CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to US Patent Publications 20020128220 and 20030166591 and PCT Publication WO 00/049937, all of which are incorporated herein by reference. This application claims priority from U.S. Provisional Application No. 60/559,324 filed Apr. 2, 2004, and U.S. Provisional Application No. 60/649,326 filed Feb. 2, 2005, which are incorporated herein by reference.

FIELD OF INVENTION

This invention relates to the field of treating cancer, more specifically to the treatment of cancer using an amount of antisense oligodeoxynucleotide effective to result in tumour-appropriate biodistribution and to result in tumour-appropriate biodistribution and to reduce the effective amount of clusterin in cancer cells with limited side effects.

BACKGROUND OF THE INVENTION

Standard treatments for cancer vary depending on type of cancer, stage and location. For local disease, excision and/or radiation may be used. For systemic disease, chemotherapy may be used, and for hormone-dependent tumours, hormone ablation therapy is an option. With the exception of surgery, treatment success is limited by toxicity and resistance.

Research during the past decade has identified several proteins that may promote progression and resistance by inhibiting apoptosis. Of special relevance to development of AI (androgen-independent) progression and hormone refractory prostate cancer (HRPC) are those survival proteins that are up-regulated after apoptotic triggers, such as androgen ablation, that function to inhibit cell death.

Clusterin is a ubiquitous protein with a diverse range of proposed activities. In prostate epithelial cells, expression of clusterin increases immediately following castration, reaching peak levels in rat prostate cells at 3 to 4 days post castration, coincident with the onset of massive cell death. These results have led some researchers to the conclusion that clusterin is a marker for cell death, and a promoter of apoptosis. On the other hand, it has been observed that Sertoli cells and some epithelial cells express high levels of clusterin without increased levels of cell death. In Sensibar et al., Cancer Research 55: 2431-2437, 1995, the authors reported on LNCaP cells transfected with a gene encoding clusterin, and watched to see if expression of this protein altered the effects of tumor necrosis factor Î± (TNFÎ±), to which LNCaP cells are very sensitive. Treatment of the transfected LNCaP cells with TNFÎ± was shown to result in a transient increase in clusterin levels for a period of a few hours, but these levels had dissipated by the time DNA fragmentation preceding cell death was observed.

Clusterin is expressed in a number of tumour types including breast (Redondo, M.; Villar, E.; Torres-Munoz, J., et al. Am J Pathol (2000) 157(2):393-9), non-small cell lung carcinoma (July, L. V.; Beraldi, E.; So, A. et al. Mol Cancer Ther (2004) 3(3):223-32.), prostate (Miyake, H.; Nelson, C.; Rennie P. S.; et al. Cancer Res. (2000) 60(1):170-6), ovarian (Hough, C. D.; Cho, K. R.; Zonderman, A. B. et al. Cancer Res. (2001) 61(10):3869-76), renal (Zellweger, T.; Miyake, H.; July, L.; et al. Can J Urol (2000) 7(3): 1018), and bladder (Miyake, H.; Gleave, M.; Kamidono, S.; Urology (2002) 59(1):150-4).

Transfection studies in vivo result in the development of a phenotype that is resistant to standard therapies. Inhibition studies result in delay in progression and the promotion of apoptosis, as well as in increased chemotherapy and radiation therapy sensitivity.

Antisense treatment of cancer is still a relatively new science. Certain aspects such as optimal dosing and route of administration are still a matter of study (Gewirtz, A. M. Curr Opin Mol Ther. (1999) 1(3):297-306; and Dean, N. M.; Bennett, C. F.; Oncogene (2003) 22(56):9087-96). Continuous infusion is a requirement of the first generation of antisense therapeutics, which are characterized by rapid clearance from plasma and do not show significant pooling in target tissue. The antisense therapeutics thus administered may not accumulate in target tissues sufficiently to down-regulate the genetic and/protein targets, particularly in solid tumours.

There is a need for therapies that have better tissue pharmacokinetics so that less frequent and inconvenient administration is required. Also, efficient delivery will reduce the risk of unwanted side effects and potential toxicities. Effective systemic therapies must therefore have excellent biodistribution to tumour sites, lymph nodes, and other common sites of metastases, to afford optimal treatment.

PCT Publication WO 00/049937 and PCT Publication WO 03/072591, which are incorporated herein by reference, describe the use of antisense therapy which reduces the expression of clusterin in certain cancers.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has now been determined that effective antisense therapy which achieves biodistribution to tumour tissue and tissues involved in metastasis (lymph nodes) and reduces the expression of clusterin with no increase in toxicity is achieved by administration of 40 to 640 mg, more preferably 300 to 640 mg clusterin antisense oligonucleotide to a patient in need of treatment for a cancer expressing clusterin. In particular, such antisense therapy can be applied in treatment of prostate cancer, lung cancer, renal cancer, bladder cancer, breast cancer, and cancers metastasizing through lymph nodes.

In accordance with one aspect of the invention, there is provided a method for treating cancer using clusterin antisense.

In accordance with another aspect of the invention, there is provided a method for providing antisense therapy which reduces the expression of clusterin to provide therapeutic benefits in the treatment of cancer including administering from 40 to 640 mg anti-clusterin antisense oligonucleotide to a patient in need of treatment for a cancer expressing clusterin.

The amount of anti-clusterin antisense oligonucleotide administered is preferably from 300 to 640 mg.

The anti-clusterin antisense oligonucleotide may have a sequence selected from among Seq. Id. Nos. 1-14, for example CAGCAGCAGAGTCTTCATCAT (SEQ ID No.: 1); ATTGTCTGAGACCGTCTGGTC (Seq. ID No.: 2); or GCTGGGCGGAGTTGGGGGCCT (Seq. ID No.: 3). The amount of anti-clusterin antisense oligonucleotide administered may be from 40 to 640 mg, or 300-640 mg. Administration of the anti-clusterin antisense oligonucleotide may be once in a seven day period, 3 times a week, or more specifically on days 1, 3 and 5, or 3, 5 and 7 of a seven day period of a treatment cycle.

The cancer being treated may be prostate cancer, bladder cancer, ovarian cancer, lung cancer, renal cancer, melanoma, and pancreatic cancer. The patient being treated may suffer from metastases or lymph node involvement. The lung cancer may be non-small cell lung cancer (NSCLC).

The method may further include the administration of a chemotherapeutic agent or agents, and the anti-clusterin antisense oligodeoxynucleotide may be administered before, after or during the chemotherapeutic agent or agents. The chemotherapeutic agents may be cisplatin and gemcitabine, taxotere, and/or paclitaxel.

The method may further include the administration of radiation therapy, instead of or along with chemotherapy. The anti-clusterin antisense oligonucleotide may be administered before, after or during the administration of radiation therapy.

The method may further include the administration of hormone ablation therapy, and the anti-clusterin antisense oligonucleotide may be administered before, after or during the hormone ablation therapy.

The method may include the anti-clusterin antisense oligonucleotide being administered in combination with hormone ablation therapy and chemotherapy.

According to another aspect of the invention, there is provided an antisense-containing pharmaceutical composition packaged in dosage unit form, said pharmaceutical composition including anti-clusterin antisense oligonucleotide, wherein the amount of anti-clusterin antisense oligonucleotide in each dosage unit is from 40 to 640 mg, or 300 to 640 mg. The antisense-containing pharmaceutical composition may have a sequence selected from among Seq. ID Nos. 1-12, and may be an injectable solution or suspension, which may further contain sodium ions.

According to another aspect of the invention, there is provided the use of an anti-clusterin antisense in the manufacture of a medicament for the treatment of cancer, wherein the medicament is formulated to deliver a dosage of 40 to 640 mg, or 300-640 mg, of the antisense to a patient. The antisense may have a sequence selected from among Seq. ID Nos. 1-12.

The medicament may contain sodium ions, and/or be in the form of an injectable solution.

The methods and pharmaceutical compositions of the invention surprisingly result in a lower than expected concentration of clusterin antisense provided to a human to achieve target tissue concentrations. Further, the methods and compositions of the invention result in surprisingly high reduction of clusterin mRNA and clusterin protein in cancer tissue in cancer patients relative to untreated patients. In particular, such extensive down-regulation was unexpected given the requirement to penetrate the blood/prostate barrier.

In accordance with another aspect of the invention, there is provided the use of clusterin antisense for the manufacture of a medicament for the treatment of cancer.

Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate embodiments of the invention:

FIG. 1 shows concentrations of anti-clusterin antisense (Seq. ID. No 1) in human prostate tissue (Tissue PK) graphically represented for doses of 40 mg, 80 mg, 160 mg, 320 mg and 480 mg;

FIG. 2 is a graphical representation of dose dependent decreases in prostate tumour clusterin expression measured by immunohistochemistry (IHC) and in situ hybridization;

FIG. 3 is a graphical representation showing IHC and computer scoring (Image Pro-Plusâ„¢) of all doses as compared to <2 month neoadjuvant hormone therapy and patients receiving no treatment;

FIG. 4 is a graphical representation showing the clusterin staining intensity of untreated prostate cancer patient cells;

FIG. 5 is a graphical representation showing dose dependent decreases in prostate tumour clusterin mRNA expression as observed by in situ hybridization (ISH), as compared to mRNA expression in patients having <2 month neoadjuvant hormone therapy;

FIG. 6 is a graphical representation showing clusterin staining intensity of untreated prostate cancer patient cells;

FIG. 7 is a graphical representation showing suppression of clusterin protein expression in lymph nodes; and

FIG. 8 is a graphical representation of the plasma pharmacokinetics resulting from a single 2 hour intravenous administration.

DETAILED DESCRIPTION OF THE INVENTION

Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of the measurement technique used in the present application to determine the value.

As used in the specification and claims of this application, the term â€œclusterinâ€ refers to the glycoprotein originally derived from rat testes, and to homologous proteins derived from other mammalian species, including humans, whether denominated as clusterin or an alternative name. The sequences of numerous clusterin species are known. For example, the sequence of human clusterin is described by Wong et al., Eur. J. Biochem. 221 (3), 917-925 (1994), and in NCBI sequence accession number NM_â€”001831. In this sequence, the coding sequence spans bases 48 to 1397.

Systematic administration of antisense clusterin oligodeoxynucleotide (ODN or ASO) in animals (including humans) bearing human prostate cancer, lung cancer such as non-small cell lung cancer (NSCLC), bladder cancer, melanoma, breast cancer, renal cancer, ovarian cancer, etc. is effective for inducing apoptosis and delaying disease progression. Systemic administration of antisense clusterin ODN is also effective in delaying progression to androgen independence. Thus, an individual suffering from a clusterin expressing tumour can be treated with chemotherapy, radiation therapy, and in case of hormone-regulated tumours, by initiating androgen-withdrawal to induce apoptotic cell death of tumour cells in the individual, and administering to the individual a composition effective to inhibit expression of clusterin by the tumor cells, thereby promoting apoptosis and/or delaying the progression of tumor cells. Furthermore, combined use of antisense clusterin plus cytotoxic or other chemotherapy (e.g. taxanes) synergistically enhances chemosensitivity in hormone refractory prostate cancer, lung cancers such as NSCLC, breast, ovarian, renal, etc.

Thus, one embodiment of the invention may further include administration of chemotherapy agents or other agents useful in cancer therapy prior to, concurrent with, or subsequent to and/or additional antisense ODNs directed at different targets in combination. For example, the antisense may be administered in conjunction with radiotherapy, surgery, hormone therapy, or chemotherapy.

Further administration may be of hormone ablating agents, or hormone blocking agents. Such agents may be administered in combination with a chemotherapeutic agent to achieve treatment goals.

A second antisense ODN which inhibits expression of an anti-apoptotic protein other than clusterin may also be administered along with the antisense clusterin ODN. For example, antisense clusterin ODN may be used in combination antisense inhibitors to bcl family members or XIAP family members.

An antisense clusterin ODN can also be combined with more conventional chemotherapy agents such as taxanes (Paclitaxelâ„¢ or Docetaxelâ„¢), mitoxanthrone, gemcitabine, cyclophosphamide, decarbazine, topoisomerase inhibitors, platinum-based chemotherapies such as cisplatin, mitoxanthrone, angiogenesis inhibitors, differentiation agents, signal transduction inhibitors, and ancillary agents in general (such as, but not limited to, Flutamideâ„¢).

More than one chemotherapeutic agent may be used in combination with antisense clusterin oligodeoxynucleotide, for example cisplatin/gemcitabine, cisplatin/paclitaxel, cisplatin/docetaxel, or carboplatin/paclitaxel combinations as described in Schiller et al; N. Eng. J. Med. 346: 92-98 (2002), or a carboplatin/gemcitabine combination.

â€œCombinationâ€ means either at the same time and frequency, or more usually, at different times and frequencies, as the ODN, as part of a single treatment plan. A chemotherapeutic agent will be administered according to the best practice known in the art, specific to that agent. For example, in one aspect, a course of treatment was composed of injections of antisense clusterin oligodeoxynucleotide (for example OGX-011 as defined below) administered over 2 hours on Days 1, 3, 5, 8, 15, 22, and 29; flutamide is administered for 4 weeks daily at 250 mg orally tid beginning on Day 1 to prevent tumour flare, and a single injection of beserelin acetate at 6.3 mg is administered subcutaneously on Day 1. In another aspect, clusterin antisense such as OGX-011 may be given as a 2 hr intravenous (IV) infusion at fixed doses starting at 480 mg weekly after 3 loading doses on days 3, 5, and 7. Cisplatin may be given at 75 mg/m2 IV on day 1, and gemcitabine at 1250 mg/m2 IV on days 1 and 8, for a maximum of 6 21-day cycles. In another schedule, clusterin antisense such a s OGX-011 may be given by 2 hr IV infusion at fixed doses starting at 40 mg weekly after loading on days 1, 3, and 5. Taxotere may be given at a standard dose, namely IV 30 mg/m²/week (w) for 5 out of 6 weeks or 75 mg/m²every 3 weeks.

Chemotherapeutic agents may therefore be used in combination with the antisense clusterin oligodeoxy nucleotide (ODN) according to an aspect of the invention, but yet be administered at different times, different dosages, and at a different frequency, than the ODN is administered.

In other embodiments of the invention, a therapeutic antibody targeted to the particular cancer being treated may be used with the anti-clusterin antisense. One example is for example trastuzumab for breast cancer.

It has also been found that antisense clusterin has beneficial effects for other cancer types. Specifically, antisense clusterin ODN enhances chemosensitivity in human renal cell cancer, a normally chemoresistant disease with no active chemotherapeutic agent having an objective response rate higher than 10%. Radiation sensitivity is also enhanced when cells expressing clusterin are treated with antisense clusterin ODN. Thus, the antisense clusterin ODNs can be used to treat a variety of cancer types in which expression of clusterin has been observed.

Antisense oligonucleotides useful in the present invention are species of oligonucleotide that interact with mRNA encoding clusterin in such a way as to reduce the effective amount of clusterin in the cell. As used in this application, the â€œeffective amount of clusterinâ€ is the amount of clusterin which is present in a form which is functional to provide anti-apoptotic protection. The effective amount of clusterin is reduced by the application of an antisense oligonucleotide complementary to the mRNA encoding clusterin, without regard for the specific mechanism by which the reduction is achieved. Specific antisense species that are useful for this purpose include CAGCAGCAGAGTCTTCATCAT (Seq. ID No. 1), ATTGTCTGAGACCGTCTGGTC (Seq. ID No.: 2), and GCTGGGCGGAGTTGGGGGCCT (Seq. ID No.: 3). A preferred antisense oligonucleotide the 21mer oligonucleotide CAGCAGCAGAGTCTTCATCAT (Seq. ID No.: 1) which is targeted to the translation initiation codon and next 6 codons of the human clusterin sequence (Genbank accession no: NM_â€”001831). Most preferably, this antisense oligonucleotide is used in the form of a 2â€²-MOE modified clusterin antisense oligonucleotide as described in U.S. patent application Ser. No. 10/080,794 filed Feb. 22, 2002. This oligonucleotide, which is referred to herein as OGX-011, has a phosphorothioate backbone throughout. The sugar moieties of nucleotides 1-4 and 18-21 (the â€œwingsâ€ ) bear 2â€²-O-methoxyethyl modifications and the remaining nucleotides (nucleotides 5-17; the â€œdeoxy gapâ€ ) are 2â€²-deoxynucleotides. Cytosines in the wings (i.e., nucleotides 1, 4 and 19) are 5-methylcytosines. Other antisense oligonucleotides that may be used in the present invention are those listed in Nos. 4 to 12 below, and those described in U.S. Pat. No. 6,383,808, which is incorporated herein by reference.

Seq.â€ƒIDâ€ƒNo.â€ƒ4gcacagcaggâ€ƒagaatcttcaâ€ƒt21 Seq.â€ƒIDâ€ƒNo.â€ƒ5gcacagcagcâ€ƒaggatcttcaâ€ƒt21 Seq.â€ƒIDâ€ƒNo.â€ƒ6tggagtctttâ€ƒgcacgcctcgâ€ƒg21 Seq.â€ƒIDâ€ƒNo.â€ƒ7ccttcagcttâ€ƒtgtctctgatâ€ƒt21 Seq.â€ƒIDâ€ƒNo.â€ƒ8agcagggagtâ€ƒcgatgcggtcâ€ƒa21 Seq.â€ƒIDâ€ƒNo.â€ƒ9atcaagctgcâ€ƒggacgatgcgâ€ƒg21 Seq.â€ƒIDâ€ƒNo.â€ƒ10gcaggcagccâ€ƒcgtggagttgâ€ƒt21 Seq.â€ƒIDâ€ƒNo.â€ƒ11ttcagctgctâ€ƒccagcaaggaâ€ƒg21 Seq.â€ƒIDâ€ƒNo.â€ƒ12aatttagggtâ€ƒtcttcctggaâ€ƒg21

Phase I pharmacokinetic and pharmacodynamic studies conducted with this 2â€²-MOE modified oligonucleotide in patients with prostate cancer prior to radical prostatectomy show that the biodistribution and down-regulated in vivo is achieved at lower concentrations than predicted from in vitro and in vivo tests, allowing broader range and decreased frequency of dosing of the therapeutic agent. Thus, in accordance with the invention, there is provided a method for providing antisense therapy which reduces the expression of cluster in the treatment of cancer including the step of administering from 40 to 640 mg clusterin antisense oligonucleotide to a patient in need of treatment for a cancer expressing Clusterin, more preferably from 300 to 640 mg. Dosages are calculated typically by patient weight, and therefore a dose range of about 1-20 mg/kg, or about 2-10 mg/kg, or about 3-7 mg/kg, or about 3-4 mg/kg could be used. This dosage is repeated at intervals as needed, clinical concept is dosing once per week with 3 loading doses during week one of cycle one only. The amount of antisense ODN administered is one that has been demonstrated to be effective in human patients to inhibit the expression of clusterin in cancer cells.

The invention further provides antisense containing pharmaceutical compositions packaged in dosage unit form, wherein the amount in each dosage unit is from 40 to 640 mg of the clusterin antisense oligonucleotide, more preferably from 300 to 640 mg. Dosage units may be, without limitation, injectable solutions or suspensions including solutions in bags for IV administration through an existing port, catheter or IV site, and powders or concentrates for preparation of injectable solutions. A formulation of particular usefulness is one including sodium salts in an aqueous solution, for use in intravenous, subcutaneous, intraperitoneal, or intratumour administration.

The invention will now be further described with reference to the following, non-limiting examples.

Example 1Enhancing Hormone Sensitivity Using Various Clusterin ASOs in Murine Model

Targeting cell survival genes up-regulated by androgen withdrawal may enhance castration-induced apoptosis and thereby prolong time to overt recurrence. ASOs corresponding to the clusterin translation initiation site were designed and shown to reduce clusterin levels in a dose-dependent and sequence-specific manner (Miyake, H.; Nelson, C.; Rennie, P. S. et al. Cancer Res. (2000) 60(1):170-6; Miyake, H.; Chi, K.; Gleave, M. E. Clinical Cancer Res (2000) 6:1655-63.

Male mice bearing Shionogi tumours were castrated 2 to 3 weeks after tumour implantation, at which time tumours were 1 to 2 cm in diameter, and randomly selected for treatment with antisense clusterin versus mismatch control oligonucleotides. Beginning 1 day after castration, 12.5 mg/kg of oligonucleotide was administered once daily by intraperitoneal injection for 40 days. Shionogi tumours regressed faster and complete regression occurred earlier in mice treated with clusterin ASO compared to controls. Furthermore, clusterin ASO treatment significantly delayed recurrence of AI tumours compared to mismatch control oligonucleotide treatment. After an observation period of 50 days post-castration, mean tumour volume in the mismatch-treated control group was 6-times that of the ASO-treated group. Dose-dependent studies using mouse clusterin ASOs in the murine Shionogi model demonstrated dose-dependent activity up to 25 mg/kg/day, but did not reveal any obvious toxicity in mice treated with up to 50 mg/kg/day. Analysis of tumours excised 3 days after castration confirmed that ASO treated tumours had lower clusterin levels and earlier evidence of PARP cleavage fragments, consistent with earlier onset of castration-induced apoptosis. Changes in clusterin mRNA levels in various normal mouse organs after ASO treatment were also evaluated. Shionogi tumours, spleen, kidney, brain and prostate were harvested 3 days post-castration for RNA extraction from mouse administered ASO or mismatch control under the same treatment schedule described above.

Murine clusterin ASO had no effect on clusterin expression levels in spleen, kidney and brain, whereas clusterin mRNA expression in tumour and prostate tissues was significantly lower after ASO administration compared to mismatch control (Miyake H (a), 2000).

Example 2Characterization of Human Clusterin ASO-OGX-011

ASO sequences directed against different sites on the human clusterin gene were synthesized and tested for their ability to decrease clusterin gene expression in human prostate cancer cell lines that overexpress clusterin (PC3 and LNCaP). The clusterin gene was â€˜walkedâ€™ using a quantitative TaqMan RT-PCR assay, which compared the potency of over 80 clusterin ASOs. An active and novel 21-mer ASO (5â€²-CAGCAGCAGAGTCTTCATCAT-3â€²; SEQ ID NO. 1), complementary to the translation initiation site of human clusterin, was capable of inhibiting clusterin mRNA expression by up to 90%. OGX-011 has this optimal sequence, and is a 2nd-generation ASO using a phosphorothioate backbone and an additional 2â€²-O-(2-methoxy)ethyl (2â€²-MOE) modification to the 2â€²-position of the carbohydrate moiety on the 4 nucleotides at the 5â€² and 3â€² ends of the molecule (gapmer).

Example 3Enhancing Chemotherapy Sensitivity Using Clusterin ASOs in Prostate Cancer

Clusterin ASOs also increased the cytotoxic effects of mitoxantrone and paclitaxel, reducing the IC50 of PC3 and Shionogi cells by 75%-90% (Miyake, H.; Rennie, P.; Nelson C. et al. (2000) Cancer Res 60:2547-54; and Miyake, H.; Chi, K.; Gleave, M. E. (2000) Clinical Cancer Res 6:1655-63). Cytotoxicity of 10-nM paclitaxel increased in a dose-dependent manner as the concentration of clusterin ASO increased. The induction of apoptosis by 10-nM paclitaxel, as demonstrated by DNA laddering and PARP cleavage, could only be seen when used with clusterin ASOs.

Synergy between clusterin ASOs and chemotherapy also occurs in vivo. The recurrence of AI Shionogi tumours is delayed longest with combined clusterin ASOs and paclitaxel compared to either agent alone. Although clusterin ASOs had no effect on the growth of established AI Shionogi or PC3 tumours, clusterin ASOs synergistically enhanced paclitaxel-induced tumour regression in both the Shionogi and human PC3 models (Miyake, H. 2000; Miyake, H, 2000, as above).

Example 4Enhancing Chemotherapy Sensitivity Using Clusterin ASOs in Renal Cancer

Clusterin may also play a role in mediating chemoresistance in renal cell carcinoma and other tumours. ASO decreased clusterin levels in human renal cell cancer Caki-2 cells in a dose-dependent and sequence-specific manner. Pretreatment of Caki-2 cells with these clusterin ASO's significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA-laddering occurred after combined treatment with OGX-011 plus paclitaxel, but not with either agent alone. In vivo administration of OGX-011 enhanced in a synergistic fashion paclitaxel-induced Caki-2 tumour regression and delayed tumour progression by 50%.

Example 5Enhancing Radiation-Sensitivity Using Human Clusterin ASOs In Vitro

Clusterin-overexpressing LNCaP cells were less sensitive to irradiation with significantly lower cell-death rates (23% after 8 Gy) compared to parental LNCaP cells (50% after 8 Gy) 3 days after irradiation (Zellweger, T.; Chi, K.; Miyake, H.; et al. Clin Cancer Res. (2002) 8(10):3276-84). Clusterin expression in PC-3 cells after radiation increased in a dose-dependent manner in vitro by 70% up to 12 Gy and in vivo by >80%. Inhibition of clusterin expression in PC-3 cells using clusterin ASO's before radiation significantly decreased PC-3 cell growth rate and plating efficiency, and enhanced radiation-induced apoptosis. In vivo administration of clusterin ASO before and after radiation significantly reduced PC-3 tumour volume by 50% at 9 weeks as compared to mismatch control oligonucleotides.

These findings support the hypothesis that clusterin acts as a cell survival protein that mediates radioresistance through the inhibition of apoptosis.

Example 6OGX-011 Pre-Clinical Data

As a class, the second-generation phosphorothioate 2â€²-O-methoxyethyl ribose gapmer antisense oligonucleotides have pharmacokinetic and toxicity profiles favorable for the intended clinical use. Pharmacokinetics of these compounds are characterized by rapid clearance from plasma following intravenous administration and broad distribution to tissues. Once distributed to tissues, these second generation ASO are cleared slowly. Tumour concentrations of an unmodified (first generation) phosphorothioate clusterin ASO corresponding to SEQ ID NO. 4 was compared to OGX-011. Using the PC3 model, animals were treated with ASO, tumours excised 24, 72, 120 and 168 hours after last dosing and tumour ASO concentration assessed by capillary gel electrophoresis. At 24 hours, OGX-011 concentration was ten times greater than that of unmodified ASO (755+261 nM vs 60+10 nM). In addition, only 10% of the unmodified phosphorothioate ASO were full length while 90% of the OGX-011 ASO detected in the tumours were full length. At 120 hours after the last treatment, the phosphorothioate ASO were not detectable, however OGX-011 remained detectable at 120 hours (632+192 nM) and 168 hours (180+14.1 nM) and also was predominantly full length (approximately 90% of detected ASO) (Zellweger, T.; Miyake, H.; Cooper, S.; et al. J Pharmacol Exp Ther (2001) 298:934-40).

In animal toxicology and pharmacokinetic studies, OGX-011 was administered by I.V. injection at doses of 1, 5, 20 and 50 mg/kg to CD-1 mice, and 1-hour I.V. infusion at 1, 3, and 10 mg/kg to rhesus monkeys. In both species, the dosing period was 4 weeks with alternate day dosing for the first 4 doses (a â€œloading periodâ€ ) and weekly thereafter (a â€œmaintenance periodâ€ ). This dosing regimen was based on existing knowledge about the long tissue half-lives of this class of compound.

No clinical signs of toxicity were observed after 4 weeks of exposure at doses up to 50 mg/kg in mice or 10 mg/kg in monkeys. Despite similar levels of tissue exposure in liver achieved in monkeys, mice were generally more sensitive to liver toxicities as indicated by elevated transaminases. Many of the toxicities observed in mice were thought to be related to a generalized mild immunostimulation, an effect of phosphorothioate oligonucleotides that is generally more prominent in rodents than in primates (Parker S L. Cancer Statistics 65: 5-27, 1996).

Genetic toxicity studies (in vitro bacterial cell gene mutation and mouse lymphoma gene mutation) were negative.

Example 7Changes in Clusterin Expression in Human Prostate Cancer Specimens after Neoadjuvant Hormone Therapy (NHT)Materials:

Buserelin acetate (Aventis). Each implant dose, in the form of two cream-filled rods, contains a total of 6.6 mg buserelin acetate (6.3 mg base) and 26.4 poly-(D,L-lactide-co-glycolide) in a 75:25 molar ratio. Both rods are implanted in one operation into the subcutaneous tissue of the lateral abdominal wall.

Flutamide is an approved commercially available non-steroid agent that specifically blocks androgen-binding receptors.

Methods and Results:

To define temporal changes in clusterin in human prostate cancer, 128 radical prostatectomy specimens from patients after 0, 3 or 8 months of NHT were stained for clusterin using immunohistochemistry (July L V, Akbari M, Zellweger T, et al. Prostate 50: 179-188, 2002). Residual foci of cancer cells from radical prostatectomy specimens treated with NHT exhibited strongly positive staining (intensity +3, +4) for clusterin in 80-90% of the surviving cancer cells compared to either absent (0) or low intensity staining (+1 to +2) in 10-20% of cancer cells in non-NHT treated specimens. Consistent with previous reports, staining for clusterin was found in the cytoplasm of luminal epithelial cells, and no nuclear staining was observed.

Western blot analyses of clusterin were performed on prostate cancer specimens from untreated patients (n=5), NHT treated patients (n=5) and two patients with androgen independent disease. To compare changes in protein expression, a normalization procedure against the cytokeratin to vimentin ratio was performed because of increases in the stromal to epithelial ratio that occur with NHT. Normalized clusterin levels were significantly higher in all treated patients compared with untreated patients, increasing 17-fold after androgen ablation. These data confirm that clusterin increases in prostate cancer cells after immediately following androgen withdrawal and support the hypothesis that clusterin serves as a protective against the apoptotic stimulus of androgen ablation.

Example 8Safety, Plasma and Tissue Pharmacokinetics, and Tissue Pharmacodynamics of OGX-011 in Prostate Cancer PatientsMaterials

Phosphorothioate 2â€²-O-methoxyethyl ribose ASO targeted to the translation initiation site of clusterin mRNA (designated OGX-011), is formulated as OGX-011 Injection, 20 mg/mL, in an isotonic, phosphate-buffered saline solution (pH 7.4) for intravenous (I.V.) administration; the drug product solution may be diluted into saline prior to I.V. administration. The formulation details are listed in Table 1.

Buserelin acetate (Aventis)â€”see above.

Flutamideâ€”see above.

TABLE 1IngredientsConcentrationOGX-01120mg/mLDibasic sodium phosphate, heptahydrate, USP14.33mg/mLMonobasic sodium phosphate, monohydrate, USP1.73mg/mLSodium chloride, USP2.70mg/mLHydrochloric Acid, NFpH adjustmentSodium Hydroxide, NFpH adjustmentWater for Injection, USPq.s. to 1.0mL

Methods:

Patients having localized prostate cancer with high-risk features and candidates for prostatectomy were enrolled to this dose escalation trial. Twenty patients were treated and evaluated.

Each course of treatment was composed of injections of OGX-011 administered over 2 hours on Days 1, 3, 5, 8, 15, 22, and 29 using fixed dose escalation plan starting at 40 mg; 4 weeks of daily flutamide 250 mg orally tid beginning on Day 1 to prevent tumour flare, and a single injection of beserelin acetate 6.3 mg subcutaneously on Day 1. Each patient received a maximum of 1 course only.

Prostatectomy was performed day 30-36. Prostate tissue concentrations of OGX-011 were determined by a fully validated ELISA method. Briefly, clusterin mRNA in human prostate tissues were determined by in situ hybridization using standardized controls including OGX-011 and human plasma (EDTA) (Biochemed), and reagents including Qiagen cutting Probe 112989cut (5â€² end biotinated, 3â€² end is labeled with digoxigenin), S1 nuclease (Life Technologies), anti-digoxigenen-Fab fragments conjugated with alkaline phosphatase (AP), AttoPhosÂ® fluorescent substrate and AttoPhosÂ® reconstitution buffer, and Reacti-Bindâ„¢ NeutrAvidinâ„¢ (Pierce) coated Nalgeneâ„¢ Nuncâ„¢ flat-bottomed polystyrene 96-well microwell plates blocked with SuperBlockÂ®. Various label-recommended buffers and salts were also used, purchased from various sources such as Fisher Scientific, Laboratoire MAT, Sigma, VWR, BDH, Pierce, and BioSource. Polypropylene containers were used to prepare and store all reference standard solutions.

Clusterin protein in human prostate tissues was also determined by:

1. IHC using visual scoring of tumour cells conducted by two independent pathologists. Specimens were graded from 0 to +3 intensity, representing the range from no staining to heavy staining and the overall percentage of cancer cells showing staining (0-100%) was indicated. The mean of the two pathologists' scores (the product of intensity and percentage of cells) was used for reporting and standard error reported; and
2. IHC using an automated scoring system of tumour cells conducted by an Image Pro-Plusâ„¢ system. The scoring system used by Image Proâ„¢ can be defined by the user to provide greater gradations and provide additional independent semi-quantitative data.

Results:

Toxicity was limited to grade 1 or 2, and included fevers, rigors, fatigue and transient AST and ALT elevations at higher doses of OGX-011. Plasma pharmokinetics analysis showed linear increases in AUC with a tÂ½ of approximately 2 hours.

The 40 mg, 80 mg and 160 mg dose cohorts achieved mean concentrations of OGX-011 that were approximately equivalent and thus not dose dependent. OGX-011 concentrations increased to a mean of 1.668:g/g (Ëœ227 nM) at 320 mg and 2.30:g/g (Ëœ313 nM) at 480 mg dosing, representing a 4.9 fold increase and 6.76 fold increase at the 320 mg and 480 mg dose, respectively.

As measured by immunohistochemistry, each of the 320 mg and 480 mg dose cohorts achieved a 30% decrease in clusterin protein expression as compared to <2 month neoadjuvant hormone therapy. The mean percent of down-regulation of clusterin protein in the 320 mg and 480 mg dose cohorts was 31.2% and 70.6%, respectively. The mean percent age of cancer cells in untreated prostate cancer patients staining intensity â€œ0â€ , or no clusterin protein, was 14.25%, and cells staining intensity â€œ3â€ (high clusterin protein) was 18.75%. Following <2 months of neoadjuvant hormone therapy, the percentage of cancer cells staining intensity 0 decreased to 1.5%, and cells staining intensity 3 increased to 48%. Surprisingly, the mean percentage of cancer cells staining intensity 0, 1, 2 and 3 in prostate cancer patients receiving 320 mg OGX-011 was restored to approximately equivalent levels to untreated controls. The mean percentage of cancer cells staining intensity 0 increased dramatically in patients treated with 480 mg of OGX-011 to 48.17%. In this same dose cohort, the percentage of cancer cells staining intensity 3 decreased dramatically to 6.56%.

IHC and computer scoring (Image Pro-Plusâ„¢) of all doses as compared to <2 month neoadjuvant hormone therapy showed a mean percent down regulation of clusterin protein in the 320 mg and 480 mg dose cohorts of 68.7% and 76.8%, respectively.

Example 9Antisense Oligonucleotide to Clusterin (OGX-011) in Combination with Gemcitabine and Cisplatin as First-Line Treatment for Patients with Advanced Non-Small Cell Lung Cancer (NSCLC)

Patients with previously untreated stage IIIB or IV NSCLC were eligible. OGX-011 was given as a 2 hr IV infusion at fixed doses starting at 480 mg weekly after 3 loading doses on days 7, 5, and 3). Cisplatin was given at 75 mg/m2 IV on day 1, and gemcitabine at 1250 mg/m2 IV on days 1 and 8. A maximum of 6 21-day cycles were performed.

Serum clusterin levels were measured at baseline and on day 1 of each cycle. Where possible, pre- and post-treatment (cycle 2, day 1) biopsies of the tumours were taken. The toxicities observed were typical for cisplatin/gemcitabine therapy alone.

The combination of OGX-011 and cisplatin/gemcitabine is more effective than either treatment alone.