EA041953B1 - COMBINATION THERAPY WITH LIPOSOMAL ANTISENSE OLIGONUCLEOTIDES - Google Patents

Description

This application claims priority advantage of U.S. Provisional Applications No.

62/487277, filed April 19, 2017, and 62/395680, filed September 16, 2016, the entire contents of which are incorporated herein by reference.

Technical field

1) The field of technology to which the invention belongs.

This invention relates generally to the field of medicine.

More specifically, it relates to the treatment of cancer with a combination therapy containing antisense oligonucleotides directed to Grb2.

2) Description of the prior art.

The American Cancer Society estimated that in 2015 there were about 6,600 patients newly diagnosed with chronic myelogenous leukemia (CML) and about 1,140 deaths from CML in the United States. The median age at diagnosis of CML is about 64 years. Nearly half of the cases are diagnosed in people aged 65 and older. This type of leukemia mostly affects adults and is rare in children. CML is differentiated into chronic, accelerated and blast phases. Most patients with CML are diagnosed with the chronic phase of the disease. If patients with CML in the chronic phase are not treated, they can quickly progress to the accelerated and blast phases (BP) of the disease.

The Philadelphia chromosome (Ph) is present in 90-95% of patients with CML. This occurs as a result of the translocation of chromosomes 9 and 22, which places the abl gene at the 3' end of the bcr gene. This genetic rearrangement is present in all patients, even those in whom the standard karyotype does not detect the Ph chromosome. This chromosomal translocation produces a chimeric gene that is expressed as the p210Bcr-Abl fusion protein in Ph+CML cells. The Bcr-Abl fusion protein has deregulated tyrosine kinase activity resulting in activation of RAS and phosphatidylinositol 3-kinase (PI3K) signaling pathways resulting in Ph+ cell malignant transformation, proliferation and survival.

Drugs known as tyrosine kinase inhibitors that target the Bcr-Abl fusion protein are the standard treatment for CML patients. This is because suppression of Bcr-Abl tyrosine kinase activity reduces the proliferation and survival of Ph+ cells. Imatinib (Gleevec®), a first-generation tyrosine kinase inhibitor, is the standard treatment for patients with chronic and accelerated phase CML. However, patients with CML in the blast phase do not respond to imatinib. In addition, resistance to imatinib has been observed in some patients with chronic phase or accelerated phase CML. A second-generation tyrosine kinase inhibitor (eg, dasatinib (Das)) is usually given to patients with CML who cannot tolerate imatinib or are resistant to imatinib. But CML patients will not respond to imatinib or second-generation tyrosine kinase inhibitors if the Bcr-Abl fusion protein adopts the T315I gene mutation. There is a need to develop more effective treatments for patients with CML who are in the accelerated or blast phase or who are resistant to tyrosine kinase inhibitors.

Brief summary of the invention

In one embodiment, provided herein are methods of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a first pharmaceutical therapy comprising a nuclease-resistant polynucleotide that hybridizes to a Grb2 nucleic acid in the patient, and a second pharmaceutical therapy comprising tyrosine kinase inhibitor Bcr-Abl. In some aspects, provided herein are compositions for use in the treatment of cancer in a patient in need thereof, said compositions comprising a first pharmaceutical therapy comprising a nuclease-resistant polynucleotide that hybridizes to a Grb2 nucleic acid in a patient and a second pharmaceutical therapy comprising tyrosine kinase inhibitor Bcr-Abl.

In some aspects, the polynucleotide hybridizes to the translation initiation site of the Grb2 nucleic acid. In some aspects, the polynucleotide is an oligonucleotide having a length of 8-50 bases. In some aspects, the polynucleotide has the sequence according to SEQ ID NO: 1. In some aspects, the polynucleotide contains P-ethoxy backbone bonds.

In some aspects, the polynucleotide is encapsulated in a liposome. In some aspects, the first pharmaceutical therapy further comprises a neutral lipid. In some aspects, the first pharmaceutical therapy is BP1001. In some aspects, the first pharmaceutical therapy is administered systemically. In some aspects, the first pharmaceutical therapy is for intra-arterial or intravenous administration. In some aspects, the first pharmaceutical therapy is administered at a dosage of about 60 mg/m ² to about 90 mg/m ² . In some aspects, the first pharmaceutical therapy is administered two to four times a week.

In some aspects, the Bcr-Abl tyrosine kinase inhibitor is dasatinib, imatinib, nilotinib, bosutinib, ponatinib, or bafetinib. In one aspect, the Bcr-Abl tyrosine kinase inhibitor is dasatinib. In some aspects, dasatinib is administered at a dosage of about 140 mg. In some aspects, dasatinib is administered once a day.

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In some aspects, the second pharmaceutical therapy is administered systemically. In some aspects, the second pharmaceutical therapy is administered orally, intra-arterially, or intravenously. In some aspects, the first pharmaceutical therapy is administered prior to the administration of the second pharmaceutical therapy.

In some aspects, the first pharmaceutical therapy and the second pharmaceutical therapy are administered simultaneously. In some aspects, the first pharmaceutical therapy and the second pharmaceutical therapy are administered in different ways.

In some aspects, the patient is human. In some aspects, the patient has previously been treated with imatinib to no avail.

In some aspects, the cancer is colorectal cancer, neuroblastoma, breast cancer, pancreatic cancer, brain cancer, lung cancer, stomach cancer, blood cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, or esophageal cancer. , cervical cancer, head and neck cancer, non-melanoma skin cancer, or glioblastoma. In some aspects, the cancer is a cancer of the blood. In some aspects, the blood cancer is chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), or myelodysplastic syndrome (MDS). In some aspects, the CML is acceleration phase CML or blast phase CML. In some aspects, the CML or AML is Bcr-Abl positive CML or Bcr-Abl positive AML. In some aspects, Bcr-Abl positive CML or Bcr-Abl positive AML is positive for the T315I Bcr-Abl mutation. In some aspects, the CML or AML is Philadelphia chromosome positive CML or Philadelphia chromosome positive AML.

In one embodiment, provided herein are methods of treating cancer or myelodysplastic syndrome (MDS) in a patient in need thereof, comprising administering to the patient an effective amount of a first pharmaceutical therapy comprising a nuclease-resistant polynucleotide that hybridizes to a Grb2 nucleic acid, and a second pharmaceutical therapy containing a cytidine analogue. In some aspects, the present invention provides compositions for use in the treatment of cancer or myelodysplastic syndrome (MDS) in a patient in need thereof, said compositions comprising an effective amount of a first pharmaceutical therapy comprising a nuclease-resistant polynucleotide that hybridizes to a Grb2 nucleic acid, and a second pharmaceutical therapy containing a cytidine analogue.

In some aspects, the polynucleotide hybridizes to the translation initiation site of the Grb2 nucleic acid. In some aspects, the polynucleotide is an oligonucleotide having a length of 8-50 bases. In some aspects, the polynucleotide has the sequence according to SEQ ID NO: 1. In some aspects, the polynucleotide contains P-ethoxy backbone bonds.

In some aspects, the polynucleotide is encapsulated in a liposome. In some aspects, the first pharmaceutical therapy further comprises a neutral lipid. In some aspects, the first pharmaceutical therapy is BP1001. In some aspects, the first pharmaceutical therapy is administered systemically. In some aspects, the first pharmaceutical therapy is for intra-arterial or intravenous administration. In some aspects, the first pharmaceutical therapy is administered at a dosage of about 60 mg/m ² to about 90 mg/m ² . In some aspects, the first pharmaceutical therapy is administered two to four times a week.

In some aspects, the cytidine analog is decitabine, cytarabine, or azacitidine. In one aspect, the cytidine analog is decitabine. In one aspect, the cytidine analog is cytarabine. In some aspects, cytarabine is administered at a dose of about 20 mg. In some aspects, cytarabine is administered twice a day. In some aspects, cytarabine is administered subcutaneously.

In some aspects, the first pharmaceutical therapy is administered prior to the administration of the second pharmaceutical therapy. In some aspects, the first pharmaceutical therapy and the second pharmaceutical therapy are administered simultaneously. In some aspects, the second pharmaceutical therapy is administered prior to the administration of the first pharmaceutical therapy. In some aspects, the first pharmaceutical therapy and the second pharmaceutical therapy are administered in different ways.

In some aspects, the patient is human.

In some aspects, the cancer is colorectal cancer, neuroblastoma, breast cancer, pancreatic cancer, brain cancer, lung cancer, stomach cancer, blood cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, or esophageal cancer. , cervical cancer, head and neck cancer, non-melanoma skin cancer, or glioblastoma. In some aspects, the cancer is a cancer of the blood. In some aspects, the blood cancer is chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), or myelodysplastic syndrome (MDS). In some aspects, the CML is acceleration phase CML or blast phase CML. In some aspects, the CML or AML is Bcr-Abl positive CML or Bcr-Abl positive AML. In some aspects, Bcr-Abl positive CML or Bcr-Abl positive AML is positive for the T315I Bcr-Abl mutation. In some aspects, the CML or AML is Philadelphia chromosome positive CML or Philadelphia chromosome positive AML.

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In one embodiment of the invention, compositions are provided comprising a population of oligonucleotides that hybridize to a GRB2 polynucleotide gene product. In some aspects, population oligonucleotides are composed of nucleoside molecules linked together via phosphate backbone bonds, wherein at least one of the phosphate backbone bonds in each oligonucleotide is a P-ethoxy backbone bond, and wherein no more than 80% of the phosphate backbone bonds in each oligonucleotide are a P-ethoxy bond backbone. In some aspects, at least one of the phosphate backbone bonds in each oligonucleotide is a phosphodiester backbone bond. In some aspects, the population oligonucleotides contain the sequence according to SEQ ID NO: 1. In various aspects, the population oligonucleotides inhibit expression of the Grb2 protein. In some aspects, the composition is lyophilized.

In some aspects, 10 to 80% of the phosphate backbone bonds are P-ethoxy backbone bonds; 20 to 80% of the phosphate backbone bonds are P-ethoxy backbone bonds; 30 to 80% of the phosphate backbone bonds are P-ethoxy backbone bonds; 40 to 80% of the phosphate backbone bonds are P-ethoxy backbone bonds; 50 to 80% of the phosphate backbone bonds are P-ethoxy backbone bonds; or 60 to 70% of the phosphate backbone bonds are P-ethoxy backbone bonds, or any range derived therefrom. In some aspects, 20 to 90% of the phosphate backbone bonds are backbone phosphate bonds; 20 to 80% of the phosphate backbone bonds are backbone phosphate bonds; 20 to 70% of the phosphate backbone bonds are backbone phosphate bonds; 20 to 60% of the phosphate backbone bonds are backbone phosphate bonds; 20 to 50% of the phosphate backbone bonds are backbone phosphate bonds; or 30 to 40% of the phosphate backbone bonds are phosphate backbone bonds or any range derived from them. In various aspects, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any the value of the phosphate bonds of the backbone in them are P-ethoxy bonds of the backbone. In various aspects, at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any value of phosphate bonds therein backbones are backbone phosphodiester bonds. In certain aspects, the backbone phosphodiester linkages are distributed across the oligonucleotides. As such, oligonucleotides are not chimeric molecules. In some aspects, the oligonucleotides do not contain a backbone phosphorothioate bond.

In some aspects, the population oligonucleotides are in the range of 7 to 30 nucleotides in size. In certain aspects, the oligonucleotides of the population have a size ranging from 12 to 25 nucleotides. In various aspects, the population oligonucleotides are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. The size range may be the average size of the oligonucleotides in the population.

In some aspects, the population oligonucleotides have an average size of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, where not more than 5, 6, 7, 8, 8, 9, 10, 11, 11, 12, 13, 14, 15, 15, 16, 17, 18, 19, 20, 20, The 21, 22, 23, or 24, respectively, phosphate backbone bonds in each oligonucleotide is the P-ethoxy backbone bond. In some aspects, the population oligonucleotides have an average size of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, where at least 2, 2, 2, 2, 3, 3, 3, 3, 4, 4, 4, 4, 4, 5, 5, 5, 5, 5, 5, 6 , 6, 6, 6, or 6, respectively, phosphate backbone bonds in each oligonucleotide is a phosphodiester backbone bond.

In some aspects, the population of oligonucleotides contains one kind of oligonucleotides. In other aspects, the population of oligonucleotides contains at least two kinds of oligonucleotides. One kind of oligonucleotide may have the same nucleotide sequence but may or may not have P-ethoxy bonds at different positions in the molecule. Thus, a population may be homogeneous in nucleotide sequence and heterogeneous in the distribution of backbone phosphodiester bonds among the population's oligonucleotides. In addition, the population may be heterogeneous in the number of P-ethoxy backbone bonds and backbone phosphodiester bonds among the oligonucleotides in the population. As a non-limiting example, a first portion of the oligonucleotides in a population may have 70% Rethoxy bonds and 30% phosphodiester bonds, while a second portion of the oligonucleotides in a population may have 60% P-ethoxy bonds and 40% phosphodiester bonds. In some aspects, the population of oligonucleotides contains antisense oligonucleotides, small interfering RNA (siRNA), miRNA (miRNA) or piwiPHK (piRNA).

In various aspects, the composition further comprises phospholipids. In some aspects, the phospholipids and oligonucleotides are present in a molar ratio of from about 5:1 to about 100:1. In some aspects, the oligonucleotides and phospholipids form an oligonucleotide-lipid complex, such as, for example, a liposome complex. In some aspects, at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98 or 99% of the liposomes are less than 5 microns in diameter. In some aspects, the population of oligonucleotides is included in the population of liposomes.

In some aspects, the phospholipids are uncharged or have a neutral charge at physiological pH. In some aspects, the phospholipids are neutral phospholipids. In certain aspects, the neutral phospholipids are phosphatidylcholines. IN

In certain aspects, the neutral phospholipids are dioleoylphosphatidylcholine. In some aspects, phospholipids essentially do not contain cholesterol.

In one embodiment of the invention, pharmaceutical compositions are provided comprising the composition of oligonucleotides and phospholipids of these embodiments of the invention and a pharmaceutically acceptable carrier. In some aspects, the composition further comprises a chemotherapeutic agent.

In one embodiment of the invention, methods are provided for reducing the level of expression of the Grb2 protein in a cell, comprising contacting the cell with an oligonucleotide composition of the present embodiment. In some aspects, the cell is a mammalian cell. In some aspects, the cell is a cancer cell.

In one embodiment of the invention, methods are provided for delivering a therapeutically effective amount of an oligonucleotide to a cell, comprising contacting the cell with a pharmaceutical composition of the present embodiment. In some aspects, the method is a method of treating hyperplasia, cancer, an autoimmune disease, or an infectious disease.

In one embodiment, the invention provides methods for treating a subject with cancer, an autoimmune disease, or an infectious disease, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of this embodiment. In some aspects, the subject is a human. In some aspects, the cancer is bladder, blood, lymphoma, pancreas, bone, bone marrow, brain, breast, colon, esophagus, stomach, head and neck, kidney, liver, lung, prostate, skin, testis, tongue, ovarian, or uterine cancer. Tumors treatable by the methods of this invention include, but are not limited to, melanoma, prostate cancer, ovarian cancer, breast cancer, breast cancer, head and neck squamous cell carcinoma, papillary renal cell carcinoma, gallbladder cancer, rectal cancer, pancreatic cancer, lung cancer, colon cancer, glioma, astrocytoma, classic Hodgkin's lymphoma and smooth muscle tumors, as well as glioblastoma cells, bone marrow stem cells, hematopoietic cells, osteoblasts, epithelial cells, fibroblasts, as well as any other tumor cells, which undergo apoptosis and cause resistance or regression of tumor cells. In some aspects, the autoimmune disease is lupus erythematosus, spondyloarthropathy, Sjögren's disease, Crohn's disease, diabetes mellitus, multiple sclerosis, or rheumatoid arthritis. In some aspects, the infectious disease is a bacterial infection, a fungal infection, a viral infection, or a parasitic infection. In some aspects, the composition is administered subcutaneously, intravenously, or intraperitoneally. In some aspects, the method further comprises administering to the subject at least a second anti-cancer therapy. In some aspects, the second cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, or cytokine therapy. In some aspects, the administration of the composition reduces the expression of the Grb2 protein in a patient.

Capture, encapsulate, and include refer to a lipid or liposome that forms an obstacle to free diffusion into solution by association with or around an agent of interest, for example, a liposome may encapsulate the agent in a lipid layer or in an aqueous compartment within or between lipid layers. In certain embodiments of the invention, the composition is contained in a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be formulated for administration to a human or patient.

In some embodiments of the invention, the lipid component has a substantially neutral charge, since it contains a neutral phospholipid or a net neutral charge. In some aspects, the neutral phospholipid may be a phosphatidylcholine, such as DOPC, egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, 1,2-diaracidoyl-sn- glycero-3-phosphocholine (DAPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl- 2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-oleoyl-2-palmitoylphosphatidylcholine (OPPC), or lysophosphatidylcholine. In other aspects, the neutral phospholipid may be a phosphatidylethanolamine, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleyl phosphatidylethanolamine (POPE), or lysophosphatidylethanolamine. In certain embodiments of the invention, the phospholipid component may contain 1, 2, 3, 4, 5, 6, 7, 8 or more species or types of neutral phospholipid. In other embodiments of the invention, the phospholipid component may contain 2, 3, 4, 5, 6 or more kinds or types of neutral phospholipids.

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In some embodiments of the invention, the lipid component may have a substantially neutral charge, since it contains a positively charged lipid and a negatively charged lipid. The lipid component may further comprise neutrally charged lipid(s) or phospholipid(s).

The positively charged lipid may be a positively charged phospholipid. The negatively charged lipid may be a negatively charged phospholipid. The negatively charged phospholipid may be a phosphatidylserine such as dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS) or brain phosphatidylserine (BPS). The negatively charged phospholipid may be a phosphatidylglycerol such as dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), or dioleylphosphatidylglycerol (DOPG). In certain embodiments of the invention, the composition further comprises cholesterol or polyethylene glycol (PEG). In other embodiments of the invention, the composition is essentially free of cholesterol. In certain embodiments of the invention, the phospholipid is a natural phospholipid. In other embodiments of the invention, the phospholipid is a synthetic phospholipid.

Liposomes can be made from one or more phospholipids, provided that the lipid material is substantially uncharged. It is important that the composition is substantially free of anionic and cationic phospholipids and cholesterol. Suitable phospholipids include phosphatidylcholines and others that are well known to those skilled in the art.

As used herein, the term substantially free in terms of a particular component is used herein to mean that none of the specified components has been purposefully formulated and/or is present only as a contaminant or in trace amounts. Thus, the total amount of said component as a result of any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of said component can be detected by standard analytical methods.

As used herein, the singular forms may also refer to the plural. As used herein in the claims(s) of the invention, when used in conjunction with the word containing, the singular forms may also denote the plural.

The use of the term or in a claim is used to mean and/or unless it is explicitly stated that it refers only to alternatives or alternatives are mutually exclusive, although the description supports a definition that refers only to alternatives and/or. As used herein, the term other may mean at least a second or more.

Throughout this application, the term about is used to indicate that the value includes an internal error change for the device, the method used to determine the value, or a change that exists among the subjects of the study.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to a person skilled in the art from this detailed description.

Brief description of graphic materials

The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments of the invention presented herein.

Fig. 1 - the concentration of the antisense oligo Grb2 in the plasma of patients. Plasma levels of BP1001 decreased exponentially by half. Solid lines were doses of 60 mg/m ² ; dotted lines were 90 mg/m ² doses.

Fig. 2 - BP1001 + low dose Ara-C reduces bone marrow blasts in patients with refractory/recurrent AML.

Fig. 3 is a diagram of the study design.

Fig. 4 is a diagram illustrating the critical role of GRB2 in tyrosine kinase signaling.

Fig. 5A-B - selective inhibition of the production of Grb2 proteins. BV173 (FIG. 5A) and ALL-1 (FIG. 5B) cells were incubated with 4-8 μM L-Grb2 antisense oligo and L-control oligo. After three days of culture, the protein containing layers were prepared and subjected to SDS-PAGE and transferred to nitrocellulose membranes. The blots were cut into sections and incubated with antibodies specific for the Grb2 protein (target) or Crk1 (control).

Fig. 6A-B show BP1001 activity in a mouse model of leukemia. Fig. 6A shows treatment

- 5,041,953 mice bearing 32Dp210 BCR-ABL-positive leukemia xenografts, c 15 mg/kg/dose

VR1001. Fig. 6B shows treatment of mice bearing 32Dp210 BCR-ABL positive leukemic cells with 5, 10, or 15 mg/kg/dose of BP1001.

Fig. 7A-C - Effect of timing and sequence of administration of BP1001 and dasatinib. Fig. 7A shows 1 hour pretreatment with BP1001 prior to dosing with dasatinib. Fig. 7B shows 1 day pre-treatment with dasatinib prior to dosing with BP1001. Fig. 7C shows 1 day pretreatment with BP1001 prior to dosing with dasatinib.

Fig. 8A-D - BP1001 enhances the induction of cell death by dasatinib. Fig. 8A shows the percentage of cells in sub-G1 phase. Fig. 8B shows the percentage of cells in G1 phase. Fig. 8C shows the percentage of cells in S phase. Fig. 8D shows the percentage of cells in G2/M phase.

Fig. 9A-B - Pre-treatment with BP1001 enhances the induction of cell death from dasatinib and nilotinib. Fig. 9A shows the percentage of cells in sub-G1 phase after pre-treatment with BP1001. Fig. 9B shows the percentage of cells in sub-G1 phase after prior treatment with a tyrosine kinase inhibitor.

Description of exemplary embodiments of the invention

Protein 2 associated with the growth factor receptor (Grb2).

The grb2 gene has been mapped to the 17q22-qter region of the human chromosome, a region that is duplicated in leukemias, which may result in increased copy number of the grb2 gene product. Since Grb2 is important for the transformation of murine hematopoietic cells and the proliferation of human leukemia cells, which express high levels of oncogenic tyrosine kinases, inhibition of Grb2 can have a significant impact on the natural history of leukemias.

Grb2 binds oncogenic tyrosine kinases to their downstream signaling molecules such as ERK and AKT, which are critical regulators of cell proliferation and survival. The Grb2 adapter protein contains one Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. Grb2 uses its SH2 domain to bind to phosphotyrosine residues found in activated tyrosine kinases such as Bcr-Abl, c-Cbl, and the epidermal growth factor receptor (EGFR), while it uses its SH3 domains to bind to proline-rich motifs, such as those found in the guanine nucleotide exchange factor, Son of Sevenless (SOS). RAS, a GTPase protein, is active when bound to GTP and inactive when bound to GDP. Guanine nucleotide exchange factors such as SOS increase the exchange of GDP for GTP at RAS. Upon growth factor stimulation, the Grb2-SOS complex is recruited to the plasma membrane, where it uses its SH2 domain to bind to growth factor-stimulated tyrosine kinase receptors. This binding allows the SOS to be in close proximity to the RAS, which is localized to the plasma membrane. Thus, it is able to stimulate RAS activity. Activation of the RAS, in turn, activates many downstream signaling pathways important for the regulation of a variety of cellular processes (Tari, 2001a).

The best known RAS signaling pathway is the mitogen-activated protein kinase (MAP) cascade. In this cascade, RAS binds to RAF in a GTP-dependent manner. This binding results in RAF activation. Activated RAF phosphorylates MEK (mitogen-activated protein kinase kinase), which in turn phosphorylates and activates ERK1,2 (extracellular regulated kinases 1,2). Activated ERK1,2 then translocates to the nucleus and activates transcription by phosphorylation of transcription factors (eg, ELK-1 and MYC). Fig. 4 illustrates the Grb2 signaling pathway. Like the Bcr-Abl oncogene, the c-Cbl proto-oncogene is constitutively phosphorylated by tyrosine in CML cells. Grb2 may mediate c-CBL-driven pathways in CML cells, as Grb2 has been shown to form stable complexes with c-Cbl. This interaction can lead to malignant transformation of myeloid progenitor cells.

By inhibiting Grb2, the Grb2-SOS complex is no longer able to exchange GDP for GTP for RAS. Consequently, the MAP kinase cascade and its subsequent transcription are downregulated (Tari 2001a).

Inhibition of Grb2 gene expression.

Several strategies have been explored to inhibit Grb2 function. One strategy involves cloning an alternatively spliced form of Grb2 that has a non-functional SH2 domain removed (Fath, 1994). The encoded Grb3-3 protein will not bind to phosphorylated EGFR because the deleted residues are integral to Grb2 binding to phosphothiosine residues; Grb3-3, however, retains functional SH3 domains. Therefore, Grb3-3 can compete with Grb2 in binding guanine nucleotide exchange factors. In a study by Fath et al. (1994), microinjection of Grb3-3 into Swiss 3T3 fibroblasts induced their apoptosis. These clones are designed to compete with Grb2 in binding guanine nucleotide exchange factors.

The second strategy uses small molecule inhibitors to prevent growth factor receptors from binding to the SH2 domain of Grb2 (Gay, 1999). These small molecular weight inhibitors of Grb2 SH2 were developed using molecular models based on X-ray structures of Grb2 in complex with phosphopeptide ligands containing the Tyr-X-Asn-X motif. These inhibitors contain

- 6 041953 elements capable of recognizing and selectively binding the SH2 domain of Grb2. The goal of these inhibitors is to reverse oncogenic transformation and prevent growth factor-induced cell motility (Gay, 1999).

A third strategy involves the use of Grb2-binding phosphopeptides (Williams, 1997). Treatment of cells with cell-permeable Grb2-binding phosphopeptides results in their association with Grb2 and inhibition of growth factor receptor binding to Grb2 (Williams, 1997). This can stop growth factor-stimulated mitogenesis.

Fourth, inhibition of Grb2 expression can be achieved using antisense oligonucleotides complementary to specific regions of the Grb2 mRNA. When antisense oligonucleotides bind to a target mRNA, a DNA-RNA hybrid is formed. Fusion formation inhibits translation of the mRNA and hence the expression of the encoded protein. If the Grb2 protein is required for cell proliferation, its inhibition can lead to growth suppression. Inhibition of Grb2 expression may also overcome drug resistance and promote chemotherapy-induced apoptosis in cancer cells.

Antisense therapy with P-ethoxy oligonucleotide against Grb2 and its inhibitory activity against CML and AML

Growth factor receptor-associated protein-2 (Grb2) is used by oncogenic tyrosine kinases such as Bcr-Abl to activate the Ras pathway. Grb2 is required by Bcr-Abl to transform fibroblasts and induce leukemia-like diseases in mice. BP1001, a liposomal antisense oligodeoxynucleotide (oligo) specific for grb2 mRNA, was designed to block Grb2 expression. The continuous Grb2 cDNA sequence is shown in SEQ ID NO: 2 and the Grb2 protein sequence is shown in SEQ ID NO: 3.

The strategy used in this study to inhibit Grb2 uses a liposome-embedded, nuclease-resistant antisense oligonucleotide specific for Grb2 mRNA. The antisense oligonucleotide drug in BP1001 is a nuclease-resistant phosphodiester analog that contains a P-ethoxy backbone. This structure was chosen one at a time with a phosphorothioate backbone because it lacks a sulfur residue. The sulfur residue in the phosphorothioate backbone was associated (influenced) with hemorrhagic diathesis.

The oligonucleotide sequence used is: 5'ATATTTGGCGATGGCTTC-3' (SEQ ID NO: 1) and is specific for the translation initiation site of human Grb2 mRNA, thereby disrupting the production of Grb2 protein. This antisense has been shown to inhibit Grb2 expression and cell growth in Bcr-Abl positive leukemic cells as well as in breast cancer cells that express high levels of HER2/neu or EGFR (Fig. 5A-B).

BP1001 is a neutral liposome coupled with a nuclease resistant hydrophobic P-ethoxy antisense oligo targeted to Grb2 mRNA. In preclinical studies, BP1001 was effective in inducing growth inhibition and reduction of ERK1,2 and Akt phosphorylation in both imatinib-sensitive and imatinib-resistant CML cell lines. In addition, BP1001 has been shown to selectively inhibit the production of the Grb2 protein in Ph+ leukemia cell lines BV173 and ALL-1, and has also been found to inhibit the proliferation of Bcr-Abl+ leukemia cell lines (ALL-1, BV173 and K562 cells) ( Table 16).

Table 16 Inhibition of Proliferation of Leukemia Cell Lines by BP1001

cell line [BP1001] µM Incubation time (days) Decrease in viability (%) 1С _5О (μM) ALL-1 0-6 5 100 4 BV173 0-10 5 70 8 K562 0-12 4 80 8 HL60* 0-12 5 0 -

* - Ph-negative leukemic cell line (Bcr-Abl negative).

Inhibition of Grb2 protein expression resulted in cell growth inhibition in Bcr-Abl+ cell lines derived from Ph+ leukemia patients, demonstrating that Grb2 plays a functional role in Bcr-Abl-induced cell proliferation and therefore plays a vital role in maintaining the oncogenic potential of Ph+ leukemia. These results indicate that downregulation of the Grb2 protein can lead to cell growth inhibition, which is regulated by the Bcr-Abl tyrosine kinase.

BP1001 was effective in increasing the survival of NOD/SCID mice bearing Bcr-Abl positive leukemia xenografts (Tari, 2007). Studies have shown an antitumor response to this agent in mice injected with 32Dp210 Bcr-Abl positive leukemia cells and subsequently treated with BP1001.

In one study, mice injected with 32Dp210 Bcr-Abl-positive leukemic cells were treated with BP1001 (15 mg/kg/dose) or control (blank DOPC liposomes) intravenously starting on the first day after tumor cell injection, twice a week for up to 6 weeks. Mice were observed daily for a state of agony/morbidity. All surviving mice were sacrificed on day 40. In mice injected with 32Dp210 Bcr-Abl positive leukemia cells, 60% of those treated with BP1001 survived at day 40 (end of study) compared to 17% of mice treated with empty liposomes. The average lifespan of treated animals was

31.6±13.1 days compared to 23.7±8.3 days in the empty liposome group (FIG. 6A).

In a second study in mice injected with 32Dp210 Bcr-Abl-positive leukemic cells, treatment groups received BP1001 at 5, 10, or 15 mg/kg/dose administered intravenously starting one day after tumor cell injection, twice a week for for up to 7 weeks. Control groups received DOPC liposomes containing random sequence oligonucleotide (15 mg/kg/dose) in the same manner. Mice were observed daily for a state of agony/morbidity. Surviving mice in the control groups were euthanized at day 41 and those in the treatment groups at day 48. At the 15 mg/kg/dose level, 80% of mice treated with BP1001 survived to day 48 (end of study), compared to 0% of mice treated with 15 mg/kg/dose of the liposomal control oligonucleotide (FIG. 6B). The average life expectancy was 44.2±8.5 versus 20.4±7.9 days in these groups, respectively. The mean lifespan of mice treated with 5 or 10 mg/kg/dose of BP1001 was 20.8±4.7 and 22.2±8.7 days, respectively. The results showed that twice-weekly treatment with BP1001 at a dose level of 15 mg/kg/dose increased survival in mice induced with 32Dp210 Bcr-Abl-positive CML compared to control oligonucleotide treatment.

In addition, a significant reduction in peripheral blasts was observed in the CML patient in the Phase I study (81% to 5%) after receiving four infusions of BP1001. The patient had a CML blast crisis characterized by the T315I mutation. These data suggest that Grb2 plays a critical role in the progression of CML. In addition, preclinical studies show that pretreatment with BP1001 enhances cytotrabin cytotoxicity in acute myeloid leukemia (AML) cells.

Pharmacology and toxicology BP1001.

Pharmacokinetic studies in rats.

Pharmacokinetic studies of BP1001 have been performed in rats after intravenous (IV) administration (Tari, 2007). Lewis rats (n=5, 400 g) were cannulated with the right femoral artery and left femoral vein and were injected intravenously with radioactively labeled ( ³² R) BP1001 at a dose of 10 mg of oligonucleotide per kg of body weight. Blood (0.3 ml) was collected through the right femoral artery at 5, 10, 20, 30, 60, 90, 120, 180 and 240 minutes after injection. After each withdrawal, the catheter was flushed with sodium heparin 1:1000 (units/ml). Whole blood samples were collected and analyzed for ^32P radioactivity by liquid scintillation as previously described (Tari, 1998; Gutierrez-Puente, 1999).

Pharmacokinetic parameters were determined using non-linear regression analysis (Rstrip; Micro Math, Inc., Salt Lake City, UT). Whole blood drug concentration data were best suited to the two-component model. It was found that the clearance of BP1001 from the blood exactly matches the two-component mathematical model (correlation r ² >0.98). The initial distribution phase occurred within the first 6 minutes after injection (t _1/2a =5.16±0.3 minutes). The half-life of the terminal phase (t _1/2 p) was 225.6±13.2 min. The immediate apparent volume of distribution (36.42±1.88 ml) was higher than the total blood volume in rats of this size. The area under the concentration curve (AUC) was 5.4±0.9 mg/ml x min and the retention time was 309.6±21.5 minutes.

The pharmacokinetic distribution of BP1001 and the high volume of distribution observed in this study were similar to those obtained with other liposomal P-ethoxyoligonucleotides previously studied (Tari, 1998; Gutierrez-Puente, 1999).

Tissue Distribution Studies of BP1001 in Mice Tissue distribution studies of BP1001 were carried out in mice following iv administration. Twenty mice (5/group) were divided into three test groups and one control group. Groups of subjects were injected intravenously through the tail vein with radioactively labeled ( ³² R) BP1001 at a dose of 20 mg of oligonucleotide per kg of body weight. Injected mice were sacrificed by CO ₂ inhalation 4,24 and 48 hours after injection for organ harvesting. Control animals were non-injected animals. Spleen, liver, kidney, heart, stomach, lung, and bone marrow tissues were collected from all animals, and tissue samples (50-100 mg) from each organ were weighed and processed. ³² R radioactivity was counted in a scintillation counter. The results were expressed as mean μg BP1001/g tissue. As expected, no radioactivity was detected in control animals. The tissue distribution of BP1001 was similar to that reported for other P-ethoxy oligonucleotides previously tested in liposomes. BP1001 accumulates in all studied tissues with the highest accumulation in the tissues of the spleen, liver and kidneys. At 4 h after injection, mean tissue concentrations of BP1001 per g of tissue were: 64 μg (spleen), 50 μg (liver), 34 μg (kidney) and ranged from 12-34 μg in other tissues. The tissue half-life was approximately 24 hours in the spleen, liver, kidneys, heart and stomach and 48 hours in the lungs. The minimum amount of BP1001 (~0.4 μg in two thighs) was found in the bone marrow,

- 8 041953 where the concentration remained relatively constant for 72 hours.

Toxicology BP1001.

Single dose toxicity study in mice.

In a BP1001 single-dose toxicity study, 15 male ICR mice (5/group) were assigned to a no-injection control group (group I) and two treatment groups (groups II and III) to receive 15 and 30 mg oligo/kg intravenously via the tail vein in as a single injection. Fifteen female ICR mice (5/group) were assigned to a no-injection control group (group IV) and two treatment groups (groups V and VI) to receive 20 and 40 mg oligo/kg, respectively, iv via the tail vein as a single injection. Animals were examined daily and blood was collected for hematological evaluation and clinical biochemistry two and six weeks after injection. Animals were euthanized after blood sampling six weeks after injection, and tissues were collected for gross and microscopic evaluation of organ toxicity. No signs of morbidity or mortality were observed. Single intravenous injections of 15-40 mg/kg of BP1001 did not cause drug-related lesions in the organs studied. A decrease in the number of white blood cells was observed two weeks after the injection of 30 and 40 mg oligo/kg. Restoration of white blood cell count was observed six weeks after injection of 30 mg oligo/kg. The mean value of the number of white blood cells was not fully restored after six weeks in animals treated with 40 mg oligo/kg, but did not differ statistically from the control (Tari, 2007).

Multiple dose toxicity study in mice.

In a multiple-dose toxicity study of BP1001 in mice, 24 ICR mice (outbred mice from the Institute for Cancer Research; 5 females/group, 3 males/group) were assigned to a no-injection control group (Group I) and two treatment groups (Group II- III) for administration of 15 and 25 mg oligo/kg intravenously via the tail vein daily for five consecutive days. Animals were examined daily and blood was collected for hematological evaluation and clinical biochemistry two and six weeks after injection. Animals were euthanized after blood sampling six weeks after injection, and tissues were collected for gross and microscopic evaluation of organ toxicity. No signs of morbidity or mortality were observed. Multiple intravenous injections of 1525 mg/kg BP1001 did not cause drug-related lesions in the organs studied. A decrease in white blood cell count was observed two weeks post-injection in the treatment groups, which was maintained six weeks post-injection. Differential white blood cell counts showed that specific white blood cell populations in treated animals did not differ from controls (Tari, 2007).

Multiple dose toxicity studies in rabbits.

A study was conducted on the potential toxicity of intravenous administration of BP1001 twice a week (slow bolus) in Dutch rabbits. BP1001 was administered over a 28 day period followed by a two week recovery phase. The design of the study was as shown in the table.

Rabbit toxicity study design

Group number Group designation Dosage level (mg/kg) Dosing concentration (mg/ml) Dosage volume (ml/kg) Number of animals* males females 1 low dose 3.75 2.5 1.5 9 9 2 Average dose 5.00 2.5 2.0 9 9 3 high dose 7.50 2.5 thirty 9 9

* - from each group, 3 male and 3 female rabbits were euthanized on study days 4, 29 and 42.

Doses were administered on study days 1, 3, 8, 10, 15, 17, 22 and 24. The first day of dosing in the study was designated study day 1. Doses were administered at approximately the same time each day. Individual dose volumes were calculated based on the last recorded body weight for each animal. Dose levels on day 1 of the study were calculated based on pre-test body weight. Based on the design of this study, there were no overt toxicological changes in clinical observations, body weight, bone marrow cytology, clinical pathology, organ weights, or microscopic findings. At autopsy, three macroscopic observations were made. Macroscopic observation of a small gallbladder was performed in group 1, male on day 4 of the study (No. 8939), and group 2, female on day 42 of the study (No. 8968). Group 1, male on the 29th day of the study (No. 8944), had a macroscopic observation of dark foci covering all lobes of the lung. These dose-independent results do not appear to be related to BP1001 administration. Based on the design of this study, there was no overt toxicity with intravenous (slow

- 9 041953 bolus) administration twice a week of 3.75, 5.00 or 7.50 mg/kg BP1001 in clinical observations, body weight, bone marrow cytology, clinical pathology, organ weight or microscopic findings.

In addition, the results showed that when BP1001 was administered to rabbits, no effect on coagulation parameters was observed.

Ames mutagenicity study BP1001 was tested in an Ames mutagenicity study. Sodium chloride (0.9%) was added to BP1001 vials containing 5 mg of the Grb2 oligonucleotide. The sample was tested against five strains of Salmonella at various concentrations in the presence and absence of the S-9 enzyme activation system. The sample was not mutagenic for the strains tested.

An in vitro mammalian chromosome aberration assay (CHO cells) BP1001 was tested in a chromosome aberration assay using CHO cells in both the absence and presence of the Aroclor-induced S9 activation system. A preliminary toxicity test was performed to determine the dose range for the analysis of chromosomal aberration. Chromosome aberration analysis was used to assess the clastogenic potential of BP1001. Based on the results of this study, it was concluded that BP1001 is negative for the induction of structural and numerical chromosome aberrations in CHO cells in both non-activated and S9-activated test systems.

Clinical studies of BP1001 in humans.

The phase I BP1001 study consisted of two parts: (A) dose escalation of BP1001 and (B) BP1001 with concomitant dose escalation of low-dose cytarabine (LDAC). For dose escalation (Part A), a standard 3+3 design was used in which successive groups of 3 or more patients with hematologic malignancies were treated with increasing doses from 5 to 135 mg/m ² BP1001 until a maximum tolerated dose (MTD) was identified. For the dose escalation portion of the study (Part B), two consecutive subgroups of AML patients were treated with BP1001 at MTD (or the highest dose tested [HTD]) and one level below MTD (or HTD) in combination with a fixed dose of LDAC for further characteristics of safety and biological effect, and to determine the recommended phase 2 dose. Both parts of the study used an open sequential design with dose escalation to assess safety, tolerability and toxicity, pharmacokinetics (PK), tumor response and antileukemic activity. A total of 39 patients participated in the study: AML (n=30), CML-BP (n=5) and MDS (n=4). Of 39 patients, 27 were evaluated; 12 did not complete a full cycle due to disease progression and were replaced as per protocol. Only one patient at 5 mg/m ² experienced dose-limiting toxicity (DOT), grade 3 mucosal inflammation, and hand-foot syndrome while taking a high dose of hydroxyurea for the treatment of proliferative CML-BP. The study drug could not be ruled out as a contributing factor, so this event was reported as DOT. After expanding to six patients, no patient developed DOT. No other drug toxicity was observed in any of the treated patients. The MTD has not been identified. The HTD for BP1001 is 90 mg/m ² .

Among the 27 patients evaluated, a median of one cycle (1-5) was entered: four received two cycles, three received three cycles, four received five cycles, and all others received one cycle. Of 21 evaluable patients in the single agent arms (Part A), 10 experienced >50% reduction in peripheral or bone marrow blasts; two had improvement in leukemia skin lesions; six had an unsustainable decrease in the number of blasts. Among the six evaluable patients treated with BP1001+LDAC combination therapy (Part B), three achieved complete remission (CR) and two achieved partial remission (PR).

Grb2 biomarker studies.

Flow cytometry was used to determine the levels of Grb2 and pErk proteins in circulating leukemia cells of patients treated with BP1001 single agent and reported as Mean Fluorescence Intensity (MFI). MFI Grb2 and pErk during treatment were compared with those at baseline. In the last sample measured (end of treatment or cycle 1 day 22), BP1001 reduced >25% Grb2 levels in 10 of 12 samples, and >25% pErk levels in 7 of 12 samples. The mean reduction in Grb2 levels was 49% (range: 28 to 91%) and pErk was 52% (range: 27 to 91%).

Pharmacokinetic studies.

Pharmacokinetic (PK) analysis was performed on plasma samples taken from refractory/relapsed patients diagnosed with AML treated with BP1001 (60 mg/m ² )+LDAC or treated with BP1001 (90 mg/m ² )+LDAC. In both groups, T _max was 1 hour after administration. C _max of both doses was 82 ng/ml. However, the plasma half-life of BP1001 was longer for the 60 mg/m ² dose than for the 90 mg/m ² dose (29.6±8.1 h versus 11.8±5.4 h). The degree of clearance of BP1001 was lower for a dose of 60 mg/m ² than for a dose of 90 mg/m ² (133±24 l/h versus 205±70 l/h). These results indicate that the 60 mg/m ² dose of BP1001 may be more beneficial than the 90 mg/m ² dose.

- 10 041953

lipids and liposomes.

The term liposomes is used herein to refer to lipid-containing vesicles having a lipid bilayer, as well as other lipid carrier particles that can capture or include antisense oligonucleotides. As such, liposome is a general term encompassing a plurality of unilamellar, multilamellar, and multivesicular lipid carriers formed by the formation of closed lipid bilayers or aggregates. In addition, liposomes may have an indefinite lamellar structure. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilayer liposomes have several lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. Lipid components self-assemble before forming closed structures and trap water and solutes between lipid bilayers (Ghosh and Bachhawat, 1991). However, this invention also encompasses compositions that have other structures in solution than the normal vesicular structure. For example, lipids may have a micellar structure or simply exist as heterogeneous aggregates of lipid molecules.

Liposomes are a form of nanoparticles that are carriers for delivering various drugs to diseased tissue. The optimal size of liposomes depends on the target tissue. In tumor tissue, the vasculature is discontinuous and pore sizes vary from 100 to 780 nm (Siwak et al., 2002). In comparison, the pore size in normal vascular endothelium is <2 nm in most tissues and 6 nm in postcapillary venules.

It is believed that negatively charged liposomes are more rapidly removed from the circulation than neutral or positively charged liposomes; however, recent studies have shown that the type of negatively charged lipid affects the rate of uptake of liposomes by the reticuloendothelial system (RES). For example, liposomes containing negatively charged lipids that are not sterically shielded (phosphatidylserine, phosphatidic acid, and phosphatidylglycerol) are cleared faster than neutral liposomes. Interestingly, cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) and cation-liposome-DNA complexes are more actively bound and internalized by angiogenic blood vessel endothelial cells via endocytosis than anionic, neutral, or sterically stabilized neutral liposomes (Thurston et al., 1998; Krasnici et al., 2003). Cationic liposomes may not be ideal delivery vehicles for tumor cells because surface interactions with tumor cells create an electrostatically derived binding site barrier effect, inhibiting further association of delivery systems with tumor spheroids (Kostarelos et al., 2004). However, neutral liposomes appear to be better able to penetrate into the tumor. Toxicity to specific liposomal drugs is also a concern. Cationic liposomes cause dose-dependent toxicity and lung inflammation by promoting the release of reactive oxygen intermediates, and this effect is more pronounced in multivalent cationic liposomes than in monovalent cationic liposomes such as DOTAP (Dokka et al., 2000). Neutral and negative liposomes do not appear to exhibit pulmonary toxicity (Guitierrez-Puente et al., 1999). Cationic liposomes, although efficient in uptake of nucleic acids, have had limited success in gene silencing in vivo, possibly due to their stable intracellular nature and consequent inability to release nucleic acid contents. Charge-neutral lipids or charge-neutralized lipid compositions, for example, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), are used herein because of their neutral properties and success in delivering antisense oligonucleotides in vivo.

The present invention provides methods and compositions for linking an oligonucleotide, such as an antisense oligonucleotide, to a lipid and/or liposome. The oligonucleotide can be incorporated into the aqueous interior of the liposome, embedded in the lipid bilayer of the liposome, attached to the liposome via a binding molecule that is associated with both the liposome and the oligonucleotide entrapped in the liposome, complexed with the liposome, dispersed in a solution containing the lipid, mixed with a lipid, combined with a lipid, suspended in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. The compositions associated with liposomes or liposomes/oligonucleotides provided herein are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or in a compressed structure. They may also simply be interspersed in the solution, possibly forming aggregates that are neither uniform in size nor shape.

Lipids.

Lipids are fatty substances that can be natural or synthetic. For example, lipids include fat droplets that naturally occur in the cytoplasm, as well as a class of compounds that are well known to those skilled in the art that contain long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. An example is the lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

The lipid compositions of this invention may contain phospholipids. In some embodiments of the invention, one type or type of phospholipid can be used in the creation of lipid compositions such as liposomes. In other embodiments of the invention, more than one species or type of phospholipid may be used.

Phospholipids include glycerophospholipids and some sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauriloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, 1,2-diarachidoyl-sn-glycero -3-phosphocholine (DAPC), 1,2-dieucosenoyl-sn-glycero-3-phosphocholine (DEPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2 -stearoylphosphatidylcholine (PSPC), 1stearoyl-2-palmitoylphosphatidylcholine (SPPC), palmitoyleoylphosphatidylcholine (POPC), 1oleoyl-2-palmitoylphosphatidylcholine (OPPC), dilauriloylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearolylphosphatidylglycerol (DPPG) , dioleoylphosphatidylglycerol (DOPG), dimyristoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid (DPPA), distearoylphosphatidic acid (DSPA), dioleoylphosphatidic acid (DOPA), dimyristoyl f osphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyleoyl phosphatidylethanolamine (POPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), brain phosphatidylserine (BPS), distearoyl sphingomyelin (DS) , brain sphingomyelin (BSP), dipalmitoyl sphingomyelin (DPSP), lysophosphatidylcholine, and lysophosphatidylethanolamine.

Phospholipids include, for example, phosphatidylcholines, phosphatidylglycerols and phosphatidylethanolamines; since phosphatidylethanolamines and phosphatidylcholines are not charged under physiological conditions (i.e., at a pH of about 7), these compounds may be particularly useful for the production of neutral liposomes. In certain embodiments, the implementation of the DOPC phospholipid is used to obtain uncharged liposomes or lipid compositions. In certain embodiments, a lipid that is not a phospholipid (eg, cholesterol) may also be used.

Phospholipids may be from natural or synthetic sources. However, phospholipids from natural sources, such as egg or soy phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin, and plant or bacterial phosphatidylethanolamine, are not used in some embodiments of the invention as the main phosphatide (i.e., constitute 50% or more total phosphatide composition) as this can lead to instability and leakage of the resulting liposomes.

neutral liposomes.

As used herein, the terms neutral liposomes or lipid composition or uncharged liposomes or lipid composition are defined as liposomes or lipid compositions having one or more lipids that give a substantially neutral net charge (substantially uncharged). In some embodiments of the invention, neutral liposomes or lipid compositions may include primarily lipids and/or phospholipids, which are themselves neutral. In certain embodiments of the invention, amphipathic lipids can be included or used to create neutral liposomes or lipid compositions. For example, a neutral liposome can be created by combining positively and negatively charged lipids so that these charges essentially cancel each other out, resulting in a substantially neutral net charge. By substantially neutral or substantially uncharged is meant that few, if any, lipids in a given population (e.g., populations of liposomes) include a charge that is not canceled by the opposite charge of another component (e.g., less than 10% of the components include an unannulled charge, more than preferably less than 5% and most preferably less than 1%). In certain embodiments of this invention, a composition can be prepared in which the lipid component of the composition is substantially neutral, but is not in the form of liposomes.

The size of liposomes varies depending on the method of synthesis. A liposome suspended in an aqueous solution is usually in the form of a spherical vesicle and may have one or more concentric layers of lipid bilayer molecules. Each layer consists of a parallel array of molecules represented by the formula XY, where X is a hydrophilic moiety and Y is a hydrophobic moiety. In an aqueous suspension, the concentric layers are arranged such that hydrophilic moieties tend to remain in contact with the aqueous phase and hydrophobic moieties tend to self-associate. For example, when aqueous phases are present in a liposome, lipid molecules can form a bilayer, known as a lamella, in an XY-YX arrangement. Lipid aggregates can form when the hydrophilic and hydrophobic portions of more than one lipid molecule become associated with each other. The size and shape of these aggregates will depend on many

- 12 041953 various variables such as the nature of the solvent and the presence of other compounds in solution.

Liposomes within the scope of this invention can be prepared in accordance with known laboratory techniques, such as, for example, the method of Bangham et al. (1965), the contents of which are incorporated herein by reference; the method of Gregoriadis (1979), the contents of which are incorporated herein by reference; the method of Deamer and Uster (1983), the contents of which are incorporated by reference; and the reverse phase evaporation method described by Szoka and Papahadjopoulos (1978). The above methods differ in their respective abilities to capture aqueous material and their respective ratios of water space to lipid.

In some embodiments, a neutral liposome may be used to deliver an oligonucleotide, such as an antisense oligonucleotide. The neutral liposome may contain one kind of oligonucleotide aimed at suppressing the translation of one gene, or the neutral liposome may contain several kinds of oligonucleotides that are aimed at suppressing the translation of several genes. In addition, the neutral liposome may also contain a chemotherapeutic agent in addition to the oligonucleotide; thus, in certain embodiments of the invention, the chemotherapeutic agent and the oligonucleotide can be delivered to a cell (eg, a human cancer cell) in the same or separate compositions.

Dried lipids or lyophilized liposomes can be dehydrated and reconstituted at an appropriate concentration with a suitable solvent (eg DPBS buffer or Hepes). The mixture can then be vigorously shaken in a vortex mixer. Liposomes can be resuspended at a suitable concentration of total phospholipid (eg, about 10-200 mM). The unencapsulated oligonucleotide can be removed by centrifugation at 29,000 g and the liposome beads washed. Alternatively, non-encapsulated oligonucleotides can be removed by dialysis against excess solvent. The amount of encapsulated oligonucleotide can be determined according to standard methods.

Inhibition of gene expression.

An inhibitory oligonucleotide can inhibit transcription or translation of a gene in a cell. The oligonucleotide may be 5 to 50 or more nucleotides in length, and in some embodiments, 7 to 30 nucleotides in length. In certain embodiments, the oligonucleotide may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 nucleotides long. The oligonucleotide may contain a nucleic acid and/or a nucleic acid analog. Typically, an inhibitory oligonucleotide will inhibit the translation of a single gene in a cell; however, in certain embodiments of the invention, the inhibitory oligonucleotide can inhibit the translation of more than one gene within the cell.

Within an oligonucleotide, the components of an oligonucleotide need not be of the same type or homogeneous throughout the type (eg, an oligonucleotide may contain a nucleotide and a nucleic acid or nucleotide analogue). In certain embodiments of this invention, the oligonucleotide may contain only one nucleic acid or nucleic acid analog. The inhibitory oligonucleotide may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more contiguous nucleobases, including all ranges in between. , which hybridize to a complementary nucleic acid to form a double-stranded structure.

Nucleic acids.

This invention provides methods and compositions for delivering an oligonucleotide through neutral liposomes. Since an oligonucleotide is composed of a nucleic acid, methods relating to nucleic acids (eg, nucleic acid production, nucleic acid modification, etc.) can also be used on the oligonucleotide.

The term nucleic acid is well known in the art. The term nucleic acid as used herein generally refers to a molecule (ie, strand) of DNA, RNA, or a derivative or analogue thereof containing a nucleic base. These definitions refer to a single-stranded or double-stranded nucleic acid. Double-stranded nucleic acids can be formed by fully complementary binding; however, in some embodiments of the invention, the double-stranded nucleic acid may be formed by partial or substantially complementary binding. As used herein, the term single-stranded nucleic acid may be denoted by the prefix oc and double-stranded nucleic acid by the prefix ds.

Nucleic bases.

As used herein, the term nucleic base refers to a heterocyclic base, such as, for example, a naturally occurring nucleic base (i.e., A, T, G, C, or U) found in at least one naturally occurring nucleic acid (i.e., DNA and RNA), as well as naturally occurring or non-naturally occurring derivative(s) and analogs of such nucleobase.

A nucleic base can typically form one or more hydrogen bonds (i.e., anneal or hybridize) to at least one naturally occurring nucleic base.

- 13 041953 base in a manner that can replace naturally occurring nucleobase pairing (eg hydrogen bonding between A and T, G and C, and A and U). The nucleobase may be contained in a nucleoside or nucleotide using any chemical or natural synthesis method described herein or known to a person skilled in the art.

Purine and/or pyrimidine nucleobase(s) encompasses naturally occurring purine and/or pyrimidine nucleobases, as well as derivative(s) and analog(s), including, but not limited to, a purine or pyrimidine substituted with one or more an alkyl, carboxyalkyl, amino, hydroxyl, halogen (ie fluoro, chloro, bromo or iodo), thiol or alkylthiol moiety. Preferred alkyl (eg, alkyl, carboxyalkyl, etc.) moieties contain from about 1, about 2, about 3, about 4, about 5 to about 6 carbon atoms. Other non-limiting examples of a purine or pyrimidine include deazapurine, 2,6diaminopurine, 5-fluorouracil, xanthine, hypoxanthine, 8-bromoguanine, 8-chloroguanine, bromtyline, 8aminoguanine, 8-hydroxyguanine, 8-methylguanine, 8-thioguanine, azaguanine, 2-aminopurine , 5-ethylcytosine, 5-methylcyosine, 5-bromouracil, 5-ethyluracil, 5-iodouracil, 5-chlorouracil, 5-propyluracil, thiouracil, 2-methyladenine, methylthioadenine, N, N-dimethyladenine, azaadenines, 8-bromadenine, 8 -hydroxyadenine, 6hydroxyaminopurine, 6-thiopurine, 4-(6-aminohexyl/cytosine) and the like. Purine and pyrimidine derivatives or analogs include, but are not limited to (abbreviation/description of modified base): ac4c/4-acetylcytidine, Mam5s2u/5-methoxyaminomethyl-2-thiouridine, Chm5u/5(carboxyhydroxylmethyl)uridine, Man q/Beta, D -mannosylqueosine, Cm/2'-O-methylcytidine, Mcm5s2u/5methoxycarbonylmethyl-2-thiouridine, Cmnm5s2u/5-carboxymethylamino-methyl-2-thioridine, Mcm5u/5methoxycarbonylmethyluridine, Cmnm5u/5-carboxymethylaminomethyluridine, Mo5u/5-methoxyuridine, D/ dihydrouridine, Ms2i6a, 2-methylthio-N6-isopentenyladenosine, Fm/2'-O-methylpsevduridine, Ms2t6a/N((9-betaI-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, Gal q/beta, D -galactosylqueosine, Mt6a/N -((9-beta-D-ribofuranosylpurin-6-yl)N-methyl-carbamoyl)threonine, Gm/2'-O-methylguanosine, Mv/uridine-5-hydroxyacetic acid methyl ester, I /inosine, o5u/uridine-5-hydroxyacetic acid (v), I6a/N6-isopentenyladenosine, Osyw/vibutoxosine, m1a/1-methyladenosine, P/pseudouridine, m1f/1methylpseudouridine, Q/queuosine, m1g/1-methylguanosine, s2c/2-thiocytidine, m1I/1-methylinosine, s2t/5methyl-2-thiouridine, m22g/2,2-dimethylguanosine, s2u/2-thiouridine, m2a/2-methyladenosine, s4u/4thiouridine, m2g/2-methylguanosine, T/5-methyluridine, m3c/3-methylcytidine, t6a/N-((9-beta-Dribofuranosylpurin-6-yl)carbamoyl)threonine, m5c/5-methylcytidine, Tm/2'-O-methyl-5-methyluridine , m6a/N6-methyladenosine, Um/2'-O-methyluridine, m7g/7-methylguanosine, Yw/vibutosine, Mam5u/5methylaminomethyluridine or X/3-(3-amino-3-carboxypropyl)uridine, (acp3)u.

Nucleosides.

As used herein, the term nucleoside refers to a single chemical entity containing a nucleobase covalently linked to a linker group of the nucleobase. A non-limiting example of a nucleobase-linker fragment is a sugar containing 5 carbon atoms (i.e., a 5-carbon sugar), including, but not limited to, deoxyribose, ribose, arabinose, or a 5-carbon sugar derivative or analog. Non-limiting examples of a 5-carbon sugar derivative or analog include 2'-fluoro-2'-deoxyribose or carbocyclic sugar, where the carbon is replaced by an oxygen atom in the sugar ring. As used herein, the term fragment generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure.

Various types of covalent attachment(s) of a nucleobase to the nucleobase of a linker moiety are known in the art. As a non-limiting example, a nucleoside containing a purine (ie, A or G) or a 7-deazapurine nucleobase typically contains a covalent attachment of the 9-position of the purine or 7-deazapurine to the 1'5 carbon sugar position. In another non-limiting example, a nucleoside containing a pyrimidine nucleobase (ie, C, T, or U) typically contains a covalent attachment of the 1 position of the pyrimidine to the 1' position of the 5-carbon sugar (Kornberg and Baker, 1992).

Nucleotides.

As used herein, the term nucleotide refers to a nucleoside further comprising a backbone bond. A backbone bond typically covalently attaches a nucleotide to another molecule containing the nucleotide or to another nucleotide to form a nucleic acid. The backbone bond in naturally occurring nucleotides typically contains a phosphate moiety (eg, a backbone phosphodiester bond) that is covalently linked to a 5-carbon sugar. Attachment of the backbone moiety usually occurs at the 3' or 5' position of the 5-carbon sugar. However, other types of attachments are known in the art, especially when the nucleotide contains derivatives or analogs of a naturally occurring 5-carbon sugar or phosphate moiety.

Nucleic acid analogues.

The nucleic acid may contain, or consist entirely of, a nucleic base derivative or analog, a linker nucleotide fragment, and/or a backbone bond, which may be present in a naturally occurring nucleic acid. As used herein, the term derivative refers to a chemically modified or altered form of a naturally occurring molecule, while the terms mimic or analog refer to a molecule that may or may not structurally resemble a naturally occurring molecule or fragment, but has similar functions. Nucleic bases, nucleoside and nucleotide analogs or derivatives are well known in the art.

Non-limiting examples of nucleosides, nucleotides, or nucleic acids containing 5-carbon sugar derivatives or analogs and/or backbone bonds are included in U.S. Patent No. 5,681,947, which describes oligonucleotides containing purine derivatives that form triple helices with and/or prevent dsDNA expression; US patent No. 5652099 and 5763167, which describe nucleic acids, including fluorescent analogs of nucleosides found in DNA or RNA, especially for use as fluorescent nucleic acid probes; US patent No. 5614617, which describes analogs of oligonucleotides with substitutions for pyrimidine rings, which have increased nuclease stability; US Pat. Nos. 5,670,663, 5,872,232, and 5,859,221, which describe modified 5-carbon sugar oligonucleotide analogs (i.e., modified 2'-deoxyfuranosyl fragments) useful in nucleic acid detection; US Pat. No. 544,6137, which describes oligonucleotides containing at least one 5-carbon sugar moiety substituted at the 4' position with a substituent other than hydrogen, which can be used in hybridization assays; US patent No. 5886165, which describes oligonucleotides with deoxyribonucleotides with 3'-5' backbone bonds and ribonucleotides with 2'-5' backbone bonds; US Pat. No. 5,714,606, which describes a modified backbone bond in which oxygen at the 3' position of the backbone bond is replaced by carbon to increase the resistance of nucleic acids to nucleases; US patent No. 5672 697, which describes oligonucleotides containing one or more 5'-methylenephosphonate backbone bonds, which increase resistance to nucleases; US Pat. Nos. 5,466,786 and 5,792,847, which describe linking a substituent fragment, which may contain a drug or label, to the 2'-carbon of an oligonucleotide to provide increased nuclease stability and the ability to deliver drugs or fragments for detection; US Pat. No. 5,223,618, which describes oligonucleotide analogs with a 2 or 3 carbon backbone linking the 4' position and the 3' position of an adjacent 5 carbon sugar moiety to enhance cellular uptake, nuclease resistance, and hybridization to target RNA; US patent No. 5470967, which describes oligonucleotides containing at least one sulfamate or sulfamide backbone bond, which are used as nucleic acid hybridization probes; US Pat. US patent No. 5858988, which describes a hydrophobic carrier agent attached to the 2'-O position of oligonucleotides to increase the permeability and stability of their membranes; US patent 5214136, which describes oligonucleotides conjugated with an anthraquinone at the 5'-end, which have increased hybridization with DNA or RNA; increased resistance to nucleases; US Pat. No. 5,700,922, which describes PNA-DNA-PNA chimeras wherein the DNA contains 2'-deoxyerythropentofuranosyl nucleotides to enhance nuclease resistance, binding affinity, and the ability to activate RNase H; US patent No. 5708154, which describes RNA associated with DNA with the formation of a DNA-RNA hybrid; US patent No. 5908845, which describes polyester nucleic acids in which one or more nucleic bases are associated with chiral carbon atoms in the polyester backbone; U.S. Patent Nos. 5,786,461, 5,891,625, 5,786,461, 5,773,571, 5,766,855, 5,736,336, 5,719,262, 5,714,331, 5,539,082 and WO 92/20702, which describe peptidic nucleic acids (PNA or peptide-based nucleic acid analog) that usually contain one or PENAM; or more than nucleotides or nucleosides that contain a nucleotide fragment, a linker nucleotide fragment that is not a 5-carbon sugar (e.g., aza nitrogens, amide and/or ureide boundaries), and/or a backbone bond that is not a backbone phosphate bond ( for example, aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfinamide, or a backbone polysulfonamide bond); and US Pat. No. 5,855,911, which describes a hydrophobic, nuclease-resistant P-ethoxy backbone bond.

Other modifications and uses of nucleic acid analogs are known in the art, and it is expected that these methods and types of nucleic acid analogs may be used with the present invention.

Preparation of nucleic acids.

The nucleic acid can be obtained by any method known to the person skilled in the art, such as chemical synthesis, enzymatic or biological production.

Non-limiting examples of a synthetic nucleic acid (e.g., a synthetic oligonucleotide) include nucleic acid prepared by in vitro chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase methods such as those described in EP 266032, incorporated herein by reference, or deoxynucleoside H- 15 041953 phosphonate intermediates as described by Froehler et al. (1986) and Pat. US No. 5705629, each of which is included in this description by reference. One or more kinds of oligonucleotides can be used in the methods of this invention. Various mechanisms for the synthesis of oligonucleotides have been described in, for example, US Pat.

Purification of nucleic acids.

Nucleic acid can be purified on polyacrylamide gels, cesium chloride gradient centrifugation, or by any other method known to those skilled in the art (see, for example, Sambrook et al. (2001), incorporated herein by reference).

In certain embodiments, this invention provides a nucleic acid that is an isolated nucleic acid. As used herein, the term isolated nucleic acid refers to a nucleic acid molecule (e.g., an RNA or DNA molecule) that has been isolated free from, or otherwise free from, a major portion of the total genomic and transcribed nucleic acids of one or more cells. In some embodiments, an isolated nucleic acid refers to a nucleic acid that has been isolated free from, or otherwise free from, mass cellular components or in vitro reaction components, such as, for example, macromolecules such as lipids or proteins, small biological molecules, and the like.

Hybridization.

As used herein, the term hybridization, hybridizable(s) or hybridizable means the formation of a two- or three-stranded molecule, or a molecule with a partial two- or three-stranded nature. As used herein, the term annealing is synonymous with the word hybridize.

As used herein, stringent condition(s) or high stringency are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s) but exclude hybridization of random sequences. Stringent conditions are not tolerant if they allow for a slight mismatch between the nucleic acid and the target strand, if any. Such conditions are well known to those skilled in the art and are preferred for applications requiring high selectivity.

Stringent conditions may include low salt and/or high temperature conditions such as providing about 0.02M to about 0.15M NaCl at temperatures from about 50°C to about 70°C. It is understood that the temperature and ionic strength of the desired stringency is determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and the presence or concentration of formamide, tetramethylammonium chloride, or other solvent(s) in the hybridization mixture.

It is also understood that these ranges, compositions, and hybridization conditions are only mentioned as non-limiting examples, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison with one or more positive or negative controls. Depending on the intended application, it is preferable to use different hybridization conditions to achieve different degrees of selectivity of the nucleic acid with respect to the target sequence. In a non-limiting example, the identification or isolation of a bound target nucleic acid that does not hybridize to the nucleic acid under stringent conditions can be achieved by hybridization at low temperature and/or high ionic strength. Such conditions are referred to as low stringency conditions or low stringency conditions, and non-limiting examples of low stringency include hybridization conducted at about 0.15 M to about 0.9 M NaCl over a temperature range of about 20°C to about 50°C. Of course, one of skill in the art can further modify the low or high stringency conditions to suit a particular application.

Method for producing a liposomal P-ethoxy antisense drug.

Antisense oligonucleotides (oligos) complementary to certain regions of the target mRNA were used to inhibit the expression of endogenous genes. When antisense oligonucleotides bind to a target mRNA, a DNA-RNA hybrid is formed. The formation of this hybrid inhibits the translation of the mRNA and hence the expression of the encoded protein. If a protein is essential for cell survival, inhibition of its expression can lead to cell death. Therefore, antisense oligonucleotides may be useful tools in anticancer and antiviral therapy.

The main barriers to the use of antisense oligonucleotides to suppress gene expression are cell instability, low cellular uptake, and poor intercellular delivery. Natural phosphodiesters are not resistant to nuclease hydrolysis; thus, high concentrations of antisense oligonucleotides are required before any inhibitory effect is observed. Modified phosphodiester analogs such as P-ethoxy have been developed to overcome this problem of nuclease hydrolysis, but they have not provided a satisfactory solution to the problem.

Cellular uptake of antisense oligonucleotides is low. To overcome this problem, physical methods such as calcium phosphate precipitation, DEAE-dextran mediation, or electroporation have been used to increase cellular uptake of oligonucleotides. These methods are difficult to reproduce and are not applicable in vivo. Cationic lipids such as lipofectin are also used to deliver oligonucleotides. An electrostatic interaction is formed between cationic lipids and negatively charged oligonucleotides, leading to the formation of a complex, which is then taken up by target cells. Because these cationic lipids do not protect oligonucleotides from nuclease cleavage and are harmful to the cell membrane, they are only useful for delivering nuclease-resistant phosphorothioates, not nuclease-cleavable phosphodiesters.

Another modified phosphodiester analogue that has been prepared is Rethoxy. The P-ethoxy antisense backbone does not have an adverse reaction to bleeding and complement activation, which are some of the toxic effects that have been reported for other antisense analogs. Modifications of P-ethoxyoligonucleotides are made in the phosphate backbone, so that the modification will not interfere with the binding of these oligonucleotides to the target mRNA. P-ethoxy oligonucleotides are prepared by adding an ethyl group to the non-bridging oxygen atom of the phosphate backbone, making these oligonucleotides uncharged compounds. Despite their resistance to nucleases, cellular uptake and intracellular delivery of Rethoxyoligonucleotides is poor because upon internalization, these oligonucleotides remain sequestered within endosomal/lysosomal vacuoles, preventing their access to the target mRNA.

P-ethoxy antisense drug.

The liposomal antisense P-ethoxy drug consists of two cGMP products, both of which have an FDA-required Certificate of Analysis with FDA-approved release criteria. Raw materials, solvents and the final medicinal product are described in this document. When manufactured, the drug product is an amber or white lyophilized crystal or powder that contains the following materials: an oligonucleotide (eg, P-ethoxy antisense drug), neutral lipids (eg, DOPC), and a surfactant (eg, polysorbate 20 ). In preparation for administration to a patient, physiological saline is added to the ampoule, at which time liposomes are formed with antisense-P-ethoxy embedded in the interior. P-ethoxy antisense drug Specific physical properties (eg, solubility and hydrophobicity, which then affect the solubility of the drug product in saline, incorporation of oligos into liposomes, and liposome particle size) of the finished product can be determined using a predetermined raw material with P- ethoxy and phosphodiester amidite should be mixed during production of the P-ethoxy antisense drug substance. Although the loss of the P-ethoxy backbone group occurs randomly during the production of oligonucleotides, resulting in phosphodiester bonds in those bonds, this loss cannot generate the preferred ratio of P-ethoxy bond: phosphodiester backbone bond within the oligonucleotide. In this case, the feed mixture with P-ethoxy and phosphodiester amidite complements the expected amount of P-ethoxy backbone deletions, thus generating an oligonucleotide with the desired ratio. Increasing the number of P-ethoxy molecules in the oligonucleotide backbone causes the molecule to become more hydrophobic (resulting in larger liposomal particles; Table 1), less polar, and less soluble (Table 2).

Methods for testing a charge-neutral hydrophobic P-ethoxy drug include mass spectrometry to determine the length distribution of oligonucleotides and assays to determine drug solubility, which for practical solubility purposes is visual inspection of a drug product reconstituted in saline. As the oligonucleotide becomes less soluble due to more backbone P-ethoxy bonds, the reconstituted solution becomes whiter while particles are being formed because the hydrophobicity becomes too high.

- 17 041953

Table 1. Particle size variability of liposome with antisense backbone composition

Ethyl backbone removal after production Particle size specifications: Cumulative distribution function Experiment Constructed antisense backbone Main Peak Composite deletion 90% value (nm) ** 50% Value (nm) 300 nm Value (%) 1 substitution 3 amidites -6 -5.67 2130 911 15.30 2 substitution 3 amidites -6 -5.67 2420 1004 15.50 3 substitution 3 amidites -6 -6.12 3682 943 15.50 4 substitution 3 amidites -7 -6, 66 3805 978 14.60 5 100% Retoxy -5 -5.66 3924 976 16.00 6 substitution 2 amidites -5 -5.32 4387 1888 11.60 7 ^a 100% Retoxy -4 -4.22 5057 1131 17.70

8 100% Retoxy -4 -4.52 5659 1359 10.00 9 ^b 100% Retoxy -4 -4.38 7571 1909 2.60 ^10th 100% Retoxy -4 -4.38 7994 1653 14.40

** - drug release criteria for 90% of liposomal particles must be less than or equal to 5000 nm, a - this batch was rejected due to poor solubility; in particular the antisense particles in the reconstituted solution, b - this lot had a lower volume of DMSO and tBA with 2 mg antisense in a 20 ml vial, which added an additional component to the increase in liposomes, c - this lot was not released because it was not met the particle size specification.

- 18 041953

Table 2. Solubility of Liposomal Particles with Antisense Backbone Composition

Ethyl backbone removal after production Solubility of drugs Expert name Designed antisense backbone Main Peak Composite removal Visual observation * * Solubility assessment 1 substitution 3 amidites -6 -5, 67 skimmed milk solution Fine 2 substitution 3 amidites -6 -5, 67 skimmed milk solution Fine 3 substitution 3 amidites -6 -6, 12 skimmed milk solution Fine 4 substitution 3 amidites -7 -6, 66 skimmed milk solution Fine

5 100% P-ethoxy -5 -5.66 skimmed milk solution Fine 6 substitution 2 amidites -5 -5.32 skimmed milk solution Fine 7 100% P-ethoxy -4 -4.52 white mortar pass t 8 ^b 100% P-ethoxy -4 -4.38 white mortar pass t ^9th 100% P-ethoxy -4 -4.38 white mortar pass t 10 ^a 100% P-ethoxy -4 -4.22 particles of white solution failure

** - if there are particles in the drug sample, the batch will be rejected, a - this batch was rejected due to poor solubility; specifically the antisense particles in the reconstituted solution, b - this lot had a lower volume of DMSO and tBA with 2 mg antisense in a 20 ml vial, which added an extra component to the increase in liposomes, c - this lot was not released because it was not met the particle size specification.

Formulation, Filtration and Lyophilization of Liposomal P-ethoxy Antisense Drug Product

One gram (1 g) of oligo pE is dissolved in DMSO at a ratio of 10 mg of oligonucleotide per 1 ml of DMSO. Then DOPC is added to tert-butyl alcohol in a ratio of 1 g DOPC to 1719 ml tert-butyl alcohol. Oligo and DOPC are combined and mixed in a ratio of 1 g of oligonucleotide to 2.67 g of DOPC. Then, 20 ml of a 0.835% (v/v) solution of polysorbate 20 was added to the mixture, resulting in a final concentration of 0.039 mg/ml. The solution is passed through a sterile filter before filling into glass vials for lyophilization.

The effect of surfactant on liposome particle size was determined by titrating the amount of surfactant (Table 3). In the absence of polysorbate 20, only 2.8% of the particles had a diameter of 300 nm or less. In the presence of 1x polysorbate 20, 12.5% of the particles had a diameter of 300 nm

- 19 041953 or less. With the addition of 3x-10x polysorbate 20, about 20% of the particles had a diameter of 300 nm or less.

Thus, increasing the amount of surfactant from 1x to 3x results in a decrease in particle size.

Table 3. Particle size variability of surfactant-containing liposomes

Particle size specifications: Cumulative distribution function Experiment Quantity surfactant Value 50% Value 90% ** Meaning 300 nm 1 Oh 5301 nm 10719 nm 2.8% 2 1x 1053 nm 4054 nm 12.5% 3 Zx 7 85 nm 2926 nm 19.1% 4 5x 721 nm 2691 nm 21.9% 5 yuh 734 nm 2937 nm 21.4%]

,.---( Note [NL1]: ) ** - The drug release criteria for 90% of the liposome particles must be less than or equal to 5000 nm.

Preparation of a liposomal P-ethoxy antisense drug for administration.

The lyophilized preparation was hydrated with normal saline (0.9%/10 mM NaCl) at a final oligo concentration of 10-5000 μM. Liposomal-P-ethoxy oligos were mixed by hand shaking.

Methods for testing a liposomal P-ethoxy antisense drug.

Visual inspection of the manufactured medicinal product: after manufacturing, a tube with a medicinal product is taken and a visual inspection is carried out. The absence of liquid is a must and then amber crystals at the bottom of the vial are acceptable, with the best result being an increase in acceptance to a white, flaky powder or appearance. White indicates a better drying process with a high surface area to mass ratio, which is very conducive to recovery for use.

Visual inspection of the reconstituted product ready for IV administration to the patient: normal saline is added to the tube containing the manufactured liposomal P-ethoxyantisense drug product and shaken to dissolve in solution with the drug crystal or powder completely dissolved. Three main observations are made: 1) that the crystal or powder is completely dissolved, 2) there are no white clumps of undissolved material, and 3) the appearance is that of milky white or skimmed milk. The bluer the appearance of the reconstituted liquid, the better, as this signals a smaller liposome particle size that reflects light in the blue spectrum.

Mass spectrometry: Mass spectrometry (mass spectrometry) is used to display the profile of various masses in a sample. When an antisense P-ethoxy material is produced, mass spectrometry is performed on the sample. The result shows the peaks of the material present on the grid, the mass of which increases on the x-axis to the right, and the relative mass content on the y-axis increases upwards. The profile from the sample is analyzed to determine the relative number of P-ethoxy backbones in the P-ethoxy sample, recognizing that the peak profile is (starting at the far right) full length material with all backbones consisting of a P-ethoxy bond, next peak moves full length from one backbone with a P-ethoxy deletion (and hence ethyl is removed, resulting in a normal backbone phosphodiester bond), and continues. The mass spectrum pattern shifted to the right represents the P-ethoxy pattern having more P-ethoxy backbones and therefore having the properties of being more hydrophobic and less soluble; and likewise, shifted to the left, having opposite effects. Inspection of the sample characterization mass spectrum chart can also be used to determine if filtration during manufacture has any undesirable reaction to the oligonucleotide composition present in the filtered drug product.

UV Testing: UV light testing is used to determine the mass of an oligonucleotide present in a sample. Oligonucleotides absorb light in the range of 260 nanometers. As a result, UV testing of the finished reconstituted drug product has come to be used as a method for determining the amount of an oligonucleotide drug substance in a drug product vial. In terms of manufacturing development and innovation, UV testing was used to determine if there were problems encountered during filtration in manufacturing, or poor solubility of the P-ethoxy antisense drug, resulting in fewer oligonucleotides in solution and therefore lower UV readings. The method will be tested and will probably become part of testing the final version of the product.

Liposome Particle Size: The finished drug tube is reconstituted and tested for liposome particle size. The result is often an approximately normal distribution having a center point, tails and means, or an approximately normal distribution of most particles and smaller secondary peaks of smaller liposome particles resulting from second order particle generation effects. It is important that the liposome particles are not too large as they can create an undesirable reaction in patients (eg, create blood flow problems in small blood vessels in the lungs). As a result, drug release criteria include that particle size testing indicates that 90% of the liposomes are 5 microns or less. In addition, smaller liposomes are preferred because they will be better taken up by cells, and secondly, smaller liposomes can penetrate into the pores of vessels, thereby allowing the liposomes to enter the interior of tumors, increasing the effectiveness of liposomal P-ethoxy antisense drug treatment.

Methods of treatment.

Certain aspects of the invention provide methods for treating a cancer patient with an oligonucleotide-lipid complex (eg, an oligonucleotide incorporated into a neutral liposome) that contains a nuclease-resistant inhibitory oligo that targets Grb2. In particular, the oligonucleotide may have a sequence that allows base pairing with a human nucleotide sequence at the Grb2 translation initiation site and thus may inhibit Grb2 expression. In some aspects, the methods further comprise administering to the patient a first line therapy, such as, for example, a tyrosine kinase inhibitor (eg, imanitib, dazanitib, nilotinib, bosutinib, ponatinib, or bafetinib) or a cytidine analog (eg, decitabine, azacitidine, or cytarabine).

Treating and treating refer to administering or applying a therapeutic agent to a subject, or performing a procedure or method of treating a subject in order to obtain a therapeutic benefit for a disease or health-related condition. For example, treatment may include administering a pharmaceutically effective amount of the oligonucleotide-lipid complex.

Subject and patient refer to human or non-human, such as primates, mammals, and vertebrates. In specific embodiments of the invention, the subject is a human.

The term therapeutic benefit or therapeutically effective as used in this application refers to anything that contributes to or improves the well-being of the subject in relation to the medical treatment of this condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, the treatment of cancer may include, for example, reducing the size of the tumor, reducing the invasiveness of the tumor, reducing the rate of cancer growth, or preventing metastasis. Treatment of cancer may also refer to prolonging the survival of a subject with cancer.

Tumors for which these treatments are useful include any type of malignant cell, such as tumors found in solid tumor, hematologic tumor, metastatic cancer, or non-metastatic cancer. Typical solid tumors may include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, caecum, esophagus, gastrointestinal tract, gums, liver, skin, stomach, testis, tongue, uterus, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, bone, lung, bladder, melanoma, prostate and breast. Exemplary hematologic tumors include bone marrow tumors, T or B cell cancers, leukemias, lymphomas, blastomas, myelomas, and the like. Additional examples of cancers that can be treated using the methods provided herein include, but are not limited to, cancer, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung and squamous cell lung cancer), peritoneal cancer, hepatocellular carcinoma, gastric or abdominal cancer (including cancer of the gastrointestinal tract and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vaginal cancer, thyroid cancer, various types of head and neck cancer, melanoma, melanoma of superficial spread, lentigo malignant melanoma, anoral lentiginous melanoma, nodular melanoma, and B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; diffuse NHL of intermediate degree; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; NHL with small non-cleaved cells of a high degree; generalized lymphadenopathy NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia), chronic lymphoblastic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myeloid leukemia.

Cancer can definitely be of the following histological type, although it is not limited to this: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell cancer; small cell cancer; papillary cancer; squamous cell carcinoma; lymphoepithelial cancer; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary pen

- 21 041953 walking cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma with adenomatous polyp; adenocarcinoma, familial intestinal polyposis; solid cancer; malignant carcinoid tumor; branchio-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; acidophilic carcinoma; oxyphilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell cancer; follicular adenocarcinoma; papillary and follicular adenocarcinoma; unencapsulated sclerosing carcinoma; cancer of the adrenal cortex; endometrial carcinoma; skin appendage cancer; apocrine adenocarcinoma; sebaceous adenocarcinoma; cerum adenocarcinoma; mucus-forming squamous cell carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; ring cell carcinoma; infiltrating ductal carcinoma; medullary cancer; lobular cancer; inflammatory cancer; Paget's disease, mammary glands; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma malignant; malignant ovarian stromal tumor; thecoma, malignant; granulosa cell tumor is malignant; androblastoma malignant; sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; malignant paraganglioma; malignant extrathoracic paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevi; melanoma of epithelioid cells; blue nevus malignant; sarcoma; fibrosarcomas; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leimiosarcoma; rhabdomyosarcoma; embryonic rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; multilyric mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma malignant; Brenner's tumor, malignant; phyllode tumor, malignant; synovial sarcoma; mesothelioma malignant; dysgerminoma; embryonic carcinoma; teratoma malignant; ovarian node, malignant; choriocarcinoma; mesonephroma malignant; hemangiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma malignant; mesenchymal chondrosarcoma; giant cell tumor of the bone; Ewing's sarcoma; tumor odontogenic, malignant; ameloblastic odontosarcoma; ameloblastoma malignant; ameloblast fibrosarcoma; pinealoma, malignant; chordoma; glioma malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillar astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; sarcoma of the cerebellum; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; neurolemmoma malignant; granular cell tumor malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's paragranuloma; malignant lymphomas, small lymphocytes; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; fungal granuloma; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative diseases of the small intestine; leukemia; lymphocytic leukemia; leukemia of plasma cells; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

In a specific embodiment, the invention provides methods of using the oligonucleotide-lipid complex, comprising contacting a population of diseased cells in vivo with a therapeutically effective amount of the oligonucleotide-lipid complex for a period of time sufficient to inhibit or reverse the disease. In one embodiment of the invention, in vivo contacting is accomplished by administering to a patient an intravenous, intraperitoneal, subcutaneous, or intratumoral injection of a therapeutically effective amount of a physiologically tolerable composition comprising an oligonucleotide-lipid complex of the present invention. The oligonucleotide-lipid complex can be administered parenterally by injection or by gradual infusion over time.

Therapeutic compositions containing the oligonucleotide-lipid complex are typically administered intravenously or subcutaneously, for example by unit dose injection. The term unit dose, when used in relation to a therapeutic composition, refers to physically discrete units suitable as a unit dose for a subject, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect in combination with the desired diluent, i. carrier or diluent.

The compositions are administered in a manner compatible with the dosage form and in a therapeutically effective amount. The amount to be administered depends on the subject being treated, the ability of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The exact amounts of active ingredient required for administration are a matter of judgement.

- 22 041953 practitioner and are individual for each person. However, suitable dosage ranges for systemic use are described herein and depend on the route of administration. Suitable regimens for initial and booster administration are also contemplated and are typical for initial administration followed by repeated doses one or more hours apart followed by an injection or another administration. Examples of multiple administrations are described herein and are particularly preferred to maintain a consistently high serum and tissue level of the polypeptide. Alternatively, a continuous intravenous infusion sufficient to maintain blood concentrations within the ranges indicated for in vivo therapy is contemplated.

It is contemplated that the method of the invention may involve administering the oligonucleotide composition systemically or locally to treat a disease, such as inhibiting tumor cell growth or killing cancer cells in cancer patients with locally advanced or metastatic cancer. They can be administered intravenously, intrathecally, subcutaneously and/or intraperitoneally. They can be administered alone or in combination with antiproliferative drugs. In one embodiment of the invention, they are administered to reduce the cancer burden in a patient prior to surgery or other procedures. Alternatively, they may be administered after surgery to ensure that any remaining cancer (eg, cancer that surgery has failed to eliminate) is killed.

A therapeutically effective amount of an oligonucleotide is a predetermined amount calculated to achieve the desired effect, i.e. inhibition of cancer cell proliferation. Thus, the dosage ranges for administering the oligonucleotides of this invention are large enough to achieve the desired effect. The dosage should not be so high as to cause unwanted side effects such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, neurological effects, and the like. Generally, the dosage will vary with the age, condition, sex, and degree of disease of the patient, and may be determined by one of skill in the art. The dosage may be adjusted by the individual physician in case of any complications.

The composition of this invention is preferably administered parenterally to the patient, for example, by intravenous, intra-arterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intrapleural or intrathecal injection, or may be used ex vivo. Preferred dosages are between 5-90 mg/m ² . Administration is preferably repeated on a schedule until the cancer disappears or regresses, and may be combined with other forms of therapy.

Pharmaceuticals.

A pharmaceutical composition containing liposomes typically includes a sterile pharmaceutically acceptable carrier or diluent such as water or saline.

Where a neutral lipid component (eg, in the form of a liposome) containing an oligonucleotide is being clinically used, it is generally useful to provide the lipid complex as a pharmaceutical composition suitable for the intended use. Typically, this will entail the preparation of a pharmaceutical composition that is substantially free of pyrogens as well as any other impurities that may be harmful to humans or animals. Appropriate buffers may also be used to render the complex stable and to allow uptake by target cells.

The phrases pharmaceutically or pharmacologically acceptable refer to molecular substances and compositions that do not cause an adverse, allergic or other adverse reaction when administered to an animal, such as a human, as the case may be. The preparation of a pharmaceutical composition that contains at least one uncharged lipid component comprising an oligonucleotide or additional active ingredient will be known to those skilled in the art in light of this disclosure, an example of which is Remington: The Science and Practice of Pharmacy, 21st, 2005, included in this document as a reference. In addition, for administration to animals (eg, humans), it should be understood that formulations must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the FDA Biological Standards Office.

As used herein, the term pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs. agents, drug stabilizers, gels, binders, fillers, disintegrants, lubricants, sweeteners, flavoring agents, colorants, such as materials and combinations thereof, as known to the person skilled in the art. A pharmaceutically acceptable carrier is preferably formulated for human administration, although in some embodiments it may be desirable to use a pharmaceutically acceptable carrier that is formulated for administration to a non-human animal but which would not be acceptable (e.g. due to government regulation) for human administration. . Except when any

- 23 041953 a conventional carrier is incompatible with the active ingredient and is intended to be used in therapeutic or pharmaceutical compositions.

The actual dosage amount of a composition of this invention administered to a patient or subject may be determined by physical and physiological factors such as body weight, severity of condition, type of disease being treated, previous or concurrent therapeutic interventions, patient idiopathy, and route of administration. In any case, the person responsible for administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

In certain embodiments of the invention, pharmaceutical compositions may include, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise, for example, between about 2% to about 75% by weight of the unit, or between about 25% to about 60%, and any range obtained therein. In other non-limiting examples, the dose may also be from about 1 microgram/kg/body weight, about 5 micrograms/kg/body weight, about 10 micrograms/kg/body weight, about 50 micrograms/kg/body weight, about 100 micrograms/kg /body weight, about 200 micrograms/kg/body weight, about 350 micrograms/kg/body weight, about 500 micrograms/kg/body weight, about 1 milligram/kg/body weight, about 5 milligrams/kg/body weight, about 10 milligrams/kg/body weight, about 50 milligrams/kg/body weight, about 100 milligrams/kg/body weight, about 200 milligrams/kg/body weight, about 350 milligrams/kg/body weight, about 500 milligrams/kg/body weight body weight, up to about 1000 mg/kg/body weight or more per administration, and any range obtained therein. In non-limiting examples of a derivable range from the numbers listed herein, the range is from about 5 µg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight etc. can be entered.

The oligonucleotide of these embodiments can be administered at a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more micrograms of nucleic acid per dose. Each dose may have a volume of 1, 10, 50, 100, 200, 500, 1000 or more μl or ml.

Solutions of the therapeutic compositions may be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Therapeutic compositions of this invention can be administered in the form of injectable compositions in the form of liquid solutions or suspensions; also can be obtained solid forms suitable for dissolution in, or suspension in liquid prior to injection. These preparations can also be emulsified. A typical composition for this purpose contains a pharmaceutically acceptable carrier. For example, the composition may contain 10 mg, 25 mg, 50 mg, or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers, and the like.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like. Intravenous vehicles include fluid and nutrient fillers. Preservatives include antimicrobial agents, antioxidants, chelating agents and inert gases. The pH and precise concentration of the various components of the pharmaceutical composition are adjusted according to well known parameters.

Therapeutic compositions of this invention may include classical pharmaceutical preparations. The introduction of therapeutic compositions according to this invention will be by any conventional route, if the target tissue is available through this route. This route includes oral, nasal, buccal, rectal, vaginal, or topical. Topical administration may be particularly useful in the treatment of skin cancer, in the prevention of chemotherapy-induced alopecia or other cutaneous hyperproliferative disorder.

Alternatively, administration may be orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous. Such compositions are usually administered as pharmaceutically acceptable compositions which include physiologically acceptable carriers, buffers or other excipients. Aerosol delivery may be used to treat lung conditions. The volume of the aerosol is between about 0.01 to 0.5 ml.

An effective amount of the therapeutic composition is determined based on the intended purpose. The term dosage unit or dosage refers to physically discrete units suitable for administration to a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired responses discussed above in connection with its administration, i. appropriate route and treatment regimen. The amount to be administered, depending on the number of treatments and the standard dose, depends on the desired protection or effect.

- 24 041953

The exact amounts of the therapeutic composition also depend on the judgment of the practitioner and are individual to each individual. Factors influencing dose include the physical and clinical condition of the patient, the route of administration, the intended goal of treatment (eg, symptomatic relief versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent.

Combined treatments.

In some embodiments of the invention, the methods of this invention include the introduction of an inhibitory oligonucleotide or an oligonucleotide capable of expressing an inhibitor of gene expression, in combination with a second or additional therapy. Methods and compositions, including combination therapy, enhance the therapeutic or protective effect and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy.

The therapeutic and prophylactic methods and compositions may be provided in a combined amount effective to achieve the desired effect, such as killing the cancer cell and/or inhibiting cell hyperproliferation. This process may include contacting cells with a gene expression inhibitor and a second therapy such as a tyrosine kinase inhibitor (eg, imatinib, nilotinib, dasatinib, bosutinib, ponatinib, or bafetinib) or a cytidine analog (eg, decitabine, cytarabine, or azacitidine). The tissue, tumor, or cell may be contacted with one or more compositions or pharmacological preparation(s) comprising one or more agents (i.e., a gene expression inhibitor or anti-cancer agent), or by contact with a tissue, tumor, and/or cell with two or more different compositions or formulations, wherein one composition provides 1) an inhibitory oligonucleotide; 2) an anti-cancer agent; or 3) both an inhibitory oligonucleotide and an anti-cancer agent. It is also contemplated that such combination therapy may be used in combination with chemotherapy, radiation therapy, surgical therapy or immunotherapy.

The inhibitory oligonucleotide may be administered before, during, after, or in various combinations regarding anti-cancer treatment. The introduction can be carried out at intervals from simultaneously minutes to days or weeks. In embodiments where the inhibitory oligonucleotide is provided to the patient separately from the anti-cancer agent, it is generally guaranteed that a significant period of time does not elapse between the time of each delivery so that the two compounds will still be able to exert a beneficial combined effect on the patient. In such cases, it is contemplated that it is possible to provide the patient with inhibitory oligonucleotide therapy and anti-cancer therapy within about 12 to 24 or 72 hours of each other, and more preferably within about 6-12 hours of each other. In some situations, it may be desirable to significantly increase the treatment period, which ranges from several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) between respective introductions.

In some embodiments of the invention, the course of treatment will last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,

69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 days or more. It is contemplated that one agent may be administered on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,

51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,

81, 82, 83, 84, 85, 86, 87, 88, 89 and/or 90, any combination thereof and other agent given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89 and/or 90, or any combination thereof. Within one day (24-hour period), the patient may be prescribed one or more doses of the drug(s). In addition, after the course of treatment, it is assumed that there is a period of time during which anti-cancer treatment is not administered. This time period may be 1, 2, 3, 4, 5, 6, 7 days and/or 1, 2, 3, 4, 5 weeks and/or 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12 months or more, depending on the condition of the patient, such as his prognosis, strength, health, etc.

Various combinations may be used. For the example below, oligonucleotide inhibitory therapy is designated A and anticancer therapy B: A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B /B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/ A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A.

The administration of any compound or therapy of this invention to a patient will follow the general protocols for administering such compounds, taking into account the toxicity of the agents, if any. Therefore, in some embodiments of the invention, there is a toxicity monitoring step that relates to combination therapy.

Treatment cycles are expected to be repeated as needed. It is also contemplated that various standard therapies as well as surgery may be used in combination with the described therapy.

- 25 041953

In specific aspects, standard therapy is contemplated to include chemotherapy, radiation therapy, immunotherapy, surgical therapy, or gene therapy, and may be used in combination with a gene expression therapy inhibitor, cancer therapy, or concurrently with a gene expression therapy inhibitor and cancer therapy, as described herein. document.

Chemotherapy.

In accordance with these embodiments, a wide range of chemotherapeutic agents can be used. The term chemotherapy refers to the use of drugs to treat cancer. A chemotherapeutic agent is used to refer to a compound or composition that is administered in the treatment of cancer. These agents or drugs are classified by the way they are active in the cell, such as whether and at what stage they affect the cell cycle. Alternatively, an agent can be characterized based on its ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic aberrations, affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamin; acetogenins (such as bulatacin and bulatacinone); camptothecin (including the synthetic analog of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs of adoselesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues of KW-2189 and CB1-TM1); eleuterobine; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trofosfamide and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediine antibiotics (eg calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); dinemycin, including dinemycin A; bisphosphonates such as clodronate; esperamycin; as well as neocarcinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carcinophyllin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-norleucine-doc, L-oxo-L (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, rhorubicin, quelamycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6azauridine, carmofur, cytarabine, decitabine, dideoxyuridine, doxifluridine, enocytabine and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid compensator such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elformitin; eliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainide; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pyrarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSC polysaccharide complex; razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anhidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Ara-C); cyclophosphamide; taxoids such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantron; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (eg CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); tyrosine kinase inhibitors such as imatinib, nilotinib, dasatinib, bosutinib, ponatinib and bafetinib; retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbyene, farnesyl protein transferase inhibitors, transferplatin, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Radiotherapy.

Other factors that cause DNA damage and are widely used include so-called γ-rays, x-rays and/or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (US Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. Most likely, all of these factors affect a wide range of DNA damage, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for x-rays varies from daily doses of 50 to 200 roentgens for extended periods of time (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and absorption by neoplastic cells.

The terms contacted and exposed in relation to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or placed in direct alignment with the target cell. For example, to achieve cell death, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

Immunotherapy.

In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and kill cancer cells. Trastuzumab (Herceptin™) is such an example. The immune effector may be, for example, an antibody specific for a marker on the surface of the tumor cell. One antibody may serve as an effector of therapy or may recruit other cells to actually kill cells. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and merely serves as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, directly or indirectly, with the target tumor cell. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic methods, ie direct cytotoxic activity and ErbB2 inhibition or reduction, would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.

Other immunotherapy may also be used as part of the silencing gene therapy combination therapy discussed above. In one aspect of immunotherapy, the tumor cell must have some marker that is targetable, ie not present on most other cells. There are many tumor markers, and any of them may be suitable for targeting in the context of this invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, bladder tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialillewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is the combination of antitumor effects with immunostimulatory effects. Immunostimulatory molecules also exist, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, IFN-gamma, chemokines such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand. It has been shown that the combination of immunostimulatory molecules as proteins or the use of gene delivery in combination with a tumor suppressor demonstrate enhanced antitumor effects. In addition, antibodies against any of these compounds can be used to target the anticancer agents discussed herein.

Examples of immunotherapy currently being researched or used are immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatics (US Pat. No. 5801005 and 5739169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy, for example, interferons α, β and γ; gene therapy for IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998), e.g. TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; US Pat. Nos. 5,830,880 and 5,846,945) and monoclonal antibodies e.g. al., 1998; US Pat. No. 5824311). It is contemplated that one or more anti-cancer therapies may be used with the silencing gene therapies described herein.

Active immunotherapy administers an antigenic peptide, polypeptide, or protein, or a composition of autologous or allogeneic tumor cells, or a vaccine, usually with a separate bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992; Mitchell et al., 1990 ; Mitchell et al., 1993).

In adaptive immunotherapy, the patient's circulating lymphocytes or tumor infiltrated lymphocytes isolated in vitro are activated by lymphokines such as IL-2 or transduced with tumor necrosis genes and reintroduced (Rosenberg et al., 1988; 1989).

Surgery.

Approximately 60% of cancer patients will undergo some type of surgery, which includes preventive, diagnostic or staging, curative and palliative surgery. Remedial surgery is a cancer treatment that

- 27 041953 can be used in combination with other therapies such as the treatment of this invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and/or alternative therapies.

Medical surgery involves resection, in which all or part of the cancerous tissue is physically removed, excised and/or destroyed. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery). In addition, it is contemplated that the present invention may be used in conjunction with the removal of superficial cancers, precancerous lesions, or random amounts of normal tissue.

When some or all of the cancer cells, tissue, or tumor is removed, a cavity can form in the body. Treatment can be achieved by perfusion, direct injection, or topical application to the area with additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments may also vary.

other agents.

It is contemplated that other agents may be used in combination with certain aspects of these embodiments of the invention to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the activation of cell surface receptors and GAP compounds, cytostatic and differentiating agents, inhibitors of cell adhesion, agents that sensitize hyperproliferative cells to apoptotic inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the antihyperproliferative effects on the adjacent population of hyperproliferative cells. In other embodiments of the invention, cytostatic or differentiating agents can be used in combination with some aspects of these embodiments of the invention to improve the antihyperproliferative efficacy of the treatment. Cell adhesion inhibitors are expected to improve the performance of these embodiments of the invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. In addition, it is contemplated that other agents that sensitize a hyperproliferative cell to apoptosis, such as the c225 antibody, may be used in combination with certain aspects of these embodiments to improve treatment efficacy.

Examples

The following examples are included to demonstrate preferred embodiments of the invention.

Those skilled in the art will appreciate that the techniques described in the following examples represent techniques discovered by the inventor to operate effectively in the practice of the invention, and thus may be considered as constituting preferred methods for its use. However, those skilled in the art, in light of this disclosure, will appreciate that many changes can be made to the specific embodiments of the invention that are described and still achieve a similar or analogous result without departing from the spirit and scope of the invention.

Example 1 Phase I study of BP1001 (antisense liposomal Grb2) in patients with hematologic malignancies.

Growth factor receptor-associated protein-2 (Grb2), essential for signaling to cancer cells, is used by oncogenic tyrosine kinases to activate Ras and ERK. BP1001 is a liposome-embedded antisense that inhibits Grb2 expression. The aim of the study is to determine the safety, maximum tolerated dose (MTD), pharmacokinetics, and antileukemic activity of BP1001 in patients with hematologic malignancies.

BP1001 drug substance and introduction.

The Grb2 antisense oligo sequence is: 5'-ATA TTT GGC GAT GGC TTC-3' (SEQ ID NO: 1), which targets codons 2-7 of the human grb2 mRNA. An antisense Grb2 oligo consisting of a P-ethoxy modification oligo was manufactured by Nitto Denko Avecia, Inc. (8560 Readingroad, Cincinnati, Ohio, 45215, USA). An antisense Grb2 oligo was incorporated into 1,2-dioleoyl-3phosphatidylcholine lipid (Avanti Polar Lipids, Alabaster, AL, USA) following a lyophilization protocol. BP1001 was prepared as a 5 mg drug powder and stored at 4°C. On the day of drug infusion, two ml of 0.9% saline was added to achieve a final concentration of BP1001 of 2.5 mg/ml. Participants were given 2 to 3 hours of intravenous (IV) infusion of BP1001 twice a week (every 3 or 4 days) for 28 days. Participants may be given up to 6 treatment cycles if they continue to benefit from the treatment.

Patient and study design. The study was designed as a standard 3+3 dose escalation study to evaluate the safety, pharmacokinetics, and efficacy of BP1001 in adults.

- 28,041,953 patients diagnosed with refractory/recurrent CML or ALL, AML or MDS with a positive Philadelphia chromosome. Groups of three patients were included at each dose level. The initial dose of BP1001 was 5 mg/m ² (group 1). If one patient developed grade 3 or higher toxicity, three more patients were added at that dose level. If two or more patients developed grade 3 or higher toxicity, that dose level was considered toxic. The maximum tolerated dose (MTD) was defined as the dose less than that which causes dose-limiting toxicity in two or more patients. Toxicity was assessed using common terminological criteria for adverse events.

National Cancer Institute (NCI CTCAE, version 3.0). If no dose-dependent toxicity (DLT) was observed, patients would be consecutively included in group 2 (10 mg/m ² ), group 3 (20 mg/m ² ), group 4 (40 mg/m ² ), group 5 (60 mg /m ² ), or group 6 (90 mg/m ² ).

After groups 1-6 had completed phase I of single agent BP1001, the safety and efficacy of the low-dose combination of BP1001 and cytarabine (Phase Ib) was studied. BP1001 was administered iv as 60 mg/m ² (group 7) or 90 mg/m ² (group 8). Patients were given three injections of BP1001 before being given 20 mg low dose cytarabine (LDAC) subcutaneously, twice daily for 10 consecutive days. This study was registered with ClinicalTrials.gov, number NCT01159028.

Assessment of peripheral blood and bone marrow. Peripheral blood evaluations were performed at baseline (within 14 days of study entry) and on days 1, 8, 15, 22 following the completion of the first cycle and as clinically indicated. Bone marrow assessment was performed at baseline (within 14 days of study entry), at the end of Cycle 1, Cycle 2, and as clinically indicated. Baseline cytogenetic data were obtained before treatment. Molecular analysis was performed to identify somatic mutations at baseline bone marrow.

flow cytometry. Peripheral blood was collected from patients in Groups 3, 4, 5, and 6 and then dosed on Cycle 1 Days 1, 8, 15, and 22 and Cycle 2 Day 1. White blood cells were isolated from peripheral blood using cell prep tubes. BD Vacutainer CPT with sodium heparin (BD Dickinson) was fixed and stored in a freezer at -80°C. Flow cytometry was used to determine the levels of Grb2 and phosphorylated Erk1,2 (pErk) in cells expressing CD33 using a validated method (Flow Contract Site Laboratory, Bottell, Washington, USA).

Pharmacokinetic sample collection and sample analysis. Blood samples for measuring the concentration of antisense Grb2 oligos were collected from patients in groups 7 and 8, before administration, 1, 2, 4, 6, 8, 24, 72 hours after the start of infusion (SOI - start-of-infusion). Urine samples for measuring BP1001 concentrations were also taken from patients in groups 7 and 8, at the start of the dose at 4 hours, 4-8 hours after administration, and 8-24 hours after administration. PK blood samples were processed to plasma and analyzed using a validated method (Charles River Laboratories, Montreal, Canada) with a lower limit of quantitation (LLOQ; 0.5 ng/mL). Urine samples were analyzed using a validated method (Charles River Laboratories, Montreal, Canada) with an LLOQ of 1.00 ng/mL.

PK parameters were assessed using Phoenix pharmacokinetic software (Certara). A non-compartmental approach compatible with the IV infusion route was used to evaluate parameters, using an estimated infusion time of 2.5 hours. All parameters were derived from individual Grb2 antisense oligo plasma concentrations from Day 1 of Cycle 1, when practicable. Cycle 1, day 1, pre-dose concentrations were <LLOQ and hence time zero was taken to be zero. The parameters were evaluated using the nominal sampling time relative to the start of the infusion. The area under the concentration-time curve (AUC) was calculated using a linear trapezoidal method with linear interpolation. The slope of the terminal elimination phase was determined using logarithmic linear regression on unweighted concentration data. Parameters based on the definition of the final exclusion phase were not reported if the coefficient of determination (R ² ) was less than 0.800 or if the AUC extrapolation to infinity (%AUCextrap) was greater than 20% of the total area.

Parameters estimated from plasma: T _max - time after dosing at which the maximum observed concentration was observed; C _max - maximum observed concentration, measured after dosing; T _1/2 visible terminal half-life; AUC( ₀ _ ₂₄ ), AUC from dosage up to 24 hours after administration; AUC( ₀ . _t ), AUC from dosing to time after dosing at which the last quantifiable concentration was observed; AUC( ₀ . _in f), calculated AUC from dosing to infinity; CL is the apparent clearance of the Grb2 antisense oligo; Vz, apparent volume of distribution of Grb2 antisense oligo.

Urine parameters were evaluated using Microsoft Excel. All parameters were derived from individual urinary Grb2 antisense oligo concentrations from day 1 of cycle 1. The parameters reported were: Total U - cumulative amount of Grb2 antisense oligo excreted in urine over the entire sampling period; CLr - renal clearance relative to plasma; percent recovered, relative amount of antisense Grb2 oligo excreted in urine compared to total drug administered.

Statistical analysis. The FK analysis as well as the generation of tables and graphs were generated by Phoenix version 1.4. Descriptive statistics (N, arithmetic mean, standard deviation) for the respective grouping and sorting variables were generated using Phoenix version 1.4 and Microsoft Excel 2007.

Research objectives. The main objectives of the phase I BP1001 study were to determine the toxicity and tolerability of increasing doses of BP1001 as single agent therapy; determining the maximum tolerated dose (MTD) of BP1001; and determining the toxicity and tolerance of BP101 in combination with low doses of cytarabine (Ara-C; phase Ib). The secondary objectives of the phase I BP1001 study were to determine the optimal biologically active dose (OBAD; defined as a 50% reduction in Grb2 expression in circulating leukemia cells); determining the in vivo pharmacokinetics of BP1001; and evaluation of tumor response.

Criteria for inclusion and exclusion. Both parts of the study used an open, sequential, escalation design to assess safety, tolerability and toxicity, PK, tumor response, and antileukemic activity. Inclusion criteria were: at least 18 years of age; have difficult-to-treat or replaced OMD, Ph+XMJI, ALL, or MDS; no anticancer therapy for at least two weeks prior to study entry (with the exception of hydroxyurea or anagrelide (24 hours), TKI 95 days) and interferon (2 weeks)); the presence of adequate liver and kidney functions (ALT <2x ULN, serum creatinine <2x ULN, serum bilirub <2x ULN); and having ECOG scores of 0-2. Exclusion criteria: the presence of serious intercurrent diseases that may affect the patient's ability to comply with the treatment program; and receiving another investigational product for more than 14 days or 5 half-lives of the previous product.

Patient characteristics. A total of 39 patients were included in the study: 13 patients (Group 1), 6 patients (Group 2), 3 patients (Group 3), 3 patients (Group 4), 3 patients (Group 5), 4 patients (Group 6), 4 patients (Group 7) and 3 patients (Group 8). Five patients were diagnosed with blast phase CML, 30 patients with AML, and 4 patients with MDS (Table 3.1). The mean age of the patients was 66 years, ranging in age from 32 to 89 years. Twenty-seven were male patients and twelve were female patients. Prior to enrollment, patients received an average of four regimens. Their average general condition status was 1.

The mean number of peripheral blood blasts was 15%, ranging from 0 to 96%. As expected, these patients had a variety of cytogenetics, which included: t (9; 22) (n=7), diploid (n=12), complex (n=10), mixed (n=13). Two patients had t(9;22)-positive CML and complex cytogenetics, while one patient had t(9;22)-positive AML.

Adverse events in evaluable patients. Of the 39 patients, 27 patients were evaluated. Twelve patients were unable to complete the full cycle due to disease progression and were replaced according to the protocol (Table 4). Of the 27 patients evaluated, 21 patients received therapy with BP1001 alone and 6 patients received combination therapy with BP1001+LDAC. As shown in Table. 8, evaluable patients had various molecular abnormalities in their leukemias, which included ASXL1 (n=1), BCR-ABL (n=2; including T315I in 1), CEBRA (n=4), DNMT3A (n=1) , FLT-ITD (n=2), IDH1/2 (n=3), JAK2 (n=2), NOTCH1 (n=1), NPM1 (n=2), NRAS (n=2), TET2 (n =2), TP53 (n=2). The characteristics of the included patients are shown in Table 1. 3.1.

Only one patient at 5 mg/m ² experienced dose-limiting toxicity (DOT), grade 3 mucosal inflammation, and hand-foot syndrome while taking a high dose of hydroxyurea for the treatment of proliferative CML-BP. The study drug could not be ruled out as a contributing factor, so this event was reported as DOT. After expanding to 6 patients, no patient developed DOT. No other drug toxicity was observed in any of the treated patients. The MTD has not been identified. The HTD for BP1001 is 90 mg/m ² .

Table 3.1. Enrolled Patient Characteristics

Characteristics All patients (n=39) ^a Phase I (n=32) ^a Phase 1b (n=7) ^a Average age (years) 66 (32-89) 63 (32-89) 72(69-85) Floor

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Man 27 22 5 Woman 12 10 2 Number of previous schemes 4 (1-8) 4 (1-8) 1 (1-4) Diagnosis at study entry AML thirty 24 6 CML (blast phase) 5 4 1 MDS 4 4 0 General state 1 (0-2) Average, leukocytes (x 10 ⁶ ml) 3 (0.2-40) mean peripheral blasts (%) 15 (0-96) Average hemoglobin (g/ml) 9 (8-11) Medium platelets (x 10 ⁶ ml) 21 (4-155) Cytogenetics t(9;22) ₇ b 6 ^b 1 Diploid 12 8 4 Mixed 13 12 1 Complex 10 9 1

^a - data are n (range) or % (range) unless otherwise indicated ^b - two patients had t(9;22)-positive CML and complex cytogenetics and one patient had t(9;22) -positive AML.

Table 4. Patients and dose per group

Group BP1001 (mg/m ² ) Ara-C Number of people treated Quantity assessed 1 5 No 13 6 2 10 No 6 3

3 20 No 3 3 4 40 No 3 3 5 60 No 3 3 6 90 No 4 3 7 60 Yes 4 3 8 90 Yes 3 3

Pharmacokinetics of the antisense oligo Grbz. The lower limit of quantification (LLOQ) of the antisense Grb2 oligo plasma concentration is 0.50 ng/mL. At the time point before the study, there was no quantifiable concentration of the antisense Grb2 oligo. Plasma levels of the antisense Grb2 oligo decreased exponentially by a factor of two (FIG. 1). The maximum plasma concentration of the antisense Grb2 oligo was observed 1 hour after infusion (FIG. 1). At 72 hours post-infusion, plasma levels of the Grb2 antisense oligo were below or slightly above the LLOQ (FIG. 1). T ₁ / ₂ in plasma of the antisense oligo Grb2 in plasma ranged from 22.2 to 37.7 h for a dose of 60 mg/m ² and from 4.64 to 14.2 h for a dose of 90 mg/m ² (Table 5) . C _max , AUC( ₀ - ₂₄ ), AUC( ₀ - _t ) and AUC( ₀ - _inf) were the same for both dose levels, despite a dose increase of 1.5 times from 60 to 90 mg / m ² (Table. 5). Vz decreased by about 1.5 times and CL increased by about 1.5 times when the dose was increased from 60 to 90 mg/m ² (Table 5).

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Table 5. Plasma concentration of BP1001 after a single IV infusion of BP1001

Subject (group) BP100 1 (mg/m ² ) (h) Stax (ng/ml) AUCq-24 (h-ng/mL) AUCo- _t (h-ng/ml) AUC ₀ -inf (h -ng / ml) Vz (L) CL (L/h) 35 (7) 60 22.2 110 419 490 513 3750 117 37(7) 37.7 69 291 404 496 6570 121 38(7) 25, 9 68 294 349 375 5980 160 Average 29, 6 82 335 414 461 5433 133 SD 8.1 24 73 71 75 1487 24 39 (8) 90 4.6 118 491 490 501 1200 180 40 (8) 14.2 106 471 580 599 3070 150 41 (8) 13.7 53 269 307 317 5600 284 Average 11.8 82 410 459 472 32 90 205 SD 5.4 24 122 139 14 2208 70

The amount of antisense Grb2 oligo excreted in urine compared to the total administered dose ranged from 0.04 to 3.23% for the 60 mg/m ² dose and from 0.36 to 4.77% for the 90 mg/m ² dose. Mean urinary concentrations of Grb2 antisense oligo were higher in the 90 mg/m ² group than in the 60 mg/m ² group (Table 6).

Table 6. BP1001 concentration in urine after a single IV infusion of BP1001

Subject (group) BP1001 (mg/m ² ) Total U (mg) CLr from plasma (ml/h) % recovered 35(7) 60 1.49 4220 1.44 37(7) 0.06 195 0.04 38(7) 4.27 14500 3.23 Average 1.94 6305 1.57 SD 2.14 7377 1.60 39(8) 90 4.27 8720 2.44 40 (8) 8.34 17700 4.77 41 (8) 0.46 8600 0.36 Average 4.36 11673 2.52 SD 3.94 5220 2.21

BP1001 reduced the levels of Grb2 and phosphorylated Erkl _r 2 (pErk). Flow cytometry was used to determine the levels of Grb2 and pErk proteins in circulating leukemia cells of patients treated with BP1001 single agent and reported as Mean Fluorescence Intensity (MFI). MFI Grb2 and pErk during treatment were compared with those at baseline (Table 7). On the last sample measured (end of treatment or cycle 1 day 22), BP1001 decreased >25% Grb2 levels in 10 of 12 samples, and >25% pErk levels in 7 of 12 samples. The mean reduction in Grb2 levels was 49% (range: 28 to 91%) and pErk was 52% (range: 27 to 91%).

Table 7 Decrease in Grb2 and pErk levels after administration of BP1001

Subject (group) BP1001 (mg/m ² ) decline Grb2 (%) Decrease pErk (%) decline Grb2 (%) Decrease pErk (%) (Day 15) (Day 22 or end of treatment) 22(3) 20 0 0 57 0 23(3) 20 0 3 28 45 24(3) 20 56 28 47 35 25(4) 40 63 82 54 91 26(4) 40 47 0 0 0 27(4) 40 NS NS 34 27 28(5) 60 0 0 thirty 54 29(5) 60 57 51 65 0 30(5) 60 54 55 43 47 31(6) 90 0 0 0 0 32(6) 90 85 54 91 63 34 (6) 90 63 42 40 0

NS=sample not collected.

Antileukemic activity of BP1001. BP1001, as a single agent therapy, reduced >50% peripheral blood blasts in 9 of 21 patients and reduced >50% bone marrow blasts in 3

- 32 041953 out of 21 patients (Table 8). According to the protocol, patients may receive treatment with BP1001 for up to 6 months if they have demonstrated stable disease (i.e., less than a 50% increase in their white blood cells during the first 4 weeks of therapy) or have had an improvement in their disease. Four patients completed 2 treatment cycles and three patients completed 5 treatment cycles (Table 8). These seven patients had various cytogenetic and molecular abnormalities such as JAK2 and NRAS mutations (Table 8).

Among the six evaluable patients who received BP1001+LDAC combination therapy, three patients received 3 treatment cycles and one patient received 5 treatment cycles (Table 8). More importantly, three patients achieved complete remission (<5% bone marrow blasts) and two patients achieved partial remission (>50% reduction in bone marrow blasts) (Table 8; Figure 2).

Table 8 Hematological experience of evaluable patients

Subject BP1001 (mg/ ^m2 ) Aga-S DX Cytogenetics Molecular anomalies Peripheral blood blasts (%) Bone marrow blasts (%) Cycles Completed Baseline Nadir Off-Rx Baseline Nadir off- Rx 1 5 No CML 7q-complex BCR-ABL T315I 93 82 97 78 ND ND <1 6* 5 No AML diploid JAK2 15 2 5 NA ND ND 5 7 5 No mds 20 complexes Negative 0 0 0 8 4 6 5 10 5 No AML diploid Negative 1 0 1 23 10 10 1 eleven 5 No CML Ph+ complex BCR-ABL Y253H, F317V 24 7 50 eleven ND ND 1 14 5 No AML DIPLOID RAS 48 5 21 33 ND ND 1 15 10 No AML diploid FLT-ITD, SEVRA 54 31 72 85 76 76 1 20 10 No AML diploid Negative 76 5 63 50 No 80 1 21 10 No AML Other Negative 71 43 74 40 38 38 2 22 20 No AML Other NRAS 1 0 1 8 3 ND 2 23 20 No mds 5q- JAK2 V617F NE NE NE NE NE NE 1** 24 20 No mds -7 Not done 0 0 0 NE NE NE 5 25 40 No AML DIPLOID Negative 10 3 19 25 No 36 2 26 40 No AML Ph+ FLT-ITD, SEVRA 16 No 80 25 No 80 1 27 40 No AML DIPLOID NPM1 exon 12 IDH1 R132H 93 No 97 87 54 ND 1 28 60 No AML Other SEVRA 96 93 98 89 88 88 1 29 60 No AML 5 and 7 complex Negative 35 7 24 28 ND ND 1 thirty 60 No AML Other IDH2 R140Q 51 17 82 72 No 92 1 31 90 No AML 8+ Negative 0 0 0 17 No 17 1 32 90 No AML 7q- NRAS 2 No 42 24 22 22 2 34 90 No AML 5q- Negative 5 No 92 66 ND ND 1 35 60 Yes AML DIPLOID Negative 0 0 0 18 2 2 1 (CR) 37 60 Yes AML 5q- TP53, NOTCH1 80 13 31 25 25 ND 1 38 60 Yes AML DIPLOID NPM1, IDH2 4 0 ND 23 2 3 5 (CR) 39 90 Yes AML Other DNMT3A, TET2, TP53 70 0 52 36 15 58 3 (PR) 40 90 Yes AML DIPLOID Negative 0 0 0 31 10 2 3 (CR) 41 90 Yes AML diploid TET2, SEVRA, ASXL1 0 0 0 18 9 14 3 (PR)

ND=Not done;

NE=BM is not estimable for differential or lower threshold due to undersampling;

No=no lower threshold or reduction in blasts, * - patient 6 was diagnosed with myelofibrosis with AML, ** - patient 23 completed cycle 1 but was withdrawn from the study due to drug problems.

Example 2 In vitro efficacy of BP1001 in combination with Das.

The clinical activity of BP1001 will be investigated in CML patients in the accelerated or blast phase. The response of patients with an accelerated phase and a blast phase to imatinib is poor or transient. These patients were often treated with other tyrosine kinase inhibitors such as dasatinib, nilotinib, bosutinib, and ponatinib. Here it was determined whether BP1001 can enhance the inhibitory effect of dasatinib, nilotinib, bosutinib and ponatinib in CML cells.

Cell culture. BV173 and K562 cells are Bcr-Abl positive cell lines derived from CML patients. They were obtained from CLS Cell Lines Service GmbH (Eppelheim, Germany) and cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum.

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Chemical substances. The sequence of the Grb2 antisense oligo is: 5'ATATTTGGCGATGGCTTC-3' (SEQ ID NO: 1). The Grb2 antisense oligo was manufactured by Avecia (Cincinnati, Ohio, USA). BP1001 was obtained by Avanti Polar Lipids (Alabaster, AL, USA) as described. Briefly, Grb2 antisense oligos were mixed with dioleoylphosphatidylcholine lipids (Avanti Polar Lipids, Alabaster, AL, USA), frozen and lyophilized. The lyophilisate was stored at 4°C. On the day of the experiment, BP1001 was hydrated with medium and added to the cells at a final concentration of 0 to 120 µg/mL. Tyrosine kinase inhibitors such as dasatinib, nilotinb, bosutinib, and ponatinib were purchased from Selleck Chemicals (Houston, TX, USA) as 10 mM DMSO stock solutions.

Cell viability analysis. BV173 and K562 cells were seeded at 5 and 15x103 cells/well, respectively, in 96-well plates in 0.1 ml of medium. Cells were incubated with BP1001 inhibitors and/or tyrosine kinase inhibitors for 4 days. Viability of untreated and treated leukemic cells was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, Wisconsin, USA). The CellTiter-Glo® Luminescent Cell Viability Assay determines the number of viable cells by quantifying the presence of ATP, an indicator of metabolically active cells.

Cell cycle analysis. After incubation with BP1001 and/or tyrosine kinase inhibitors for 4 days, BV173 cells were harvested and washed with phosphate buffer (PBS). Cells were then fixed and permeabilized for 1 h at 4° C. with 1X fixation/permeabilization buffer (Ebioscience, San Diego, CA, USA). After permeabilization, cells were washed and incubated with 7-aminoactinomycin D (7-AAD, Biolegend, San Diego, CA, USA) in the dark at 4° C. for about 30 minutes. Flow cytometry data collection was obtained using BD FACSDiva™ software (BD Biosciences, San Jose, CA, USA). The flow cytometer was set to collect 20,000 events. Relative percentages in different phases of the cell cycle (sub-G1, G1, S, G2/M) were recorded.

Pretreatment with BP1001 enhanced dasatinib inhibition. First, it was determined whether BP1001 could enhance the inhibition of dasatinib in CML cell lines. To determine whether the timing of BP1001 addition could affect dasatinib activity, co-incubation of BP1001 and dasatinib was performed in different sequences: (1) BP1001 was added to cells 1 hour prior to dasatinib addition (to mimic same-day treatment); (2) BP1001 was added to cells 1 day after cells were treated with dasatinib; (3) BP1001 was added to cells the day before cell treatment with dasatinib.

Co-incubation sequence (1): BP1001 and dasatinib were added on the same day. K562 and BV173 cells were incubated with BP1001 (0-120 μg/ml) for 1 hour prior to treatment with 1 nM dasatinib. After four days, the growth inhibition effects were determined. In the absence and presence of 120 μg/ml BP1001, dasatinib reduced K562 viability by 25% and 15%, respectively (FIG. 7A). In the absence and presence of 120 μg/ml BP1001, dasatinib reduced BV173 viability by 40% and 49%, respectively (FIG. 7A). These data show that when CML cells were treated with BP1001 and dasatinib on the same day, BP1001 had minimal effect on dasatinib inhibition.

Co-incubation sequence (2): dasatinib added before BP1001. CML cell lines were treated with dasatinib for 1 day followed by treatment with BP1001. Cell viability was determined after 3 days. Dasatinib reduced the viability of K562 by 12% (Figure 7B). The addition of BP1001 at a concentration of 190 μg/ml reduced the viability of K562 to 50% (Fig. 7B). However, when BP1001 was added at a concentration of 120 μg/ml to dasatinib, the viability of K562 was reduced by 25% (Fig. 7B). Dasatinib reduced the viability of BV173 by 6% (Fig. 7B). The addition of 120 μg/ml BP1001 reduced the viability of BV173 by 34% (FIG. 7B). BP1001 added 1 day after dasatinib treatment further reduced the viability of BV173. But it is not clear whether BP1001 added 1 day after treatment with dasatinib can further reduce the viability of K562.

Co-incubation sequence (3): BP1001 added before dasatinib. CML cell lines were incubated with BP1001 for 1 day prior to dasatinib treatment. Dasatinib alone reduced the viability of K562 by 25% (Fig. 7C). When 120 μg/ml BP1001 was added to dasatinib, K562 viability was reduced by 44% (FIG. 7C). In the absence and presence of 120 μg/ml BP1001, dasatinib reduced BV173 viability by 10% and 68%, respectively (FIG. 7C). These data show that 1 day pretreatment with BP1001 enhanced dasatinib inhibition in both CML cell lines.

BP1001 enhanced the induction of cell death by dasatinib. We then determined the mechanism by which BP1001 enhanced dasatinib inhibition. BV173 cells were incubated with BP1001 for 1 day prior to dasatinib treatment. Three days later, cells were harvested and processed for flow cytometry. The main mechanism of inhibition of the combination of BP1001 + dasatinib was the induction of cell death, as evidenced by an increase in the percentage of cells in the sub-G1 phase.

- 34 041953 (Fig. 8A-D). The percentage of cells in the sub-Gl phase after treatment with BP1001, dasatinib, and the combination was 3.1, 10.6, and 28.0, respectively (FIGS. 8A-O).

Pretreatment with BP1001 selectively enhanced the induction of cell death by dasatinib and nilotinib. Flow cytometry was used to determine the effect of BP1001 pretreatment on nilotinib, bosutinib, and ponatinib. In FIG. 3 shows that BP1001 pretreatment enhanced the inhibition of dasatinib and nilotinib. The percentage of cells in the sub-G1 phase was increased from 30.7 to 40.6 for dasatinib and from 26.4 to 32 for nilotinib (Figure 9A). However, BP1001 pretreatment had no effect on bosutinib or ponatinib inhibition (FIG. 9A). The percentage of cells treated with bosutinib and the combination of BP1001+6 osutini6 in the sub-G1 phase was 34.3 and 36.4, respectively, while for ponatinib and the combination of BP1001+ponatinib was 34.0 and 36.0%, respectively (Fig. 9A) . These data indicate that BP1001 pretreatment selectively enhanced the inhibition of dasatinib and nilotinib.

Interestingly, a reduction in cell death was observed when cells were treated with BP1001 after tyrosine kinase inhibitors. The percentage of cells in the sub-G1 phase was reduced from 64.1 to 51.8 for dasatinib, from 65.0 to 57.1 for nilotinib, from 66.1 to 48.6 for bosutinib, and from 71.1 to 51.7 for ponatinib (Fig. 9B).

Example 3 Phase Ib/IIa clinical trial to evaluate the safety, pharmacokinetics and efficacy of BP1001 in combination with dasatinib (Das) in patients with Ph+ CML in the accelerated or blast phase.

Rationale for the study. The Phase Ib study will include participants with Ph+ CML in either the acceleration phase or the blast phase. Participants will receive BP1001 plus Das. This is an open, simple clinical study. Participants who are eligible for treatment in the Phase Ib study will receive BP1001 (60 mg/m ² for group 1 or 90 mg/m ² for group 2) and the same dose level of Das (140 mg). Once the dose has been determined for phase IIa, all participants who are eligible for treatment in phase IIa will receive the same dose levels for both BP1001 and Das. There will be no randomization for this study.

Each treatment cycle will be 28 days. BP1001 will be administered for approximately 1-3 hours every 2-4 days (+/-1 day of allowable interval) starting on cycle day 1 for a total of eight injections in each treatment cycle. On or about cycle day 5 and at least 24 hours after completion of the second dose of BP1001, participants will begin a single oral dose of 140 mg Das per day and continue every day thereafter without interruption. Repeat cycles under this regimen will be given every 4 weeks until there is progression, relapse, intolerance, the participant completes 6 cycles of treatment, or the participant/investigator requests an interruption of the study. When participants complete the dosing portion of the study, they will be followed for approximately 6 months until either death or study completion, whichever comes first.

Rationale for the study: Choice of dose. Preliminary data indicate that Grb2 reduction (and potential benefit from leukemia) can be achieved in participants with BP1001 at HTD (90 mg/m ² ) and one level below HTD (60 mg/m ² ), and that safety/tolerability did not differ significantly between the two doses. Based on this, the safety and tolerability of the combination of 60 or 90 mg/m ² BP1001 with Das will be studied.

In the Phase Ib study, BP1001 will be given intravenously (IV) twice a week (every 2-4 days) with a tolerance window of +/-1 day, for approximately 1-3 hours, for a total of eight doses given over 28- daily cycle. The starting dose will be 60 mg/m ² with the first dose of BP1001 on day 1 of each 28 day cycle. At least 24 hours after completion of the second dose of BP1001, participants will begin a daily dose of 140 mg Das and continue each day thereafter without interruption. Das dosing may begin as early as day 5 if started at least 24 hours after completion of the second dose of BP1001. Three patients will receive BP1001 at each dose level for 28 days in the absence of DLT. Dose escalation in this part of the study will continue until class 3 DLT is detected.

The next group of patients will be entered into the study after three patients from the previous group have completed 28 days of treatment without evidence of DLT. After that, the dose increase will reach 90 mg/m ² . If one patient experiences DLT, this group will be expanded with 3 additional patients. Any cohort in which >2 of the 3-6 patients treated with DLT is considered to have exceeded the MTD and the lower dose cohort itself will be selected for further study. All patients will be assessed for toxicity at the end of treatment (28 days) using the NCI CTCAE criteria. Repeat cycles under this regimen will be given every 4 weeks until there is progression, relapse, intolerance, the participant completes 6 cycles of treatment, or the participant/investigator requests an interruption of the study.

The Phase IIa treatment schedule will be identical to the Phase Ib study. However, all participants will be given the same dose of BP1001 (as determined in the phase Ib study) and 140 mg of Das.

Rationale for the study: study design.

The efficacy and safety of BP1001 will be evaluated in combination with Das, a therapeutic regimen well established in the treatment of Ph+ CML patients in the accelerated or blast phase. Based on our preclinical studies, it is predicted that

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BP1001 in combination with Das will benefit patients with accelerated or blast phase CML who typically do not respond to first-line imatinib therapy. The study design is presented graphically in Fig. 3.

This study will use a simple open design to evaluate the safety profile, pharmacokinetics, and efficacy of BP1001 in combination with Das. The phase Ib study uses an open sequential escalation design to evaluate safety, tolerability and toxicity, tumor response and antileukemic activity.

A standard 3+3 regimen will be used in which consecutive groups of CML patients are treated with BP1001 at the maximum tolerated dose (MTD) or HTD if MTD is undetermined and 1 level below MTD (or HTD) in combination with a fixed dose of Das for safety characterization and biological effect, as well as to determine the recommended phase IIa dose.

Three patients will receive BP1001 at each dose level for 28 days in the absence of DLT. The next group of patients will be entered into the study after 3 patients from the previous group have completed 28 days of treatment without signs of DLT. After that, the dose increase will reach 90 mg/m ² . If one patient experiences DLT, this group will be expanded with 3 additional patients. Any group in which >2 of the 3-6 patients treated with DLT will be considered to have exceeded the MTD and the lower dose group will be selected for further study. All patients will be assessed for toxicity at the end of treatment (28 days) using the NCI CTCAE criteria.

Patients may receive an extension of treatment. Repeat cycles under this regimen will be given every 4 weeks until there is progression, relapse, intolerance, the participant completes 6 cycles of treatment, or the participant/investigator requests an interruption of the study.

Phase IIa is an open label single dose level study. All participants who are eligible to receive treatment during the study will receive the same dose level for BP1001 (HTD level or 1 level below HTD) as defined in the Phase Ib study and Das (140 mg). There will be no randomization for this study.

The Phase IIa study will compare the efficacy of BP1001 in combination with Das to historical response rates documented for Das alone and evaluate the pharmacokinetics of BP1001 when administered alone or in combination with Das.

Approximately 40 eligible participants will receive the combination of BP1001 with Das in phase IIa as follows. After eligibility testing during the screening period, participants will receive their initial dose of BP1001. The dose will be determined in phase Ib based on the safety and tolerability of the combination of BP1001 with Das. BP1001 will be given at C1D1 and every 2-4 days thereafter for a total of 8 doses over a 28 day cycle. On or about day 5 of the cycle and at least 24 hours after completion of the second dose of BP1001, participants will begin the once-daily oral dose of 140 mg Das and continue every day thereafter without interruption.

Participants who have completed at least 3 cycles of treatment will be assessed on their response. The target response rate is 35%. The Bayesian approach of Tull, Simon, Estey will be implemented to monitor futility. The following futility stop rule will be applied at group size 5 starting with the 5th patient: prob{p(RR)>0.35)<0.03, where p(RR) denotes response rate. That is, the study will be prematurely terminated due to futility if, during the study, we determine that there is less than a 3% chance that the RR is greater than 35%.

A Bayesian approach will also be applied to monitor toxicity for enrolled patients, where toxicity is defined as any non-hematological toxicity, defined as DLT, that is at least possibly related to treatment. Toxicity, referred to as TOX, will be controlled by Bayesian stop limits calculated from beta-binomial distributions. We take p(TOX)~beta (0.6, 1.4) as a priori values. The study will be stopped due to toxicity if Pr(p(TOX)>0.30|data)>0.88. That is, we will stop the trial to enroll new patients if, at any time during the course of the study, we determine that there is more than an 88% chance that the toxicity level is greater than 30%.

Patients may receive an extension of treatment. Repeat cycles under this regimen will be given every 4 weeks until there is progression, relapse, intolerance, the participant completes 6 cycles of treatment, or the participant/investigator requests an interruption of the study.

Research objectives. The primary goal of the phase Ib study is to determine the dose-dependent toxicity (DLT) and maximum tolerated dose (MTD) of BP1001 in combination with Das. The main objectives of the phase IIa study are to evaluate the pharmacokinetics (PK) and efficacy of the combination of BP1001 and Das.

The following secondary objectives of this study: to evaluate the safety of BP1001 in combination with Das; determine if the combination of BP1001 and Das provides greater efficacy (hematological response, cytogenetic response, or molecular response) than Das alone (by historical comparison); assess the pharmacokinetics (PK) of BP1001 when taken alone or in combination with Das; evaluate overall survival, time to response, and duration of response.

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The research objective of this study will be to assess the correlation of treatment response with mutations in the abl domain of the kinase.

Study population: Inclusion criteria. At the time of screening, participants must meet all of the following criteria to be considered eligible for study participation:

1) Adults >18 years of age.

Women must not be of childbearing potential, be surgically sterile, postmenopausal, or use adequate methods of contraception during the study and for 30 days after the last dose of study drug or Das.

Men must agree to use an adequate method of contraception during the study and for at least 30 days after the last dose of study drug or Das.

Histologically confirmed diagnosis of Ph+ CML, in the acceleration or blast phase. The imperious phase is defined as >30% blasts in peripheral blood or bone marrow, or the presence of extramedullary disease, excluding the liver or spleen. One of the following parameters is required to meet the criteria for CML in the acceleration phase:

Blasts in peripheral blood or bone marrow >15%.

Promyelocytes and blasts in peripheral blood or bone marrow >30%.

Basophils RV or VM >20%.

Thrombocytopenia <100x10 ³ /ml, not as a result of therapy.

Cytogenetic clonal evolution.

Adequate liver and kidney function, as defined by:

aspartate transaminase (ACT) and alanine transaminase (ALT) < 2.5 times the upper limit of normal (ULN); and total bilirubin < 1.5 times ULN; And

Estimated glomerular filtration rate (eGFR) not less than 50 ml/min; the Cockcroft-Gault formula will be used to determine eGFR when a blood urea nitrogen (BUN) and creatinine test is performed at baseline. A combination of serum creatinine eGFR and BUN will be used to assess the patient's renal function to ensure safety.

Cockcroft-Gault equation: Estimated creatinine clearance = [(140 - age)xtotal body weight]/(serum creatinine72)

Multiply by 0.85 for women.

Documented general condition of the Eastern United Oncology Group (ECOG) 0, 1, or 2 (Table 9).

Recovery from the effects of any previous surgery, radiation therapy, or anticancer treatment (with the exception of alopecia).

Willingness and ability to provide written informed consent.

Table 9. General ECOG Status

Class ECOG 0 Fully active, able to do everything as before the disease without restrictions 1 Limited in physically strenuous activities but treated as an outpatient and able to perform light or sedentary work such as housework, office work 2 Treated as an outpatient and capable of self-care, but unable to perform any work. And about 50% of waking hours are actively spent 3 Able to limited self-care, confined to bed or chair for more than 50% of waking hours 4 Completely incapable. Cannot self-service. Completely chained to a bed or chair 5 Dead

Study population: exclusion criteria. During screening, participants who meet any of the following criteria will be excluded from the study.

Patients with the T315I mutation will not be excluded, but their response will be analyzed separately.

One other primary malignancy other than CML in the past 2 years, excluding non-melanoma skin cancer or cervical cancer in situ.

Known active leptomeningeal leukemia requiring intrathecal therapy.

Note: Patients with a history of CNS disease may be eligible based on at least two consecutive documented negative CSF assessments prior to screening.

Isolated extramedullary leukemia that does not meet criteria for bone marrow leukemia.

Received any cancer therapy within 14 days of starting BP1001 treatment except hydroxyurea or anagrelide or TKI (within 2 days).

Uncontrolled active, untreated, or progressive infection.

Receipt of any investigational agent within 14 days or 5 half-lives from initiation of BP1001.

Women who are pregnant, have a positive pregnancy test, or are breastfeeding during the screening period, or who intend to become pregnant or breastfeed during the study or within 30 days of the last dose of study drug.

Previous exposure to VR1001.

Patients with a history of intolerance to Das or for whom Das may not be appropriate.

Serious intercurrent medical or psychiatric illness that may prevent the participant from completing the study.

Active/chronic hepatitis B infection (based on a positive surface antigen [HBsAg]), hepatitis C infection (based on a positive antibody [HCV Ab]), or human immunodeficiency virus (HIV-1 or HIV-2 based on a positive antibody).

The presence of comorbid conditions that may compromise the participant's ability to tolerate investigational treatment or interfere with any aspect of the conduct of the study or the interpretation of results. This includes, but is not limited to, unstable or uncontrolled angina, NYHA class III or IV congestive heart failure, uncontrolled and sustained hypertension, clinically significant cardiac dysrhythmia, or clinically significant ECG abnormality (e.g., QTcF >470 ms ).

In the past 6 months, any of the following has occurred: pleural effusion, myocardial infarction, unstable angina, coronary/peripheral artery bypass, acute cerebrovascular accident, or transient ischemic attack.

Uncontrolled seizure disorder (i.e. seizures within the last 2 months).

Unable or unwilling to communicate or cooperate with investigator or follow protocol for any reason.

Study Treatment: BP1001 dose and administration. BP1001 is supplied as a preservative-free sterile white lyophilized powder for intravenous reconstitution and is supplied in 20 ml vials. Each 20 ml single use vial of BP1001 contains 5 mg of Grb2 oligonucleotide and 13.35 mg of DOPC. Dosing calculations are based on the Grb2 oligonucleotide. Each vial of BP1001 must be diluted in 2 ml of saline (0.9% sodium chloride) for injection to give a concentration of 5.0 mg/2.0 ml of BP1001 (2.5 mg/ml). Reconstituted BP1001 is stable for approximately 6 hours after reconstitution; therefore, the IV infusion should be completed no longer than approximately 6 hours after recovery.

In the phase Ib study, the dose of BP1001 to be used is 60 mg/m ² (group 1) or 90 mg/m ² (group 2). BP1001 will be given as an IV infusion on days 1, 4, 8, 11, 15, 18, 22 and 25 (+/-1 day allowance) of each 28 day cycle. For treatment cycle 1, participants must receive both doses of BP1001 (for days 1 and 4) in order to transition to Das dosing and continue the study. Participants who do not receive both doses of BP1001 prior to the start of the Das dose may be excluded from the dosing portion of the study and enter the follow-up portion of the study.

The dose amount for BP1001 will be based on baseline (at screening) body surface area (BSA); however, doses will be adjusted for participants who experience a weight change of >10% from baseline. Actual weight will be used to calculate BSA, except for participants weighing over 100kg; the dose will be calculated on a 100 kg basis for these subjects.

The IV infusion will be administered over approximately 1-3 hours and should not be given as an IV infusion or IV bolus. The infusion will be through a dedicated central or peripheral IV line and cannot be done at the same time as other medications.

Investigational treatment: dasatinib dose and administration. Dasatinib (Das) should be given orally, once a day, starting around day 5 of each 28-day cycle. Das dosing may begin as early as day 5 of each cycle if started at least 24 hours after completion of the second dose of BP1001.

Once treatment has started Das should be continued without interruption. The dose of Das used in this study is 140 mg (each dose).

Concomitant drugs. During the first 4 weeks of therapy, hydroxyurea will be allowed. Anagrelide will also be allowed at any time to manage thrombocytosis >500K. The following drugs are not allowed during the treatment period: any anti-cancer agents (other than Das, hydroxyurea, or anagrelide) for the treatment of the disease under study or other malignancies.

- 38 041953

Timetable and description of procedures. Participants will undergo screening procedures as shown in Table 1. 10.

Table 10. Schedule of events and assessments

EVENT / EVALUATION Treatment period cycle day -14 up to 1 1 4 8 eleven 15 18 22 25 28/EOT Tolerance (in days) 0 0 +1 +1 +1 +1 +1 +1 +1 ±1 Informed Consent X Demography X Disease history X X ^a Physical examination (PE) X ^{b xc'd Xc} General condition of ECOG X Height, weight and BSA X ^Xe Vital Signs X X X X X X X X X X ECG (at least 12 leads) X X Chest radiograph X Serology for HBV, HCV, HIV X SHS (at times, incl. blast %) X X ^f X ^d X X ^d x9 Coagulation profile X X ^d Blood chemistry X X ^f X ^d X X ^d X Analysis of urine X X ^d X

Bone Marrow Assessment X X ^h Pregnancy test (serum) ^f ' ¹ X Pregnancy test (urine) ^f ' ¹ X Acceptance Criteria Confirmation X X ^d , f Introduction ^BP1001j X X X X X X X X Das ^k introduction X -- — Plasma FC VR1001 ^{1 Xd'f Xd'f} Recording Concomitant Medications _X ------------------------------------------------- -----------------------------------------X Recording unwanted phenomena X------------------------------------------------- -----------------------------------------X

a - updated, based on history given in SCR, b - physical examination should include a complete symptom review and a complete examination, c - symptom-oriented brief PE only, d - cycle 1 only; Samples will be collected pre-dose (within 30 minutes pre-dose) and then 1, 2, 4, 6, 8 and 24 hours post-dose. Post-dose samples can be taken +/- 15 minutes of their indicated time, e is weight only, f is blood samples to be taken prior to initiation of any study drug or Das drug, g is hematological response assessed at the end of each cycle, h is bone marrow assessment, including cytogenetic and molecular responses, will be performed at the end of cycles 1, 2, 4, 6, and even cycles thereafter, i - women of childbearing potential only, j - BP1001 is administered intravenously (IV) by infusion twice a day week (every 2-4 days) with an interval of ±1 day, k - Das is administered without interruption; orally once a day, at least 24 hours after completion of the second dose of BP1001, l - multiple PK estimates. Samples should be collected at the following time points: pre-dose, 1, 2, 4, 6, 8 and 24 hours post-dose. Post-dose samples can be ±15 min.

- 39 041953

The initial follow-up visit should take place within 30 days (+/-7 days) of stopping treatment cycles. The initial observation visit will include: Physical examination; SHS with differential and platelets, including blasts %; blood chemistry; Recording concomitant medications; Recording of adverse events.

Long-term follow-up visits should occur every 8 weeks (+/-1 week) from the 30-day follow-up visit, up to 12 months from the start of therapy or until study closure or initiation of other therapy or death.

Efficiency Endpoints. Preliminary efficacy will be assessed by evaluating remission and objective response using the following response criteria and measurements. Additional efficacy endpoints will include time to response, duration of response, and overall survival.

Criteria for hematological response (HR) are presented in table. 11. Confirmed HR is obtained when all criteria are met at least 28 days after they were first met.

Table 11 Hematological response criteria

Answer Criteria Main Answer Peripheral blood leukocytes < ULN upper limit of norm Platelets <450,000 x10 ⁹ /l No blasts or promyelocytes in peripheral blood < 5% myelocytes plus metamyelocytes in peripheral blood Peripheral blood basophils <20% No extramedullary involvement including splenomegaly or hepatomegaly < 5% blasts in bone marrow Partial Peripheral blood leukocytes <

answer institutional ULN Platelets > 450,000 x10 ⁹ /l, but < 50% to therapeutic level No blasts or promyelocytes in peripheral blood > 5% myelocytes and metamyelocytes in peripheral blood Peripheral blood basophils <20% Splenomegaly or hepatomegaly, if present, must be < 50% to therapeutic level progressive disease Advancement of the patient's acceleration phase to the blast phase For patients with a blast phase, doubling the number of peripheral leukocytes at least twice within 28 days to > 20,000/l.c For any patient death by disease stable disease Patient does not meet criteria for hematologic response or progressive disease

Cytogenetically, the response is classified according to the suppression of the Philadelphia chromosome (Ph) by cytogenetics (or FISH if cytogenetic analysis is not informative, for example, not enough metaphases), as shown in Table. 12. Primary cytogenetic response is a combination of complete and partial response.

- 40 041953

Table 12. Cytogenetic response criteria

Answer Criteria No cytogenetic response Ph positive > 95% Minor cytogenetic response Ph positive 36-65% Partial cytogenetic answer Ph positive 1-35% Complete cytogenetic response Ph positive 0%

The molecular response is classified according to the criteria given in Table. 13. Table 13. Molecular response criteria

Answer Criteria Basic BCR-ABL/ABL ratio < 0.1% (International scale - IS-) MR4 BCR-ABL/ABL < 0.01% IS MR4.5 BCR-ABL/ABL <0.0032% IS

Safety Report: Adverse Events (AEs). Each participant who receives study drug will be closely monitored for AEs. An AE is any adverse medical event associated with the use of drugs in humans, whether or not it is thought to be drug related. Adverse events include any signs, symptoms, diseases, or diagnoses that appear or worsen during the course of the study. The AE may be an intercurrent illness, injury, or any concomitant deterioration in the health of the participant. All AEs occurring at any time from the moment consent is obtained until the end of treatment will be recorded regardless of the etiology of the event. CTCAE NCI v4.03 will be used to assess AEs. The CTCAE can be found online at evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14 QuickReference 5x7.pdf.

Adverse events are classified as serious and non-serious for regulatory reporting purposes. Note that the definition of serious does not necessarily depend on the severity of the event. All AEs that do not meet the severity criteria are considered non-serious.

Any AE occurring at any dose (including overdose), regardless of the relationship to the investigational medicinal product, will be classified as a serious adverse event (SAE) when (1) it results in death (i.e. the AE caused or resulted in death , the participant was at risk of death at the time of the event; this does not refer to an event that would hypothetically lead to death if it were more severe); (2) it was life-threatening (i.e. the AE put the person at immediate risk of death, not that the AE could hypothetically lead to death if it were more severe); (3) required hospitalization or prolonged hospitalization beyond the expected length of stay (i.e. hospitalizations for elective care and elective medical/surgical procedures are not SAEs by this criterion); (4) incapacitation (ie, the AE resulted in a significant reduction in the participant's ability to perform activities of daily living); (5) it resulted in a congenital anomaly or birth defect (i.e., an adverse finding in the child or fetus of the participant exposed to the study drug before conception or during pregnancy); or (6) it was a medically significant condition (i.e. the AE did not meet any of the major criteria above but, based on the relevant medical judgment, could have put the participant at risk or required medical or surgical intervention to prevent 1 of listed serious outcomes of these criteria [i.e. intensive care in the emergency department or at home for allergic bronchospasm; blood dyscrasias or seizures that do not result in hospitalization]).

Causality with BP1001 IP or Das for all AEs will be assessed as follows: (1) Definitely related - AEs clearly related to the study regimen; (2) likely related-AEs for which there is a possibility of a causal relationship for the schema under study; (3) possibly related - AEs for which there are comorbidities, medications, or other considerations, but for which it is possible that the AE may have been caused by the study regimen; or (4) unrelated-AEs that are considered to be clearly not causally related to the schema under study, or for which there is a clear alternative explanation.

An unexpected AE is any AE that is not identified by nature, severity or frequency as a known consequence of either BP1001 or Das. Unexpected, as used in this definition, refers to an AE that has not been previously observed, and not in terms of experience that is not expected from the pharmacological properties of a pharmaceutical product.

In table. 14 shows the most common AEs for BP1001 monotherapy, as well as AEs with

- 41 041953 intensity grade 3 or greater (based on Common Terminology Criteria for Adverse Events [CTCAE], National Cancer Institute [NCI] Version 4.0) in study 2003-0578, represented by treatment group, disease diagnosis at study entry, and organ system class (SOC)/preferred term (PT) as specified in the Regulatory Dictionary of Medicine (MedDRA version 18.0).

Table 14 Most common adverse events (>grade 3) by treatment group, disease, SOC, and RT

Number of patients (N=39) System Class Organ (Events/ Patients) Preferred term Monotherapy -BP1001 mg/ ^m2 Combination therapy BP1001 mp/m ² +LDAC Diagnosis at study entry 5 (n=13 ) 10 (n=6) 20 (n=3 ) 40 (n=3) 60 (n=3) 90 (n=4) 60 (n=4) 90 (n=3) oml (n= 30) hmm l(n =5 ) md s (p =4 ) Patients with AE (91/28) 10 4 1 3 1 4 3 2 23 4 1 Metabolic and metabolic disorders (19/12) 12 3 0 1 0 3 0 0 8 4 0 hypokalemia 1 1 0 1 0 1 0 0 3 1 0 Tumor lysis syndrome 3 0 0 0 0 0 0 0 1 2 0 Acidosis 2 1 0 0 0 0 0 0 3 0 0 hypocalcemia 1 0 0 0 0 1 0 0 1 1 0 Respiratory failure 2 1 0 0 0 0 0 0 3 0 0 hypoglycemia 2 0 0 0 0 0 0 0 1 1 0 Hyponatremia 1 0 0 0 0 1 0 0 1 1 0 Disorders of the blood and lymphatic system (12/11) 5 2 1 1 0 1 2 0 10 2 0 Leukocytosis 3 2 1 1 0 1 0 0 6 2 0 hypovolemia 0 0 0 0 0 0 1 0 1 0 0

- 42 041953

Febrile neutropenia 2 1 ^a 0 0 0 0 1 0 3 0 0 Infections and infections (9/9) 4 3 0 0 0 2 1 0 10 1 0 Pneumonia 0 2 0 0 0 2 0 0 4 0 0 Sepsis 4 1 0 0 0 0 4 1 0 Bilateral lung infection 0 0 0 0 0 0 1 0 1 0 0 Infection 0 0 0 0 0 0 1 0 1 0 0 Respiratory, thoracic and mediastinal disorders (7/7) 2 3 0 1 0 1 3 1 10 1 0 Dyspnea 0 0 0 1 0 1 0 1 3 0 0 hypoxia 1 1 0 0 0 0 2 0 4 0 0 Pleural effusions 1 0 1 0 0 Respiratory distress 1 2 0 0 0 0 0 0 2 1 0 General Disorders and Injection Site Conditions (5/5) 3 1 0 0 0 1 1 0 4 2 0 Asthenia 1 0 0 0 0 1 0 0 1 1 0 Multiple organ failure 2 1 0 0 0 0 0 0 2 1 0 Lower limb edema 0 0 0 0 0 0 1 0 1 0 0 Renal and urinary disorders (4/4) 3 0 0 1 0 0 0 0 3 1 0 Acute kidney injury 3 0 0 1 0 0 0 0 3 1 0 Heart disorders (3/3) 3 0 0 0 0 0 0 0 1 1 1 Heart failure 3 0 0 0 0 0 0 0 1 1 1 Surveys (3/2) 1 0 0 0 0 1 0 0 2 0 0 The number of platelets has decreased 1 0 0 0 0 1 ^a 0 0 2 0 0 Vascular disorders (2/2) 2 0 0 0 0 0 1 0 3 0 0 Hypotension 2 0 0 0 0 0 1 0 3 0 0 Hepatobiliary disorders (2/2) 1 1 0 0 0 0 0 0 2 0 0 Liver failure 1 1 0 0 0 0 0 0 2 0 0

Neoplasms benign, malignant and unspecified (including cysts and polyps) (2/2) 0 1 0 0 1 0 0 0 2 0 0 Gastrointestinal Disorders 0 0 0 0 0 0 1 0 1 0 0 Abdominal pain 0 0 0 0 0 0 1 0 1 0 0 Mental disorders 0 0 0 0 0 0 0 1 1 0 0 hallucinations 0 0 0 0 0 0 0 1 1 0 0 Disorders of the musculoskeletal system and connective tissue 0 0 0 0 0 0 1 1 0 0 Backache 0 0 0 0 0 0 0 1 1 0 0 Diseases of the reproductive system and breast 0 0 0 0 0 0 1 0 1 0 0 Pain in the penis 0 0 0 0 0 0 1 0 1 0 0 Eye disorders 0 0 0 0 0 0 1 0 1 0 0 Cataract 0 0 0 0 0 0 1 0 1 0 0 AML progression 10 4 1 3 3 2 1 1 19 5 2

In table. 15 provides detailed data on the frequency of all treatment with monotherapy with unwanted

- 43 041953 BP1001 events in study 2003-0578, SOC/PT, incidence and intensity (based on NCI CTCAE).

Table 15. Adverse events assessed as related to study drug BP1001

Organ System Class (Events/Patients) Preferred term Patient ID AND I Dose of monotherapy BP1001 mg/ ^m2 Dose combination therapy BP1001 mt/m ²⁺ LDAC Class Dose limiting toxicity (DLT) Disorders of the blood and lymphatic system (1/1) coagulopathy eleven 15 1 να 1 1 No Gastrointestinal disorders (4/1) Diarrhea 1 5 ΝΑ 1 No Mucositis 1 2 No Mucositis 1 4 yes ^a Nausea 1 2 No General disorders and conditions of the injection site (4/1) Edema (limbs) eleven 15 1 να 12 | No Fever (hyperthermia) 1 1 No Multiple organ failure 1 5 No Pain 1 2 No Infections and infections (1/1) Increased blood creatinine 10 5 ΝΑ 2 No Metabolic and metabolic disorders (6/1) Hyperkalemia 1 5 ΝΑ 2 No hypocalcemia 1 2 ^b No hypocalcemia 1 Z ^b No hypokalemia 1 5 ΝΑ 3 No Musculoskeletal and connective tissue disorders (1/D Pain in the limb eleven \ 15 1 vα 1 1 2 v | No Mental disorders (1/1) Confusion 1 5 ΝΑ 1 No Syndrome of palmar plantar erythrodysesthesia (hands and feet) 1 5 ΝΑ 3 yes ^a Itching 1 1 No

^a - since these events were assessed as possibly related to study drug BP1001 and were Grade 3, they were rated as

DLT. Both events occurred in patient 001 at the 5 mg/m dose level (Group 1). According to the protocol, the group was expanded to 6 evaluable patients. At this time, all 6 patients were examined without recurrence of any of these events in any treated patient, ^b - two (2) cases of this event were reported for this patient, at the same class level.

Possible risks and side effects. Dasatinib (Das) and BP1001 may cause a decrease in the number of blood cells (erythrocytes, platelets and leukocytes). Known side effects of Das include fever, skin rash, headache, fatigue, diarrhea, nausea, shortness of breath, and muscle pain. Based on animal and human studies, BP1001 may cause hand-foot syndrome, blisters/mouth ulcers, and low white blood cell counts.

All methods described and claimed herein can be made and performed without undue experimentation in light of this description. While the compositions and methods of this invention have been described in terms of the preferred embodiments of the invention, it should be apparent to those skilled in the art that changes described herein may be applied to the methods and steps or sequence of steps of the described method without departing from the concept. , essence and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can replace the agents described herein while the same or similar results are achieved. All such similar substitutions and modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the invention as defined in the appended claims.

- 44 041953

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Claims (45)

Tari AM, Gutierrez-Puente Y, Stephens C, et. al. Liposomeincorporated Grb2 antisense oligodeoxynucleotide increases the survival of mice bearing bcr-abl-positive leukemia xenografts. (2007) Int J Oncol 31: 1243-1250. Tari AM, Lopez-Berestein G. Cellular uptake of antisense oligonucleotides. Curr Opin Invest Drugs, 2001; 2, 1450-1453. Tari AM, Lopez-Berestein G. (2001) GRB2: a pivotal protein in signal transduction. Semin Oncol 28: 142-147. Tari AM, Stephens C, Rosenblum M, Lopez-Berestein G. Pharmacokinetics, tissue distribution, and safety of P-ethoxy oligonucleotides incorporated in liposomes. J Liposome Res, 1998; 8, 251-264. Tari AM, Hung M-C, Li K, Lopez-Berestein G. Growth inhibition of breast cancer cells by Grb2 downregulation is correlated with inactivation of mitogen-activated protein kinase in EGFR, but not in ErbB2, cells. Oncogene, 1999; 18, 1325-1332. Tari AM, Arlinghaus R, Lopez-Berestein G. Inhibition of Grb2 and Crkl proteins results in growth inhibition of Philadelphia chromosome positive leukemia cells. Biochem Biophys ResCommun, 1997; 235, 383-388. Thall PF, Simon RM, Estey EH. (1995) Bayesian sequential monitoring designs for single-arm clinical trials with multiple outcomes. Statistics in medicine 14: 357-379. Tilly H, Castaigne S, Bordessoule D, Casassus P, Le Prize PY, Tertian G, Desablens B, Henry-Amar M, Degos L. Low-dose cytarabine versus intensive chemotherapy in the treatment of acute nonlymphocytic leukemia in the elderly. J Clin Oncol, 1990; 8(2), 272-279. Vardiman JW, Thiele J, Arber DA, et. al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. (2009) Blood 114: 937-951. Williams E, Dunican D, Green P et al, Selective inhibition of growth factor-stimulated mitogenesis by a cell-permeable Grb2-binding peptide. J Biol Chern, 1997; 272, 22349-22354. CLAIM 1. A method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a first pharmaceutical composition containing a nuclease-resistant polynucleotide that hybridizes to a Grb2 nucleic acid in a patient, and a second pharmaceutical composition containing a Bcr-Abl tyrosine kinase inhibitor, wherein the inhibitor the Bcr-Abl tyrosine kinase is dasatinib or nilotinib, wherein the first pharmaceutical composition is administered prior to the administration of the second pharmaceutical composition. 2. The method of claim 1, wherein said polynucleotide hybridizes to a translation initiation site of a Grb2 nucleic acid. 3. The method according to claim 1 or 2, wherein said polynucleotide is an oligonucleotide having a length of 8-50 bases. 4. The method according to any one of claims 1 to 3, wherein said polynucleotide has the sequence according to SEQ ID NO: 1. 5. The method of any one of claims 1 to 4, wherein said polynucleotide contains P-ethoxy backbone bonds. 6. The method according to any one of claims 1 to 5, wherein said polynucleotide is encapsulated in a liposome. 7. The method of claim 1 wherein the first pharmaceutical composition further comprises a neutral lipid. 8. The method of claim 1 wherein said first pharmaceutical composition is administered systemically. 9. The method of claim 1, wherein said first pharmaceutical composition is for intra-arterial or intravenous administration. - 49 041953 10. The method of claim 7 wherein said first pharmaceutical composition is administered at a dosage of about 60 to about 90 mg/m ² . 11. The method of claim 7 wherein said first pharmaceutical composition is administered two to four times a week. 12. The method of claim 1 wherein the Bcr-Abl tyrosine kinase inhibitor is dasatinib. 13. The method of claim 12 wherein dasatinib is administered at a dosage of about 140 mg. 14. The method of claim 12 wherein dasatinib is administered once a day. 15. The method of claim 1 wherein said second pharmaceutical composition is administered systemically. 16. The method of claim 15, wherein said second pharmaceutical composition is administered orally, intra-arterially, or intravenously. 17. The method of claim 1, wherein said first pharmaceutical composition is administered at least one day prior to said second pharmaceutical composition being administered. 18. The method of claim 1, wherein said first pharmaceutical composition and said second pharmaceutical composition are administered by different routes. 19. The method of claim 1 wherein the patient is a human. 20. The method of claim 1 wherein the cancer is colorectal cancer, neuroblastoma, breast cancer, pancreatic cancer, brain cancer, lung cancer, stomach cancer, blood cancer, skin cancer, testicular cancer, prostate cancer, cancer ovarian cancer, liver or esophageal cancer, cervical cancer, head and neck cancer, non-melanoma skin cancer, or glioblastoma. 21. The method of claim 1 wherein the cancer is blood cancer. 22. The method of claim 21, wherein the blood cancer is chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), or myelodysplastic syndrome (MDS). 23. The method of claim 22 wherein the CML is acceleration phase CML or blast phase CML. 24. The method of claim 22 wherein the CML or AML is Bcr-Abl positive CML or Bcr-Abl positive AML. 25. The method of claim 24, wherein the Bcr-Abl positive CML or Bcr-Abl positive AML is positive for the T315I Bcr-Abl mutation. 26. The method of claim 22 wherein the CML or AML is Philadelphia chromosome positive CML or Philadelphia chromosome positive AML. 27. The method of claim 1 wherein the patient has previously failed treatment with imatinib. 28. A method of treating cancer or myelodysplastic syndrome (MDS) in a patient in need thereof, comprising administering to the patient an effective amount of a first pharmaceutical composition containing a nuclease-resistant polynucleotide that hybridizes to a Grb2 nucleic acid and a second pharmaceutical composition containing a cytidine analogue. 29. The method of claim 28, wherein said polynucleotide hybridizes to a translation initiation site of a Grb2 nucleic acid. 30. The method according to claim 28 or 29, wherein said polynucleotide is an oligonucleotide having a length of 8-50 bases. 31. The method according to any one of claims 28-30, wherein said polynucleotide has the sequence according to SEQ ID NO: 1. 32. The method of any one of claims 28-31, wherein said polynucleotide contains P-ethoxy backbone bonds. 33. The method according to any one of paragraphs. 28-32, wherein said polynucleotide is encapsulated in a liposome. 34. The method of claim 28, wherein said first pharmaceutical composition further comprises a neutral lipid. 35. The method of claim 28 wherein said first pharmaceutical composition is administered systemically. 36. The method of claim 28, wherein said first pharmaceutical composition is for intra-arterial or intravenous administration. 37. The method of claim 34 wherein said first pharmaceutical composition is administered at a dosage of about 60 to about 90 mg/m ² . 38. The method of claim 34 wherein said first pharmaceutical composition is administered two to four times a week. 39. The method of claim 1, wherein the cytidine analogue is decitabine, cytarabine, or azacitidine. 40. The method of claim 39 wherein the cytidine analog is decitabine. 41. The method of claim 39 wherein the cytidine analog is cytarabine. 42. The method of claim 41 wherein cytarabine is administered at a dosage of about 20 mg. 43. The method of claim 41 wherein cytarabine is administered twice a day. 44. The method of claim 41 wherein cytarabine is administered subcutaneously. 45. The method of claim 28 wherein said first pharmaceutical composition is administered prior to said second pharmaceutical composition being administered.