melanomas, and hematopoietic neoplasias preferentially disable the tumor-suppressive action of TGFP by losing the tumor-suppressive arm of the signaling pathway. In lung adenocarcinomas and squamous cell carcinomas the loss of the TGFBRII results in aggressive tumor growth and reduced survival. Malkoski SP. et al. Clin. Cancer Res. 18:2173-83 (2012). Instead of targeting the tumor cells, the nanoparticle preparations described herein are fabricated to target the myeloid derived phagocytic cells that can function with direct anti-tumor effects such as 1) engulfing tumor cells directly or 2) by priming anti-tumor immune responses through antigen presentation as antigen presenting cells (APC). Preserving these functions in the tumor microenvironment is independent of the loss of the tumor-suppressive arm of the tumor cells.

[0035] Elevated plasma levels of TGF have been found for several cancer types including colorectal, prostate, bladder, breast, pancreatic, or renal cancers and on myeloma and lymphoma. High levels of TGFpi immunostaining in infiltrating breast carcinoma has long been associated with metastasis. Dalai BI. et al. Am. J. Pathol. 143:381-9 (1993). It is now generally accepted that high levels of TGFP in the tumor microenvironment influence primary tumor cells toward metastatic potential. Padua D. (2008). In support of this is ER^~ breast tumors, where low expression of TGFBRII is associated with a favorable outcome. Buck MB. et al. Clin. Cancer Res. 10:491-8 (2004). In mice, where TGFP levels are increased by radiation or chemotherapy the use of blockers prevents lung metastasis. Biswas S. et al. J. Clin. Invest. 117: 1305-13 (2007). Studies of the role of TGFP in murine metastases have shown various effects pro-metastatic or anti-metastatic depending on the context. The bottom line is that metastatic cells need to have motility and extravasation to seed distal tumors and this is associated with high levels of TGFp. In this regard, TGFP stimulates expression of ANGPTL4 which functions to disrupt vascular endothelial cell junctions, to increase permeability of lung capillary walls and facilitates seeding of pulmonary metastases. Since the elevation of TGF levels is immunosuppressive to macrophages and dendritic cells, the targeting of these specific cells with the deliverty of TGF inhibitors through the formulations disclosed herein is intended to reverse these effects.

[0036] In addition to the role of TGFP in local tumor invasion, growing evidence implicates TGFP in the promotion of distal metastasis. Metastasis proceeds through a series of steps whereby cancer cells enter the circulatory system, disseminate to distal capillary beds, enter a parenchyma by extravasation, adapt to the new host microenvironment, and eventually grow into lethal tumor colonies in those distal organs. Fidler IJ. Nat. Rev. Cancer 3:453-8 (2003); Gupta GP. et al. Cell 127:679-95 (2006). Metastasis follows characteristic organ distribution patterns that reflect distinct colonization aptitudes of cancer cells from different origins, different tumor-efferent circulation patterns, and distinct compatibilities between disseminated cells and the organ that they encounter. Beyond the proliferative, survival, and invasive functions of a malignant state, metastasis requires extravasation and colonization functions that come into play once malignant cells disseminate. Such functions may be acquired in the primary tumor but become selected mainly during seeding and colonization distal metastases. Studies in model systems have described a broad range of potential and sometimes contradictory TGF effects on metastasis.

[0037] Bone metastases are a significant problem in late stage breast cancer patients. Following their mobilization into marrow, cancer cells trigger osteoclasts to release which further influences cytokine release which which in turn enhances metastatic invasiveness. Kingsley L.A. et al. Mol. Cancer Ther. 6:2609-17 (2007). Two genes that modulate bone metastases in ER^" breast cancer cells are interleukin-11 (IL-11) and connective tissue growth factor (CTGF). These are TGF target genes. CTGF is an extracellular mediator of invasion and angiogenesis, whereas IL-11 stimulates the production of the osteoclastogenic factors RANKL and GM-CSF in osteoblasts. Induction of IL-11 and CTGF expression by TGF is mediated by the Smad pathway (Kang Y. et al. Proc Natl Acad Sci U S A. 102: 13909- 14 (2005)) and has been confirmed in malignant cells isolated from patients with metastatic breast cancer. Gomis RR. et al. Cancer Cell 10:203-14 (2006). TGFP also induces IL-10 and IL-6 expression where IL-10 provides positive feedback for TGFP expression. In ER^~ breast tumors that are positive for both the TGFP gene response signature and lung metastasis signature (LMS) are associated with the highest risk of relapse through lung metastases. Minn AJ. et al. Nature 436:518-24 (2005). Patients with these signatures showing enhanced function may be selective candidates for TGFP blocking therapy.

[0038] It is surmised from clinical data that TGFP acts as a tumor-derived

immunosuppressor, an inducer of tumor mitogens, a promoter of carcinoma invasion, and a trigger of prometastatic cytokine secretion, all of which contribute to interest in TGFP as a therapeutic target. Blocking TGFP signaling by overexpressing the inhibitor Smad7 or a dominant-negative form of the TGFP receptor inhibits the formation of osteolytic metastases by human breast cancer (Yin J.J. et al. J. Clin. Invest. 103: 197-206 (1999)), melanoma (Javelaud D. et al. Cancer Res. 67:2317-24 (2007)) and renal carcinoma cell line xenografts. (Kominsky SL. et al. J. Bone Miner. Res. 22:37-44 (2007). One of the mediators of TGFP osteolytic action is parathyroid hormone-related protein (PTHrP). [Kingsley LA. (2007)]. TGFP stimulates PTHrP secretion without appearing to increase PTHrP mRNA levels.

PTHrP stimulates the production of RANK ligand (RANKL) in osteoblasts, which in turn promotes the differentiation of osteoclast precursors and bone resorption. Administration of anti-PTHrP neutralizing antibodies inhibits TGFP-dependent osteolytic bone metastasis in mice. The role of TGF in metastatic colony expansion may not be limited to bone metastasis. A majority of metastases to lung, liver, and brain in breast cancer patients stain positive for phospho-Smad2, suggesting a widespread activation of this pathway in metastasis by locally released TGF . In breast cancer cells that have entered the pulmonary parenchyma, TGF may facilitate tumor reinitiation through an aberrant induction of ID1 expression. Padua D. (2008).

[0039] Inhibitors of the TGFP pathway developed to date encompass several classes. They include antisense oligonucleotides, inhibitors of ligand-receptor interactions such as anti-TGFp antibodies (Morris JC. et al. PLoS One 9:e90353 (2014)), anti-receptor antibodies, TGFP-trapping receptor ectodomain proteins, and small-molecule inhibitors that target TGFP receptor kinases. A few anti-TGFp compounds have shown efficacy in preclinical studies and several of these compounds are being evaluated in clinical trials. Arteaga CL. Curr. Opin. Genet. Dev. 16:30-7 (2006); Bierie B. et al. Nat. Rev. Cancer 6:506-20 (2006). Clinical trials for each of these inhibitor classes have been initiated not only against cancers (glioma, melanoma, breast cancer) but also against fibrosis, scarring, and other conditions that result from excessive TGFP activity. Blockers of TGFP signalling may potentially enhance enhance the progression of premalignant lesions, undermining potential clinical benefit especially for patients with TGFP promoted tumor growth. However, systemic

administration of TGFP blockers has not been reported to increase spontaneous tumor development in animal models with one possible exception. Prolonged treatment with an antibody (1D11) that binds to all three isoforms of TGFp, was shown to give rise to carcinomas. Such lesions are believed to be derived from pre-malignant foci that normally remain suppressed in the absence of antibody. Indeed, when antibody treatment is discontinued, the carcinomas resolve.

[0040] Therapeutic targeting of the TGFP pathway in tumors such as glioma, melanoma, and renal cell carcinoma is based on the rationale that TGFP exerts strong

immunosuppressive effects in these tumors. Thus, blocking TGFP function may empower the immune system against tumors. Blocking TGFP action may also have additional tumor- specific benefits. For example, TGFP inhibition in gliomas may curtail the production of autocrine survival factors, such as PDGF. Blocking TGFP in ER^~ breast cancer, on the other hand, might prevent primary or metastatic tumors from seeding and reseeding metastasis. Nam JS. et al. Cancer Res. 68:3835-43 (2008). Finally, in osteolytic bone metastasis, blocking TGFP might interrupt the cycle of TGFP-induced osteoclastogenic factors and halt tumor growth. Inhibition of TGF may lead to chronic inflammatory and autoimmune reactions, but this has not been observed in preclinical or clinical trials of systemic TGF blockers. It is also possible that targeting TGF receptor function may contribute to alternatively enhanced activity through compensatory mechanisms by other activators of the Smad pathway, reminicent to what occurs in patients with inactivating mutations in TGFBRI or TGFBR11. Loeys BL. et al. N. Engl. J. Med. 355:788-98 (2006).

[0041] Progress in delineating the protumorigenic effects and mechanisms of TGF in specific tumor types, in different stages of cancer progression and in specific cell types in the tumor microenvironment may aid in determining when and how anti-TGF targeted therapy might be feasible. The use of nanoparticle formulations as demonstrated herein to target the specific myeloid derived population of macrophages and dendritic cells to promote their antitumor function as a generalized approach to cancer treatment is promising.

[0042] For additional insights into the biology and function of TGF see: Zou Z. et al. Protein Expr. Purif. 37:265-72 (2004); Annes JP. et al. J. Cell Set ; 116:217-24 (2003); Puthawala K. et al. Am. J. Respir. Crit. Care Med. 177:82-90 (2008); Valcourt U. et al. Mol. Biol. Cell 16: 1987-2002 (2005); Takatori H. et al. Mod. Rheumatol. 18:533-41 (2008); Manel N. et al. Nat. Immunol. 9:641-9 (2008).

[0043] It has been indicated that systemic elevation of TGF is common to cancers and that blocking TGF together with Adenosine Receptors in combination may overcome pro- tumor effects and immunosuppression:

1. Cancers have elevated levels of TGF and adenosine that contribute to

immunosuppression and pro-tumor effects.

2. The systemic administration of an anti-TGF antibody (1D11) that inhibits TGF i, TGF 2 and TGF 3 was shown to enhance an anti-cancer vaccine Lonning S. et al. Curr. Pharm. Biotechnol. 12:2176-89 (2011)) in preclinical studies.

3. Anti-TGF approaches have potential for the clinic. Arteaga CL. (2006); Bierie B. et al. (2006); Wrzesinski SH. et al. Clin. Cancer Res. 13:5262-70 (2007).

4. Deletion of adenosine receptors slows tumor growth.

5. Antagonists of the Adenosine A2A receptor inhibit tumor growth.

[0044] Nanoparticles herein comprising adenosine antagonists mediate effective antitumor responses. The nanoparticles developed herein have facilitated delivery and uptake into APCs of anti-immunosuppressive small molecules to engender systemic anti-tumor immune responses. It has been indicated that transient suppression of TGF would be sufficient for protective tumor immunity through reduction of Tregs. Conroy H. et al. Cancer Immunol Immunother. 2012;61(3):425-31. The nanoparticles herein may provide a "tumor vaccine approach" through delivery of the combination of TGF inhibitor and adenosine antagonist into even one or a small number of tumors resulting in systemic immune surveillance response in metastatic tumors such as breast cancer. This concept is supported by data described the Examples shown herein below.

[0045] In some embodiments there are provided nanoparticle compositions comprising an adenosine receptor antagonist, a permeation enhancer, a TGF inhibitor, and a poly(lactic-co- glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.

[0046] "Adenosine receptor antagonist" refers to any of the adenosine antagonist subtypes, and includes any combinations of subtypes. The Examples below show results with selective A2A receptor antagonists. However, those skilled in the art will appreciate that A2A antagonists may still have some inhibitory effect on the other adenosine receptors and vice versa. Thus, nanoparticle formulations with A2B antagonists or the combination of A2A and A2B antagonists may be combined with TGF .

[0047] In some embodiments, the adenosine receptor antagonist comprises an A₂A-type antagonist and/or an A₂B-type antagonist. In some embodiments, the adenosine receptor antagonist comprises A2A-type antagonist. In some embodiments, the A2A-type antagonist comprises SCH58621 of formula I:

[0048] In some embodiments, the adenosine receptor antagonist is present in an amount in a range from about 0.02% to about 0.5% by total weight of the composition, including any subrange in between such as 0.05 to 0.25% by total weight of the composition, or 0.1 to 0.2% by total weight of the composition.

In some embodiments, the TGF-β inhibitor is selected from, but not limited to the group consisting of SB431542 (4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2- yl]benzamide), SB525334 ( 6-[2-(l,l-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-lH-imidazol- 4-yl]quinoxaline), Ki26894 (Kirin Brewery Company, Gunma, Japan, (Ehata et al Cancer Sci 98): 127-133), LY364947 (4-[3-(2-Pyridinyl)-lH-pyrazol-4-yl]-quinoline), SD-208 (2-(5- Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine), SD-093 (2-(2-fluorophenyl)-N- pyridin-4-ylpyrido[2,3-d]pyrimidi -4-amine) (US Patent No. 6,476,031):

, SM16 (4-(5-(benzo[d][l,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-lH-imidazol-2- yl)bicyclo[2.2.2]octane-l-carboxamide), Ly2109761(4-[2-[4-(2-pyridin-2-yl-5,6-dihydro-4H- pyrrolo[l,2-b]pyrazol-3-yl)quinolin-7-yl]oxyethyl]morpholine), Ly2157299 (2-(6-methyl- pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro -4H-pyrrolo[l,2-b]pyrazole

monohydrate), K02288(3-[6-amino-5-(3,4,5-trimethoxy-phenyl)-pyridin-3-yl]-plienol), SB505124 (2-[4-(l,3-Benzodioxol-5-yl)-2-(l, l-dimethylethyl)-lH-imidazol-5-yl]-6-methyl- pyridine), LDN-193189 ( 4-(6-(4-(piperazin-l-yl) phenyl) pyrazolo[l,5-a]pyrimidin-3- yl)quinoline hydrochloride), GW788388 ( 4-[4-[3-(2-Pyridinyl)-lH-pyrazol-4-yl]-2- pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), Ly580276 (3-(4-fluorophenyl)-2-(6- methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole), EW-7203 (3-((5- ([l,2,4]triazolo[l,5-a]pyridin-6-yl)-4-(6-methylpyridin-2-yl)thiazol-2- ylamino)methyl)benzonitrile), EW-7195 (3-[methyl-[5-(6-methylpyridin-2-yl)-4- ([l,2,4]triazolo[l,5-a]pyridin-6-yl) H-imidazol-2-yl]amino]benzonitrile), EW-7197 (N-[[4- ([l,2,4]triazolo[l,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-lH-imidazol2-yl]methyl]-2- fluoroaniline), YR-290 (N-phenylacetyl-l,3,4,9-tetrahydro-l H -beta-carboline), A 83-01(3- (6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)- lH-pyrazole- 1 -carbothioamide), D4476 ( 4-[4-(2,3-Dihydro-l,4-benzodioxin-6-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]benzamide), RepSox[alternatively E-616452, SJN 2511] ( 2-(3-(6-Methylpyridine-2-yl)-lH-pyrazol-4-yl)- 1,5-naphthyridine), R268712 (4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-lH-pyrazol-4- yl]phenyl]-lH-pyrazole-l-ethanol) (or mixtures or combinations thereof, or and

pharmaceutically acceptable salts thereof.

[0049] In some embodiments, the TGF inhibitor comprises SB431542 of formula II:

[0050] In some embodiments, the TGF inhibitor is present in an amount in a range from about 0.02% to about 0.5% by total weight of the composition.

[0051] Nanoparticle compositions here comprise a permeation enhancer. A "permeation enhancer" is a compound that aids in delivery of the nanoparticles into the target cells. That is, they enhance the ability of the nanoparticles to cross the cell membrane and enter the cell. In some embodiments, the permeation enhancer is selected from the group consisting of chitosan material, a fatty acid, a bile salt, a salt of fusidic acid, a polyoxyethylenesorbitan, a sodium lauryl sulfate, polyoxyethylene-9-lauryl ether (LAURETH™-9), EDTA, citric acid, a salicylate, a caprylic glyceride, a capric glyceride, sodium caprylate, sodium caprate, sodium laurate, sodium glycyrrhetinate, dipotassium glycyrrhizinate, glycyrrhetinic acid hydrogen succinate, a disodium salt, an acylcarnitine, a cyclodextrin, a phospholipid, and mixtures or combinations thereof.

[0052] In some embodiments, the permeation enhancer comprises chitosan. In some embodiments, chitosan or othe permeation enhancer is present in an amount in a range from about 1 % to about 20% by total weight of the composition. In some embodiments, the nanoparticle compositions have a zeta potential in a range from about -30 mvolts to about +30 mvolts, such zeta potential being modifiable by altering the amount of permeation enhancer chitosan.

[0053] Other polymer matrices may perform a similar function as PLGA, which is primarily used for the property of being a biodegradable polymer. Thus, in accordance with embodiments herein other biodegradable polymers suitable as matrices for the nanoparticles include, without limitation, a polyester, a lactic acid polymer, homopolymers of lactic acid or glycolic acid (e.g., poly lactic acid (PLA), poly gly colic acid (PGA)), poly-s-caprolactone (PCL), poly(anhydrides), poly(amides), poly(urethanes), poly(carbonates), poly(acetals), poly (ortho-esters), poly(glycolide-co-trimethylene carbonate), poly(dioxanone),

poly(phosphoesters), poly(phosphazenes), poly(cyanoacrylate), poly(ethylene oxide), poly(propylene oxide), poly(N-isopropylacrylamide) (PNIPAAm), poly(2- (diethylamino)ethyl methacrylate) (PDEAEMA), poly(2-aminoethyl methacrylate)

(PAEMA), 2 (dimethylamino)ethyl methacrylate (DMAEMA), polyethylene glycol) (PEG), N-(2-hydroxypropyl)methacrylamide (HPMA), poly(beta-benzyl-L-aspartate) (PBLA), poly(hydroxybutyrate-co valerate), derivatives thereof, and mixtures or combinations thereof.

[0054] In some embodiments, a size of the nanoparticle is in a range from about 50 nm to about 2,000 nm. In some embodiments, a size of the nanoparticle is in a range from about 50 nm to about 1,000 nm. In some embodiments, a size of the nanoparticle is in a range from about 100 nm to about 1,000 nm. In some embodiments, a size of the nanoparticle is in a range from about 200 nm to about 1,000 nm. In some embodiments, a size of the nanoparticle is in a range from about 50 nm to about 500 nm. In some embodiments, a size of the nanoparticle is in a range from about 100 nm to about 500 nm. In some embodiments, more than 50% of the nanoparticles have a size in a range with from about 400 nm to about 800 nm. Consistent with the term "nanoparticle"as used in the art, such term refers to a particle having an average diameter of about 0.5 nm to about 1 micron. In some embodiments, the nanoparticle has an average diameter of about 5 nm to about 950 nm, about 50 nm to about 900 nm, about 100 nm to about 800 nm, about 150 nm to about 750 nm, about 200 nm to about 700 nm, about 300 nm to about 600 nm, or about 400 nm to about 500 nm.

[0055] In some embodiments, the nanoparticles may comprise a dye. Such dyes may include, without limitation, lipophilic tracer dyes such as DiD dye (l,l '-dioctadecyl- 3,3, 3", 3"- tetramethylindodicarbocyanine), DiO dye (3,3'- dioctadecyloxacarbocyanine), DiA dye (4-(4- (dihexadecylamino)styryl)-N- methylpyridinium ), Dil dye ((2Z)-2-[(E)-3-(3,3-dimethyl-l- octadecylindol-l-ium-2- yl)prop-2-enylidene]-3,3-dimethyl-l-octadecylindole; perchlorate; CAS No. 41085- 99-8) , and DiR dye (l,l '-dioctadecyl-3,3,3',3'- tetramethylindotricarbocyanine), which are commercially available from Life Technologies. The dyes described herein can have various emission wavelengths. One of skill in the art will appreciate that the dyes described herein have various purposes including but not limited to particle identification, size determination, tracking, and quantification in vitro and in vivo.

[0056] In some embodiments, nanoparticle compositions herein have been developed to selectively block both the adenosine and TGF-β (TGF-beta I, TGF-beta II)

immunosuppressive pathways through nanoparticle delivery and uptake into antigen presenting cells. The combination of these pathway inhibitors may be more effective than either inhibitor alone as a tumor therapeutic. The combined nanoparticle formulation of TGF and Adenosine pathway inhibitors may be of general use as an immunologically based cancer therapy with the potential to induce protective anti-tumor immunity.

[0057] In some embodiments, the adenosine receptor antagonist is present in an amount in a range from about 0.02% to about 0.5% by total weight of the composition.

In some embodiments, the TGF-β inhibitor is selected from, but not limited to the group consisting of SB431542 (4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2- yljbenzamide), SB525334 ( 6-[2-(l,l-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-lH-imidazol-

4-yl]quinoxaline), Ki26894 (Kirin Brewery Company, Gunma, Japan).

127-133), LY364947 (4-[3-(2-Pyridinyl)-lH-pyrazol-4-yl]-quinoline), SD-208 ( 2-(5-

Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine), SD-093 ( 2-(2-fluorophenyl)-N- pyridin-4-ylpyrido[2,3-d]pyrimidi -4-amine) ( ref: US Patent No 6,476,031 (to Scios, Inc.)):

, SM16 (4-(5-(benzo[d][l,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-lH-imidazol-2- yl)bicyclo[2.2.2]octane-l-carboxamide), Ly2109761(4-[2-[4-(2-pyridin-2-yl-5,6-dihydro-4H- pyrrolo[l,2-b]pyrazol-3-yl)quinolin-7-yl]oxyethyl]morpholine), Ly2157299 (2-(6-methyl- pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro -4H-pyrrolo[l,2-b]pyrazole

monohydrate), K02288(3-[6-amino-5-(3,4,5-trimethoxy-phenyl)-pyridin-3-yl]-phenol), SB505124 (2-[4-(l,3-Benzodioxol-5-yl)-2-(l, l-dimethylethyl)-lH-imidazol-5-yl]-6-methyl- pyridine), LDN-193189 ( 4-(6-(4-(piperazin-l-yl) phenyl) pyrazolo[l,5-a]pyrimidin-3- yl)quinoline hydrochloride), GW788388 ( 4-[4-[3-(2-Pyridinyl)-lH-pyrazol-4-yl]-2- pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), Ly580276 (3-(4-fluorophenyl)-2-(6- methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole), EW-7203 (3-((5- ([l,2,4]triazolo[l,5-a]pyridin-6-yl)-4-(6-methylpyridin-2-yl)thiazol-2- ylamino)methyl)benzonitrile), EW-7195 (3-[methyl-[5-(6-methylpyridin-2-yl)-4- ([l,2,4]triazolo[l,5-a]pyridin-6-yl) H-imidazol-2-yl]amino]benzonitrile), EW-7197 (N-[[4- ([l,2,4]triazolo[l,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-lH-imidazol2-yl]methyl]-2- fluoroaniline), YR-290 (N-phenylacetyl-l,3,4,9-tetrahydro-l H -beta-carboline), A 83-01(3- (6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)- lH-pyrazole- 1 -carbothioamide), D4476 ( 4-[4-(2,3-Dihydro-l,4-benzodioxin-6-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]benzamide), RepSox[alternatively E-616452, SJN 2511] ( 2-(3-(6-Methylpyridine-2-yl)-lH-pyrazol-4-yl)- 1,5-naphthyridine), R268712 (4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-lH-pyrazol-4- yl]phenyl]-lH-pyrazole-l-ethanol) (or mixtures or combinations thereof, or and

pharmaceutically acceptable salts thereof.

[0058] In some embodiments, nanoparticle compositions may also comprise a targeting moiety. The targeting moiety may be selected from the group consisting of a tumor-targeting moiety, a viral-specific moiety, a bacteria-specific moiety, and a cell- targeting moiety. The targeting moiety may be a cell-targeting moiety and may be selected from the group consisting of a phagocytic cell-targeting moiety, a natural killer cell-targeting moiety, a T-cell targeting moiety, a B-cell targeting moiety, a glial cell targeting moiety, a myeloid cell targeting moiety, an epithelial cell targeting moiety, a macrophage-targeting moiety, a tumor cell-targeting moiety, and a dendritic cell-targeting moiety.

[0059] In some embodiments the macrophage or dendritic cell targeting moiety is chitosan.

[0060] The nanoparticle compositions herein may form part of a pharmaceutical composition comprising the nanoparticles described herein. Such pharmaceutical

compositions may be formulated for parenteral, intravenous, intradermal, subcutaneous, oral, inhalation, transdermal, transmucosal, rectal and intratumoral administration.

[0061] In some embodiments, there are provided methods of treating a disease or disorder characterized by immunosuppression comprising administering to a mammalian subject a nanoparticle composition comprising an adenosine receptor antagonist, a TGF inhibitor a permeation enhancer, and poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition. In some such embodiments the disease or disorder may be an inflammatory condition or an autoimmune condition.

[0062] In some embodiments, there are provided methods of treating cancer comprising administering to a mammalian subject a nanoparticle composition comprising an adenosine receptor antagonist, a TGF inhibitor a permeation enhancer, and poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.

[0063] Neoplasia, tumor, cancer and malignancy treatable in accordance with the methods herein include solid cellular mass, hematopoietic cells, or a carcinoma, sarcoma (e.g. lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma,

rhabdomyosarcoma or fibrosarcoma), lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma, mesothelioma, reticuloendothelial, lymphatic or haematopoietic (e.g., myeloma, lymphoma or leukemia) neoplasia, tumor, cancer or malignancy.

[0064] Neoplasia, tumor, cancer and malignancy treatable in accordance with the methods herein can be present in or affect a lung (small cell lung or non-small cell lung cancer), thyroid, head or neck, nasopharynx, throat, nose or sinuses, brain, spine, breast, adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genito-urinary tract (uterus, ovary, cervix, endometrial, bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood, muscle, skin or stem cell neoplasia, tumor, cancer, or malignancy.

[0065] As described above, adenosine receptor and TGF pathways are implicated in the activity of either the purinergic pathway involving adenosine signaling and/or TGF signaling. The use of small molecule inhibitors inhibits the immunosuppressive signals of these pathways and forms the basis for treatment of a variety of conditions including, without limitation, cancer.

[0066] In some embodiments, methods directed to cancer treatment may further comprise administering a chemotherapeutic agent to the subject. Exemplary chemotherapeutic agent classes useful with the nanoparticle compositions include, without limitation, anthracyclines, platinum drugs, intercalating chemotherapeutic agents, topoisomerase poisons,

cyclophosphamide drugs, and mixtures thereof.

[0067] Specific exemplary chemotherapeutic agents include, without limitation, daunomycin, Cytoxan, cytarabine, melphalan, adriamycin, colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferons, camptothecin and derivatives thereof, phenesterine, taxanes and derivatives thereof (e.g., paclitaxel and derivatives thereof, taxotere and derivatives thereof), topetecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab-5801, Irinotecan, HKP, Ortataxel, gemcitabine, HERCEPTIN®, vinorelbine, DOXIL®, capecitabine, ALIMTA®, AVASTIN®, VELCADE®, TARCEVA®, NEULASTA®, Lapatinib, Sorafenib, derivatives thereof, and other chemotherapeutic agents known in the art. In some embodiments, the chemotherapeutic agent is also composition comprising nanoparticles, either separately or together with the A2A anatagonist and TGF inhibitor. [0068] In some embodiments, the chemotherapeutic agent may be an antagonist of other factors that are involved in tumor growth, such as EGFR, ErbB2, ErbB3, ErbB4, or TNF. In some such embodiments, it may be beneficial to also administer one or more cytokines to the individual. In some some embodiments, the therapeutic agent is a growth inhibitory agent. In some embodiments, methods designed for the treatment of cancer may also further comprise administering radiation therapy.

[0069] In some embodiments, methods herein employ an adenosine receptor antagonist that comprises SCH58621 of formula I:

the TGF inhibitor co

the permeablizing agent comprises chitosan or derivatives of chitosan.

[0070] Although embodiments herein describe nanoparticle compositions in which the adenosine receptor antagonist and TFG inhibitor are present in the same nanoparticle matrix, it is also possible to deliver them in separate nanoparticles. Thus, in some embodiments, there are provided methods of treating a disease or disorder characterized by immunosuppression comprising administering to a mammalian subject a first nanoparticle composition comprising an adenosine receptor antagonist and co-administering a second nanoparticle composition comprising a TGF inhibitor, wherein the first and second nanoparticle compositions each comprise a permeation enhancer and a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix.

[0071] In some such embodiments, both administering and co-administering steps are peformed simultaneously. In some such embodiments, the first nanoparticle and second nanoparticle are disposed in a single oral capsule. In other embodiments, the first

nanoparticle composition is disposed in a first oral capsule and the second nanoparticle composition is disposed in a second oral capsule. Oral delivery is not limited to capsule delivery and this is merely exemplary. Other oral adminstration routes may include tablets, syrups, and the like.

In some embodiments, the administering and co-administering steps are performed intravenously. In some embodiments, the administering and co-administering steps are performed subcutaneously. In some embodiments the administering and co-administering steps are performed by intratumoral injection. In another aspect, the disclosure provides a pharmaceutical composition comprising the particles described herein. In some embodiments of all aspects, the composition is formulated for parenteral, intravenous, intradermal, subcutaneous, oral, inhalation, transdermal, transmucosal, rectal, intrathecal and intratumoral administration. In some embodiments in the adjuvant can be delivered p.o, i.p. s.c, i.v., i.t. (intrathecal) into spinal fluid, sublingual, lung inhalation, nasal administration, suppositories, eye drops or other means of administration.

[0072] In some embodiments, there are provided uses of a nanoparticle compositions in the manufacture of a medicament for the treatment of cancer, an inflammatory condition, or an autoimmune condition, the nanoparticle composition comprising an adenosine receptor A2A antagonist, an adenosine receptor A2B antagonist, a TGF-beta inhibitor, a permeabilizing agent; and a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.

Examples

EXAMPLE 1

[0073] Nanoparticle (NP) formulations were fabricated using 50:50 poly (lactide-co- glycolide) (PLGA 50:50) and chitosan (15% wt:wt) to enhance both mucosal penetration and uptake into phagocytic cells, specifically macrophages and dendritic cells. The concentration of adenosine inhibitor SCH58621 in this Example was 1 microgram per milligram of PLGA polymer. Tumor injections were done using 100 microliter volumes per injection at an overall concentration of 65 nM SCH58261 based on the amount of drug within the nanoparticles being administered (see Figure 2).

[0074] Anti-tumor effects of an exemplary nanoparticle composition were assessed by directly injecting tumors with NPs containing an A2A antagonist alone (SCH58261 (the nanoparticle-drug composition designated "SCH-NP" at 65 nM)). Such treatment resulted in inhibition of the growth of syngeneic 4T1 breast cancer in Balb/c mice as disclosed by Hammerl D. et al., "Intratumoral injection of microparticles containing the A2A receptor antagonist SCH58261 slow tumor growth and metastasis more effectively than system drug administration," Abstract in Purinergic Signal. 1019 (2016). "Tumor cells were injected into both right and left breast fat pads and allowed to grow for 5 days. Only one of the two tumors [right breast pad tumor] was treated by injecting it intratumorally with NPS embedded with the A2A antagonist. [The other tumor was untreated]. Both the intratumorally treated and untreated contralateral tumor slowed in growth and in some of the mice the tumors were completely inhibited. SCH-NP inhibited the highly metastatic potential of these cells as shown by the dramatic decrease of lung metastases. Intratumoral injections of free drug, without nanoparticle matrix, were much less effective than the injection of SCH-NP [due to rapid diffusion out from the tumor]. APC activation was observed in the draining lymph node on the intratumorally injected side but not on the tissue treated side. Treg cell numbers were reduced and CTL activation was observed in both tumors and their draining lymph nodes." Effective targeting of dendritic cells and macrophages (myeloid derived cells) was disclosed.

[0075] Figures 2A and 2B show the effects of NPs containing SCH58261 (NP-SCH) on the growth and metastasis of treated and untreated 4T1 breast cancer cells. Balb/cJ mice were injected in mammary fat pads on both sides with 75,000 4Tl-luciferase breast cancer cells. Intratumoral injections were done with 100 ml PBS, PLGA/chitosan/DID particle controls (NP), 65 nM NP-SCH (65 nM = 6e5 particles in 100 ml) at 1 week intervals starting on day 5, into the right tumor only. SCH concentrations were calculated as if the drug were free in solution in a volume of 100. Figure 2A shows tumor progression in injected tumors and Figure 2B shows tumor progression in uninjected contralateral tumors. In Figures 2 A and 2B, the NP-SCH were significantly slowed in tumor growth.

[0076] As indicated in Figure 2B, the treatment was effective even in the uninjected contralateral tumors indicating a systemic treatment effect possibly through drug distribution to the contralateral tumor or by a systemic immunostimulation that was initiated near the injected tumor. EXAMPLE 2 Manufacture of Nanoparticles Comprising a Combination of an

Adenosine A2A Receptor Antagonist and a TGF-beta Inhibitor.

[0077] PLGA-particles were prepared by modification of the method of Ravi Kumar. Ravi Kumar MN. et al. Biomaterials 25: 1771-1777 (2004) to include an adenosine antagonist and a TGF-beta inhibitor. Solutions of 5 mg/mL each of SCH58261, an A2A receptor antagonist (Baraldi PG. et al. J. Med. Chem. 37: 4329-4337 (1994).), and SB431542, a TGF- beta inhibitor (Inman GJ. et al. Mol. Pharmacol. 62: 65-74 (2002).), were made in DMSO. The amount of 200 mg of PLGA (50:50) was dissolved in 10 mL of ethyl acetate after which was added 0.05 mL of each drug solution alone or in combination and 0.1 mL of DiD dye (Invitrogen) in DMSO. An aqueous solution was prepared by mixing 9 mL sterile water (SW) with 15 mg of chitosan and 15 microliters of acetic acid. After dissolving the chitosan, 1 mL of 1% Poly- vinyl Alcohol (PVA) in SW was added. The ethyl acetate solution was poured into the aqueous solution and vigorously mixed by high-speed vortexing for 3 minutes.

Twenty mL of 0.1% PVA in SW was added while continuing to vortex. The suspension was transferred to a beaker on a magnetic stirrer and an additional 60 mL of 0.1 % PVA/SW was added and stirred overnight to evaporate the ethyl acetate. The microparticle suspension was centrifuged at 70 x g for 1 minute and the supernatant collected and then centrifuged at 1000 x g for 10 minutes. The pellet was taken up in 0.1 M citrate pH 3.3, aliquoted and stored at - 20°C (stock).

[0078] Microparticle characterization. An aliquot of 250 microliters of the final suspension (stock) was vacuum dried in a tared 1.5 mL microtube and the dry weight of the nanoparticles was determined. The dried nanoparticles were dissolved in 1 mL of DMSO. To determine the drug concentration, the sample was centrifuged at 1000 x g for 1 min and the absorbances at 285 and 336 nm were determined on the supernatant. The concentration of SCH58261 was calculated by dividing the absorbance at 285 nm by 0.061 to obtain the concentration in μg/mL and multiplied by 4 to determine the stock concentration of drug. The concentration of SB431542 was determined by dividing the absorbance at 336 nm by 0.0482 and multiplied by 4 to obtain the stock concentration in μg/mL. HPLC was performed by adding 2 volumes of Ethanol:Water:Glacial Acetic Acid (20:75:5::vol:vol:vol) (EWA) to 1 volume of the DMSO solution and then mixed by shaking. The precipitates were removed by centrifugation at 10,000 x g for 5 min and 50 microliters of the supernatant was run on an Agilent Zorbax GF250 column equilibrated to 30°C at a flow rate of 1 niL/min using EWA as a mobile phase. Dilutions of the stock were then made to the appropriate nM concentrations of drug in 5mM Phosphate buffer, 0.9% saline pH 6.8. The ratio of drug to polymer was generally 0.8-1 g/mg polymer. The particle size and charge was determined by

Nanocomposix using a Malvern Nanosight unit and a Malvern Zetasizer, repectively, after diluting the stock nanoparticles in water to 0.1 mg/mL of polymer (Figures 3 and 4).

[0079] Figure 3 shows the averaged size distribution of nanoparticles comprising SCH58261 and SB431542. The stock nanoparticles of Lot 16005 were diluted in water to 0.1 mg/mL and size measurements were performed using Nanosight technology (Nanocomposix, San Diego, CA). The mean particle size was 533.4 nm + 118.2 nm. The particle concentration was 3.19 x 10⁹ + 3.28 x 10⁸ particles/mL.

[0080] Figure 4 shows the zeta potential measurement of nanoparticles. The stock nanoparticles of Lot 16005 were diluted in sterile water to 0.1 mg/mL of polymer and the zeta potential was measured using a Malvern Zetasizer (Nanocomposix, San Diego, CA). A plot of the particle counts vs apparent zeta potential is shown. The zeta potential was measured to be 18.4 + 5.89 mV.

[0081] Figures 5A/5B show the UV spectra of SCH58261 and SB431542, respectively. The nanoparticles (0.250 mL of stock) were dried and dissolved in DMSO and centrifuged at 1000 x g for 1 minute. The UV spectra were obtained on the supernatants using a reference of non-drug containing nanoparticles prepared in the same way. The Top Panel shows the UV spectra of nanoparticles containing each individual drug in an overlay of SB431542 (dotted line) and SCH58261 (solid line). SCH58261 has strong absorbance at 285 nm in a region where SB431542 is transparent while SB438542 absorbs at 336 in a region where SCH58261 is transparent. The bottom panel shows the UV spectrum nanoparticles (Lot24b) containing the combination of both SCH58261 and SB431542.

[0082] FIGURE 6 shows an HPLC trace of SB431542 (left panel) and SCH58261 (right panel) using an Agilent Zorbax GF-250 9.4 x 250 mm column equilibrated to 30°C. Stock drug solutions of 5 mg/mL in DMSO were diluted 1:500 in Ethanol:Water:Glacial Acetic Acid (20:75:5)(EWA) and run at 1 niL/min in EWA as the mobile phase. The retention times for SB431542 and SCH58261 were 9.178 and 16.011 min respectively.

[0083] Figure 7 shows an HPLC trace of an extract from nanoparticles embedded with both SCH58261 (peak retention time of 15.884 minutes) and SB431542 (peak retention time of 9.288 minutes). Particles were vacuum dried and dissolved in DMSO. Two volumes of Ethanol:Water:Glacial Acetic Acid (20:75:5::vol:vol:vol) (EWA) were added to 1 volume of the DMSO solution and then mixed by shaking. The precipitates were removed by centrifugation at 10,000 x g for 5 min and the supernantant collected and filtered through a 0.2 PTFE filter. The amount of 50 microliters of the supernatant was run using an Agilent Zorbax GF250, 9.4 x 250 mm column equilibrated to 30°C at a flow rate of 1 niL/min. EWA was used as the mobile phase. The retention times of SB431542 and SCH58261 (labeled peaks at 9.288 and 15.884 minutes, respectively) were consistent with the original stocks as shown in FIGURE 5.

[0084] Figures 8A/8B indicate the enhanced anti-tumor activity of nanoparticles containing both an adenosine A2A receptor antagonist and a TGF-beta inhibitor. The amount of 5 x 10⁴ Lewis Lung Carcinoma cells was injected into both flanks of syngeneic C57/B16 mice. When tumors volumes reached 60 mm³ on day 10, nanoparticle-drug preparations (100 nM drug or BLANK) were injected into the tumors in the left flanks (Injected Tumor) while no injections were made into tumors on the right flanks (Uninjected Tumor). Two additional injections were performed on days 14 and 17. Tumor growth was monitored by caliper measurements over the indicated number of days. The nanoparticle preparations were TI- BLANK, a non-drug containing control; TI-07B, nanoparticles containing SB431542 (a TGF- beta inhibitor); TI-07H, nanoparticles containing SCH58261 (an adenosine A2A receptor antagonist) and TI-07, nanoparticles containing both SB431542 and SCH58261 (the combination of a TGF-beta inhibitor and an A2A receptor antagonist). The combination formulation, TI-07, resulted in enhanced anti-cancer effects regarding tumor growth. As in Figures 2A/B, it is remarkable that the untreated tumors on the contralateral side still responded to the treatment as effectively as the intratumorally injected side. This was true for the nanoparaticle composition with both the A2A antagonist and the TGFD inhibitor, which performed the best. Figure 8A and 8B show a slowing of the tumor growth with TI-07 (asterisks *) starting at day 14 (day of 2nd injection). A slowing of tumor growth in Figure 8B (asterisk *) is consistent with a generalized anti-tumor immune response.

[0085] Figure 9 shows a bar graph tumor volumes for TI-07 and controls on day 18 of Figure 6. Lewis Lung Carcinoma tumors were generated as described in Figure 6 with two tumors per mouse, one tumor on the left and one tumor on the right flank of each mouse. The left flank tumors of each mouse were injected while the right flank tumors were uninjected. Plotted are the average tumor volumes of the TI-07 injected tumors (left flanks) and uninjected tumors (right flanks) on day 18 of Figure 8A and 8B, respectively: only the left flank tumors were injected with the combination of adenosine A2A receptor antagonist (SCH58261, 100 nM) and TGF-beta inhibitor (SB431542, 20 nM) (n=10); Control: only the left flank tumors were injected with Blank nanoparticles (n=7). The p values were obtained using the Student's T-test (Microsoft Excel, 2016); Arrayl= TI-07; Array2= Control;

Type=2; Tails=2. The data show significant suppression of tumor growth with TI-07 the combination of SB431542, the TGFb inhibitor, and SCH58261, the adensonine A2A receptor antagonist, compared to the controls.

Claims

What is claimed is:

1. A nanoparticle composition comprising:

an adenosine receptor antagonist;

a permeation enhancer;

a TGF inhibitor; and

a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.

2. The nanoparticle composition of claim 1, wherein the adenosine receptor antagonist

comprises an A_2A receptor-type antagonist and/or an A₂B receptor-type antagonist.

3. The nanoparticle composition of claim 1, wherein the adenosine receptor antagonist comprises A2A receptor-type antagonist.

4. The nanoparticle composition of claim 3, wherein the A -type antagonist comprises SCH58621 of formula I:

5. The nanoparticle composition of any one of claims 1 to 4, wherein the adenosine receptor antagonist is present in an amount in a range from about 0.02% to about 0.5% by total weight of the composition.

6. The nanoparticle composition of any one of claims 1 to 5, wherein the TGF-β

inhibitor is selected from, but not limited to the group consisting of SB431542, SB25334, Ki26894, LY364937, SD-208, SD093, SM16, Ly2109761, Ly2157299, K02288, SB505124, Ly2157299, LDN-193189, GW788388, Ly580276, EW-7203, EW-7195, EW-7197, YR-290, A 83-01, D4476, RepSox, R268712 or mixtures or combinations thereof, or and pharmaceutically acceptable salts thereof.

7. The nanoparticle composition of claim 6, wherein the TGF inhibitor comprises SB431542 of

8. The nanoparticle composition of claim 6, wherein the TGF inhibitor is present in an amount in a range from about 0.02% to about 0.5% by total weight of the

composition.

9. The nanoparticle composition of any one of claims 1 to 8, wherein the permeation enhancer comprises chitosan.

10. The nanoparticle composition of claim 9, wherein chitosan is present in an amount in a range from about 1 % to about 20% by total weight of the composition.

11. The nanoparticle composition of any one of claims 1 to 10, wherein a size of the nanoparticle is in a range from about 50 nm to about 2,000 nm.

12. The nanoparticle composition of any one of claims 1 to 10, wherein more than 50% of the nanoparticles have a size in a range with from about 400 nm to about 800 nm.

13. The nanoparticle of any one of claims 1 to 12, having a zeta potential in a range from about -30 mvolts to about +30 mvolts.

14. The nanoparticle of any one of claims 1 to 13, further comprising a dye.

15. A method of treating a disease or disorder characterized by immunosuppression

comprising:

administering to a mammalian subject a nanoparticle composition comprising: an adenosine receptor antagonist;

a TGF inhibitor

a permeation enhancer; and

a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.

16. The method of claim 15, further comprising administering a chemotherapeutic agent.

17. The method of claim 15, further comprising administering radiation therapy.

18. The method of claim 15, 16 ,17 or 18 wherein the adenosine receptor antagonist comprises SCH58621 of formula I:

the TGF-β inhibitor comprises SB431542 of formula II:

the permeation enhancer comprises chitosan or derivatives of chitosan.

19. The method of any one of claims 15 to 20, wherein the disease or disorder is selected from the group consisting of cancer, an inflammatory condition, and an autoimmune condition.

20. A method of treating cancer or a disease or disorder characterized by

immunosuppression comprising:

administering to a mammalian subject a first nanoparticle composition comprising an adenosine receptor antagonist; and

co-administering a second nanoparticle composition comprising a TGF inhibitor;

wherein the first and second nanoparticle compositions each comprise a permeation enhancer and a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix.

21. The method of claim 20, wherein both administering and co-administering steps are peformed simultaneously.

22. The method of claim 21, wherein the first nanoparticle and second nanoparticle are disposed in a single oral capsule.

23. The method of claim 20 or 21, wherein the first nanoparticle composition is disposed in a first oral capsule and the second nanoparticle composition is disposed in a second oral capsule.

24. The method of claim 20 or 21, wherein the administering and co-administering steps are performed intravenously.

25. The method of claim 20 or 21, wherein the administering and co-administering steps are performed subcutaenously.

26. A method of treating cancer comprising:

administering to a mammalian subject a nanoparticle composition comprising: an adenosine receptor antagonist;

a TGF inhibitor;

a permeation enhancer; and

a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.

27. The method of claim 26, further comprising administering a chemotherapeutic agent.

28. The method of claim 26, further comprising administering radiation therapy.

29. The use of a nanoparticle composition in the manufacture of a medicament for the treatment of cancer, an inflammatory condition, or an autoimmune condition, the nanoparticle composition comprising:

an adenosine receptor A2A antagonist,

a TGF-beta inhibitor,

a permeation enhancer; and

a poly(lactic-co-glycolic acid) (PLGA) copolymer as a matrix for the nanoparticle composition.