WO2009033284A1

WO2009033284A1 - Inhibitors of collagen biosynthesis as anti-tumor agents

Info

Publication number: WO2009033284A1
Application number: PCT/CA2008/001614
Authority: WO
Inventors: Brian Lichty; Vettai Ananthanarayanan
Original assignee: McMaster University
Current assignee: McMaster University
Priority date: 2007-09-14
Filing date: 2008-09-12
Publication date: 2009-03-19
Anticipated expiration: 2010-03-14

Abstract

A method of treating cancer by inhibiting collagen formation is provided. In particular, small molecules that inhibit Hsp47 activity are used to either inhibit the proliferation of tumor cells or to kill tumor cells.

Description

Inhibitors of Collagen Biosynthesis as Anti-Tumor Agents

FIELD OF INVENTION

[0001 ] The present invention relates to an antitumor agent and methods of using the agent to prevent or treat a tumor. In particular, agents and methods that interfere with collagen synthesis are provided.

BACKGROUND OF THE INVENTION

[0002] Hsp47 is a collagen-specific chaperone that binds to nascent procollagen polypeptide strands as they transit through the ER and enter the Golgi apparatus. In the low-pH environment of the Golgi, Hsp47 dissociates from the now triple-helical procollagen molecule Procollagen polymerizes into dense aggregates s in the Golgi (Lamande and Bateman, 1999) before its secretion into the extracellular matrix where mature collagen is produced by higher order assembly and cross-linking (Bateman J. F., Lamande S. R., Ramshaw JAM. (1996).

[0003] Hsp47 is critical to proper collagen synthesis. The absence of Hsp47 leads to premature polymerization of collagen fibrils in the ER and this leads to a stress response that induces apoptosis and the subsequent death of the cell (Marutani et al., 2004). Thus, inhibition of Hsp47 function in cells expressing collagen leads to aberrant collagen secretion and can lead to the death of the secreting cell.

[0004] As collagen is a relatively stable protein with a long half-life in vivo, the ongoing production of collagen in an adult is very low. Inhibition of Hsp47 function was therefore predicted to have minimal effects on normal tissues (where collagen production if negligible) but should limit collagen production in pathological states such as fibrosis.

[0005] The present invention addresses the need for new cancer therapies based on inhibition of collagen formation.

SUMMARY OF THE INVENTION

[0006] The invention comprises strategies that target Hsp47 function to treat cancer. The invention relates to various strategies to target Hsp47 expression and/or function within tumours to impair the growth of the tumour or to kill tumorous cells. The invention encompasses methods and compositions that can be directed to both malignant cells and supporting tumour stroma.

[0007] In one aspect of the invention, compositions comprising agents that inhibit Hsp47 are provided. The compositions are preferably used for the treatment of cancer.

[0008] In a preferred embodiment of the invention a small molecule inhibitor of Hsp47 function is used to impair tumour growth. Collagen secreting cells within the tumour are targeted. This leads to impaired function of these cells and as a consequence, to cell death.

[0009] In a preferred embodiment of the invention, the inhibitor has a structure selected from the following formulae:

, where Ri is amine or substituted amine, R₂ is nitro and

R3 is alkyloxy or halogen.

, where R-i is halogen and R₂ is alkylcarboxylate.

, where R₁ is cyclohexyl or thienyl, R₂ is cyanyl and

where X is halogen and Y is alkyl. [0010] In a preferred embodiment of this invention the inhibitor is selected from the group consisting of:

[0011] In a particularly preferred embodiment of this invention, the compound is compound 1 (also known as RF03420).

[0012] In another aspect of the invention, methods for inhibiting Hsp47 function are provided. By inhibiting the function of this protein one can impair collagen production within a growing tumour and impede tumour growth and/or kill cells within a tumourous mass or metastases. Potential target cells are any cells expressing Hsp47 and secreting collagen.

[0013] In a preferred embodiment of the invention, the tumor is selected from the group consisting of lung carcinoma, breast carcinoma, prostate carcinoma, colon adenocarcinoma, cervical carcinoma, endometrial carcinoma, ovarian carcinoma, bladder carcinoma, Wilm's tumor, fibrosarcoma, osteosarcoma, melanoma, synovial sarcoma, epidermoid carcinoma, pancreatic carcinoma, endocrine system carcinoma, astrocytoma, oligodendroglioma, menigioma, neuroblastoma, glioblastoma, ependyoma, Schwannoma, neurofibrosarcoma, neuroblastoma, and medullablastoma.

[0014] In a further preferred embodiment of the invention, the target cells are malignant cells expressing Hsp47 and collagen.

[0015] In another preferred embodiment of the invention, the target cells are malignant stromal fibroblasts expressing Hsp47 and collagen.

[0016] In yet another preferred embodiment of this invention these target cells are m endothelial cells expressing Hsp47 and collagen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

FIGURE 1 illustrates relative Hsp47 expression levels for several cell lines;

FIGURE 2 shows graphically the viability of cultured cells following exposure to an Hsp47 inhibitor;

FIGURE 3 illustrates the effect of topical treatment with an Hsp47 inhibitor on subcutaneous CT26 tumours;

FIGURE 4 demonstrates systemic treatment of breast carcinoma in a xenograft model;

FIGURE 5 illustrates the effect of peri-tumoural treatment in a subcutaneous colon cancer model;

FIGURE 6 is a series of photomicrographs showing immunohistochemical staining of CT26 tumours; FIGURE 7 is a series of photomicrographs at a higher magnification illustrating immunohistochemical staining of Hsp47on CT26 tumours;

FIGURE 8 illustrates immunohistochemical staining of normal tissues;

FIGURE 9 illustrates immunohistochemical staining of MDA MB 231 tumours;

DETAILED DESCRIPTION

[0018] Collagen synthesis is associated with solid tumour growth. Proper collagen synthesis and secretion requires Hsp47 function. By inhibiting Hsp47 function one can impair collagen synthesis and impair tumour growth. Inhibition of Hsp47 function in collagen secreting cells is very stressful to these cells and can kill them. In a growing tumour, collagen secretion can be mediated by malignant cells, normal stromal cells or both. Inhibition of Hsp47 function in these cells can inhibit tumour growth and/or kill the cells leading to tumour destruction. The terms "inhibition of Hsp47 function" and "Hsp47 inhibitor" are used herein to refer to methods and compositions that inhibit Hsp47 expression, function and/or interaction with collagen. Since collagen turnover is virtually nil in normal adult cells but is excessive in tumor cells, impairing collagen synthesis by inactivation of Hsp47 by the said inhibitors would not cause any side effects, unlike the case with other available cancer treatments. Hsp47 is a novel therapeutic target for the treatment of cancer.

[0019] According to the invention, a method of treating a tumor by administering a composition comprising a Hsp47 inhibitor is provided. Different types of Hsp47 inhibitors, such as a small molecule, siRNA, shRNA, intrabody, antibody and peptide are encompassed.

[0020] The Hsp47 inhibitor may be administered in combination with other therapeutic agents, either at the same time or sequentially. The additional therapeutic agent may be a chemotherapeutic agent. Other examples of additional agents that can be combined with the Hsp47 inhibitor include regulators of the unfolded protein response (UPR), endoplasmic reticulum-associated protein destruction (ERAD) response, the proteasome and heatshock protein function and combinations thereof. Compositions comprising a Hsp47 inhibitor are provided. A composition comprising an inhibitor and an additional anti-tumor agent are encompassed. For example, compositions comprising together or sequentially an oncolytic virus and/or an anti-tumor pharmaceutical and methods for their use are also encompassed.

[0021] Various types of tumors, including but not limited to, lung carcinoma, breast carcinoma, prostate carcinoma, colon adenocarcinoma, cervical carcinoma, endometrial carcinoma, ovarian carcinoma, bladder carcinoma, Wilm's tumor, fibrosarcoma, osteosarcoma, melanoma, synovial sarcoma, epidermoid carcinoma, pancreatic carcinoma, endocrine system carcinoma, astrocytoma, oligodendroglioma, menigioma, neuroblastoma, glioblastoma, ependyoma, Schwannoma, neurofibrosarcoma, neuroblastoma, and medullablastoma can be treated using the compositions and methods of the invention.

[0022] The composition comprising the Hsp47 inhibitor may be administered via parenteral, oral, rectal, vaginal, topical, intranasal, inhalation, buccal, or ophthalmic administration. The administration may be via intravenous injection, subcutaneous injection, intraperitoneal injection, intra-arterial injection, intramuscular injection, intralesional injection into the tumor, intralesional injection adjacent to the tumor, intravenous infusion, and/or intra-arterial infusion.

[0023] The route of administration and the dosage to be administered can be determined based on the type of tumor being treated and patient characteristics, such as weight, age, sex, etc.

[0024] Several inhibitors of collagen synthesis have been identified (Thomson, C.A., Atkinson, H. M., and Ananthanarayanan, V.S. (2005)). These include:

[0025] These inhibitors were tested for their ability to inhibit Hsp47 and for their ability to induce regression or slow growth of a tumor. Exemplary results using some of these inhibitors are shown in the attached figures where it can be seen that treatment with an Hsp47 inhibitor is an effective therapy for the treatment of cancer.

[0026] Figure 1 illustrates that tumour cells can express high levels of Hsp47 in some cases and Figures 6 and 7 demonstrate that tumour stromal fibroblasts can also express high levels in vivo. These results indicate that two types of tumours can be targeted by agents that inhibit Hsp47 activity: Firstly, tumours where the malignant cells themselves are high expressors of collagen and Hsp47 can be targeted. This is exemplified by, but not limited to, breast carcinoma as demonstrated by the results using human breast carcinoma cell line MDA MB 231. In addition, tumours that induce a strong fibrotic response can be targeted. This is be exemplified by, but not limited to, pancreatic tumours wherein stromal fibroblasts are induced to express copious collagen and other extracellular matrix components. The Hsp47 inhibitor compounds identified have a high potential for arresting fibrosis. [0027] The Hsp47 inhibitor compounds were tested for their ability to inhibit collagen production in several cell lines. Cell lines that express Hsp47 are more sensitive to inhibitor compounds, than a cell line that does not express Hsp47. While the human neuronal cell line HEK 293, and its derivatives such as 293T, do not express collagen or Hsp47, a number of other tumour cell lines do. As illustrated in Figure 1 , when treated with the Hsp47 inhibitor RF03420, the growth of cells expressing Hsp47 was inhibited to a greater degree than were the 293T cells. 293T cells do not express the target protein as shown in Figure 2.

[0028] The Hsp47 inhibitor, RF03420, was shown to inhibit tumour growth in vivo in a syngeneic colon carcinoma model when applied topically as shown in Figure 3 and when applied peritumourally as shown in Figure 5. In addition, this compound was shown to inhibit tumour growth in a breast carcinoma xenograft model when delivered systemically as illustrated in Figure 4. The highest expression of Hsp47 within the colon carcinoma model was seen in stromal fibroblasts near the surface of the tumour mass. Weaker expression was seen within the tumour cells themselves as shown in Figure 6 and 7. Within the breast carcinoma tumours both the tumour cells and the supporting stroma expressed the target as seen in Figure 9. Figure 8 illustrates normal, tumour-free tissues from an adult mouse display undetectable expression of Hsp47.

[0029] The above disclosure generally describes the present invention. It is believed that one of ordinary skill in the art can, using the preceding description, make and use the compositions and practice the methods of the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely to illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Other generic configurations will be apparent to one skilled in the art. All reference documents referred to herein are hereby incorporated by reference. EXAMPLES

[0030] Although specific terms have been used in these examples, such terms are intended in a descriptive sense and not for purposes of limitation. Methods of microbiology, molecular biology and chemistry referred to but not explicitly described in the disclosure and these examples are reported in the scientific literature and are well known to those skilled in the art.

Example 1. Relative Hsp47 expression levels for several cell lines.

[0031] Quantitative western blots were performed comparing Hsp47 expression levels for several cell lines. Western blots were probed with an anti-Hsp47 monoclonal antibody (colligin m16.10A1 ; Stressgen Biotechnologies, Victoria, BC), which was then detected using a chicken anti-mouse Alexa Fluor 680 secondary antibody by scanning the blot on a LiCor Odyssey instrument. The blots were then stripped and reprobed with a polyclonal anti-GAPDH antibody (AbCam) followed by an chicken anti-rabbit Alexa Fluor 680 secondary antibody and again the blots were scanned on a LiCor Odyssey. The fluorescent intensity was determined using LiCor software and plotted as relative to the GAPDH signal for each cell line. The results are shown in Figure 1 for the following cell lines: 293T (human neuronal), MDA-MB- 231 (human breast carcinoma), U2OS (human osteosarcoma), L929 (murine fibroblast), CT26 (murine colon carcinoma), B16 (murine melanoma).

Example 2. Viability of Cultured Cells Following Exposure to the Hsp47 Inhibitor RF03420.

[0032] Cells were plated in 96 well plates and then exposed to a dose range of the Hsp47 inhibitor compound RF03420. All wells received equivalent volumes of DMSO containing a concentration range of inhibitor. Following 48 hours (or as indicated) the cells were subjected to an MTS assay as per the manufacturer's directions (Promega) to measure metabolic activity. The results are shown in Figure 2. Data is plotted as relative to cells treated with DMSO alone. Each data point represents the average for four wells. The legend is as follows: A and B) MDA-MB-231 versus 293T, two independent experiments. C) CT26 versus 293T D) 293T time course E) L929 time course F) MDA MB 231 time course G) U2OS time course. Example 3. Topical Treatment of Subcutaneous CT26 Tumours.

[0033] Balb/c mice were engrafted with 1x10⁶ CT26 subcutaneously. Nine days post- engraftment treatment with Compound 1 (RF03420, 250μM in DMSO, 5 mice), Compound 2 (RH01393, 250μM in DMSO, 5 mice) or DMSO (4 mice) was started consisting of 2x daily topical administration of HSP47 inhibitor on weekdays and 1x daily topical administration on weekends. The DMSO solutions were applied topically by painting the surface of the tumours with a small paintbrush. Treatment was continued until the tumour volume endpoint was reached as per institution prescribed animal utilization protocol. The results are shown in Figure 3. Two RF03420 treated mice were long-term survivors.

Example 4. Systemic Treatment of Breast Carcinoma in a Xenograft Model.

[0034] Rag2^"/7γ_c ^'/" mice were engrafted with 2x10⁶ MDA-MB231 subcutaneously. Seven days post engraftment treatment was started consisting of daily intraperitoneal injections of HSP47 inhibitor or vehicle. δOμl of 12mM Compound 1 (RF03420) in DMSO was diluted to 10Oul with DMSO and then this was mixed with 30OuI of saline just prior to injection (5 mice). 14ml of 7OmM Compound 2 (RH01393) in DMSO was diluted to 100μl of DMSO and the mixed with 300 ml of saline for injection (4 mice). The DMSO group received 100μl of DMSO mixed with 300μl of saline (5 mice). Mice were monitored and euthanized once endpoint was reached as per our animal use protocol. The results are shown in Figure 4 as A) Tumour volumes on last day of treatment and B) Survival proportions.

Example 5. Peri-tumoural Treatment in a Subcutaneous Colon Cancer Model.

[0035] Balb/c mice were engrafted with 8x10⁵ CT26 cells subcutaneously. Five days post engraftment treatment began with daily peritumoural subcutaneous injections of 100 μM HSP47 inhibitor (RF03420, 5 mice) in 1%DMSO or with vehicle (4 mice). Mice were monitored and euthanized once endpoint was reached as per our animal use protocol. The results are shown in Figure 5 as A) Tumour volumes and B) survival curves were recorded.

40 Example 6. lmmunohistochemical staining of CT26 Tumours.

[0036] CT26 Tumours were removed from BaIb-C mice, fixed in 10% formalin (Sigma) for 72-90 hours at 4°C, paraffin embedded and sectioned at 4μm. Antigen retrieval was performed on the tissue using citrate buffer, pH 6.0 and then stained with OC-HSP47 monoclonal antibody (Stressgen) at a dilution of 1 :1000 using an ARK kit as per the manufacturers protocol (DAKO) and counterstained with hematoxalin/eosin (H&E). Strong staining was seen in fibroblasts, primarily near the surface of the tumour (spindle-shaped cells in upper left panel) and in tumour cells within the tumour (diffuse brown staining in cuboidal cells in lower left panel). The results are shown in Figure 6.

Example 7. Higher Magnification of HSP47 lmmunohistochemical Staining of CT26 tumours.

[0037] The immunohistological sections were examined under higher magnification and the results are shown in Figure 7wherein 7A) Shows staining of fibroblasts near surface of tumour and 7B) shows staining of tumour cells within core of tumour.

Example 8. lmmunohistochemical Staining of Normal Tissues.

[0038] Various normal tissues were stained for tumour markers and display virtually no staining for Hsp47. The results are shown in Figure 8.

Example 9. lmmunohistochemical staining of MDA MB 231 Tumours.

[0039] MDA MB 231 tumours were removed from RAG2 -/- gamma chain -/- mice, fixed in 10% formalin (Sigma) for 72-90 hours at 4°C, paraffin embedded and sectioned at 4μm. Antigen retrieval was performed on the tissue using citrate buffer, pH 6.0 and then stained with α-HSP47 monoclonal antibody (Stressgen) at a dilution of 1 :1000 using an ARK kit as per the manufacturers protocol (DAKO) and counterstained with hematoxalin/eosin (H&E). The results are shown in Figure 9 according to the following legend: A & B) High power view of stained sections; C & D) Lower power view of stained sections; E & F) No primary antibody controls. Strong staining is seen in spindle-shaped fibroblasts near the tumour surface along with strong staining in cuboidal tumour cells throughout this tumour. [0040] One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

REFERENCES:

Bachem, M. G., Schunemann, M., Ramadani, M., Siech, M., Beger, H., Buck, A., Zhou, S., Schmid-Kotsas, A., and Adler, G. (2005). Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology 128, 907-921.

Bateman J. F., Larnande S. R. and Ramshaw J.A.M. (1996) Matrix 2, 22-27. Desmouliere, A., Guyot, C, and Gabbiani, G. (2004). The stroma reaction myofibroblast: a key player in the control of tumor cell behavior. The International journal of developmental biology 48, 509-517. Kenny, P.A., Lee, G.Y., and Bissell, M.J. (2007). Targeting the tumor microenvironment. Front Biosci 12, 3468-3474.

Lamande, S. R., and Bateman, J. F. (1999). Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones. Seminars in cell & developmental biology 10, 455-464.

Maitra, A., lacobuzio-Donahue, C, Rahman, A., Sohn, T.A., Argani, P., Meyer, R., Yeo, CJ. , Cameron, J. L., Goggins, M., Kern, S. E., et al. (2002). lmmunohistochemical validation of a novel epithelial and a novel stromal marker of pancreatic ductal adenocarcinoma identified by global expression microarrays: sea urchin fascin homolog and heat shock protein 47. American journal of clinical pathology 118, 52-59.

Marutani, T., Yamamoto, A., Nagai, N., Kubota, H., and Nagata, K. (2004). Accumulation of type IV collagen in dilated ER leads to apoptosis in Hsp47- knockout mouse embryos via induction of CHOP. Journal of cell science 117, 5913-5922.

Nagai, N., Hosokawa, M., Itohara, S., Adachi, E., Matsushita, T., Hosokawa, N., and Nagata, K. (2000). Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. The Journal of cell biology 150, 1499-1506.

Nagata, K. (1996). Hsp47: a collagen-specific molecular chaperone. Trends in biochemical sciences 21, 22-26.

Nagata, K. (1998). Expression and function of heat shock protein 47: a collagen- specific molecular chaperone in the endoplasmic reticulum. Matrix Biol 16, 379- 386.

Nan, A., Ghandehari, H., Hebert, C, Siavash, H., Nikitakis, N., Reynolds, M., and Sauk, J.J. (2005). Water-soluble polymers for targeted drug delivery to human squamous carcinoma of head and neck. Journal of drug targeting 13, 189-197. Praia, D.A., and Kuperwasser, C. (2005). Stroma: tumor agonist or antagonist. Cell cycle (Georgetown, Tex 4, 1022-1025.

Satoh, M., Hirayoshi, K., Yokota, S., Hosokawa, N., and Nagata, K. (1996). Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen. The Journal of cell biology 733, 469-483.

42 Sauk, J.J., Smith, T., Norris, K., and Ferreira, L. (1994). Hsp47 and the translation-translocation machinery cooperate in the production of alpha 1(1) chains of type I procollagen. The Journal of biological chemistry 269, 3941-3946.

Schneiderhan, W., Diaz, F., Fundel, M., Zhou, S., Siech, M., Hasel, C, Moller, P.,

Gschwend, J. E., Seufferlein, T., Gress, T., et al. (2007). Pancreatic stellate cells are an important source of MMP-2 in human pancreatic cancer and accelerate tumor progression in a murine xenograft model and CAM assay. Journal of cell science 120, 512-519.

Smith, T., Ferreira, L.R., Hebert, C, Norris, K., and Sauk, J.J. (1995). Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum. The Journal of biological chemistry

270, 18323-18328.

Thomson, C.A., and Ananthanarayanan, V.S. (2000). Structure-function studies on hsp47: pH-dependent inhibition of collagen fibril formation in vitro. The

Biochemical journal 349 Pt 3, 877-883.

Thomson, C.A., Atkinson, H. M., and Ananthanarayanan, V.S. (2005).

Identification of small molecule chemical inhibitors of the collagen-specific chaperone Hsp47. Journal of medicinal chemistry 48, 1680-1684.

Yen, T.W., Aardal, N. P., Bronner, M. P., Thorning, D.R., Savard, C.E., Lee, S. P., and Bell, R.H., Jr. (2002). Myofibroblasts are responsible for the desmoplastic reaction surrounding human pancreatic carcinomas. Surgery 131, 129-134.

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Claims

WHAT IS CLAIMED IS:

1. A method for inducing regression of a tumor in a subject comprising administering to the subject a therapeutically effective amount of an inhibitor of Hsp 47 function.

2. The method according to claim 1 , wherein the inhibitor has a structure selected from the following formulae:

, where Ri is amine or substituted amine, R₂ is nitro and

R3 is alkyloxy or halogen.

, where R₁ is halogen and R₂ is alkylcarboxylate.

, where R₁ is cyclohexyl or thienyl, R₂ is cyanyl and

where X is halogen and Y is alkyl.

3. The method according to claim 1 , wherein the inhibitor is selected from the group consisting of:

4+

4. The method of claim 3 wherein the compound is compound 1 (RF03420)

5. The method according to claim 1 , wherein the tumor is selected from the group consisting of lung carcinoma, breast carcinoma, prostate carcinoma, colon adenocarcinoma, cervical carcinoma, endometrial carcinoma, ovarian carcinoma, bladder carcinoma, Wilm's tumor, fibrosarcoma, osteosarcoma, melanoma, synovial sarcoma, epidermoid carcinoma, pancreatic carcinoma, endocrine system carcinoma, astrocytoma, oligodendroglioma, menigioma, neuroblastoma, glioblastoma, ependyoma, Schwannoma, neurofibrosarcoma, neuroblastoma, and medullablastoma.

6. The method according to claim 1 , wherein the tumor is a tumor that expresses high levels of collagen.

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7. A method according to claim 6 wherein the tumor is breast cancer.

8. The method according to claim 1 wherein the tumor is a tumor that induces a fibrotic response.

9. The method according to claim 8 wherein the tumor is pancreatic cancer.

10. The method according to claim 1 , wherein the mode of administering the pharmaceutical composition is selected from the group consisting of parenteral, oral, rectal, vaginal, topical, intranasal, inhalation, buccal, or ophthalmic administration.

11.The method according to claim 10, wherein mode of administering the pharmaceutical composition is selected from the group consisting of intravenous injection, subcutaneous injection, intraperitoneal injection, intra-arterial injection, intramuscular injection, intralesional injection into the tumor, intralesional injection adjacent to the tumor, intravenous infusion, and intra-arterial infusion.

12. The method according to claim 5, wherein the mode of administering the pharmaceutical composition is by intravenous infusion.

13. The method according to claim 1 wherein the composition further comprises an additional anti-tumor agent.

14. The method according to claim 14 wherein the additional anti-tumor agent is a chemotherapy agent.

15. The method according to claim 14 wherein the additional anti-tumor agent is selected from the group consisting of regulators of the unfolded protein response (UPR), endoplasmic reticulum-associated protein destruction (ERAD) response, the proteasome and heatshock protein function and combinations thereof.

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16.An anticancer composition comprising an inhibitor of Hsp47 and a pharmaceutically acceptable carrier.

17.An anticancer composition according to claim 17 further comprising at least one addition anti-tumor agent.

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