HK1169676B - Lipid formulated compositions and methods for inhibiting expression of eg5 and vegf genes - Google Patents

Abstract

This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA) in a lipid formulation, and methods of using the compositions to inhibit the expression of the Human kinesin family member 11 (Eg5) and Vascular Endothelial Growth Factor (VEGF), and methods of using the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.

Description

Lipid formulated compositions and methods for inhibiting expression of Eg5 and VEGF genes

Technical Field

The present invention relates to lipid formulated compositions containing double-stranded ribonucleic acid (dsRNA), and their use in mediating RNA interference to inhibit expression of a combination of genes, such as Eg5 and Vascular Endothelial Growth Factor (VEGF) genes. The dsRNA is formulated as a lipid formulation and may include a lipoprotein, such as apolipoprotein E. The invention also includes the use of the composition in the treatment of pathological processes mediated by Eg5 and VEGF expression, such as cancer.

Cross Reference to Related Applications

The present application claims the benefit of united states provisional application serial No. 61/159,788 filed on 12/3/2009, united states provisional application serial No. 61/231,579 filed on 5/8/2009, and united states provisional application serial No. 61/285,947 filed on 11/12/2009, all of which are hereby incorporated by reference in their entirety for all purposes.

Sequence listing reference

This application includes a sequence listing electronically submitted in the 2010 at XX month XX, date with a text file named 16564US _ sequencing. The sequence listing is incorporated by reference.

Background

The maintenance of a population of cells within an organism is determined by the cellular processes of cell division and programmed cell death. Within normal cells, the cellular events associated with the initiation and completion of each process are highly regulated. In proliferative diseases such as cancer, one or both of these processes may be disturbed. For example, cancer cells may lose their regulation of the cell division cycle (checkpoint control) through mutation, either by overexpression of a positive regulator or loss of a negative regulator.

Alternatively, cancer cells may lose the ability to undergo programmed cell death by overexpression of a negative regulator. Therefore, there is a need to develop new chemotherapeutic drugs that can restore checkpoint control and programmed cell death processes of cancer cells.

One approach to treating human cancer is to target proteins essential for cell cycle progression. In order for the cell cycle to progress from one stage to the next, certain prerequisite events must be completed. There are checkpoints within the cell cycle that perform the proper sequence of events and phases. One such checkpoint is the spindle checkpoint which occurs during the metaphase stage of mitosis. Small molecules that target proteins with essential functions in mitosis can trigger spindle checkpoints to block mitosis in cells. Among the small molecules that block mitosis, those that show clinically anti-tumor activity also induce apoptosis-a morphological change associated with programmed cell death. An effective chemotherapy for the treatment of cancer may therefore be a therapy that induces checkpoint control and programmed cell death. Unfortunately, few compounds are effective at controlling these processes inside cells. Most compounds that cause mitotic arrest and apoptosis are known as tubulin binding agents. These compounds alter the dynamic instability of microtubules and indirectly alter the function/structure of the mitotic spindle, thus causing mitotic arrest. Since most of these compounds specifically target tubulin, which is a component of all microtubules, they can also affect one or more of many normal cellular processes in which microtubules have an effect. Thus, there remains a need for agents that more specifically target proteins associated with proliferating cells.

Eg5 is one of several kinesin-like motor proteins that focus on the mitotic spindle and is known to be required for the formation and/or function of bipolar mitotic spindles. Recently small molecules that interfere with the bipolarity of the mitotic spindle have been reported (Mayer, T.U. et al 1999.Science 286(5441)971-4, incorporated herein by reference). More specifically, the small molecules induce the formation of abnormal mitotic spindles in which a single star array of microtubules emanates from a central pair of centrosomes, with chromosomes bound to the distal ends of the microtubules. Due to the single star array, this small molecule is called "monastractin" (monastrol). This single star array phenotype has previously been observed in Eg5 kinetin immunodepleted mitotic cells. This distinctive monarch array phenotype facilitates monarch identification as a potential inhibitor of Eg 5. In fact, monascin has also been shown to inhibit the Eg5 motor-driven motility of microtubules in vitro assays. The Eg inhibitor monascin had no significant effect on the associated kinesin dynamics or on the dynamics responsible for intracellular golgi motility. Cells showing the single star array phenotype were arrested in the M-phase of the cell cycle by immunodepletion of Eg5 or inhibition of Eg 5. However, mitotic arrest induced by either immunodepletion or inhibition of Eg5 is transient (Kapoor, t.m., 2000.J Cell Biol 150(5) 975-80). Both the monosomic array phenotype and cell cycle arrest in mitosis induced by monosomicin are reversible. The cells recover to form a normal bipolar mitotic spindle to complete mitosis and continue the cell cycle and normal cell proliferation. These data suggest that Eg5 inhibitors that induce transient mitotic arrest may not be effective in treating cancer cell proliferation. Nevertheless, the discovery that monascin causes mitotic arrest is attractive, and there is a need for further research and identification of compounds that can be used to modulate Eg5 motor protein in a manner effective in the treatment of human cancer. There is also a need to investigate the use of these compounds in combination with other antineoplastic agents.

VEGF (vascular endothelial growth factor, also known as vascular permeability factor, VPF) is a multifunctional cytokine that stimulates angiogenesis, epithelial cell proliferation, and endothelial cell survival. VEGF can be produced by a variety of tissues, and its overexpression or abnormal expression can lead to a variety of disorders, including cancer and retinopathies, such as age-related macular degeneration and other angiogenic disorders.

Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619(Fire et al) discloses the use of dsRNA of at least 25 nucleotides in length to inhibit gene expression in nematodes. dsRNA has also been shown to degrade target RNA in other organisms including plants (see, e.g., WO 99/53050, Waterhouse et al; and WO99/61631, Heifetz et al), Drosophila (see, e.g., Yang, D., et al, Curr. biol. (2000) 10: 1191-1200), and mammals (see, e.g., WO 00/44895, Limmer; and DE 10100586.5, Kreutzer et al). This natural mechanism has now been the focus of developing a new class of pharmaceutical formulations for the treatment of diseases caused by abnormal or deleterious regulation of genes.

Brief description of the invention

The present invention provides compositions and methods for inhibiting the expression of human Eg5/KSP and VEGF genes in cells using lipid formulated compositions comprising dsRNA.

The compositions of the invention comprise a nucleic acid lipid particle comprising a first double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a human kinesin family member 11(Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of a human VEGF in a cell. The nucleic acid lipid particle comprises a lipid formulation comprising 45-65 mol% of a cationic lipid, 5 mol% to about 10 mol% of a non-cationic lipid, 25-40 mol% of a sterol, and 0.5-5 mol% of a PEG or PEG-modified lipid. A first dsRNA targeting Eg5/KSP comprises a first sense strand and a first antisense strand, and the first sense strand has a first sequence, the first antisense strand having a sequence identical to SEQ ID NO: 1311 (5'-UCGAGAAUCUAAACUAACU-3'), wherein the first sequence and the second sequence are complementary, and wherein the first dsRNA is 15 to 30 base pairs in length. The second dsRNA comprises a second sense strand having a third sequence and a second antisense strand having a sequence identical to SEQ ID NO: 1538 (5'-GCACAUAGGAGAGAUGAGCUU-3'), wherein the third sequence and the fourth sequence are complementary, and wherein the second dsRNA is 15 to 30 base pairs in length.

In one embodiment, the cationic lipid of the composition has formula a, wherein formula a is:

wherein R1 and R2 are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, R3 and R4 are independently lower alkyl or R3 and R4 may be taken together to form an optionally substituted heterocyclic ring.

In other embodiments, the cationic lipid is XTC (2, 2-dioleyl-4-dimethylaminoethyl- [1, 3] -dioxolane). In a related embodiment, the cationic lipid is XTC, the non-cationic lipid is DSPC, the sterol is cholesterol, and the PEG lipid has PEG-DMG. In another related embodiment, the cationic lipid is XTC and the formulation is selected from the group consisting of:

in another embodiment, the cationic lipid of the composition is ALNY-100((3aR, 5s, 6aS) -N, N-dimethyl-2, 2-bis ((9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3 aH-cyclopenta [ d ] [1, 3] dioxol-5-amine)). In other embodiments, the cationic lipid is ALNY-100 and the formulation comprises:

in other embodiments, the cationic lipid is MC3(4- (dimethylamino) butanoic acid (6Z, 9Z, 28Z, 31Z) -thirty-seven-carbon-6, 9, 28, 31-tetraen-19-yl ester)). In a related embodiment, the cationic lipid is MC3 and the lipid formulation is selected from the group consisting of:

In another embodiment, the first dsRNA comprises a sequence consisting of SEQ ID NO: 1534 (5'-UCGAGAAUCUAAACUAACUTT-3') and a sense strand consisting of SEQ id no: 1535 (5'-AGUUAGUUUAGAUUCCUGATT-3'), and the second dsRNA comprises an antisense strand consisting of SEQ ID NO: 1536 (5'-GCACAUAGGAGAGAUGAGCUU-3') and a sense strand consisting of SEQ id no: 1537 (5'-AAGCUCAUCUCUCCUAUGUGCUG-3'). In yet another embodiment, each strand is modified as described below to include 2' -O-methyl ribonucleotides (represented by the lower case "c" or "u") and phosphorothioates (represented by the lower case "s"): the first dsRNA comprises a sequence consisting of SEQ ID NO: 1240(5 '-ucgagaaucuaaacutst-3') and a sense strand consisting of SEQ id no: 1241 (5' -AGUuAGUuAGAUUCUCGATST); the second dsRNA comprises a sequence consisting of SEQ ID NO: 1242(5 '-gcacauauaaggagagagugagcusu-3') and a sense strand consisting of SEQ ID NO: 1243(5 '-AAGCUcAUCUCUCUCCuAuGuGCusG-3').

In other embodiments, the first and second dsRNA comprise at least one modified nucleotide. In some embodiments, the modified nucleotide is selected from the group consisting of: 2 '-O-methyl modified nucleotides, nucleotides having a 5' -phosphorothioate group, and terminal nucleotides linked to a cholesteryl derivative or dodecanoic acid didecanamide group. In another embodiment, the modified nucleotide is selected from the group consisting of: 2 ' -deoxy-2 ' -fluoro modified nucleotides, 2 ' -deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2 ' -amino modified nucleotides, 2 ' -alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base containing nucleotides. In yet another embodiment, the first and second dsRNA each comprise at least one 2 '-O-methyl modified nucleotide and at least one nucleotide having a 5' -phosphorothioate group.

In some embodiments, each dsRNA is 19-23 bases in length. In another embodiment, each strand of each dsRNA is 21-23 bases in length. In yet another embodiment, each strand of the first dsRNA is 21 bases in length, the sense strand of the second dsRNA is 21 bases in length and the antisense strand of the second dsRNA is 23 bases in length. In other embodiments, the first and second dsRNA are present in an equimolar ratio. In one embodiment, the composition further comprises Sorafenib (Sorafenib). In another embodiment, the composition further comprises a lipoprotein. In another embodiment, the composition further comprises apolipoprotein E (ApoE).

In another embodiment, the composition inhibits the expression of Eg5 by at least 40% when contacted with a cell expressing Eg 5. In yet another embodiment, the composition inhibits VEGF expression by at least 40% when contacted with a cell expressing VEGF. In other embodiments, administration of the composition to a cell reduces the expression of Eg5 and VEGF in the cell. In a related embodiment, the composition is administered in nM concentration. In another embodiment, administration of the composition to a cell increases the formation of a monomer in the cell.

In other embodiments, administration of the composition to a mammal results in at least one effect selected from the group consisting of: preventing tumor growth, reducing tumor growth, or prolonging the survival of a mammal. In some embodiments, the effect is determined using at least one assay selected from the group consisting of: body weight determination, organ weight determination, visual inspection, mRNA analysis, serum AFP analysis, and survival monitoring.

The invention also provides methods for inhibiting the expression of Eg5/KSP and VEGF in a cell. The method comprises the step of administering a composition of the invention to the cell. The invention also provides methods for inhibiting tumor growth, reducing tumor growth, or prolonging the survival of a mammal in need of treatment for cancer. The method comprises the step of administering to the mammal a composition of the present invention. In one embodiment, the mammal has liver cancer. In another embodiment, the mammal is a human having liver cancer. In some embodiments, a dose containing 0.25mg/kg to 4mg/kg dsRNA is administered to the mammal. In other embodiments, the dsRNA is administered to a human at a dose of about 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg/kg.

In yet another embodiment, the present invention provides a method for reducing tumor growth in a mammal in need of treatment for cancer, the method comprising administering to the mammal a composition of the present invention, the method reducing tumor growth by at least 20%. In another embodiment, the method reduces KSP expression by at least 60%.

Drawings

Fig. 1 is a graph showing the percentage of liver weight by body weight after SNALP-siRNA administration to a Hep3B mouse model.

Fig. 2A is a graph showing the effect of PBS on the body weight of a Hep3B mouse model.

FIG. 2B is a graph showing the effect of SNALP-siRNA (VEGF/KSP) on body weight in a Hep3B mouse model.

FIG. 2C is a graph showing the effect of SNALP-siRNA (KSP/luciferase) on body weight in a Hep3B mouse model.

FIG. 2D is a graph showing the effect of SNALP-siRNA (VEGF/luciferase) on body weight of a Hep3B mouse model.

Fig. 3 is a graph showing the effect of SNALP-siRNA on body weight of a Hep3B mouse model.

Fig. 4 is a graph showing body weight of untreated control animals.

FIG. 5 is a graph showing the effect of control luciferase-SNALP siRNA on body weight of a Hep3B mouse model.

FIG. 6 is a graph showing the effect of VSP-SNALP siRNA on body weight of a Hep3B mouse model.

Fig. 7A is a graph showing the effect of SNALP-siRNA on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.

Fig. 7B is a graph showing the effect of SNALP-siRNA on serum AFP levels measured by serum ELISA in a Hep3B mouse model.

Fig. 8 is a graph showing the effect of SNALP-siRNA on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.

Fig. 9 is a graph showing the effect of SNALP-siRNA on human KSP levels normalized to human GAPDH levels in a Hep3B mouse model.

Fig. 10 is a graph showing the effect of SNALP-siRNA on human VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.

Fig. 11A is a graph showing the effect of SNALP-siRNA on mouse VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.

Fig. 11B is a set of graphs showing the effect of SNALP-siRNA on human GAPDH levels and serum AFP levels in a Hep3B mouse model.

FIG. 12A is a graph showing the effect of PBS, luciferase and ALN-VSP on tumor KSP as determined by percentage relative to hKSP mRNA in a Hep3B mouse model.

FIG. 12B is a graph showing the effect of PBS, luciferase and SNALP-VSP on tumor VEGF as determined by percentage relative to hVEGF mRNA in a Hep3B mouse model.

Fig. 12C is a graph showing the effect of PBS, luciferase and SNALP-VSP on GAPDH levels in a Hep3B mouse model, where GAPDH levels are determined as a percentage of hGAPDH mRNA.

Fig. 13A is a graph showing the effect of SNALP si-RNA on survival in mice bearing liver tumors. Treatment was started 18 days after tumor cell inoculation.

Fig. 13B is a graph showing the effect of SNALP siRNA on survival of mice with liver tumors. Treatment was started 26 days after tumor cell inoculation.

FIG. 14 is a graph showing the effect of SNALP-siRNA on serum alpha-fetoprotein (AFP) levels.

FIG. 15A is an image of H & E stained sections of tumor-bearing animals (three weeks after implantation of Hep3B cells) dosed with 2mg/kg SNALP-VSP. Twenty-four hours later, tumor-bearing liver lobes were processed for histological analysis. Arrows indicate single stars.

FIG. 15B is an image of H & E stained sections of tumor-bearing animals (three weeks after implantation of Hep3B cells) dosed with 2mg/kg SNALP-Luc. Twenty-four hours later, tumor-bearing liver lobes were processed for histological analysis.

Fig. 16 is a graph showing the effect of SNALP formulated siRNA and sorafenib administration on survival.

FIG. 17 is a flow chart of an in-line mixing process.

FIG. 18 is a graph showing the effect of treatment of VSP formulated with LNP-08 on KSP and VEGF expression in mouse intrahepatic Hep3B tumors.

FIG. 19 illustrates the chemical structures of PEG-DSG and PEG-C-DSA.

Figure 20 illustrates the structure of cationic lipids ALNY-100, MC3 and XTC.

FIG. 21 is a graph showing the effect of treatment of VSPs formulated with SNALP-1955(Luc), ALN-VSP02, SNALP-T-VSPLNP11, and LNP-12 on KSP and VEGF expression in mouse intrahepatic Hep3B tumors.

FIG. 22 is a set of graphs comparing the effect on KSP and VEGF expression in Hep3B tumors in mouse liver following treatment with LNP08-Luc, ALN-VSP02 and LNP-08 and LNP08-C18 formulated VSP.

Detailed Description

The present invention provides compositions and methods for inhibiting the expression of the Eg5 gene and the VEGF gene in a cell or mammal using dsRNA. The dsRNA is encapsulated in a lipid nucleic acid particle. The invention also provides compositions and methods for treating pathological conditions and diseases in mammals caused by the expression of the Eg5 gene and the VEGF gene, such as liver cancer. The dsRNA controls sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

The following detailed description discloses how to make and use compositions comprising dsRNA to inhibit the expression of Eg5 gene and VEGF gene, respectively, as well as compositions and methods for treating diseases and disorders (e.g., cancer) caused by the expression of these genes. Pharmaceutical compositions featured by the invention include a dsRNA comprising an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, typically 19-24 nucleotides in length, and which is substantially complementary to at least a portion of an RNA transcript of the Eg5 gene, and a pharmaceutically acceptable carrier. Compositions featured by the invention also include dsrnas that contain an antisense strand comprising a region of complementarity that is less than 30 nucleotides in length, typically 19-24 nucleotides in length, and that is substantially complementary to at least a portion of an RNA transcript of a VEGF gene.

Accordingly, certain aspects of the present invention provide pharmaceutical compositions comprising Eg5 and vegfp dsrna together with a pharmaceutically acceptable carrier, methods of inhibiting expression of Eg5 gene and VEGF gene, respectively, using the compositions, and methods of treating diseases caused by expression of Eg5 and VEGF gene using the pharmaceutical compositions.

I. Definition of

For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. In the event of a significant difference between the usage of terms in other parts of the specification and their definitions provided in this section, the definition in this section controls.

"G", "C", "A" and "U" each generally represent a nucleotide comprising guanine, cytosine, adenine and uracil as bases, respectively. "T" and "dT" are used interchangeably herein to mean deoxyribonucleotides wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it is to be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, or a surrogate substituent group, as described in detail below. It will be appreciated by those skilled in the art that guanine, cytosine, adenine and uracil may be substituted with other groups without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such a substituent group. For example, without limitation, a nucleotide comprising inosine as its base may form a base pair with a nucleotide comprising adenine, cytosine, or uracil. Thus, nucleotides comprising uracil, guanine or adenine in the nucleotide sequence of the present invention may be replaced by nucleotides comprising inosine, for example. In another example, adenine and cytosine wherever in the oligonucleotide may be replaced by guanine and uracil, respectively, to form G-U wobble base pairing with the target mRNA. Sequences containing such substituent groups are embodiments of the present invention.

As used herein, "Eg 5" refers to human kinesin family member 11, also known as KIF11, Eg5, HKSP, KNSL1, or TRIP 5. The Eg5 sequence can be regarded as NCBI GeneID: 3832. HGNC ID: HGNC: 6388 and Ref Seq ID number: NM _ 004523. The terms "Eg 5" and "KSP" and "Eg 5/KSP" are used interchangeably.

As used herein, "VEGF," also known as vascular permeability factor, is an angiogenic growth factor. VEGF is a human dimeric 45kDa glycoprotein that exists in at least three different isoforms. The VEGF isoforms are expressed in endothelial cells. The VEGF gene contains 8 exons and expresses a 189 amino acid isoform of the protein. The 165 amino acid isoform lacks the residues encoded by exon 6, while the 121 amino acid isoform lacks the residues encoded by exons 6 and 7. VEGF145 is an isoform predicted to contain 145 amino acids and to lack exon 7. VEGF can act on endothelial cells by binding to endothelial tyrosine kinase receptors such as Flt-1(VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed in endothelial cells and is associated with the differentiation of granulocytes and angiogenesis. The third receptor, VEGFR-3, is involved in lymphogenesis.

The various isoforms have different biological activities and clinical significance. For example, VEGF145 induces angiogenesis and, like VEGF189 (but unlike VEGF165), VEGF145 binds effectively to the extracellular matrix through a mechanism that is independent of extracellular matrix-associated heparin sulfate. VEGF shows activity in vitro as an endothelial cell mitogen and chemoattractant and induces vascular permeability and angiogenesis in vivo. VEGF is secreted by a variety of cancer cell types and promotes tumor growth by inducing growth of tumor-associated vasculature. Inhibition of VEGF function has been shown to limit the growth of primary experimental tumors and the incidence of metastases in immunocompromised mice. Various dsRNAs directed to VEGF are described in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, which are incorporated herein by reference in their entirety.

As used herein, "target sequence" means a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Eg5/KSP and/or VEGF genes, including mRNA which is an RNA processing product of the primary transcription product.

As used herein, the term "strand comprising a sequence" means an oligonucleotide comprising a series of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

As used herein, unless otherwise specified, the term "complementary," when used to describe the relationship of a first nucleotide sequence to a second nucleotide sequence, means the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as understood by those skilled in the art. For example, such conditions may be stringent conditions, wherein stringent conditions may include: hybridization was carried out for 12 to 16 hours at 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA, 50 ℃ or 70 ℃ and then washed. Other conditions may be used, such as physiologically relevant conditions that may be encountered in vivo. Depending on the final application of the hybridizing nucleotide, one skilled in the art can determine the set of conditions that is best suited for the test of complementarity of the two sequences.

The term "complementary" includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of the first and second nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" with respect to one another. However, when a first sequence is said to be "substantially complementary" with respect to a second sequence of the invention, the two sequences may be fully complementary, or they may form one or more, but typically no more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under conditions most relevant to their end use. However, when two oligonucleotides are designed to form one or more single stranded overhangs upon hybridization, such overhangs will not be considered mismatches in determining complementarity. For example, a dsRNA containing one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, which dsRNA may still be referred to as "fully complementary" for the purposes of the present invention when the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide.

As used herein, the term "complementary" sequences may also include, or be formed entirely of, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, so long as the above requirements with respect to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G: U Wobble or Hoogstein base pairs.

The terms "complementary", "fully complementary" and "substantially complementary" of the invention may be used with respect to base pairing between the sense strand and the antisense strand of a dsRNA or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) "refers to a polynucleotide that is substantially complementary to a contiguous portion of a messenger RNA of interest (e.g., encoding Eg5/KSP and/or VEGF) that includes a 5 'untranslated region (UTR), an Open Reading Frame (ORF), or a 3' UTR. For example, a polynucleotide is complementary to at least a portion of an Eg5mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding Eg 5.

As used herein, the term "double-stranded RNA" or "dsRNA" refers to a duplex structure containing two antiparallel and substantially complementary nucleic acid strands as defined above. Typically, most of the nucleotides of each strand are ribonucleotides, but as detailed in the present invention, each or both strands may also comprise at least one non-ribonucleotide, such as a deoxyribonucleotide and/or a modified nucleotide. In addition, as used herein, "dsRNA" may include chemical modifications of ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. For the purposes of the present specification and claims, any such modification (as used in siRNA type molecules) is covered by "dsRNA".

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule and are thus connected by a continuous series of nucleotides between the 3 'end of one strand and the 5' end of the respective other strand forming a duplex structure, the strand to which the RNA is connected is referred to as a "hairpin loop". A linked structure is referred to as a "linker" when two strands are covalently linked by means other than a contiguous series of nucleotides between the 3 'end of one strand and the 5' end of the corresponding other strand forming the duplex structure. The RNA strands may have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides of the shortest strand of the dsRNA minus any overhang present in the duplex. In addition to duplex structure, the dsRNA may comprise one or more nucleotide overhangs. Typically, most of the nucleotides of each strand are ribonucleotides, but as detailed in the present invention, each or both strands may also comprise at least one non-ribonucleotide, such as a deoxyribonucleotide and/or a modified nucleotide. In addition, as used herein, "dsRNA" may include chemical modifications of ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. For the purposes of the present specification and claims, any such modification (as used for siRNA-like molecules) is covered by "dsRNA".

As used herein, "nucleotide overhang" means an unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when the 3 'end of one strand of the dsRNA extends beyond the 5' end of the other strand (or vice versa). By "blunt" or "blunt-ended" is meant that the end of the dsRNA has no unpaired nucleotides, i.e., no nucleotide overhang. A "blunt-ended" dsRNA is a dsRNA that is double-stranded throughout its length, i.e., there are no nucleotide overhangs at either end of the molecule. In some embodiments, the dsRNA may have a nucleotide overhang at one end of the duplex and a blunt end at the other end.

The term "antisense strand" means a strand of dsRNA comprising a region substantially complementary to a target sequence. As used herein, the term "complementary region" means a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence as defined herein). When the complementary region and the target sequence are not completely complementary, mismatches may be present in the interior or terminal regions of the molecule. Typically, the majority of tolerance mismatches are located in terminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5 'and/or 3' end.

As used herein, the term "sense strand" means a dsRNA strand that includes a region that is substantially complementary to a region of the antisense strand.

When referring to dsRNA, "introduced into a cell" means promoting uptake or absorption into the cell, as understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unassisted diffusion or active cellular processes or through adjuvants or devices. The meaning of the term is not limited to cells in vitro. dsRNA may also be "introduced into a cell," where the cell is part of a living organism. In this case, introducing the cell will include delivery to the organism. For example, for in vivo delivery, the dsRNA may be injected into a tissue site or administered systemically. Introduction of cells in vitro includes methods known in the art, such as electroporation and lipofection.

The terms "silence" and "inhibit expression", "down-regulate expression", "prevent expression", and the like, when they relate to the Eg5 and/or VEGF gene, refer to at least partial inhibition of expression of the Eg5 gene, as indicated by a reduction in the amount of Eg5mRNA and/or VEGF mRNA that is isolatable from a first cell or group of cells in which the Eg5 and/or VEGF gene is transcribed, and which has been treated such that expression of the Eg5 and/or VEGF gene is inhibited as compared to a second cell or group of cells (control cells) that is substantially the same as the first cell or group of cells, but which has not been so treated. The degree of inhibition is generally expressed by the following equation:

Alternatively, the degree of inhibition may be given by a decrease in a parameter functionally related to the expression of Eg5 and/or VEGF genes, e.g., the amount of protein encoded by Eg5 and/or VEGF genes produced by the cells, or the number of cells exhibiting a certain phenotype, e.g., apoptosis. In principle, target gene silencing can be determined in any cell expressing the target (either constitutively or by genetic engineering) and by any appropriate assay. However, when a reference is required, the assays provided in the examples below will serve as such reference in order to determine whether a given dsRNA inhibits the expression of the Eg5 gene to some extent and is therefore encompassed by the present invention.

For example, in certain instances, expression of the Eg5 gene (or VEGF gene) is inhibited by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a double-stranded oligonucleotide of the invention. In some embodiments, Eg5 and/or VEGF gene is inhibited by at least about 60%, 70%, or 80% by administering a double-stranded oligonucleotide of the invention. In other embodiments, the Eg5 and/or VEGF gene is inhibited by at least about 85%, 90%, or 95% by administering a double stranded oligonucleotide of the invention. The following tables and examples provide expression inhibition values using various concentrations of Eg5 and/or VEGF dsRNA molecules.

As used herein, in the context of Eg5 expression (or VEGF expression), the terms "treat (verb)", "treat (noun)" and the like refer to the alleviation or slowing of pathological processes mediated by Eg5 and/or VEGF expression. In the context of the present invention, the terms "treat", "treating", "treatment" and the like, insofar as they relate to any of the other disorders described below (other than pathological processes mediated by Eg5 and/or VEGF expression), refer to alleviating or slowing at least one symptom associated with such a disorder, or delaying or reversing the development of such a disorder, for example delaying the development of liver cancer.

As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" mean an amount that provides a therapeutic benefit in treating, preventing or controlling a pathological process mediated by Eg5 and/or VEGF expression or an overt symptom mediated by Eg5 and/or VEGF expression. The specific therapeutically effective amount can be readily determined by the ordinarily skilled practitioner and may vary depending on factors known in the art such as the type of pathological process mediated by Eg5 and/or VEGF expression, the patient's medical history and age, the stage of pathological process mediated by Eg5 and/or VEGF expression, and the administration of other agents that counteract the pathological process mediated by Eg5 and/or VEGF expression.

As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of dsRNA and a pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to an amount of RNA effective to produce a predetermined pharmacological, therapeutic, or prophylactic result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 25%, a therapeutically effective amount of a drug for treating the disease or condition is an amount necessary to result in a reduction in the parameter by at least 25%.

The term "pharmaceutically acceptable carrier" refers to a carrier used to administer a therapeutic agent. As described in more detail below, such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to: pharmaceutically acceptable excipients, such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, colorants, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binders may include starch and gelatin, while lubricants (if present) are typically magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

As used herein, a "transformed cell" is a cell into which a vector has been introduced, the cell expressing a dsRNA molecule from the vector.

II. Double-stranded ribonucleic acid (dsRNA)

As described in more detail herein, the invention provides a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the Eg5 and/or VEGF gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a portion of a mRNA formed in the expression of the Eg5 and/or VEGF gene, and wherein said region of complementarity is less than 30 nucleotides in length, typically 19-24 nucleotides in length, and wherein expression of the Eg5 and/or VEGF gene is inhibited upon contact of the dsRNA with a cell expressing the Eg5 and/or VEGF gene. The dsRNA of the invention may also comprise one or more single-stranded nucleotide overhangs.

The dsRNA can be synthesized by standard methods known in the art as described below, for example by using an automated DNA synthesizer commercially available, for example, from the company Biosearch, Applied Biosystems, Inc. The dsRNA comprises two strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity which is substantially complementary, usually fully complementary, to a target sequence derived from an mRNA sequence formed during expression of the Eg5 and/or VEGF gene, and the other strand (the sense strand) comprises a region of complementarity to the antisense strand, such that when bound under appropriate conditions, the two strands hybridize to form a duplex structure. Typically, the duplex is 15 to 30, or 25 to 30, or 18 to 25, or 19 to 24, or 19 to 21, or 19, 20, or 21 base pairs in length. In one embodiment, the duplex is 19 base pairs in length. In another embodiment, the duplex is 21 base pairs in length. When two different siRNAs are used in combination, the duplex lengths may be the same or different.

Each strand of the dsRNA of the invention is typically 15 to 30, or 18 to 25, or 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In other embodiments, each strand is 25-30 base pairs in length. The individual strand lengths of the duplexes may be the same or different. When two different siRNAs are used in combination, each strand length of each siRNA may be the same or different. For example, a composition may contain a dsRNA targeting Eg5 having a 21 nucleotide sense strand and a 21 nucleotide antisense strand, and a second dsRNA targeting VEGF having a 21 nucleotide sense strand and a 23 nucleotide antisense strand.

The dsRNA of the invention may comprise one or more single stranded overhangs of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, typically 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides, each located at the 3 'end and 5' end of the sense strand. In other embodiments, the sense strand of the dsRNA has an overhang of 1-10 nucleotides, each at the 3 'end and 5' end of the antisense strand.

Surprisingly, the inhibitory properties of dsRNA with an overhang of at least one nucleotide may be superior to blunt-ended counterparts. In some embodiments, the presence of an overhang of only one nucleotide enhances the interference activity of the dsRNA without affecting its overall stability. dsRNA with only one overhang has been shown to be particularly stable and effective in vivo as well as in a variety of cells, cell culture media, blood and serum. Typically, the single stranded overhang is located at the 3 'end of the antisense strand, or alternatively, at the 3' end of the sense strand. The dsRNA may also have a blunt end, typically located at the 5' end of the antisense strand. Such dsRNA may have improved stability and inhibitory activity, thus allowing for administration at low doses, i.e., less than 5mg/kg of recipient body weight/day. Typically, the antisense strand of the dsRNA has a nucleotide overhang at the 3 'end, and the 5' end is blunt-ended. In another embodiment, one or more nucleotides in the overhang are replaced with a nucleoside phosphorothioate.

As described in more detail herein, the compositions of the invention comprise a first dsRNA targeting Eg5 and a second dsRNA targeting VEGF. The first and second dsrnas may have the same overhang configuration, e.g., the number of nucleotide overhangs on each strand, or each dsRNA may be specifically configured differently. In one embodiment, a first dsRNA targeting Eg5 comprises a 2 nucleotide overhang at the 3 'end of each strand, and a second dsRNA targeting VEGF comprises a 2 nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5 'end of the antisense strand (e.g., the 3' end of the sense strand).

In one embodiment, the Eg5 gene targeted by the dsRNA of the invention is the human Eg5 gene. In one embodiment, the antisense strand of the dsRNA targeting Eg5 comprises at least 15 contiguous nucleotides of one of the antisense sequences of tables 1-3. In a specific embodiment, the first sequence of the dsRNA is selected from one of the sense strands of tables 1-3, and the second sequence is selected from the antisense sequences of tables 1-3. Alternative antisense agents that target elsewhere in the target sequences provided in tables 1-3 can be readily determined using the target sequence and flanking Eg5 sequences. In some embodiments, a dsRNA targeting Eg5 will comprise at least two nucleotide sequences selected from the sequences provided in tables 1-3. One of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to an mRNA sequence produced in the expression of the Eg5 gene. Likewise, the dsRNA will comprise two oligonucleotides, wherein one oligonucleotide is described as the sense strand in tables 1-3 and the second oligonucleotide is described as the antisense strand in tables 1-3.

In embodiments using a second dsRNA targeting VEGF, such agents are illustrated in the examples, tables 4a and 4b, and co-pending U.S. Pat. Nos. 11/078,073 and 11/340,080 (incorporated herein by reference). In one embodiment, the VEGF-targeting dsRNA has an antisense strand complementary to at least 15 contiguous nucleotides of a VEGF target sequence described in table 4 a. In other embodiments, the VEGF-targeting dsRNA comprises one of the antisense sequences of table 4b, or one of the sense sequences of table 4b, or one of the duplexes (sense and antisense strands) of table 4 b.

It is well understood by those skilled in the art that dsRNAs containing duplex structures of 20 to 23, especially 21, base pairs have proven to be particularly effective in inducing RNA interference (Elbashir et al, EMBO 2001, 20: 6877-6888). However, it has also been found that shorter or longer dsrnas may also be effective. In the embodiments described above, the dsRNA of the invention may comprise at least one strand of minimum length of 21nt by virtue of the properties of the oligonucleotide sequences provided in tables 1-3. It is reasonable to expect that shorter dsrnas containing only a few nucleotides minus one of the sequences of tables 1-3 at one or both ends may be similarly effective compared to the dsRNA described above. Accordingly, the invention relates to a dsRNA comprising a partial sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides from one of the sequences of tables 1-3 and having an ability to inhibit expression of the Eg5 gene in a FACS assay as described below which differs from a dsRNA comprising the full sequence by no more than 5, 10, 15, 20, 25 or 30% inhibition. In addition, dsrnas that cleave the target sequences provided in tables 1-3 can be readily prepared using the Eg5 sequence and the provided target sequences. Other dsrnas targeting VEGF can be designed in a similar manner using the sequences disclosed in tables 4a and 4b, examples and co-pending U.S. serial nos. 11/078,073 and 11/340,080 (incorporated herein by reference).

In addition, the RNAi agents provided in tables 1-3 recognize sites in Eg5mRNA that are susceptible to RNAi-based cleavage. Thus, the invention also includes RNAi agents, e.g., targeting dsRNA within a sequence targeted by one of the agents of the invention. As used herein, a second RNAi agent is said to be targeted within the sequence of a first RNAi agent if it cleaves a messenger anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such second agents typically consist of at least 15 contiguous nucleotides from one of the sequences provided in tables 1-3, which are contiguous with other nucleotide sequences from the region contiguous with the selected sequence in the Eg5 gene. For example, SEQ ID NO: 1 form a single stranded reagent of 21 nucleotides based on one of the sequences provided in tables 1-3. Other RNAi agents, for example, VEGF-targeting dsRNA, can be designed in a similar manner using the sequences disclosed in tables 4a and 4b, examples, and co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080 (incorporated herein by reference).

The dsRNA of the invention may comprise one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention comprises no more than 3 mismatches. If the antisense strand of the dsRNA contains a mismatch with the target sequence, it is preferred that the mismatch region is not located in the center of the complementary region. If the antisense strand of the dsRNA contains a mismatch to the target sequence, it is preferred that the mismatch is limited to 5 nucleotides from either end, e.g., 5, 4, 3, 2 or 1 nucleotides from the 5 'or 3' end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand complementary to the Eg5 gene region, typically the dsRNA does not contain any mismatch within the central 13 nucleotides. The methods described herein can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the Eg5 gene. The effectiveness of dsRNA with mismatches in inhibiting the expression of the Eg5 gene is important, particularly if polymorphic sequence variations are known in particular regions of complementarity in the Eg5 gene within a population.

Decoration

In yet another embodiment, the dsRNA is chemically modified to increase stability. The nucleic acids of the invention may be synthesized and/or modified by methods well known in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L., et al (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is incorporated herein by reference. Specific examples of preferred dsRNA compounds for use in the invention include dsRNA containing a modified backbone or no natural internucleoside linkages. As defined herein, dsRNA containing a modified backbone includes those that retain a phosphorus atom in the backbone and those that do not contain a phosphorus atom in the backbone. For the purposes of this specification, and as once referred to in the art, modified dsrnas which do not contain a phosphorus atom in the internucleoside backbone can also be considered oligonucleosides.

Preferred modified dsRNA backbones include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester; methyl and other alkyl phosphates including 3' -alkylene phosphates and chiral phosphates, phosphonites; phosphoramidates, including 3 ' -phosphoramidates and aminoalkyl phosphoramidates with normal 3 ' -5 ' linkages, thiocarbonylaminophosphates, thiocarbonylalkylphosphates, thiocarbonylalkylphosphate triesters, and boranophosphates (boranophosphates), 2 ' -5 ' linked analogs of these, and those with opposite polarity in which adjacent pairs of nucleoside units are 3 ' -5 ' to 5 ' -3 ' or 2 ' -5 ' to 5 ' -2 ' linkages. Various salts, mixed salts and free acid forms are also included.

Typical U.S. patents that teach the preparation of the above-described phosphorus-containing linkages include, but are not limited to, U.S. patent nos.3,687,808; 4,469,863; 4,476,301, respectively; 5,023,243; 5,177,195, respectively; 5,188,897, respectively; 5,264,423; 5,276,019; 5,278,302; 5,286,717, respectively; 5,321,131, respectively; 5,399,676, respectively; 5,405,939, respectively; 5,453,496, respectively; 5,455,233, respectively; 5,466,677, respectively; 5,476,925, respectively; 5,519,126, respectively; 5,536,821, respectively; 5,541,316, respectively; 5,550,111, respectively; 5,563,253, respectively; 5,571,799, respectively; 5,587,361, respectively; and 5,625,050, each of which is incorporated herein by reference.

Preferred modified dsRNA backbones, excluding the phosphorus atom, have backbones formed of short alkyl chains or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These backbones include those having morpholino linkages (formed in part from the sugar portion of the nucleoside); a siloxane backbone; sulfide, sulfoxide and sulfone backbones; formyl and thiocarbonyl backbones; methylene formyl and thiocarbonyl backbones; an olefin-containing backbone; a sulfamate backbone; methylene imino and methylene hydrazo backbones; sulfonate and sulfonamide backbones; a skeleton of an amide skeleton; and other backbones with mixed N, O, S and CH2 components.

Typical U.S. patents that teach the preparation of the above-described oligonucleotides include, but are not limited to, U.S. patent nos.5,034,506; 5,166,315, respectively; 5,185,444, respectively; 5,214,134, respectively; 5,216,141, respectively; 5,235,033, respectively; 5,64,562; 5,264,564, respectively; 5,405,938, respectively; 5,434,257, respectively; 5,466,677, respectively; 5,470,967, respectively; 5,489,677; 5,541,307, respectively; 5,561,225, respectively; 5,596,086, respectively; 5,602,240; 5,608,046, respectively; 5,610,289, respectively; 5,618,704, respectively; 5,623,070, respectively; 5,663,312, respectively; 5,633,360, respectively; 5,677,437, respectively; and 5,677,439, each of which is incorporated herein by reference.

In other preferred dsRNA mimetics, both the sugar and internucleoside linkages of the nucleotide units, i.e. the backbone, are replaced by new groups. The base units are retained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, the dsRNA mimetic that has been shown to have excellent hybridization properties, is known as Peptide Nucleic Acid (PNA). In PNA compounds, the sugar backbone of dsRNA is replaced by an amide containing a backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. Typical U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. patent nos.5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.

The most preferred practice of the inventionBy means of dsRNA having a phosphorothioate backbone and an oligonucleoside with a heteroatom backbone, in particular- -CH of the above-cited U.S. Pat. No.5,489,677₂--NH--CH₂--、--CH₂--N(CH₃)--O--CH₂- - - [ named methylene (methylimino) or MMI skeleton]、--CH₂--O--N(CH₃)--CH₂--、--CH₂--N(CH₃)--N(CH₃)--CH₂- - -and- -N (CH)₃)--CH₂--CH₂- - - - [ wherein the natural phosphodiester backbone is represented by- -O- -P- -O- -CH₂--]And the amide backbone of U.S. Pat. No.5,602,240, cited above. Also preferred are dsrnas having the morpholino backbone structure of U.S. patent No.5,034,506 cited above.

The modified dsRNA may also comprise one or more substituted sugar moieties. Preferred dsrnas comprise at the 2' position one of: OH; f; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁To C₁₀Alkyl or C₂To C₁₀Alkenyl and alkynyl groups. Particularly preferred is O [ (CH)₂)_nO]_mCH₃、O(CH₂)_nOCH₃、O(CH₂)_nNH₂、O(CH₂)_nCH₃、O(CH₂)_nONH₂And O (CH)₂)_nON[(CH₂)_nCH₃)]₂Wherein n and m are from 1 to about 10. Other preferred dsrnas include one of the following at the 2' position: c₁To C₁₀Lower alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH₃、OCN、Cl、Br、CN、CF₃、OCF₃、SOCH₃、SO₂CH₃、ONO₂、NO₂、N₃、NH₂Heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalating group, for improving dsRNA pharmacokinetics A group of lipids or a group for improving pharmacodynamic properties of dsRNA, and other substituents having similar properties. Preferred modifications include 2 '-methoxyethoxy (2' -O- -CH)₂CH₂OCH₃Also known as 2 '-O- (2-methoxyethyl) or 2' -MOE) (Martin et al, Helv. Chim. acta, 1995, 78, 486-504), i.e. alkoxy-alkoxy groups. Other preferred modifications include 2' -dimethylaminoxyethoxy, i.e., O (CH)₂)₂ON(CH₃)₂The group, also known as 2 ' -DMAOE, as described in the examples below, and 2 ' -dimethylaminoethoxyethoxy (also known in the art as 2 ' -O-dimethylaminoethoxyethyl or 2 ' -DMAEOE), i.e., 2 ' -O- -CH₂--O--CH₂--N(CH₂)₂Also described in the following examples.

Other preferred modifications include 2 '-methoxy (2' -OCH)₃) 2 '-Aminopropoxy (2' -OCH)₂CH₂CH₂NH₂) And 2 '-fluoro (2' -F). Similar modifications can also be made at other positions of the dsRNA, particularly at the 3 'position of the sugar on the 3' terminal nucleotide or in 2 '-5' linked dsRNAs to neutralize the 5 'position of the 5' terminal nucleotide. The dsRNA may also replace the pentofuranosyl sugar with a glycomimetic, such as a cyclobutyl moiety. Typical U.S. patents that teach such modified sugar structures include, but are not limited to, U.S. patent nos.4,981,957; 5,118,800, respectively; 5,319,080, respectively; 5,359,044, respectively; 5,393,878, respectively; 5,446,137, respectively; 5,466,786, respectively; 5,514,785, respectively; 5,519,134, respectively; 5,567,811, respectively; 5,576,427, respectively; 5,591,722, respectively; 5,597,909, respectively; 5,610,300, respectively; 5,627,053, respectively; 5,639,873, respectively; 5,646,265, respectively; 5,658,873, respectively; 5,670,633, respectively; and 5,700,920, some of which are commonly owned by the present application, and each of which is incorporated by reference herein in its entirety.

dsRNA may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in The sense Encyclopedia of Polymer Science And Engineering, pp 858-859, Kroschwitz, J.L, ed.John Wiley & Sons, 1990, Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613, And those disclosed in Sanghvi, Y.S., Chapter 15, DsRNA Research And Applications, pp 289-302, crook, S.T. And Lebleu, B.E., Ed, Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These nucleobases include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. The 5-methylcytosine substituent has been shown to increase nucleic acid duplex stability by 0.6-1.2 degrees celsius (Sanghvi, y.s., crook, s.t. and Lebleu, b., eds., DsRNA research Applications, CRC Press, Boca Raton, 1993, pages 276-278), which is currently the preferred base substituent, especially when combined with 2' -O-methoxyethyl sugar modification.

Exemplary U.S. patents that teach the preparation of certain of the above-cited modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-cited U.S. patent nos. 3,687,808 and 4,845,205; 5,130, 30; 5,134,066, respectively; 5,175,273, respectively; 5,367,066, respectively; 5,432,272; 5,457,187, respectively; 5,459,255; 5,484,908, respectively; 5,502,177, respectively; 5,525,711, respectively; 5,552,540, respectively; 5,587,469, respectively; 5,594,121, 5,596,091; 5,614,617, respectively; and 5,681,941, each of which is incorporated herein by reference, and U.S. patent No.5,750,692, which is also incorporated herein by reference.

Conjugates

Another modification of the dsRNA of the invention comprises one or more moieties or conjugates that increase the activity, cellular distribution or cellular uptake of the dsRNA chemically linked to the dsRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al, proc.natl.acid.sci.usa, 199, 86, 6553-6556), cholic acid (Manoharan et al, biorg.med.chem.let., 199441053-1060); thioethers, such as emerald-S-triphenylmethanethiol (Manohara et al, Ann.N.Y.Acad.Sci., 1992, 660, 306-309; Manohara et al, Bio rg.Med.chem.Let., 1993, 3, 2765-2770), mercaptocholesterol (Oberhauser et al, Nucl.acids Res., 1992, 20, 533-538); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al, EMBO J, 1991, 10, 1111-1118; Kabanov et al, FEBS Lett., 1990, 259, 327-330; Svinarchuk et al, Biochimie, 1993, 75, 49-54); phospholipids, such as dihexadecyl-rac-glycerol or triethyl-ammonium 1, 2-di-O-hexadecyl-rac-propanetrioxy-3-H phosphate (manohara et al, tetrahedron lett., 1995, 36, 3651-3654; Shea et al, nucleic acids res., 1990, 18, 3777-3783); polyamine or polyethylene glycol chains (Manoharan et al, Nucleotides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al, biochim. biophysis.acta, 1995, 1264, 229-237) or an octadecylamine or hexylamine-carbonyl hydroxycholesterol moiety (Crooke et al, j. pharmacol. exp.ther., 1996, 277, 923-937).

Typical U.S. patents that teach the preparation of these dsRNA conjugates include, but are not limited to, U.S. patent nos.4,828,979; 4,948,882, respectively; 5,218,105; 5,525,465, respectively; 5,541,313, respectively; 5,545,730, respectively; 5,552,538, respectively; 5,578,717, 5,580,731; 5,591,584, respectively; 5,109,124, respectively; 5,118,802, respectively; 5,138,045; 5,414,077, respectively; 5,486,603, respectively; 5,512,439, respectively; 5,578,718, respectively; 5,608,046, respectively; 4,587,044, respectively; 4,605,735, respectively; 4,667,025, respectively; 4,762,779, respectively; 4,789,737, respectively; 4,824,941, respectively; 4,835,263, respectively; 4,876,335, respectively; 4,904,582, respectively; 4,958,013, respectively; 5,082,830; 5,112,963, respectively; 5,214,136, respectively; 5,082,830; 5,112,963, respectively; 5,214,136, respectively; 5,245,022, respectively; 5,254,469, respectively; 5,258,506, respectively; 5,262,536, respectively; 5,272,250, respectively; 5,292,873, respectively; 5,317,098, respectively; 5,371,241, 5,391,723; 5,416,203, 5,451, 463; 5,510,475, respectively; 5,512,667, respectively; 5,514,785, respectively; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726, respectively; 5,597,696; 5,599,923, respectively; 5,599,928 and 5,688,941, each of which is incorporated by reference herein.

It is not necessary to make uniform modifications at all positions of a given compound, and in fact more than one such modification may be combined in a single compound or even in a single nucleoside within a dsRNA. The invention also includes dsRNA compounds that are chimeric compounds. In the context of the present invention, a "chimeric" dsRNA compound or "chimera" is a dsRNA compound, in particular dsRNA, comprising two or more chemically distinct regions each consisting of at least one monomeric unit (i.e. nucleotides in the case of dsRNA compounds). These dsrnas typically comprise at least one region in which the dsRNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake and/or increased binding affinity to the target nucleic acid. Other regions of the dsRNA may serve as substrates for enzymes capable of cleaving RNA: DNA or RNA: RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA-DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, greatly enhancing the effect of dsRNA in inhibiting gene expression. Thus, when chimeric dsrnas are used, similar results can generally be obtained with shorter dsrnas as compared to phosphorothioate deoxydsrnas that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis, if necessary, and related nucleic acid hybridization techniques known in the art.

In some cases, the dsRNA may be modified by non-ligand groups. Many non-ligand molecules have been conjugated to dsRNA to enhance dsRNA activity, cellular distribution or cellular uptake, and procedures for such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86: 6553), cholic acid (Manohara et al, bioorg. Med. chem. Lett., 1994, 4: 1053); thioethers, such as hexyl-S-triphenylmethanethiol (Manohara et al, Ann.N.Y.Acad.Sci., 1992, 660: 306; Manohara et al, bioorg.Med.chem.Let., 1993, 3: 2765), mercaptocholesterol (Oberhauser et al, Nucl.AcidsRs., 1992, 20: 533); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al, EMBO J., 1991, 10: 111; Kabanov et al, FEBSLett., 1990, 259: 327; Svinarchuk et al, Biochimie, 1993, 75: 49); phospholipids, such as dicetyl-rac-glycerol or triethylammonium 1, 2-di-O-hexadecyl-rac-propanetrioxy-3-H-phosphate (Manohara et al, Tetrahedron Lett., 1995, 36: 3651; Shea et al, Nucl. acids Res., 1990, 18: 3777); polyamine or polyethylene glycol chains (Manohara et al, Nucleotides & Nucleotides, 1995, 14: 969), or adamantane acetic acid (Manohara et al, Tetrahedron Lett., 1995, 36: 3651), a palmityl moiety (Mishra et al, Biochim. Biophys. acta, 1995, 1264: 229) or an octadecylamine or hexylamine-carbonyl hydroxycholesterol moiety (crook et al, J.Pharmacol. Exp.Ther., 1996, 277: 923). Typical us patents teaching the preparation of such dsRNA conjugates are listed above. Typical conjugation schemes involve the synthesis of dsRNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with a molecule conjugated with a suitable coupling agent or activating agent. The conjugation reaction can be performed with the dsRNA still bound to the solid support or after cleaving the dsRNA in the solution phase. Purification of the dsRNA conjugate by HPLC typically results in a pure conjugate.

In some cases, the ligand may be multifunctional and/or the dsRNA may be conjugated to more than one ligand. For example, dsRNA may be conjugated to one ligand to enhance uptake and to a second ligand to improve release.

Vector encoded siRNA agents

In another aspect of the invention, the Eg5 and VEGF-specific dsRNA molecules expressed by the transcription unit are inserted into a DNA or RNA vector (see, e.g., Couture, A, et al, TIG. (1996), 12: 5-10; Skelern, A., et al, International PCT publication No. WO00/22113, Conrad, International PCT publication No. WO 00/22114, and Conrad, U.S. Pat. No.6, 054, 299). These transgenes may be introduced as linear constructs, circular plasmids, or viral vectors, which may be incorporated and inherited as transgenes integrated into the host genome. The transgene can also be constructed to be inherited as an extrachromosomal plasmid (Gassmann, et al, Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

The individual strands of the dsRNA can be transcribed by promoters located on two separate expression vectors and co-transfected into the target cell. Alternatively, each individual strand of the dsRNA may be transcribed by a promoter that is both located on the same expression plasmid. In a preferred embodiment, the dsRNA may be represented as inverted repeats joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

Recombinant dsRNA expression vectors are typically DNA plasmids or viral vectors. Viral vectors expressing dsRNA can be constructed based on, but are not limited to, the following viruses: adeno-associated virus (for review see Muzyzka, et al, curr. topics. micro. immunol. (1992) 158: 97-129)); adenoviruses (see, e.g., Berkner, et al, BioTechniques (1998) 6: 616), Rosenfeld et al (1991, Science 252: 431-434), and Rosenfeld et al (1992), Cell 68: 143-155)); or alphaviruses, as well as other viruses known in the art. Retroviruses have been used to introduce a variety of genes into a number of different cell types in vitro and/or in vivo, including epithelial cells (see, e.g., Eglitis, et al, Science (1985) 230: 1395-1398; Danos and Mulligan, Proc. NatI.Acad. Sci.USA (1998) 85: 6460-6464; Wilson et al, 1988, Proc. Natl. Acad. Sci.USA 85: 3014-3018; Armentano, et al, 1990, Proc. Natl. Acad. Sci.USA 87: 61416145; Huber et al, 1991, Proc. Natl. Acad. Sci.USA 88: 8039-8043; Ferry et al, 1991, Proc. Natl. Acad. Sci. USA 88: 8377-8381; Chudhowury et al, 1991, Sci. Sci.254: 1985-1992; WO 10876, USA 10858: 19832; PCT application No. WO 10835: 19832; WO 10835; WO 10873/1992; WO patent application No. 10832; WO 10835; USA) 10832; WO 10892; USA). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be prepared by transfecting the recombinant retroviral genome into an appropriate packaging cell line, such as PA317 and Psi-CRIP (Comette et al, 1991, Human Gene therapy 2: 5-10; Cone et al, 1984, Proc. Natl. Acad. Sci. USA 81: 6349). Recombinant adenoviral vectors can be used to infect a variety of cells and tissues in a susceptible host (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al, 1992, j. infection Disease, 166: 769) and have the advantage of not requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequence of the dsRNA molecule to be expressed may be used, for example, derived from Adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., Lentivirus (LV), rhabdovirus, murine leukemia virus); vectors for herpes viruses and the like. The tropism of a viral vector can be modified by pseudotyping the vector and envelope proteins or other surface antigens from other viruses or optionally by substituting different viral capsid proteins.

For example, the lentiviral vectors of the invention can pseudotype with surface proteins from Vesicular Stomatitis Virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be targeted to different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is referred to as AAV 2/2. The serotype 2 capsid gene in the AAV2/2 vector can be replaced with a serotype 5 capsid gene to produce an AAV2/5 vector. Techniques for constructing AAV vectors expressing different capsid protein serotypes are within the purview of those skilled in the art; see, for example, Rabinowitz J E et al (2002), J Virol 76: 791-801, which are hereby incorporated by reference in their entirety.

The selection of recombinant viral vectors suitable for use in the present invention, methods for inserting nucleic acid sequences for expressing dsRNA into the vector, and methods of delivering viral vectors into a desired cell are within the purview of those skilled in the art. See, e.g., Dornburg R (1995), Gene therapy.2: 301-310; eglitis M A (1988), Biotechniques 6: 608-614; miller A D (1990), Hum Gene therapy.1: 5-14; anderson W F (1998), Nature 392: 25-30; and Rubinson D a et al, nat. genet.33: 401-406, which are all hereby incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single stranded RNA molecules from a recombinant AAV vector, e.g., containing a U6 or H1 RNA promoter, or a Cytomegalovirus (CMV) promoter.

Suitable AV vectors for expressing the dsRNA of the invention, methods for constructing recombinant AV vectors and methods for delivering the vectors into target cells are described in Xia H et al (2002), nat. biotech.20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in SamulskiR et al (1987), j.virol.61: 3096-3101; fisher K J et al (1996), J.Virol, 70: 520-532; samulski R et al (1989), J.Virol.63: 3822-3826; U.S. Pat. No.5,252,479; pat. No.5,139,941; international patent application No. wo 94/13788; and international patent application No. wo 93/24641, which are incorporated herein by reference in their entirety.

The promoter driving expression of the dsRNA of the invention in a DNA plasmid or viral vector may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or typically an RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, such as the T7 promoter, provided that the expression plasmid also encodes the T7RNA polymerase required for transcription from the T7 promoter. The promoter may also direct transgene expression to the pancreas (see, for example, the insulin regulatory sequence for pancreas (Bucchini et al, 1986, Proc. Natl. Acad. Sci. USA 83: 2511-2515)).

In addition, the expression of transgenes can be precisely regulated, for example, by using inducible regulatory sequences and expression systems, such as those sensitive to certain physiological regulators, e.g., circulating glucose levels or hormones (Dochery et al, 1994, FASEB J.8: 20-24). Such inducible expression systems, suitable for controlling transgene expression in a cell or mammal, include regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl- β -D1-thiogalactopyranoside (EPTG). One skilled in the art will be able to select the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.

Typically, a recombinant vector capable of expressing a dsRNA molecule is delivered as described below and maintained in the target cell. Alternatively, viral vectors can be used which provide for transient expression of the dsRNA molecule. Such a carrier can be repeatedly administered as necessary. Once expressed, the dsRNA binds to the target RNA and modulates its function or expression. delivery of the dsRNA expression vector can be systemic, e.g., via intravenous or intramuscular administration, by administration to target cells explanted from the patient and then reintroduced into the patient, or by any other means capable of introducing the desired target cells.

DsRNA expression DNA plasmids are commonly used as cationic lipid vectors (e.g., Oligofectamine) or non-cationic lipid-based vectors (e.g., Transit-TKO)^TM) The complex of (a) is transfected into a target cell. The invention also relates to multiple lipofections for dsRNA-mediated inhibition, and to a method for producing such a lipofectionThe inhibition was directed to different regions of a single EG5 gene (or VEGF gene) or multiple EG5 genes (or VEGF genes) over a week or more. The successful introduction of the vectors of the invention into the host cell can be monitored by using various known methods. For example, transient transfection may be signaled using a reporter gene, e.g., a fluorescent marker, such as Green Fluorescent Protein (GFP). Markers that provide resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance, to transfected cells can be used to ensure stable transfection of cells ex vivo.

Eg 5-specific dsRNA molecules and VEGF-specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a patient, for example, by intravenous injection, topical administration (see U.S. Pat. No. 5,328,470), or stereotactic injection (see, e.g., Chen et al (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical formulation of the gene therapy vector may comprise the gene therapy vector in an acceptable diluent, or may comprise a slow release matrix embedding the gene delivery vehicle. Alternatively, if an intact gene delivery vector, such as a retroviral vector, can be prepared intact from recombinant cells, the pharmaceutical preparation may comprise one or more cells producing the gene delivery system.

Pharmaceutical compositions comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositions comprising the dsrnas described herein and a pharmaceutically acceptable carrier, and methods of administering the pharmaceutical compositions. The pharmaceutical compositions containing the dsRNA are useful for treating diseases or disorders associated with the expression or activity of the Eg5/KSP and/or VEGF genes, such as pathological processes mediated by Eg5/KSP and/or VEGF expression, e.g., liver cancer. Such pharmaceutical compositions are formulated based on the mode of delivery.

Dosage form

The pharmaceutical compositions featured in this invention are administered in a dosage sufficient to inhibit the expression of the Eg5/KSP and/or VEGF genes. In general, suitable dosages of dsRNA are in the range of 0.01 to 200.0 milligrams (mg) per kilogram (kg) of recipient body weight per day, usually 1 to 50mg per kilogram of body weight per day. For example, the dsRNA may be administered at 0.01mg/kg, 0.05mg/kg, 0.5mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 3mg/kg, 5.0mg/kg, 10mg/kg, 20mg/kg, 30mg/kg, 40mg/kg or 50mg/kg per single dose.

The pharmaceutical composition may be administered once a day, or the dsRNA may be administered in two, three or more sub-doses at appropriate intervals throughout the day. The effect of a single dose on Eg5/KSP and/or VEGF levels is sustained such that subsequent doses are administered at no more than 7 day intervals or at no more than 1, 2, 3 or 4 week intervals.

In some embodiments, the dsRNA is administered using continuous infusion, or is delivered via a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be reduced accordingly to reach the total daily dose. The dosage units may also be mixed for delivery over several days, for example, using common sustained release formulations that provide sustained release of the dsRNA over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering agents to a specific site, e.g., for use with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding plurality of daily doses.

One skilled in the art will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to: the severity of the disease or condition, previous treatments, the overall health and/or age of the subject, and other existing diseases. In addition, treating a subject with a therapeutically effective amount of the composition can include a single treatment or a series of treatments. The effective dose and in vivo half-life of the individual dsRNA involved in the invention can be estimated according to in vivo assays using routine methods or using appropriate animal models described elsewhere in the invention.

Mouse genetic advances have generated many mouse models for studying various human diseases, such as pathological processes mediated by Eg5/KSP and/or VEGF expression. This model is useful for in vivo testing of dsRNA, as well as for determining therapeutically effective doses. Suitable mouse models are, for example, mice containing plasmids expressing human Eg5/KSP and/or VEGF. Another suitable mouse model is a transgenic mouse carrying a transgene expressing human Eg5/KSP and/or VEGF.

Toxicity and therapeutic efficacy of such compounds can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals for determining LD50 (the dose at which 50% of the population dies) and ED50 (the dose at which 50% of the population is therapeutically effective). The dose ratio of toxic to therapeutic effect is the therapeutic index and can be expressed as the ratio LD50/ED 50. Compounds with high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for human use. The dosage of the compositions featured in this invention is generally within the circulating concentration range that includes the ED50 but with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, a therapeutically effective dose can be estimated initially from cell culture assays. The dose can be formulated in animal models to achieve a circulating plasma concentration range for the compound and, if appropriate, for the polypeptide product of the target sequence (e.g., to achieve a reduced polypeptide concentration), which includes IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. This information can be used to more accurately determine useful doses in humans. For example, plasma levels can be determined by high performance liquid chromatography.

In addition to their administration as discussed above, the dsrnas featured in the invention may be administered in combination with other known agents effective in the treatment of pathological processes mediated by target gene expression. In any event, the practitioner can adjust the dosage and timing of dsRNA administration based on observed results measured using efficacy criteria known in the art or described herein.

Administration of drugs

The pharmaceutical compositions of the present invention may be administered in a variety of ways depending on whether local or systemic treatment is desired and depending on the area to be treated. Administration can be topical, pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer), intratracheal, intranasal, epidermal and transdermal as well as subcutaneous, oral or parenteral, e.g., subcutaneous.

Typically, when treating mammals suffering from hyperlipidaemia, dsRNA molecules are administered systemically via parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular administration. For example, dsRNA conjugated or unconjugated or formulated to contain liposomes or not may be administered intravenously to a patient. To this end, the dsRNA molecules can be formulated into compositions, such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oily matrices. Such solutions may also contain buffers, diluents and other suitable additives. For parenteral, intrathecal or intraventricular administration, the dsRNA molecules may be formulated into compositions, such as sterile aqueous solutions, which may also contain buffers, diluents and other suitable additives (e.g., penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers). The present invention describes the formulation in more detail.

The dsRNA may be delivered in a manner that targets a particular tissue, such as the liver (e.g., hepatocytes of the liver).

Preparation

Pharmaceutical formulations of the invention which may conveniently be presented in unit dosage form may be prepared according to conventional methods well known in the pharmaceutical industry. Such techniques include the step of mixing the active ingredient with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, nonaqueous or mixed media. Aqueous suspensions may also contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. The suspension may also contain a stabilizer.

The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be produced from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. In one aspect, when treating liver diseases such as hyperlipidemia, the formulation is a liver-targeting formulation.

Additionally, dsrnas targeting EG5/KSP and/or VEGF genes can be formulated into compositions containing the dsRNA mixed, encapsulated, conjugated or otherwise linked with other molecules, molecular structures or mixtures of nucleic acids. For example, compositions containing one or more dsRNA agents targeting EG5/KSP and/or VEGF genes may comprise other therapeutic agents, such as other cancer therapeutic agents or one or more dsRNA compounds targeting non-EG 5/KSP and/or VEGF genes.

Oral, parenteral, topical and biological formulations

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or aqueous or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, an oral formulation is one in which the dsrnas featured in the present invention are administered together with one or more penetration enhancers, surfactants, and chelating agents. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycocholic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24, 25-dihydro-fusidate and sodium glycerodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, didecanoic acid, tridecanoic acid, glycerol monooleate, glycerol dilaurate, glycerol 1-monodecanoate, 1-dodecylazacycloheptan-2-one, acyl carnitines, acyl cholines, monoglycerides, diglycerides, or pharmaceutically acceptable salts (e.g., sodium salts) thereof. In some embodiments, a permeation enhancer combination is used, for example, a fatty acid/salt and bile acid/salt combination. A typical combination is the sodium salt of lauric acid, capric acid and UDCA. Other penetration enhancers include polyoxyethylene-9-lauryl ester, polyoxyethylene-20-cetyl ester. dsRNA according to features of the invention can be delivered orally in particulate form comprising spray-dried particles or in particulate form comprising complexed micro-or nanoparticles. The dsRNA complexing agent comprises a polyamino acid; a polyimine; a polyacrylate; polyalkylacrylates, polyoxetanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG), and starch; a polyalkylcyanoacrylate; DEAE-derived polyimine, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethyl chitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polymercaptyldiethylaminomethyl ethylene P (TDAE), polyaminostyrene (e.g., para-amino), poly (methyl cyanoacrylate), poly (ethyl cyanoacrylate), poly (butyl cyanoacrylate), poly (isobutyl cyanoacrylate), poly (hexyl cyanoacrylate), DEAE-methacrylate, DEAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethacrylates, polyhexamethylene acrylate, poly (D, L-lactic acid), poly (DL-lactic acid-co-glycolic acid (PLGA), alginate and polyethylene glycol (PEG) formulations for oral administration of dsRNA and their preparation are described in detail in US Patents 6,887,906, PLGA. dsRNA and their preparation, U.S. patent publication No.20030027780 and U.S. patent No.6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. It may be necessary or desirable to employ conventional pharmaceutical carriers, aqueous solutions, powders or oily bases, thickeners and the like. Suitable topical formulations include those in which a compound of the invention features a combination of a topical delivery agent such as a lipid, liposome, fatty acid ester, steroid, chelating agent and surfactant. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidydope ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltrimethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The dsrnas featured in the invention may also be encapsulated within liposomes or may form complexes therewith, particularly with cationic liposomes. Alternatively, the dsRNA may be complexed with a lipid, particularly a cationic lipid. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, arachidic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, dicaprate, tricaprate, glycerol monooleate, glycerol dilaurate, glycerol 1-monodecanoate, 1-dodecylazacycloheptan-2-one, acyl carnitines, acyl cholines, or C1-10 alkyl esters (e.g., isopropyl myristate), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. patent No.6,747,014, which is incorporated herein by reference. In addition, the dsRNA molecules can be administered to a mammal as a biological or non-biological means, for example, as described in U.S. patent No.6,271,359. Abiotic delivery can be accomplished by a variety of methods, including but not limited to: (1) by using the present invention The dsRNA acid molecules provided load the liposomes and (2) complex the dsRNA molecules with the lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposomes may be composed of cationic and neutral lipids that are commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, but are not limited to lipofectin, lipofectamine, lipofectace, and DOTAP. Methods of forming liposomes are known in the art. For example, the liposome composition may be formed from lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, dimyristoyl phosphatidylglycerol, or dioleoylphosphatidylethanolamine. A number of lipophilic agents are commercially available, including Lipofectin^TM(Invitrogen/Life Technologies, Carlsbad, Calif.) and Effectene^TM(Qiagen, Valencia, Calif.). In addition, commercially available cationic lipids such as DDAB or DOTAP can be used to optimize the systemic delivery method, each of which can be mixed with a neutral lipid such as DOPE or cholesterol. In some cases, liposomes such as those described by Templeton et al (Nature Biotechnology, 15: 647-652(1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to accomplish in vivo and in vitro delivery (Boletta et al, J.Am Soc.Nephrol.7: 1728 (1996)). Additional information on the use of liposomes to deliver nucleic acids can be found in U.S. Pat. No.6,271,359, PCT publication WO 96/40964, and Morrissey, D. et al 2005.Nat Biotechnol.23 (8): 1002-7.

Biological delivery can be achieved by a variety of methods, including but not limited to the use of viral vectors. For example, viral vectors (e.g., adenovirus and herpes viral vectors) can be used to deliver dsRNA molecules to hepatocytes. Standard molecular biology techniques can be used to introduce one or more dsrnas provided by the invention into one of many different viral vectors previously developed to deliver nucleic acids to cells. The resulting viral vectors can be used to deliver one or more dsrnas to a cell by, for example, infection.

Liposome formulations

In addition to microemulsions, a number of organized surface active structures have been studied and used in pharmaceutical formulations. Including monolayers, micelles, bilayers, and vesicles. Vesicles (e.g., liposomes) are of great interest because of the specificity and persistence of action they provide in drug delivery. As used herein, the term "liposome" refers to a vesicle composed of amphipathic lipids arranged in a spherical bilayer or multiple spherical bilayers.

Liposomes are unilamellar or multilamellar vesicles having a membrane formed by a lipophilic material and an aqueous interior. The aqueous portion comprises the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes, although they do not fuse effectively with the cell wall, are taken up by macrophages in the body.

In order to penetrate intact mammalian skin, lipid vesicles must penetrate a series of pores less than 50nm in diameter under the influence of a suitable transdermal gradient. Therefore, it is desirable to use liposomes that are highly deformable and capable of penetrating such pores.

Other advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can bind many water and lipid soluble drugs; and liposomes can protect drugs encapsulated in their internal compartments from metabolism and degradation (Rosoff, in pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, inc., New York, n.y., vol.1, page 245). Important factors to consider in the preparation of liposome formulations are the lipid surface charge, the vesicle size and the aqueous volume of the liposome.

Liposomes are capable of transferring and delivering active ingredients to the site of action. Because the liposome membrane is similar in structure to a biological membrane, when the liposome is applied to tissue, the liposome begins to fuse with the cell membrane, and the liposome contents flow into the cells where the activator may act, due to liposome fusion and cell progression.

Liposomal formulations have been the focus of extensive research as a means of delivery for many drugs. There is increasing evidence that liposomes have several advantages over other formulations for topical administration. Such advantages include a reduction in side effects associated with high systemic absorption of the administered drug, an increase in accumulation of the administered drug on the desired target, and the ability to administer a variety of hydrophilic and hydrophobic drugs into the skin.

Several reports detail the ability of liposomes to deliver agents containing high molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high molecular weight DNA have been administered to the skin. Most applications result in targeting the epidermis.

Liposomes fall into two broad categories. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. Positively charged DNA/liposome complexes bind to the negatively charged cell surface and are internalized within the endosome. Due to the acidic pH within the endosome, liposomes burst, releasing their contents into the cytoplasm (Wang et al, biochem. biophysis. res. commun., 1987, 147, 980-.

The pH-sensitive or negatively charged liposomes capture the DNA rather than complex it. Since both DNA and lipids carry similar charges, repulsion occurs rather than complex formation. However, some DNA is trapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding thymidine kinase genes to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al, Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposome composition includes phospholipids other than naturally-derived lecithins. For example, a neutral liposome composition can be formed from dimyristoyl lecithin (DMPC) or dipalmitoyl lecithin (DPPC). Anionic liposome compositions are typically formed from dimyristoyl phosphatidylglycerol, whereas anionic gene fusion liposomes are formed primarily from phosphatidylethanolamine (DOPE). Another liposome composition is formed from lecithin (PC) such as soybean PC and egg PC. The other type is formed by a mixture of phospholipids and/or lecithins and/or cholesterol.

Several studies evaluated the topical delivery of liposomal pharmaceutical formulations to the skin. Application of interferon-containing liposomes to guinea pig skin results in a reduction in skin herpes sores, whereas delivery of interferon via other means (e.g., as a solution or as an emulsion) is ineffective (Weiner et al, Journal of drug targeting, 1992, 2, 405-. In addition, other studies have tested the effect of administering interferon as part of a liposomal formulation and using an aqueous system, concluding that liposomal formulations are superior to aqueous administration (du Plessis et al, Antiviral Research, 1992, 18, 259-.

Nonionic liposomal systems, particularly systems comprising nonionic surfactants and cholesterol, were also investigated to determine their effectiveness in delivering drugs into the skin. Contains Novasome^TMI (glyceryl dilaurate/Cholesterol/polyoxyethylene-10-stearyl ether) and Novasome^TMII (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) nonionic liposome formulations were used to deliver cyclosporin a into the dermis of mouse skin. The results show that this nonionic liposome system is effective in promoting cyclosporin a deposition into different layers of the skin (Hu et al, s.t.p.pharma.sci., 1994, 4, 6, 466).

Liposomes also include "sterically stabilized" liposomes, as that term is used herein to mean liposomes containing one or more specific lipids which, when incorporated into the liposome, result in an increased circulation duration as compared to liposomes lacking such specific lipids. Examples of sterically stabilized liposomes are liposomes in which a part (A) of the vesicle-forming lipid fraction of the liposome comprises one or more glycolipids, such as monosialoganglioside G_M1Or (B) those derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes comprising gangliosides, sphingomyelin, or PEG-derivatized lipids, these The increased circulating half-life of sterically stabilized liposomes results from a decreased uptake into the reticuloendothelial system (RES) cells (Allen et al, FEBS Letters, 1987, 223, 42; Wu et al, Cancer Research, 1993, 53, 3765).

Various liposomes containing one or more glycolipids are known in the art. Papahadjoulos et al (ann.n.y.acad.sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings are detailed by Gabizon et al (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO88/04924 to Allen et al disclose liposomes containing (1) sphingomyelin and (2) the ganglioside GM1 or galactocerebroside sulfate. U.S. Pat. No.5,543,152 (Webb et al) discloses liposomes containing sphingomyelin. Liposomes containing 1, 2-sn-dimyristoyl lecithin are disclosed in WO97/13499(Lim et al).

Many liposomes containing lipids derivatized with one or more hydrophilic polymers and methods for their preparation are known in the art. Sunamoto et al (Bull. chem. Soc. Jpn., 1980, 53, 2778) describe compositions containing the nonionic detergent 2C _1215GThe liposome of (1), which contains a PEG moiety. Illum et al (FEBS Lett., 1984, 167, 79) noted that hydrophilic coated polystyrene particles containing polymeric ethylene glycol resulted in a significantly increased blood half-life. Synthetic phospholipids modified by conjugation to carboxyl groups of polyglycols (e.g., PEG) are described by Sears (U.S. patent nos.4,426,330 and 4,534,899). Klibanov et al (FEBS Lett., 1990, 268, 235) describe experiments demonstrating that liposomes containing Phosphatidylethanolamine (PE) derivatized with PEG or stearate PEG significantly increase blood circulation half-life. Blume et al (Biochimica et Biophysica Acta, 1990, 1029, 91) extended this study to other PEG-derivatized phospholipids, for example, DSPE-PEG formed by combining Distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their outer surface are described in Fisher's European patent Nos. EP 0445131B 1 and WO 90/04384. Liposome compositions comprising 1-20 mole percent PEG-derivatized PE and methods of use thereofDescribed by Woodle et al (U.S. Pat. Nos.5,013,556 and 5,356,633) and Martin et al (U.S. Pat. No.5,213,804 and European patent No. EP 0496813B 1), among others. Liposomes containing many other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No.5,225,212(Martin et al) and WO 94/20073(Zalipsky et al). Liposomes containing PEG-modified ceramide lipids are described in WO 96/10391(Choi et a 1). U.S. Pat. No.5,540,935(Miyazaki et al) and U.S. Pat. No.5,556,948(Tagawa et al) describe PEG-containing liposomes whose surface can be further derivatized with a functional moiety.

Many liposomes containing nucleic acids are known in the art. WO 96/40062 to Thiierry et al discloses a method for encapsulating high molecular weight nucleic acids into liposomes. U.S. patent No.5,264,221 to Tagawa et al discloses protein-bound liposomes and states that the contents of such liposomes may include dsRNA. U.S. Pat. No.5,665,710 to Rahman et al describes certain methods for encapsulating oligodeoxyribonucleotides into liposomes. WO 97/04787 to Love et al discloses liposomes containing dsRNA targeted to the raf gene.

Transfersomes are another class of liposomes, and they are highly deformable lipid aggregates, attractive candidates for drug delivery vehicles. The carriers may be described as lipid droplets which are so highly deformable that they can easily penetrate pores smaller than the droplets. Transfersomes are suitable for the environment in which they are used, for example, they are self-optimizing (suitable for the shape of skin pores), self-healing, often reaching their target without breaking, and generally self-loading. To prepare the transfersomes, a surface edge-activating agent, typically a surfactant, may be added to standard liposome compositions. Transfersomes are used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common method of classifying and classifying many different classes of surfactants, including natural and synthetic, is by the use of the hydrophilic/lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for classifying the different surfactants used in the formulation (Rieger, in pharmaceutical Dosage Forms, Marcel Dekker, inc., New York, n.y., 1988, p.285).

If the surfactant molecule is non-ionic, it is classified as a non-ionic surfactant. Nonionic surfactants are widely used in pharmaceuticals and cosmetics and can be used over a wide range of pH values. Typically, their HLB value ranges from 2 to about 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glycerol esters, polyglycerol esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as ethoxylated fatty alcohols, propoxylated alcohols and ethoxy/propoxy block copolymers are also included in this class. Polyoxyethylene surfactants are the most commonly used members of the class of nonionic surfactants.

When the surfactant molecule is dissolved or dispersed in water, it carries a negative charge, and the surfactant is classified as an anionic surfactant. Anionic surfactants include carboxylic acid esters, such as soaps; acyl lactylates; an amino acid amide; sulfates, such as alkyl sulfates and ethoxylated alkyl sulfates; sulfonates such as alkylbenzenesulfonates, acylisethionates, acyltaurates and sulfosuccinates and phosphates. The most important members of the class of anionic surfactants are alkyl sulfates and soaps.

When a surfactant molecule is dissolved or dispersed in water, it carries a positive charge, and the surfactant is classified as a cationic surfactant. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most common members of this class of surfactants.

Surfactants are classified as amphoteric surfactants if the surfactant molecule is capable of carrying a positive or negative charge. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phospholipids.

The use of surfactants in pharmaceuticals, formulations and emulsions is described (Rieger, in pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, page 285).

Nucleic acid lipid particles

In one embodiment, the dsRNA featured in the invention is fully encapsulated in a lipid formulation, e.g., to form a nucleic acid-lipid particle. Typically, the nucleic acid-lipid particle comprises a cationic lipid, a non-cationic lipid, a sterol, and a lipid (e.g., a PEG-lipid conjugate) that prevents aggregation of the particle. Nucleic acid-lipid particles are very useful for systemic application because they exhibit extended circulation duration following intravenous (i.v.) injection and accumulate at a distal site (e.g., a site physically separate from the site of administration). In addition, when present in the nucleic acid-lipid particles of the invention, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed in, for example, U.S. patent nos.5,976,567; 5,981,501, respectively; 6,534,484, respectively; 6,586,410, respectively; 6,815,432, respectively; and PCT publication No. wo 96/40964.

The nucleic acid-lipid particle may also include one or more other lipids and/or components such as cholesterol. Other lipids may be included in the liposome composition for a variety of purposes, for example, to prevent oxidation of the lipid or to bind the ligand to the liposome surface. Any of a variety of lipids may be present, including amphiphilic, neutral, cationic, and anionic lipids. Such lipids may be used alone or in combination. Specific examples of other lipid components that may be present are described.

Other components that may be present in the nucleic acid-lipid particle include bilayer stabilizing components, such as polyamide oligomers (see, e.g., U.S. Pat. No.6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to phosphatidylethanolamine and PEG conjugated to ceramides (see, e.g., U.S. Pat. No.5,885,613).

The nucleic acid-lipid particle may include one or more of a second amino lipid or a cationic lipid, a neutral lipid, a sterol, and a lipid selected to reduce aggregation of the lipid particle during formation, which may result from steric stabilization of the particle that prevents charge-induced aggregation during formation.

For example, nucleic acid-lipid particles include SPLP, pSPLP, and SNALP. The term "SNALP" refers to a stable nucleic acid-lipid particle containing SPLP. The term "SPLP" refers to a nucleic acid-lipid particle that encapsulates plasmid DNA within a lipid vesicle. SPLP includes "pSPLP" which includes the encapsulated condensing agent-nucleic acid complex set forth in PCT publication No. wo 00/03683.

The particles of the present invention generally have an average diameter of from about 50nm to about 150nm, more typically from about 60nm to about 130nm, more typically from about 70nm to about 110nm, and most typically from about 70nm to about 90nm, and are substantially non-toxic.

In one embodiment, the ratio of lipid to drug (mass/mass ratio) is from about 1: 1 to about 50: 1, from about 1: 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or from about 6: 1 to about 9: 1, or about 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, or 33: 1.

Cationic lipids

The nucleic acid-lipid particles of the invention typically comprise a cationic lipid. For example, the cationic lipid can be N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (I- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), N- (I- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxy) propylamine (DODMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-dilinolylenyloxy-N, N-dimethylaminopropane (DLenDMA), 1, 2-dioleyiidenocarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-dioleyloxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dioleyloxy-3-morpholinopropane (DLin-MA), 1, 2-dioleoyloxy-3-dimethylaminopropane (DLInDAP), 1, 2-dioleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-oleoyl-2-linoleoxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleyloxy-3-trimethylaminopropane chloride (DLin-TMA. Cl), 1, 2-dioleyloxy-3-trimethylaminopropane chloride salt (DLin-TAP. Cl), 1, 2-dioleyloxy-3- (N-methylpiperazin-1-yl) propane (DLin-MPZ) or 3- (N, N-dioleylamino) -1, 2-propanediol (DLINAP), 3- (N, N-dioleylamino) -1, 2-propanediol (DOAP), 1, 2-dioleyloxy-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA), 1, 2-diinyloxy-N, N-dimethylaminopropane (DLinDMA), 2, 2-dioleyl-4-dimethylaminomethyl- [1, 3] -dioxolane (DLin-K-DMA) or an analogue thereof, (3aR, 5s, 6aS) -N, N-dimethyl-2, 2-bis ((9Z, 12Z) -octadec-9, 12-dienyl) tetrahydro-3 aH-cyclopenta [ d ] [1, 3] dioxol-5-amine (ALNY-100), 4- (dimethylamino) butanoic acid (6Z, 9Z, 28Z, 31Z) -thirty-seven-carbon-6, 9, 28, 31-tetraen-19-yl ester (MC3), or mixtures thereof.

In addition to those specifically described above, other cationic lipids that carry a net positive charge at approximately physiological pH may also be included in the lipid particles of the present invention. Such cationic lipids include, but are not limited to, N-dioleyl-N, N-dimethylammonium chloride ("DODAC"); n- (2, 3-dioleyloxy) propyl-N, N-triethylammonium chloride ("DOTMA"); n, N-distearyl-N, N-dimethylammonium bromide ("DDAB"); n- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride ("DOTAP"); 1, 2-dioleyloxy-3-trimethylaminopropane chloride salt ("dotap. cl"); 3 β - (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol ("DC-Chol"); n- (1- (2, 3-dioleyloxy) propyl) -N-2- (spermimido) ethyl) -N, N-dimethylammonium trifluoroacetate ("DOSPA"); dioctadecylamidoglycylcarboxyptamine ("DOGS"); 1, 2-dioleoyl-sn-3-phosphatidylethanolamine ("DOPE"), 1, 2-dioleoyl-3-dimethylammoniopropane ("DODAP"); n, N-dimethyl-2, 3-dioleyloxy) propylamine ("DODMA") and N- (1, 2-dimyristoyloxypropan-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). In addition, a number of commercial preparations of cationic lipids may be used, such as LIPOFECTIN (including DOTMA core DOPE, available from GIBCO/BRL) and LIPOFECTIAMINE (including DOSPA and DOPE, available from GIBCO/BRL). In a specific embodiment, the cationic lipid is an amino lipid.

As used herein, the term "amino lipid" is meant to include lipids having one or two fatty acids or fatty alkyl chains and an amino head group (including alkylamino or dialkylamino groups) that can be protonated to form cationic lipids at physiological pH.

Other amino lipids include lipids having selective fatty acid groups and other dialkylamino groups, including lipids in which the alkyl substituents are different (e.g., N-ethyl-N-methylamino-, N-propyl-N-ethylamino-, etc.). For R in the formula¹¹And R¹²Are both long chain alkyl or acyl groups, which may be the same or different. In general, amino lipids with less saturated acyl chains are easier to control in size, especially for filter sterilization purposes, the complex must be below about 0.3 micron in size. Preferably comprising a carbon chain length of C₁₄To C₂₂Amino lipids of unsaturated fatty acids of (a). Other scaffolds may also be used to separate the amino and fatty acid or fatty alkyl moieties of amino lipids. Suitable scaffolds are known to those skilled in the art.

In certain embodiments, the amino or cationic lipids of the present invention have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH7.4) and neutral at a second pH, preferably at or above physiological pH. It will of course be understood that the addition or removal of protons as a function of pH is an equilibrium process and that reference to a charged or neutral lipid refers to the nature of the predominant species, and does not require that all lipids be present in a charged or neutral form. The present invention does not exclude the use of lipids having more than one protonatable or deprotonatable group, or zwitterionic lipids.

In certain embodiments, the pKa of the protonatable groups in the inventive protonatable lipids is from about 4 to about 11. The most preferred pKa is from about 4 to about 7, since these lipids will be cationic at the lower pH formulation stage, while the particle surface will be largely (but not completely) neutralized at physiological pH of about pH 7.4. One advantage of this pKa is that at least some of the nucleic acid attached to the outer surface of the particle loses its electrostatic interaction at physiological pH and can be removed by simple dialysis; thus greatly reducing the susceptibility of the particles to removal.

An example of a cationic lipid is 1, 2-di-linolenyloxy-N, N-dimethylaminopropane (DLinDMA). The synthesis and preparation of nucleic acid-lipid particles including DlinDMA is described in international application PCT/CA2009/00496 filed 4, 15, 2009.

In one embodiment, the cationic lipid XTC (2, 2-dioleyl-4-dimethylaminoethyl- [1, 3] -dioxolane) is used to prepare nucleic acid-lipid particles. The synthesis of XTC is described in U.S. provisional patent application No. 61/107,998 filed on 23/10/2008, which is incorporated herein by reference.

In another embodiment, the cationic lipid MC3(4- (dimethylamino) butanoic acid (6Z, 9Z, 28Z, 31Z) -thirty-seven-carbon-6, 9, 28, 31-tetraen-19-yl ester) (e.g., DLin-M-C3-DMA) is used to make nucleic acid-lipid particles. The synthesis of MC3 and formulations containing MC3 is described, for example, in U.S. provisional serial No.61/244,834, filed on day 9 and 22 of 2009, and U.S. provisional serial No.61/185,800, filed on day 6 and 10 of 2009, which are incorporated herein by reference.

In another embodiment, the cationic lipid ALNY-100((3aR, 5s, 6aS) -N, N-dimethyl-2, 2-bis ((9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3 aH-cyclopenta [ d ] [1, 3] dioxol-5-amine)) is used for preparing the nucleic acid-lipid particles. The synthesis of ALNY-100 is described in international patent application PCT/US09/63933 filed 11/10/2009, which is incorporated herein by reference.

FIG. 20 illustrates the structure of ALNY-100, MC3 and XTC.

The cationic lipid may comprise from about 20 mol% to about 70 mol% or about 45-65 mol% or about 40 mol% of the total lipid present in the particle.

Non-cationic lipids

The nucleic acid-lipid particles of the invention may comprise a non-cationic lipid. The non-cationic lipid may be an anionic lipid or a neutral lipid. Examples include, but are not limited to: distearoyl lecithin (DSPC), dioleoyl lecithin (DOPC), dipalmitoyl lecithin (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl lecithin (POPC), Palmitoyl Oleoyl Phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexylamine-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), Cholesterol or mixtures thereof.

Anionic lipids suitable for use in the lipid particles of the present invention include, but are not limited to, phosphatidylglycerol, diphosphatidylglycerol, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups attached to neutral lipids.

When present in the lipid particle, the neutral lipid can be any of a variety of lipid species that exist in the form of uncharged or neutral zwitterions at physiological pH. Such lipids include, for example, diacyl lecithin, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cerebrumPhospholipids and cerebrosides. The choice of neutral lipids for use in the particles described herein is generally guided by, for example, considering liposome size and stability of the liposomes in the bloodstream. Preferably, the neutral lipid component is a lipid having two acyl groups (i.e., diacyl lecithin and diacyl phosphatidylethanolamine). Lipids having a variety of acyl chain groups of varying chain length and saturation are commercially available and can be isolated or synthesized by well-known techniques. In one group of embodiments, it is preferred to have a carbon chain length of C ₁₄To C₂₂The saturated fatty acid lipid of (4). In another set of embodiments, a composition comprising a carbon chain length of C is used₁₄To C₂₂Of mono-or di-unsaturated fatty acids. In addition, lipids containing a mixture of saturated and unsaturated fatty acid chains may be used. Preferably, the neutral lipid used in the present invention is DOPE, DSPC, POPC or any related lecithin. Neutral lipids for use in the present invention may also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids with other headgroups such as serine and inositol.

In one embodiment, the non-cationic lipid is distearoyl lecithin (DSPC). In another embodiment, the non-cationic lipid is dipalmitoyl lecithin (DPPC).

If cholesterol is included, the non-cationic lipid may comprise from about 5 mol% to about 90 mol%, from about 5 mol% to about 10 mol%, or about 58 mol% of the total lipid present in the particle.

Conjugated lipids

Conjugated lipids may be used for the nucleic acid-lipid particles to prevent aggregation, including polyethylene glycol (PEG) -modified lipids, monosialoganglioside Gm1, and polyamide oligomers ("PAOs"), for example (described in U.S. Pat. No.6,320,017). Other compounds containing uncharged, hydrophilic steric hindering moieties, which prevent aggregation during formulation, such as PEG, Gm1 or ATTA, may also be linked to the lipids used in the methods and compositions of the invention. ATTA-lipids are described, for example, in U.S. Pat. No.6,320,017, and PEG-lipid conjugates are described, for example, in U.S. Pat. Nos.5,820,873, 5,534,499 and 5,885,613. Typically, the concentration of the lipid component selected to reduce aggregation is about 1 to 15% (mole percent of lipid).

Specific examples of PEG-modified lipids (or lipid-polyoxyethylene conjugates) that can be used in the present invention may have a variety of "anchoring" lipid moieties to immobilize the PEG moiety to the surface of the lipid vesicle. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), which are described in co-pending USSN 08/486,214, incorporated herein by reference, PEG-modified dialkylamines, and PEG-modified 1, 2-diacyloxypropane-3-amine. Particularly preferred are PEG-modified diglycerides and dialkylglycerols.

In embodiments where the sterically larger moiety, such as PEG or ATTA, is conjugated to a lipid anchor, the choice of lipid anchor depends on the type of form in which the conjugate is conjugated to the lipid particle. It is well known that mePEG (mw2000) -distearoylphosphatidylethanolamine (PEG-DSPE) will remain conjugated to liposomes until the particles are cleared from circulation, perhaps on the order of days. Other conjugates, such as PEG- -CerC20 have similar retention capabilities. However, upon exposure to serum, PEG-CerC14 was rapidly exchanged out of the preparation, T in some experiments _1/2Less than 60 minutes. As described in U.S. patent application SN 08/486,214, at least three characteristics affect the exchange rate: the length of the acyl chain, the saturation of the acyl chain and the size of the steric hindrance head group. Compounds having suitable variations of these characteristics may be used in the present invention. In some therapeutic applications, it is preferred that the PEG-modified lipid rapidly detach from the nucleic acid-lipid particle in vivo, whereby the PEG-modified lipid will have a relatively short lipid anchor. In other therapeutic applications, it is preferred that the nucleic acid-lipid particles have a long plasma circulation time, whereby the PEG-modified lipid will have a relatively long lipid anchor. Typical lipid anchors include a length of about C₁₄To about C₂₂Preferably about C₁₄To about C₁₆Those of (a).In some embodiments, a PEG moiety, e.g., mPEG-NH₂Is about 1000, 2000, 5000, 10000, 15000 or 20000 daltons.

It should be noted that the compounds that prevent aggregation do not necessarily require lipid conjugation to function properly. Free PEG or free ATTA in solution may be sufficient to prevent aggregation. If the particles are stable after preparation into a formulation, the PEG or ATTA can be removed by dialysis prior to administration to a subject.

The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethylene glycol (PEG) -lipid, including, but not limited to, PEG-Diglyceride (DAG), PEG-Dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. For example, the PEG-DAA conjugate can be PEG-dilauryloxypropyl (Ci)₂) PEG-dimyristyloxypropyl (Ci)₄) PEG-dipalmitoyloxypropyl (Ci)₆) Or PEG-distearyloxypropyl (Ci)₈). Other conjugated lipids include polyethylene glycol-dimyristate glyceride (C14-PEG or PEG-C14, wherein the average molecular weight of PEG is 2000Da) (PEG-DMG); (R) -2, 3-dioctadecyloxy) propyl 1- (methoxypoly (ethylene glycol) 2000) propylcarbamate) (PEG-DSG); PEG-carbamoyl-1, 2-dimyridyloxypropylamine, wherein the average molecular weight of PEG is 2000Da (PEG-cDMA); n-acetylgalactosamine- ((R) -2, 3-dioctadecyloxy) propyl 1- (methoxypoly (ethylene glycol) 2000) propylcarbamate)) (GalNAc-PEG-DSG); and polyethylene glycol-dipalmitoyl glyceride (PEG-DPG).

In one embodiment the conjugated lipid is PEG-DMG. In another embodiment the conjugated lipid is PEG-cDMA. In yet another embodiment the conjugated lipid is PEG-DPG. Or the conjugated lipid is GalNAc-PEG-DSG.

The conjugated lipid that prevents aggregation of the particles may be 0 mol% to about 20 mol% or about 0.5 to about 5.0 mol% or about 2 mol% of the total lipid present in the particles.

When present, the sterol component of the lipid mixture may be any sterol typically used in the art of liposome, lipid vesicle or lipid particle formulation. The preferred sterol is cholesterol.

In some embodiments, the nucleic acid-lipid particle further comprises a sterol, such as cholesterol, for example from about 10 mol% to about 60 mol% or from about 25 to about 40 mol% or about 48% of the total lipid present in the particle.

Lipoprotein

In one embodiment, the formulation of the invention further comprises an apolipoprotein. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoproteins and variants and fragments thereof known to those skilled in the art, as well as to apolipoprotein agonists, analogs or fragments thereof, as described below.

Suitable apolipoproteins include, but are not limited to, ApoA-I, ApoA-II, ApoA-IV, ApoA-V, and ApoE, and active polymorphs, isoforms, variants and mutants and fragments or truncated forms thereof. In certain embodiments, the apolipoprotein is a thiol-containing apolipoprotein. "thiol-containing apolipoprotein" refers to an apolipoprotein, variant, fragment or isoform that contains at least one cysteine residue. The most commonly used thiol-containing apolipoprotein is ApoA-I Milano (ApoA-I) containing a cysteine residue _M) And ApoA-I Paris (ApoA-I)_P) (Jia et al, 2002, biochem. biophysis. res. comm.297: 206-13; bielicki and Oda, 2002, Biochemistry 41: 2089-96). ApoA-II, ApoE2 and ApoE3 are also thiol-containing apolipoproteins. Isolated ApoE and/or active fragments and polypeptide analogs thereof (including recombinantly produced forms thereof) are described in U.S. patent nos.5,672,685; 5,525,472, respectively; 5,473,039, respectively; 5,182,364, respectively; 5,177,189, respectively; 5,168,045, respectively; 5,116,739, all disclosures of which are incorporated herein by reference. ApoE3 is described in Weisgraber, et al, "Human E aproprotein geneticity: cysteine-converting in the amino acid sequence of the apo-Eisoforms, "J.biol.chem. (1981) 256: 9077-containing 9083 and Rall, et al, "structural library for receiver binding kinetics of apolipoproteinE from type IIIhyperlipoproteinemic subjects, "proc.nat.acad.sci. (1982) 79: 4696-.

In certain embodiments, the apolipoprotein can be in its mature form, its preproapoprotein form, or its preproapoprotein form. It is also possible within the scope of the present invention to use both the original ApoA-I and the mature ApoA-I (Duverger et al, 1996, Arterioscler.Thromb.Vasc. biol.16 (12): 1424-29), ApoA-I Milano (Klon et al, 2000, Biophys.J.79 (3) 1679-87; France schini et al, 1985, J.biol.chem.260: 1632-35), ApoA-I Paris (Daum et al, 1999, J.mol.Med.77: 614-22), ApoA-II (Shelness et al, 1985, J.biol.chem.260 (14): 8637-46; Shelness et al, 1984, J.biol.chem.259 (15): 9929-35), ApoA-IV (verger et al, 1991, J.chem.2. J.892. 1984, J.biol.259 (15): 9929-35), ApoA-IV (Duchen.201, 1985, Biochem.201, 1983, Biochem.93, 1983, ISO-93, and Biochem et al (93).

In certain embodiments, the apolipoprotein can be a fragment, variant, or isoform of an apolipoprotein. The term "fragment" refers to any apolipoprotein having an amino acid sequence shorter than that of the native apolipoprotein, and which fragment retains the activity, including lipid binding, of the native apolipoprotein. "variant" refers to a substitution or alteration of the amino acid sequence of an apolipoprotein which does not abrogate the activity of the native apolipoprotein, including lipid binding properties (e.g., addition or deletion of amino acid residues). Thus, a variant may comprise a protein or peptide having an amino acid sequence substantially identical to a native apolipoprotein provided by the present invention, wherein one or more amino acid residues are conservatively substituted with a chemically similar amino acid. Examples of conservative substitutions include the substitution of at least one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another. Likewise, for example, the present invention relates to substitutions between at least one hydrophilic residue such as arginine and lysine, glutamine and asparagine, and glycine and serine (see U.S. Pat. nos.6,004,925, 6,037,323 and 6,046,166). The term "isoform" refers to products which have the same, more or partial function and similar, identical or partial sequence and which may or may not be the same gene and proteins which are usually tissue-specific (see Weisgraber 1990, J.Lipid Res.31 (8): 1503-11; Hixson and Powers 1991, J.Lipid Res.32 (9): 1529-35; Lackner et al, 1985, J.biol.chem.260 (2): 703-6; Hoeg et al, 1986, J.biol.chem.261 (9): 3911-4; Gordon et al, 1984, J.biol.chem.259 (1): 468-74; Powell et al, 1987, Cell 50 (6): 831-40; Aviram et al, 1998, Arterioscsci.Va.18 (10): 7-24; Dis.1618; Cell 50; Cell 75-40; Aviram et al, 1998, Inc.42; Aviram.42; Metrang.42, J.29-42; Metrang.42, J.29; Ne.7, 1984; Ne.32, J.28; Ne.29; Ne.7, J.7, Biol.9; Ne.7, J.7, 1988; Ne.7, J.7, Biol.9; Ne.7, Biol.9; Ne.9; Ne.7, Ne.9; Ne, 1980, j.biol.chem.255 (21): 10464-71; dyer et al, 1995, j.lipid res.36 (1): 80-8 parts of; sacre et al, 2003, FEBS Lett.540 (1-3): 181-7; weers, et al, 2003, biophysis. chem.100 (1-3): 481-92; gong et al, 2002, j.biol.chem.277 (33): 29919-26; ohta et al, 1984, j.biol.chem.259 (23): 14888-93 and U.S. Pat. No.6,372,886).

In certain embodiments, the methods and compositions of the invention include chimeric constructs using apolipoproteins. For example, chimeric constructs of apolipoproteins may consist of an apolipoprotein domain having a high lipid binding capacity associated with an apolipoprotein domain comprising ischemia reperfusion protective properties. The chimeric construct of the apolipoprotein may be a construct including a separation region within the apolipoprotein (i.e., a homologous construct) or the chimeric construct may be a construct including a separation region between different apolipoproteins (i.e., a heterostructure). Compositions containing the chimeric constructs may also include fragments that are variants of apolipoproteins or that are designed to have specific properties (e.g., lipid binding, receptor binding, enzyme activation, antioxidant or reduction-oxidative properties) (see Weisgraber 1990, J.Lipid Res.31 (8): 1503-11; Hixson and Powers 1991, J.Lipid Res.32 (9): 1529-35; Lackner et al, 1985, J.biol.chem.260 (2): 703-6; Hoeg et al, 1986, J.biol.chem.261 (9): 3911-4; Gordon et al, 1984, J.biol.chem.259 (1): 468-74; Powell et al, 1987, 50 (6): 831-40; Aviram et al, 1998, Arterioscscler.Thromb.Va.18 (10-1617; 1987; 50; 19. cell.275; Aviram.42; Aviram et al, 1988; Avermen.42; Metrang.42; Metrang.43; 1989; see, biol chem.260 (4): 2258-64; widler et al, 1980, j.biol.chem.255 (21): 10464-71; dyer et al, 1995, j.lipid res.36 (1): 80-8 parts of; sorenson et al, 1999, arierioscler, thromb, vasc, biol.19 (9): 2214-25; palgunachari 1996, arierioscler, biob, vasc, biol.16 (2): 328-38: thurberg et al, j.biol.chem.271 (11): 6062-70; dyer 1991, j.biol.chem.266 (23): 150009-15; hill 1998, j.biol.chem.273 (47): 30979-84).

The apolipoproteins used in the present invention also include recombinant, synthetic, semi-synthetic or purified apolipoproteins. The methods used in the present invention for obtaining apolipoproteins or equivalents thereof are well known in the art. For example, apolipoproteins can be isolated from plasma or natural products, e.g., by density gradient centrifugation or immunoaffinity chromatography, or prepared by recombinant DNA techniques or synthetically, semisynthetically as known in the art (see, e.g., Mulugeta et al, 1998, J.Chromatogr.798 (1-2): 83-90; Chung et al, 1980, J.Lipid Res.21 (3): 284-91; Cheung et al, 1987, J.Lipid Res.28 (8): 913-29; Persson, et al, 1998, J.Chromatogr.711: 97-109; U.S.Pat.Nos.5, 528, 5, 05596, 5,876,968 and 5,721,114; and PCT publications WO86/04920 and WO 87/02062).

The apolipoproteins used in the present invention also include apolipoprotein agonists, such as the mimetic ApoA-I, ApoA-I Milano (ApoA-I)_M)、ApoA-I Paris(ApoA-I_P) ApoA-II, ApoA-IV and ApoE. For example, the apolipoprotein can be any of the apolipoproteins described in U.S. Pat. Nos.6,004,925, 6,037,323, 6,046,166 and 5,840,688, the contents of which are incorporated by reference The formula is incorporated herein.

The apolipoprotein agonist peptide or peptide analog can be synthesized or prepared using any peptide synthesis technique known in the art, including, for example, the techniques described in U.S. Pat. nos.6,004,925, 6,037,323 and 6,046,166. For example, the peptide may be prepared using the solid phase synthesis technique first described by Merrifield (1963, J.Am.chem.Soc.85: 2149-. Other Peptide Synthesis techniques can be found in Peptide Synthesis by Bodanszky et al, John Wiley & Sons, 2d Ed. (1976) and other references readily available to those skilled in the art. A summary of polypeptide synthesis techniques can be found in Stuart and Young's Solid Phase peptide synthesis, Pierce chemical company, Rockford, Ill., (1984). Peptides can also be synthesized by solution methods as described in The Proteins, Vol.II, 3d Ed., Neurath et al, eds., p.105-237, Academic Press, New York, N.Y. (1976). Suitable protecting Groups for the different peptide syntheses are described in the above-mentioned documents and also in McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973). The peptides of the invention may also be prepared by chemical or enzymatic cleavage of a larger portion of, for example, apolipoprotein a-I.

In certain embodiments, the apolipoprotein can be an apolipoprotein mixture. In one embodiment, the apolipoproteins may be a homogeneous mixture, i.e., a single species of apolipoprotein. In another embodiment, the apolipoproteins may be a heterogeneous mixture of apolipoproteins, i.e., a mixture of two or more different apolipoproteins. Embodiments of the xenogenic mixture of apolipoproteins may for example comprise a mixture of apolipoproteins of animal origin and apolipoproteins of semisynthetic origin. In certain embodiments, the heterogeneous mixture may comprise, for example, a mixture of ApoA-I and ApoA-I Milano. In certain embodiments, the heterogeneous mixture may comprise, for example, a mixture of ApoA-I Milano and ApoA-I Paris. Suitable mixtures for use in the methods and compositions of the present invention will be apparent to those skilled in the art.

If the apolipoprotein is obtained from a natural source, it may be obtained from a plant or animal source. If the apolipoprotein is obtained from an animal source, the apolipoprotein can be from any species. In certain embodiments, the apolipoprotein can be obtained from an animal source. In certain embodiments, the apolipoprotein can be obtained from a human source. In a preferred embodiment of the invention, the apolipoprotein is derived from the same species as the individual to whom the apolipoprotein is administered.

Other Components

In many embodiments, amphipathic lipids are included in the lipid particles of the present invention. By "amphipathic lipid" is meant any suitable material wherein the hydrophobic part of the lipid material is directed towards the hydrophobic phase and the hydrophilic part is directed towards the aqueous phase. Such compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids. Typical phospholipids include sphingomyelin, lecithin, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysolecithin, lysophosphatidylethanolamine, dipalmitoyl oleoyl phosphatidylcholine, dioleoyl lecithin, distearoyl lecithin or dioleyl lecithin. Other phosphorus-free compounds may also be used, such as sphingolipids, glycosphingolipids, diglycerides and beta-acyloxy acids. Such amphiphilic lipids can be readily mixed with other lipids such as triglycerides and sterols.

Also suitable for inclusion in the lipid particle of the invention are programmable fusogenic lipids. Such lipid particles have a tendency to fuse with the cell membrane and deliver their payload until a given signaling event occurs. This allows the lipid particles to be more evenly distributed after injection into the organism or lesion, which then begins to fuse with the cells. The signaling event may be, for example, a change in pH, temperature, ionic environment, or time. In the latter case, the fusion retarding or "capping" component, e.g., the ATTA-lipid conjugate or the PEG-lipid conjugate, may simply be exchanged out of the lipid particle membrane over time. Typical lipid anchors include a length of about C ₁₄To about C₂₂Preferably about C₁₄To about C₁₆Those of (a). In some embodiments, a PEG moiety (e.g., mPEG-NH)₂) Is about 1000, 2000, 5000, 10000, 15000 or 20000 daltons.

The lipid particle conjugated to the nucleic acid agent may also include a targeting moiety, e.g., a targeting moiety that specifically targets a cell type or tissue. Targeting of lipid particles using a variety of targeting moieties such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin), and monoclonal antibodies has been previously described (see, e.g., U.S. Pat. nos.4,957,773 and 4,603,044). The targeting moiety may comprise the entire protein or a fragment thereof. The targeting mechanism typically requires that the targeting agent be located on the surface of the lipid particle in such a way that the targeting moiety is available to interact with the target (e.g., a cell surface receptor). A variety of different targeting agents and methods are known and available in the art, including those described in Sapra, p.and Allen, TM, prog.lipid res.42 (5): 439-62 (2003); and Abra, RM et al, j.liposome res.12: 1-3, (2002).

Surface coatings of lipid particles, i.e., Liposomes, together with hydrophilic polymer chains, such as polyethylene glycol (PEG) chains, have been proposed for targeting (Allen, et al, Biochimica et Biophysica Acta 1237: 99-108 (1995); Defees, et al, Journal of the American chemistry society 118: 6101-, et al, Journal of liposome Research 2: 321- + 334 (1992); kirpotin et al, FEBS Letters 388: 115-118(1996)).

Standard methods of coupling targeting agents can be used. For example, phosphatidylethanolamine, which can be activated for attachment of targeting agents, or derivatized lipophilic compounds, such as lipid-derivatized bleomycin, can be used. Liposomes targeted to antibodies can be constructed, for example, using Liposomes that bind protein A (see, Rennessen, et al, J.Bio.Chem., 265: 16337-16342(1990) and Leonetti, et al, Proc.Natl.Acad.Sci. (USA), 87: 2448-2451 (1990). other examples of antibody conjugates are disclosed in U.S. Pat. No.6,027,726, the teachings of which are incorporated herein by reference.

Preparation of nucleic acid-lipid particles

In one embodiment, the nucleic acid-lipid particle formulation of the present invention is prepared via an extrusion process or an in-line mixing process.

Extrusion (also known as pre-forming or batch) is a process in which empty liposomes (i.e., without nucleic acid) are first prepared and then the nucleic acid is added to the empty liposomes. Extrusion of the liposomal composition through a small pore polycarbonate membrane or a non-uniform ceramic membrane results in a relatively well-defined particle size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired liposome complex particle size distribution is achieved. The liposomes can be continuously extruded through a small pore membrane to achieve a gradual reduction in liposome size. In some cases, the formed lipid-nucleic acid composition can be used without the need for sizing. These methods are disclosed in US 5,008,050; US 4,927,637; US 4,737,323; biochim Biophys acta.1979 Oct 19; 557(1): 9-23; biochim Biophys acta.1980 Oct 2; 601(3): 559-7; biochim Biophys acta.1986 Jun 13; 858(1): 161-8; and Biochim.Biophys.acta 1985812, 55-65, which are incorporated herein by reference in their entirety.

The in-line mixing method is a method of adding a lipid and a nucleic acid together into a mixing chamber. The mixing chamber may be a simple T-connector or any other mixing chamber known to those skilled in the art. These processes are disclosed in U.S. Pat. nos.6,534,018 and US 6,855,277; U.S. publication No. 2007/0042031 and Pharmaceuticals Research, Vol.22, No.3, Mar.2005, p.362-372, which are incorporated herein by reference in their entirety.

It is also understood that the formulations of the present invention may be prepared by any method known to those skilled in the art.

Characterization of nucleic acid-lipid particles

Formulations prepared by standard or non-extrusion methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. They should be white clear solutions free of aggregates or precipitates. The particle size and particle size distribution of the lipid-nanoparticles can be determined by light scattering, for example using Malvern zetasizer Nano ZS (Malvern, USA). The size of the particles should be about 20-300nm, e.g. 40-100 nm. The particle size distribution should be monomodal. The total siRNA concentration in the formulation was evaluated using a dye exclusion assay, as well as the capture fraction. The formulated siRNA samples may be incubated with RNA-binding dyes such as Ribogreen (molecular probes) in the presence or absence of a surfactant that disrupts the formulation, such as 0.5% Triton-X100. The total siRNA in the formulation can be determined relative to a standard curve from the signal emitted in the surfactant-containing sample. The capture fraction was determined by subtracting the "free" siRNA content (determined from the signal in the absence of surfactant) from the total siRNA content. The percentage of captured siRNA is typically > 85%. In one embodiment, the formulation of the invention is at least 75%, at least 80%, or at least 90% captured.

For nucleic acid-lipid particle formulations, the particle size is at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 110nm, and at least 120 nm. Suitable ranges are generally from about at least 50nm to about at least 110nm, from about at least 60nm to about at least 100nm, or from about at least 80nm to about at least 90 nm.

Nucleic acid-lipid particle preparation

LNP01

An example of a synthetic nucleic acid-lipid particle is as follows. Nucleic acid-lipid particles were synthesized using the lipid ND 98.4 HCl (MW1487) (formula 1), cholesterol (Sigma-Aldrich), and PEG-ceramide C16(Avanti Polarlipids). The nucleic acid-lipid particles are sometimes referred to as LNP01 particles. Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; cholesterol, 25mg/ml, PEG-ceramide C16, 100 mg/ml. Stock solutions of ND98, cholesterol, and PEG-ceramide C16 may then be combined, for example, in a molar ratio of 42: 48: 10. The combined lipid solution is mixed with the aqueous siRNA (e.g., in sodium acetate at pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Once mixed, lipid-siRNA nanoparticles typically form spontaneously. Depending on the desired particle size distribution, the resulting nanoparticle mixture may be extruded through a polycarbonate membrane (e.g., 100nm cut-off), for example, using a thermal barrier extruder, such as a Lipex extruder (northern lipids, Inc). In some cases, the extrusion step may be omitted. Ethanol removal and concurrent buffer exchange can be achieved by, for example, dialysis or tangential flow filtration. The buffer can be exchanged for, for example, Phosphate Buffered Saline (PBS) at about pH 7, e.g., at about pH 6.9, at about pH 7.0, at about pH 7.1, at about pH 7.2, at about pH 7.3, or at about pH 7.4.

LNP01 formulations are described, for example, in international application publication No. wo2008/042973, which is incorporated herein by reference.

Other typical nucleic acid-lipid particle formulations are described in the following table. It is understood that the names of the nucleic acid-lipid particles in the table are not limiting. For example, as used herein, the term SNALP means a formulation that includes the cationic lipid DLinDMA.

XTC-containing formulations are described, for example, in U.S. provisional serial No. 61/239,686 filed on 9/3 in 2009, which is incorporated herein by reference.

Formulations containing MC3 are described, for example, in U.S. provisional serial No. 61/244,834 filed on day 9/22 2009 and U.S. provisional serial No. 61/185,800 filed on day 6/10 2009, which are incorporated herein by reference.

Formulations containing ALNY-100 are described, for example, in international patent application PCT/US09/63933 filed 11/10/2009, which is incorporated herein by reference.

Other exemplary formulations are described in tables 25 and 26. Lipid refers to a cationic lipid.

Table 25: composition of typical nucleic acid-lipid particles prepared via extrusion method (mol%)

Table 26: compositions of typical nucleic acid-lipid particles prepared via in-line mixing process

Synthesis of cationic lipids

Any of the compounds used in the nucleic acid-lipid particles of the present invention, such as cationic lipids, etc., can be prepared by known organic synthesis techniques, including the methods described in more detail in the examples. Unless otherwise indicated, all substituents are defined below.

"alkyl" refers to straight or branched chain, acyclic or cyclic, saturated aliphatic hydrocarbons containing from 1 to 24 carbon atoms. Typical saturated straight chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; and saturated branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Typical saturated cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; and unsaturated cyclic alkyl groups include cyclopentenyl and cyclohexenyl, and the like.

"alkenyl" refers to an alkyl group as defined above that contains at least one double bond between adjacent carbon atoms. Alkenyl includes cis and trans isomers. Typical straight and branched alkenyl groups include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2, 3-dimethyl-2-butenyl, and the like.

"alkynyl" refers to any alkyl or alkenyl group as defined above that further contains at least one triple bond between adjacent carbon atoms. Typical straight and branched alkynyl groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

"acyl" refers to any alkyl, alkenyl, or alkynyl group substituted at the carbon at the point of attachment with an oxy group, as defined below. For example, -C (═ O) alkyl, -C (═ O) alkenyl, and-C (═ O) alkynyl are acyl groups.

"heterocycle" means a 5-to 7-membered monocyclic, or 7-to 10-membered bicyclic, heterocyclic ring which may be saturated, unsaturated, or aromatic, and which contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and which nitrogen and sulfur heteroatoms may be optionally oxidized, and which nitrogen heteroatoms may be optionally quaternized, and which heterocyclic ring also includes any bicyclic ring in which the above heterocyclic ring is fused to a benzene ring. The heterocyclic ring may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactam, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl, tetrahydrothiopyranyl, and the like.

The terms "optionally substituted alkyl", "optionally substitutedThe terms "alkenyl", "optionally substituted alkynyl", "optionally substituted acyl" and "optionally substituted heterocycle" refer to when substituted, at least one hydrogen atom is replaced by a substituent. In the case of an oxygen substituent (═ O) two hydrogen atoms are replaced. In this regard, substituents include oxygen, halogen, heterocycle, -CN, -OR^x、-NR^xR^y、-NR^xC(＝O)R^y、-NR^xSO₂R^y、-C(＝O)R^x、-C(＝O)OR^x、-C(＝O)NR^xR^y、-SO_nR^xand-SO_nNR^xR^yWherein n is 0, 1 or 2, R^xAnd R^yThe same or different, independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of the following groups: oxygen, halogen, -OH, -CN, alkyl, -OR^xHeterocyclic radical, -NR^xR^y、-NR^xC(＝O)R^y、-NR^xSO₂R^y、-C(＝O)R^x、-C(＝O)OR^x、-C(＝O)NR^xR^y、-SO_nR^xand-SO_nNR^xR^y。

"halogen" refers to fluorine, chlorine, bromine and iodine.

In some embodiments, the methods of the present invention may require the use of protecting groups. Protecting group methods are well known to those skilled in the art (see, e.g., Protective Groups in organic Synthesis, Green, T.W. et al, Wiley-Interscience, New York City, 1999). In short, a protecting group within the context of the present invention is any group that reduces or eliminates the reactivity of an undesired functional group. Protecting groups may be added to the functional groups to shield them from reactivity during certain reactions, and then removed to reveal the original functional groups. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the reactivity of an unwanted alcohol functional group. Protecting groups may be added and removed using techniques well known in the art.

A compound of the formula ABecome into

In one embodiment, the nucleic acid-lipid particles of the invention are formulated with a cationic lipid of formula a:

wherein R1 and R2 are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 may together form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2, 2-dioleyl-4-dimethylaminoethyl- [1, 3] -dioxolane). Generally, the lipids of formula a above can be prepared by the following reaction schemes 1 or 2, wherein all substituents are as defined above unless otherwise indicated.

Scheme 1

Lipid A can be prepared according to scheme 1, wherein R₁And R₂Independently is alkyl, alkenyl or alkynyl, each of which may be optionally substituted, and R₃And R₄Independently is lower alkyl or R₃And R₄May be taken together to form an optionally substituted heterocyclic ring. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 1 and 2 produced ketal 3. Treatment of ketal 3 with amine 4 produces lipids of formula a. Lipids of formula a can be converted to the corresponding ammonium salts with organic salts of formula 5, wherein X is an anionic counterion selected from halogen, hydroxide, phosphate, sulfate, and the like.

Scheme 2

Alternatively, the ketone 1 starting material can be prepared according to scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 6 and 7 produces ketone 1. The conversion of ketone 1 to the corresponding lipid of formula a is described in scheme 1.

Synthesis of MC3

DLin-M-C3-DMA (i.e., 4- (dimethylamino) butanoic acid (6Z, 9Z, 28Z, 31Z) -thirty-seven carbon-6, 9, 28, 31-tetraen-19-yl ester)) was prepared as follows. A solution of (6Z, 9Z, 28Z, 31Z) -thirty-seven-carbon-6, 9, 28, 31-tetraen-19-ol (0.53g), 4-N, N-dimethylaminobutyric acid hydrochloride (0.51g), 4-N, N-dimethylaminopyridine (0.61g) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.53g) in methylene chloride (5mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid and then with dilute aqueous sodium bicarbonate. The organic portion was dried over anhydrous magnesium sulfate, filtered and the solvent removed on a rotary evaporator. The residue was passed through a silica gel column (20g) eluting with a gradient of 1-5% methanol in dichloromethane. The fractions containing the purified product were combined and the solvent was removed to give a colorless oil (0.54 g).

Synthesis of ALNY-100

The synthesis of ketal 519[ ALNY-100-100] was performed using scheme 3 below:

515 synthesis:

to a stirred suspension of LiAlH4(3.74g, 0.09852mol) in 200ml anhydrous THF in two-necked RBF (1L) under nitrogen atmosphere at 0 deg.C was slowly added 514(10g, 0.04926mol) in 70ml THF. After complete addition, the reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reactionMonitored by TLC. After the reaction was complete (monitored by TLC), the mixture was cooled to 0 ℃ and saturated Na was added carefully₂SO₄The solution was quenched. The reaction mixture was stirred at room temperature for 4h and filtered. The residue was washed appropriately with THF. The filtrate and washings were mixed and diluted with 400ml dioxane and 26ml concentrated HCl and stirred at room temperature for 20 minutes. Volatiles were removed under vacuum to give 515 as the hydrochloride salt as a white solid. Yield: 7.12g, 1H-NMR (DMSO, 400 MHz): 9.34 (width, 2H), 5.68(s, 2H), 3.74(m, 1H), 2.66-2.60(m, 2H), 2.50-2.45(m, 5H).

516 synthesis:

to a stirred solution of compound 515 in 100mL of anhydrous DCM in 250mL of a two-necked RBF was added NEt3(37.2mL, 0.2669mol) and cooled to 0 ℃ under nitrogen. After the N- (benzyloxy-carbonyloxy) -succinimide (20g, 0.08007mol) in 50mL of anhydrous DCM was slowly added, the reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3h, monitored by TLC), 1N HCl solution (1X100mL) and saturated NaHCO were used ₃The mixture was washed successively with solution (1 × 50 mL). Then using anhydrous Na₂SO₄The organic layer was dried and the solvent was evaporated to give a crude material which, after purification by silica gel column chromatography, gave 516 as a sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): 7.36-7.27(m, 5H), 5.69(s, 2H), 5.12(s, 2H), 4.96(br., 1H)2.74(s, 3H), 2.60(m, 2H), 2.30-2.25(m, 2H). LC-MS [ M + H ]]-232.3(96.94％)。

517A and 517B:

cyclopentene 516(5g, 0.02164mol) was dissolved in a single necked 500mLRBF in 220mL of acetone and water (10: 1) and to this were added N-methylmorpholine-N-oxide (7.6g, 0.06492mol) and 4.2mL of a 7.6% solution of OsO4(0.275g, 0.00108mol) in tert-butanol at room temperature. After the reaction was complete (about 3h), by adding solid Na₂SO₃The mixture was quenched and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300mL) and water (2 × 100mL) followed by saturated NaHCO₃(1X50mL) solution, water (1X30mL) and finallyBrine (1 × 50mL) wash. With anhydrous Na₂SO₄The organic phase was dried and the solvent was removed in vacuo. Purification of the crude material by silica gel column chromatography gave a mixture of diastereomers, which was separated by preparative HPLC. Yield: 6g of crude product.

517A-Peak-1 (white solid), 5.13g (96%). 1H-NMR (DMSO, 400 MHz): 7.39-7.31(M, 5H), 5.04(s, 2H), 4.78-4.73(M, 1H), 4.48-4.47(d, 2H), 3.94-3.93(M, 2H), 2.71(s, 3H), 1.72-1.67(M, 4H), LC-MS- [ M + H ] -266.3, [ M + NH4+ ] -283.5 present, HPLC-97.86%. Stereochemistry was confirmed by X-ray.

518 Synthesis:

compound 518(1.2g, 41%) was obtained as a colorless oil using a method analogous to that described for the synthesis of compound 505. 1H-NMR (CDCl3, 400 MHz): 7.35-7.33(m, 4H), 7.30-7.27(m, 1H), 5.37-5.27(m, 8H), 5.12(s, 2H), 4.75(m, 1H), 4.58-4.57(m, 2H), 2.78-2.74(m, 7H), 2.06-2.00(m, 8H), 1.96-1.91(m, 2H), 1.62(m, 4H), 1.48(m, 2H), 1.37-1.25(br m, 36H), 0.87(m, 6H) HPLC-98.65%.

General procedure for synthesis of compound 519:

a solution of compound 518(1eq) in hexane (15mL) was added dropwise to an ice-cold solution of LAH in THF (1M, 2 eq). After complete addition, the mixture was heated at 40 ℃ for 0.5h and then cooled again on an ice bath. With saturated Na₂SO₄The aqueous solution was carefully hydrolyzed and then filtered through Celite (Celite) to reduce to oil. Column chromatography afforded purified 519(1.3g, 68%) as a colorless oil. 13C NMR 130.2, 130.1(x2), 127.9(x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9(x2), 29.7, 29.6(x2), 29.5(x3), 29.3(x2), 27.2(x3), 25.6, 24.5, 23.3, 226, 14.1; electrospray MS (+ ve): molecular weight (M + H) + calcd for C44H80NO2, 654.6, found 654.6.

Therapeutic agent-lipid particle compositions and formulations

The invention includes compositions comprising the lipid particle of the invention and an active agent, wherein the active agent is associated with the lipid particle. In a specific embodiment, the active agent is a therapeutic agent. In particular embodiments, the active agent is encapsulated within the aqueous interior of the lipid particle. In other embodiments, the active agent is present in one or more lipid layers of the lipid particle. In other embodiments, the active agent is associated with the outer or inner lipid surface of the lipid particle.

As used herein, "fully encapsulated" refers to nucleic acids in particles that are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA. In a fully encapsulated system, preferably less than 25% of the particulate nucleic acid is degraded in a treatment that typically degrades 100% of the free nucleic acid, more preferably less than 10% and most preferably less than 5% of the particulate nucleic acid is degraded. Alternatively, the complete encapsulation may be by OligreenAnd (4) testing and measuring. OligreenIs an ultrasensitive fluorescent nucleic acid stain (available from Invitrogen Corporation, Carlsbad, CA) for quantifying oligonucleotides and single-stranded DNA in solution. Complete encapsulation also suggests that the particles are serum stable, i.e., they do not rapidly break down into their constituent parts once administered in vivo.

Active agents useful in the present invention include any molecule or compound that is capable of exerting a desired effect on a cell, tissue, organ or subject. This effect may be biological, physiological or cosmetic, for example. The active agent can be any kind of molecule or compound, including, for example, nucleic acids, peptides, and polypeptides, including, for example, antibodies, such as polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibody, recombinant human antibody and Primatized^TMAntibodies, cytokines, growth factors, apoptosis factors, differentiation-inducing factors, cell surface receptors and their ligands; laserA peptide; and small molecules, including organic small molecules or compounds.

In one embodiment, the active agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives may be therapeutically active themselves, or they may be prodrugs which become active upon further modification. Thus, in one embodiment, the therapeutic agent derivative retains some or all of its therapeutic activity as compared to the unmodified agent, while in another embodiment, the therapeutic agent derivative lacks therapeutic activity.

In various embodiments, the therapeutic agent includes any therapeutically effective agent or drug, such as anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, birth control drugs, antipyretics, vasodilators, antiangiogenic agents, cytovascular agents (cytostatic agents), signal transduction inhibitors, cardiovascular agents (e.g., antiarrhythmics), vasoconstrictors, hormones, and steroids.

In certain embodiments, the therapeutic agent is an oncology drug, which may also be referred to as an antineoplastic, anticancer, oncology, antineoplastic agent, or the like. Examples of oncological drugs which may be used in the present invention include, but are not limited to, Adriamycin, Levolysin, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, amitrazole, Bexarotene, bicNU, bleomycin, intravenous Marilan, oral Marilan, Capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, Cyclosporine A, fluorouracil, cytarabine, daunorubicin, canceroid, daunorubicin, dexamethasone, dexrazoxane, polyenic taxol, Adriamycin, DTIC, epirubicin, estramustine, Etoposide phosphate, Etoposide and VP-16, exemestane, FK506, Fludarabine, Fluorouracil, 5-Gemcitabine, Tozar-Gezar, Zolmicin-Gemcitabine, Zusamivir, allopurin, doxycycline, 5-Gemcitabine, doxycycline, and doxycycline, Goserelin acetate, hydroxyurea, daunorubicin, ifosfamide, imatinib mesylate, interferon, irinotecan (Camptostar, CPT-111), letrozole, leucovorin, clarithrone, leuprorelin, levamisole, alitretinoin, megestrol, melphalan, L-PAM, thioethyl sulfonate, methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone, mechlorethamine, taxol, disodium pamidronate, methoxypolyethylene glycol succinamide adenosine deaminase, pentostatin, porfimer sodium, prednisone, B cell monoclonal antibodies, streptomycin, STI-571, tamoxifen, taxotere, temozolomide, teniposide, VM-26, topotecan (Hycamtin), toremifene, tretinoin, ATRA, valrubicin, vinblastine, vincristine, vinblastine, VP16 and vinorelbine. Other examples of oncological drugs which may be used in the present invention are ellipticine and ellipticine analogues or derivatives, epothilones, intracellular kinase inhibitors and camptothecins.

Other formulations

Emulsion formulation

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another in the form of droplets generally exceeding 0.1 μm in diameter (Idson, in Pharmaceutical delivery Forms, Lieberman, Rieger and ers (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, p.199; Rosoff, in Pharmaceutical delivery Forms, Lieberman, Rieger and Bank (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, 245, Block in Pharmaceutical delivery Forms, Lieberman, Rieger and Bank (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, p.245; Block in Pharmaceutical delivery Forms, Lieger and Bank (Eds.), 1988, Marcel Dekker, York, N.Y., N.2, U.S., Inc., Pa., Inc., U.S.S.S.S.3, Inc., Japan, Inc., U.S.S.3, Inc., 2, U.S.S.S.3, Inc., U.S.S.S.S.S.S.S.S.S.S.S.A. Emulsions are generally two-phase systems comprising two immiscible liquid phases intimately mixed and dispersed in each other. In general, emulsions may be of the water-in-oil (w /) or oil-in-water (o/w) variety. When the aqueous phase is finely dispersed and dispersed as fine droplets in a bulk oil phase, the resulting composition is referred to as a water-in-oil (w/o) emulsion. Alternatively, when the oil phase is finely dispersed and dispersed as fine droplets in a bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o/w) emulsion. In addition to the dispersed phase, the emulsion may contain other components, and the active agent may be present as a solution in the aqueous phase, the oil phase, or as a separate phase itself. Pharmaceutical excipients such as emulsifiers, stabilizers, colorants and antioxidants may also be present in the emulsion if desired. The drug emulsion may also be a multiple emulsion consisting of two or more phases, for example, an oil-in-water-in-oil (o/w/o) emulsion and a water-in-oil-in-water (w/o/w) emulsion. Such complex formulations generally provide certain advantages not found with simple two-phase emulsions. In a multiple emulsion, the individual oil droplets of the o/w emulsion surround the water droplets to make up the w/o/w emulsion. Also, a system of water droplets surrounding oil droplets, stably present in an oil continuous phase, provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Generally, the dispersed or discontinuous phase of the emulsion is well dispersed in the external or continuous phase and the form is maintained by the viscosity of the emulsifier or formulation. Any phase of the emulsion may be semi-solid or solid, as is the case with emulsion-type ointment bases and creams. Other methods of stabilizing emulsions require the use of emulsifiers, which may be added to either phase of the emulsion. Emulsifiers can be broadly divided into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption matrices and finely divided solids (Idson, in pharmaceutical dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, inc., New York, n.y., volume 1, p.199).

Synthetic surfactants, also known as surfactants, have a wide range of applicability in emulsion formulations and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Bank (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Bank (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, Vol.1, page 199). Surfactants are typically amphiphilic and contain both hydrophilic and hydrophobic portions. The ratio of hydrophilicity to hydrophobicity of a surfactant is called the hydrophilic/lipophilic balance (HLB), which is an important tool in classifying and selecting surfactants in the preparation of formulations. Surfactants can be classified into different types based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, page 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and gum arabic. The absorbent matrices are hydrophilic so that they can absorb water to form a w/o emulsion while still retaining their semi-solid viscosity, e.g., anhydrous lanolin and hydrophilic petrolatum. Finely divided solids are also used as good emulsifiers, especially in combination with surfactants, and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, palygorskite, hectorite, kaolin, montmorillonite, colloidal aluminium and magnesium aluminium silicates, pigments and non-polar solids such as carbon or glycerol tristearate.

A variety of non-emulsifying materials may also be included in the emulsion formulation and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrocolloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Bank (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, page 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, page 199).

Hydrocolloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., gum arabic, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomers, cellulose ethers, and carboxyvinyl polymers). These materials are dispersed in or swell in water to form colloidal solutions which stabilize emulsions by forming strong interfacial films around dispersed phase droplets and by increasing the viscosity of the external phase.

Preservatives are often added to these formulations because emulsions typically contain many ingredients that readily support microbial growth, such as carbohydrates, proteins, sterols, and phospholipids. Common preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, parabens, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. The antioxidant used may be a radical scavenger, such as tocopherol, alkyl gallate, butyl hydroxyanisole, butyl hydroxytoluene; or reducing agents, such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid and lecithin.

The use of emulsion formulations via the cutaneous, oral and parenteral routes and their preparation have been documented (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and dbanker (Eds.), 1988, Marcel Dekker, inc., New York, n.y., vol.1, page 199). Emulsion formulations for oral delivery have been widely used due to ease of formulation and efficacy in terms of absorption and bioavailability (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Bank (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, p.245; Idson, in Pharmaceutical Dosage Forms, Liebeman, Rieger and Bank (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, p.199). Mineral oil based laxatives, oil soluble vitamins and high fat nutritional formulations are materials that are usually administered orally as o/w emulsions.

In one embodiment of the invention, the dsRNA and nucleic acid composition is formulated as a microemulsion. Microemulsions can be defined as water, oil and amphiphilic systems which are single optically isotropic and thermodynamically stable liquid solutions (Rosoff, in Pharmaceutical delivery Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, inc., New York, n.y., vol.1, page 245). Microemulsions are generally systems prepared by the following method: the oil is first dispersed in an aqueous surfactant solution and then a sufficient amount of a fourth component, typically a medium chain length alcohol, is added to form a clear system. Microemulsions are therefore also referred to as thermodynamically stable, isotropic transparent dispersions of two immiscible liquids which are stabilized by an interfacial film of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and aggregations systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are typically prepared by combining three to five components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether a microemulsion is of the water-in-oil (w/o) or oil-in-water (o/w) type depends on the nature of the oil and surfactant used and the structural and geometric assembly of the polar head and hydrocarbon tail of the surfactant molecule (Schott, in Remington's pharmaceutical sciences, Mack Publishing co., Easton, Pa., 1985, p.271).

The phenomenological approach of using phase diagrams has been extensively studied by those skilled in the art and has gained comprehensive knowledge of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Bank (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, p.245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Bank (Eds.),. 8, Marcel Dekk, Inc., New York, N.Y., Vol.1, p.335). Microemulsions have the advantage of dissolving water-insoluble drugs in spontaneously formed thermodynamically stable droplet formulations compared to conventional emulsions.

Surfactants used to prepare the microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij96, polyoxyethylene oleyl ether, polyglycerol esters of fatty acids, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with co-surfactants. Due to the gaps created between the surfactant molecules, co-surfactants (typically short chain alcohols such as ethanol, 1-propanol and 1-butanol) are suitable for increasing interfacial fluidity by penetrating into the surfactant film and thus creating an abnormal film. However, microemulsions may be prepared without the use of co-surfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. Generally, the aqueous phase can be, but is not limited to, water, aqueous drug solutions, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, the following materials: such as Captex 300, Captex355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-and triglycerides, fatty acid glycerides of polyoxyethylene, fatty alcohols, polyglycolyzed glycerides, saturated polyglycolyzed C8-C10 glycerides, vegetable oils and silicone oils.

Microemulsions are of particular interest in terms of drug dissolution and enhanced drug absorption. Lipid-based microemulsions (o/w and w/o) have been proposed to improve the oral bioavailability of drugs, including peptides (Constantinides et al, Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, meth. find. exp. clin. pharmacol., 1993, 13, 205). Microemulsions have the following advantages: improved drug dissolution, avoidance of drug hydrolysis by enzymes, possible increased drug absorption due to surfactant-induced changes in membrane fluidity and permeability, ease of manufacture, ease of oral administration over solid dosage forms, improved clinical efficacy and reduced toxicity (Constantides et al, pharmaceutical research, 1994, 11, 1385; Ho et al, J.pharm.Sci., 1996, 85, 138-143). Microemulsions may generally form spontaneously when the components of the microemulsion are mixed together at ambient temperature. This may be particularly advantageous when formulating thermolabile drug peptides or dsrnas. Microemulsions are also effective in transdermal delivery of active ingredients in cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of dsRNA and nucleic acids from the gastrointestinal tract, as well as improved local cellular uptake of dsRNA and nucleic acids.

The microemulsions of the present invention may also contain other components and additives, such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of the formulation and to increase the absorption of the dsRNA and nucleic acids of the present invention. Penetration enhancers for microemulsions of the present invention may be divided into one of five major classes-surfactants, fatty acids, bile salts, chelating agents and non-chelating non-surfactants (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of these categories has been discussed above.

Penetration enhancer

In one embodiment, the present invention uses a variety of permeation enhancers to allow for the efficient delivery of nucleic acids, particularly dsRNA, to the skin of an animal. Most drugs exist in solution in ionized or non-ionized form. However, generally only lipid soluble or lipophilic drugs readily permeate cell membranes. It has been found that even non-lipophilic drugs can permeate cell membranes if the membrane to be permeated is treated with a permeation enhancer. In addition to helping the diffusion of non-lipophilic drugs across cell membranes, permeation enhancers also increase the permeability of lipophilic drugs.

Penetration enhancers can be divided into one of five broad categories, namely surfactants, fatty acids, bile salts, chelators, and non-chelating non-surfactants (Lee et al, clinical Reviews in therapeutic Drug carriers Systems, 1991, p.92). Each of the above categories of penetration enhancers is described in detail below.

Surfactant (b): according to the invention, a surfactant is a chemical entity that, when dissolved in an aqueous solution, is capable of reducing the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in an increased absorption of dsRNA through the mucosa. In addition to bile salts and fatty acids, such penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (Lee et al, clinical Reviews in Therapeutic drug carriers Systems, 1991, p.92); and perfluorinated chemical emulsions, such as FC-43. Takahashi et al, j.pharm.pharmacol, 1988, 40, 252).

Fatty acid: various fatty acids and derivatives thereof as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-capric acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, capric acid, tricaprinic acid, glycerol monooleate (1-monooleyl-rac-glycerol), dilaurin glycerol, caprylic acid, arachidonic acid, glycerol 1-monodecanoate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C thereof_1-10Alkyl esters (e.g., methyl, isopropyl, and t-butyl) and mono-and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al, Critical Reviews in Therapeutic drug carriers Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic drug carriers Systems, 1990, 7, 1-33; El Hariri et al, J.pharm.Pharmacol., 1992, 44, 651-654).

Bile salt: the physiological effects of bile include promoting The dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The pharmacological basis of Therapeutics, 9th Ed., Hardman et al eds., McGraw-Hill, New York, 1996, pp.934-935). Various natural bile salts and their synthetic derivatives are used as penetration enhancers. The term "bile salt" thus includes any naturally occurring bile component as well as any synthetic derivative thereof. Suitable bile salts include, for example, cholic acid (or a pharmaceutically acceptable sodium salt thereof, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycocholic acid (sodium glycocholate), glycolic acid (sodium glycolate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24, 25-dihydro-fusidate (STDHF), sodium glycerodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (Lee et al, clinical Reviews In therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaero, ed., Mackblising, Pushing Co, Pa., 1990, Mitsushi-3, 1990; Muishi, critical reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; yamamoto et al, j.pharm.exp.ther., 1992, 263, 25; yamashita et al, j.pharm.sci., 1990, 79, 579-583).

Chelating agent: a chelating agent as used herein may be defined as a compound that removes metal ions from solution by forming a complex with the metal ions, resulting in increased absorption of the dsRNA through the mucosa. When used as penetration enhancers in the present invention, chelators also have the additional advantage of acting as DNase inhibitors, being inhibited by chelators since most of the characterised DNA nucleases require catalysis by divalent metal ions (Jarrett, j. chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium Ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanillic acid salts), N-acyl derivatives of collagen, N-acyl derivatives of laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines) (Lee et al, Critical Reviews in Therapeutic Drug carriers Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug carriers Systems, 1990, 7, 1-33; Buur et al, J.Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: as used herein, a non-chelating non-surfactant penetration enhancer compound can be defined as a compound that does not exhibit significant chelator or surfactant activity, but still enhances the uptake of dsRNA through the trophic mucosa (Muranishi, clinical reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Such penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl-and 1-alkenyl azacyclo-alkanone derivatives (Lee et al, clinical Reviews in Therapeutic Drug carriers Systems, 1991, page 92); and non-steroidal anti-inflammatory drugs such as diclofenac, indomethacin, and phenylbutazone (Yamashita et al, j. pharm. pharmacol., 1987, 39, 621-626).

Agents that increase uptake of dsRNA at the cellular level may also be added to the pharmaceutical and other compositions of the invention. For example, cationic lipids are known, such as liposomes (lipofectins) (Junichi et al, u.s.pat. No.5,705,188); a cationic glycerol derivative; and polycationic molecules such as polylysine (Lollo et al, PCT application WO 97/30731) can enhance cellular uptake of dsRNA.

Other agents may be used to increase the permeability of the administered nucleic acid, including glycols, such as ethylene glycol and propylene glycol; pyrroles, such as 2-pyrrole; (ii) azone; and terpenes such as limonene and montelukast.

Carrier

The dsRNA of the invention can be formulated in a pharmaceutically acceptable carrier or diluent. A "pharmaceutically acceptable carrier" (also referred to herein as an "excipient") is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert medium. Pharmaceutically acceptable carriers can be liquid or solid and can be selected according to the intended mode of administration to provide the desired volume, compatibility, and other relevant transport and chemical properties. Typical pharmaceutically acceptable carriers include, for example, but are not limited to: water; a salt solution; a binder (e.g., polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol or sodium acetate); disintegrants (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

Certain compositions of the present invention also incorporate a carrier compound in their formulation. As used herein, a "carrier compound" or "carrier" can mean a nucleic acid or analog thereof that is inert (i.e., not biologically active itself), but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of the biologically active nucleic acid, e.g., by degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of nucleic acid and carrier compound (usually the latter in excess) can result in a significant reduction in the amount of nucleic acid recovered in the liver, kidney or other external circulating reservoirs, possibly due to competition of the carrier compound and nucleic acid for the co-receptor. For example, when co-administered with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4' isothiocyanato-stilbene-2, 2-disulfonic acid, recovery of a portion of phosphorothioate dsRNA in liver tissue can be reduced (Miyao et al, DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al, DsRNA & Nucl. acid drug Dev., 1996, 6, 177-183).

Excipient

In contrast to carrier compounds, "pharmaceutical carriers" or "excipients" are pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert medium for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected in the intended mode of administration to provide the desired bulk, compatibility, etc. when combined with the nucleic acid and other components of a given pharmaceutical composition. Typical drug carriers include, but are not limited to: binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, dibasic calcium phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

The compositions of the invention may also be formulated using pharmaceutically acceptable organic or inorganic excipients that do not deleteriously react with the nucleic acid and are suitable for non-parenteral administration. Suitable pharmaceutically acceptable carriers include, but are not limited to: water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of nucleic acids in liquid or solid oily matrices. The solution may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients which do not deleteriously react with nucleic acids and which are suitable for non-parenteral administration may also be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to: water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, and the like.

Other Components

The compositions of the present invention may also contain other auxiliary components commonly used in pharmaceutical compositions at usage levels established in the art. Thus, for example, the compositions may contain other compatible pharmaceutically active substances, e.g., antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain other materials useful in physically formulating various dosage forms of the compositions of the present invention, e.g., colorants, flavors, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, should not unduly interfere with the biological activity of the components of the compositions of the present invention. If desired, the formulations can be sterilized and mixed with auxiliary agents, for example, lubricants which do not deleteriously react with the nucleic acids of the formulations, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors and/or aromatic substances, etc.

Aqueous suspensions may contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. The suspension may also contain a stabilizer.

Combination therapy

In one aspect, the compositions of the invention are useful in combination therapy. The term "combination therapy" includes combinations of other biologically active ingredients (such as, but not limited to, a second and different anti-neoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation therapy) when administered to a subject compound. For example, the compounds of the present invention may be used in combination with other pharmaceutically active compounds, preferably compounds that enhance the effects of the compounds of the present invention. The compounds of the invention may be administered simultaneously (as a single formulation or separate formulations) or sequentially with other drug therapies. In general, combination therapy contemplates administration of two or more drugs during a single cycle or course of treatment.

In one aspect of the invention, the subject compounds can be administered in combination with one or more separate agents to modulate protein kinases associated with a variety of disease conditions. Examples of such kinases may include, but are not limited to: serine/threonine specific kinases, receptor tyrosine specific kinases, and non-receptor tyrosine specific kinases. Serine/threonine kinases include mitogen-activated protein kinase (MAPK), meiosis-specific kinase (MEK), RAF and aurora kinases. Examples of receptor kinase families include Epidermal Growth Factor Receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB2, ErbB3, ErbB4, Xmrk, DER, Let23), Fibroblast Growth Factor (FGF) receptor (e.g., FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R), hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA, SEX), insulin receptor (e.g., IGFI-R), Eph (e.g., CEK5, CEK8, EBK, ECK, EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g., Mer/Nyk, Rse); RET; platelet Derived Growth Factor Receptors (PDGFRs) (e.g., PDGF α -R, PDG β -R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1, FLT3/FLK 2/STK-1). The family of non-receptor tyrosine kinases includes, but is not limited to: BCR-ABL (e.g., p 43) ^ablARG); BTK (e.g., ITK/EMT, TEC); CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK, and SYK.

In another aspect of the invention, the subject compounds may be administered in combination with one or more agents that modulate a non-kinase biological target or process. Such targets include Histone Deacetylases (HDACs), DNA methyltransferases (DNMTs), heat shock proteins (e.g., HSP90), and proteosomes.

In one embodiment, the subject compounds may be combined with anti-neoplastic agents (e.g., small molecules, monoclonal antibodies, antisense RNAs, and fusion proteins) that inhibit one or more biological targets, such as vorinostat (Zolinza), tarceva, iressa, lapatinib (Tykerb), gleevec, sotan, dasatinib (Sprycel), Nexavar, sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, trastuzumab (Herceptin), cetuximab (Erbitux), AG24322, PD 3201, ZD6474, PD 59 184322, Obatodax, ABT737, and e 788. The therapeutic efficacy of such a combination is enhanced relative to that achieved using any of the agents alone, and the appearance of anti-mutation variants can be inhibited or delayed.

In certain preferred embodiments, the compounds of the present invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents include many of the treatments in the field of oncology. These agents are administered at various stages of the disease to shrink the tumor, destroy residual cancer cells left after surgery, induce remission, maintain remission, and/or alleviate symptoms associated with the cancer or its treatment. Examples of such agents include, but are not limited to: alkylating agents, such as mustard gas derivatives (nitrogen mustards, cyclophosphamide, chlorambucil, melphalan, ifosfamide), ethyleneimines (thiophosphine triamide, hexamethyl pyrimethanil), alkyl sulfonates (malilan), hydrazines and triazines (hexamethyl pyrimethanil, toluidine, dacarbazine and temozolomide), nitrosoureas (carmustine, lomustine and streptozotocin), ifosfamide and metal salts (carboplatin, cisplatin, oxaliplatin); plant alkaloids such as podophyllotoxins (etoposide and etoposide), taxanes (paclitaxel, docetaxel), vinca alkaloids (vincristine, vinblastine, desacetylvinblastide and vinorelbine) and camptothecin analogs (irinotecan, topotecan); antitumor antibiotics, such as chromomycin (dactinomycin and mithramycin), anthracyclines (adriamycin, daunorubicin, epirubicin, mitoxantrone, valrubicin, and idarubicin), and other antibiotics, such as mitomycin, actinomycin, and bleomycin; antimetabolites, such as folate antagonists (methotrexate, pemetrexed, raltitrexed, pteridine), pyrimidine antagonists (5-fluorouracil, floxuridine, fluorouracil, capecitabine, and gemcitabine), purine antagonists (6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitors (cladribine, fludarabine, mercaptopurine, clofarabine, thioguanine, nelarabine, and pentostatin); topoisomerase inhibitors, such as topoisomerase I inhibitors (irinotecan, topotecan) and topoisomerase II inhibitors (amsacrine, etoposide phosphate, epipodophyllotoxin thiophenoside); monoclonal antibodies (alemtuzumab, gemtuzumab ozogamicin, rituximab, trastuzumab, ibritumomab tiuxetan, cetuximab, panitumumab, tositumomab, bevacizumab); and other antineoplastic agents, such as ribonucleotide reductase inhibitors (hydroxyurea); corticoid inhibitors (mitotane); enzymes (asparaginase and pegapase); anti-microtubule agents (estramustine); and retinoids (bexarotene, isotretinoin, tretinoin (ATRA)). In certain preferred embodiments, the compounds of the present invention are administered in combination with a chemoprotectant. Chemoprotectants protect the body or minimize the side effects of chemotherapy. Examples of such agents include, but are not limited to: amifostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are used in combination with radiation therapy. Radiation is typically delivered internally (radioactive material is implanted near the cancer site) or externally by machines using photon (x-ray or gamma-ray) or particulate radiation. When combination therapy also includes radiation therapy, the radiation therapy can be administered at any appropriate time so long as the beneficial effect is obtained from the combined action of the therapeutic agent and the radiation therapy. For example, where appropriate, beneficial effects may still be obtained when radiation therapy is temporarily removed from the administered therapeutic agent, perhaps days or even weeks.

It is understood that the compounds of the present invention may be used in combination with immunotherapeutic agents. One form of immunotherapy is by administering vaccines at a site remote from the tumorThe vaccine composition thereby generates an active systemic tumor-specific immune response of host origin. Various types of vaccines have been proposed, including isolated tumor-antigen vaccines and anti-idiotype vaccines. Another approach is to use tumor cells from the subject to be treated, or derivatives of such cells (as described by Schirrmacher et al (1995) J. cancer Res. Clin. Oncol.121: 487). U.S. patent No.5,484,596 to Hanna jr et al claims a method for treating resectable tumors to prevent recurrence or metastasis comprising surgically removing the tumor, dispersing the cells with collagenase, irradiating the cells and using about 10 ⁷At least three successive doses of each cell are inoculated into the patient.

It will be appreciated that the compounds of the invention may be advantageously used in combination with one or more adjunctive therapeutic agents. Examples of suitable agents for adjunctive therapy include steroids such as corticosteroids (amcinolone, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol acetate, clobetasol butyrate, clobetasol 17-propionate, cortisone, deflazacort, desoximetasone, diflucortolone valerate, dexamethasone sodium phosphate, prednisolone, furoate, fluocinonide acetate, hastellide, hydrocortisone butyrate, hydrocortisone sodium succinate, hydrocortisone valerate, prednisolone, dehydrocortisol, triamcinolone acetonide and halobetasol propionate); 5HTi agonists, such as triptan (e.g., sumatriptan or nolatin); adenosine Al agonists; an EP ligand; NMDA modulators, such as glycine antagonists; sodium channel blockers (e.g., lamotrigine); substance P antagonists (e.g., NKi antagonists); cannabinoids; paracetamol or phenacetin; 5 a lipoxygenase inhibitor; a leukotriene receptor antagonist; DMARDs (e.g., methotrexate); gabapentin and related compounds; tricyclic antidepressants (e.g., amitriptyline); neurocyte stabilizing antiepileptic drugs; mono-aminergic uptake inhibitors (e.g., venlafaxine); a matrix metalloproteinase inhibitor; nitric Oxide Synthase (NOS) inhibitors, such as iNOS or nNOS inhibitors; inhibitors of tumor necrosis factor release or action; antibody therapy, such as monoclonal antibody therapy; antiviral agents, such as nucleoside inhibitors (e.g., lamivudine) or immune system modulators (e.g., interferon); opioid analgesics; a local anesthetic; stimulants, including caffeine; h2-antagonists (e.g., amifurol); proton pump inhibitors (e.g., omeprazole); antacids (e.g., aluminum or magnesium hydroxide); antiflatulent agents (e.g., dimethicone); decongestants (e.g., neosynephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, naphazoline, xylometazoline, hexahydromethamphetamine, or levomethamphetamine); antitussives (e.g., codeine, hydrocodone, caramiphene, viscapine, or dextromethorphan); diuretic agents; or a sertraline or non-sertraline antihistamine.

The compounds of the invention may be administered with sirnas targeting other genes. For example, a compound of the invention may be administered with an siRNA targeting the c-Myc gene. In one embodiment, AD-12115 may be administered with the c-Myc siRNA. Examples of c-Myc targeted siRNAs are disclosed in U.S. patent application Ser. No. 12/373,039, which is incorporated herein by reference.

Methods of treating diseases caused by expression of Eg5 and VEGF genes

The invention particularly relates to the use of a composition comprising at least two dsrnas (one targeting the Eg5 gene and one targeting the VEGF gene) in the treatment of cancer, for example liver cancer, for inhibiting tumor growth and tumor metastasis. For example, a composition, e.g., a pharmaceutical composition, can be used to treat a solid tumor, e.g., an intrahepatic tumor as may occur in liver cancer. Compositions containing dsRNA targeting Eg5 and dsRNA targeting VEGF can also be used for the treatment of other tumors and cancers, such as breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm tumor, multiple myeloma and for the treatment of skin cancer, melanoid tumors, for the treatment of lymphomas and blood cancers. The invention also relates to the use of a composition comprising Eg5dsRNA and VEGF dsRNA to inhibit the accumulation of ascites and pleural effusion in different types of cancer, such as liver cancer, breast cancer, lung cancer, head cancer, neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm tumor, multiple myeloma, skin cancer, melanoma, lymphoma and blood cancer. Because of the inhibitory effects on Eg5 and VEGF expression, the composition of the present invention or the pharmaceutical composition prepared therefrom can improve quality of life.

In one embodiment, a patient having a tumor associated with AFP expression, or a tumor that secretes AFP, such as a liver cancer or teratoma, is treated. In certain embodiments, the patient has malignant teratoma, endoblastoma (yolk sac cancer), neuroblastoma, hepatoblastoma, hepatocellular carcinoma, testicular cancer, or ovarian cancer.

The invention also relates to the use of the dsRNA or a pharmaceutical composition thereof, e.g. for the treatment of cancer or for the prevention of tumor metastasis, in combination with other drugs and/or other therapeutic methods, e.g. in combination with known drugs and/or known therapeutic methods (e.g. methods currently used for the treatment of cancer and/or for the prevention of tumor metastasis).

The invention can also be practiced by including a combination of a particular RNAi agent and other anti-cancer chemotherapeutic agents, e.g., any conventional chemotherapeutic agent. The combination of the specific binding agent and the additional agent may potentiate the chemotherapeutic method. Many chemotherapeutic methods capable of being combined with the methods of the invention will be present in the brain of practitioners in the art. Any chemotherapeutic agent may be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compounds of the present invention may be administered with antibiotics such as adriamycin and other anthracycline analogs, nitrogen mustard such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, tacroline and natural and synthetic derivatives thereof, and the like. As another example, for mixed tumors, such as adenomas of the breast, wherein the tumor comprises gonadotropin-dependent and gonadotropin-independent cells, the compounds may be administered with leuprolide or a synthetic peptide analog of sex hormone-blocking drug (LH-RH). Other anti-tumor approaches include the use of tetracycline compounds and another form of therapy, e.g., surgery, radiation, etc., also referred to herein as "the adjuvant anti-tumor form". Thus, the method of the present invention can be used with such conventional methods, advantageously reducing side effects and enhancing efficacy.

Methods for inhibiting expression of Eg5 gene and VEGF gene

In yet another aspect, the invention provides methods for inhibiting the expression of the Eg5 gene and the VEGF gene in a mammal. The methods comprise administering a featured composition of the invention to a mammal, thereby silencing the expression of the target Eg5 gene and the target VEGF gene.

In one embodiment, a method for inhibiting Eg5 gene expression and VEGF gene expression comprises administering to a mammal in need of treatment a composition comprising two different dsRNA molecules, wherein the nucleotide sequence of one dsRNA molecule is complementary to at least a portion of an RNA transcript of the Eg5 gene and the nucleotide sequence of the other dsRNA molecule is complementary to at least a portion of an RNA transcript of the VEGF gene. When the organism in need of treatment is a mammal, such as a human, the composition can be administered by any method known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In a preferred embodiment, the composition is administered by intravenous infusion or injection.

Method for preparing lipid particles

The methods of the compositions of the present invention employ certain cationic lipids, the synthesis, preparation and characterization of which are described below and in the accompanying examples. In addition, the present invention provides methods of making lipid particles, including those associated with therapeutic agents, such as nucleic acids. In the methods described herein, the lipid mixture is mixed with an aqueous nucleic acid buffer to prepare an intermediate mixture comprising nucleic acids encapsulated in lipid particles, wherein the encapsulated nucleic acids are present at a nucleic acid/lipid ratio of about 3 wt% to about 25 wt%, preferably 5 to 15 wt%. The size of the intermediate mixture can optionally be adjusted to obtain lipid-encapsulated nucleic acid particles in which the lipid fraction is unilamellar vesicles, preferably having a diameter of 30 to 150nm, more preferably about 40 to 90 nm. The pH is then increased to neutralize at least a portion of the surface charge on the lipid-nucleic acid particle, thereby providing an at least partially surface-neutralized lipid-encapsulated nucleic acid composition.

As described above, several of these cationic lipids are amino lipids, which are charged at a pH below the pKa of the amino group and are substantially neutral at a pH above the pKa. These cationic lipids are referred to as titratable cationic lipids and can be used in the formulations of the present invention by using a two-step process. First, lipid vesicles are formed at lower pH with titratable cationic lipids and other vesicle components in the presence of nucleic acids. In this manner, the vesicles will encapsulate and capture nucleic acids. Second, the surface charge of the newly formed vesicles is neutralized by raising the pH of the medium to a level above the pKa of the titratable cationic lipid present, i.e., to a physiological pH or higher. Particularly advantageous aspects of the method include the ease of removal of any surface-adsorbed nucleic acid, and the resulting nucleic acid delivery medium having a neutral surface. Liposomes or lipid particles with a neutral surface are expected to avoid rapid clearance from the circulation and to avoid certain toxicities associated with cationic liposome formulations. Additional details regarding these uses of such titratable cationic lipids in nucleic acid-lipid particle formulations are provided in U.S. patent 6,287,591 and U.S. patent 6,858,225, which are incorporated herein by reference.

It is also noted that vesicles formed in this manner provide formulations with uniform vesicle size and high levels of nucleic acid. In addition, the size of the vesicles ranges from about 30 to about 150nm, preferably from about 30 to about 90 nm.

Without being bound to any particular theory, it is believed that the very high efficiency of nucleic acid encapsulation is a result of electrostatic interactions at low pH. At acidic pH (e.g., pH4.0), the vesicle surface is charged and binds to a portion of the nucleic acid through electrostatic interaction. When an exogenous acidic buffer replaces a more neutral buffer (e.g., ph7.5), the surface of the lipid particle or liposome is neutralized, thereby removing any extraneous nucleic acids. More detailed information on the formulation method is provided in various publications (e.g., U.S. Pat. No. 6,287,591 and U.S. Pat. No. 6,858,225).

In view of the above, the present invention provides a method of preparing a lipid/nucleic acid formulation. In the methods described herein, the lipid mixture is mixed with a buffered aqueous solution of nucleic acids to produce an intermediate mixture containing nucleic acids encapsulated in lipid particles, e.g., wherein the encapsulated nucleic acids are present at a nucleic acid/lipid ratio of about 10 wt% to about 20 wt%. The size of the intermediate mixture can optionally be adjusted to obtain lipid-encapsulated nucleic acid particles in which the lipid fraction is unilamellar vesicles, preferably having a diameter of 30 to 150nm, more preferably about 40 to 90 nm. The pH is then increased to neutralize at least a portion of the surface charge on the lipid-nucleic acid particle, thereby providing an at least partially surface-neutralized lipid-encapsulated nucleic acid composition.

In certain embodiments, the lipid mixture comprises at least two lipid components: the first amino lipid component of the invention is selected from lipids having a pKa such that at a pH below said pKa said lipid is cationic and at a pH above said pKa said lipid is neutral, and the second lipid component is selected from lipids which prevent aggregation of the particle during lipid-nucleic acid particle formation. In a specific embodiment, the amino lipid is the novel cationic lipid of the invention.

In preparing the nucleic acid-lipid particles of the present invention, the lipid mixture is typically a solution of lipids in an organic solvent. The lipid mixture can then be dried to form a film or lyophilized to form a powder, and then hydrated with an aqueous buffer to form liposomes. Alternatively, in a preferred method, the lipid mixture may be dissolved in a water-soluble alcohol, such as ethanol, and the ethanol solution added to the aqueous buffer, resulting in spontaneous liposome formation. In the most preferred embodiment, the alcohol is used in a commercially available form. For example, absolute ethanol (100%) or 95% ethanol is used, the remainder being water. This process is described in more detail in us patent 5,976,567.

According to the invention, the lipid mixture is mixed with an aqueous buffer solution which may comprise nucleic acids. The aqueous buffer solution is typically a solution in which the pH of the buffer is less than the pKa of the protonatable lipid in the lipid mixture. Examples of suitable buffers include citrate, phosphate, acetate, and MES. A particularly preferred buffer is a citrate buffer. Preferred buffers have anions in the range of 1-1000mM, depending on the chemistry of the encapsulated nucleic acid, and optimizing buffer concentration may be important to achieve high loading levels (see, e.g., U.S. patent 6,287,591 and U.S. patent 6,858,225). Alternatively, pure water acidified to pH5-6 with chloride, sulfate, or the like may be used. In this case, it is appropriate to add 5% glucose, or another non-ionic solute, which balances the osmotic pressure across the membrane of the particle when dialyzing the particle to remove ethanol, raise the pH, or mix with a pharmaceutically acceptable carrier, such as physiological saline. The amount of nucleic acid in the buffer may vary, but is generally from about 0.01mg/mL to about 200mg/mL, more preferably from about 0.5mg/mL to about 50 mg/mL.

The mixture of lipid and aqueous buffer solution of therapeutic nucleic acid is mixed to provide an intermediate mixture. The intermediate mixture is typically a lipid particle mixture with encapsulated nucleic acids. Alternatively, the intermediate mixture may also comprise a portion of nucleic acids attached to the surface of the lipid particle (liposome or lipid vesicle) due to ionic attraction of the negatively charged nucleic acids and the positively charged lipids on the surface of the lipid particle (the amino lipids or other lipids that make up the protonatable first lipid component are positively charged in a buffer at a pH less than the pKa of the protonatable groups on the lipid). In one set of preferred embodiments, the lipid mixture is an alcohol solution of lipids, and the volume of each solution is adjusted such that, once combined, the resulting alcohol content is from about 20% by volume to about 45% by volume. The method of combining the mixtures may include any kind of treatment, generally depending on the scale of the formulation prepared. For example, when the total volume is about 10-20mL or less, the solutions can be mixed in a test tube and stirred together using a vortex stirrer. Large scale processing can be carried out in glassware of appropriate production scale.

Optionally, the size of the lipid-encapsulated therapeutic agent (e.g., nucleic acid) complex prepared by mixing the lipid mixture and the buffered aqueous solution of the therapeutic agent (nucleic acid) can be adjusted to obtain a desired size range and relatively narrow lipid particle size distribution. Preferably, the compositions provided herein have an average diameter of from about 70 to about 200nm, more preferably from about 90 to about 130 nm. Several techniques can be used to adjust the liposomes to the desired size. One method of resizing is described in U.S. patent No.4,737,323, which is incorporated herein by reference. Sonication of the liposome suspension by bath or probe sonication results in a gradual reduction in size, resulting in Small Unilamellar Vesicles (SUVs) of less than about 0.05 microns in size. Homogenization is another method that relies on shear energy to break down larger liposomes into smaller liposomes. In a typical homogenization process, the multilamellar vesicles are recirculated through a standard emulsion homogenizer until a selected liposome size is observed, typically about 0.1 to 0.5 microns. In both methods, the particle size distribution can be monitored by a conventional laser beam particle size analyzer. In certain methods of the invention, extrusion is used to obtain uniform vesicle size.

The liposome composition is extruded through a small pore polycarbonate membrane or an asymmetric ceramic membrane to obtain a relatively well defined particle size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired liposome complex particle size distribution is achieved. The liposomes can be continuously extruded through a small pore membrane to achieve a gradual reduction in liposome size. In some cases, the formed lipid-nucleic acid composition can be used without resizing.

In particular embodiments, the methods of the invention further comprise the step of neutralizing at least some of the surface charge on the lipid portion of the lipid-nucleic acid composition. By at least partially neutralizing the surface charge, the unencapsulated nucleic acids are released from the surface of the lipid particle and can be removed from the composition using conventional methods. Preferably, unencapsulated and surface-adsorbed nucleic acids are removed from the resulting composition by displacing the buffer solution. For example, replacement of citrate buffer (pH about 4.0, used to form the composition) with HEPES-buffered saline (HBS, pH about 7.5) solution results in the release of neutralized nucleic acids from the surface of the liposomes. The released nucleic acids can then be removed by standard methods by chromatography and then converted to a buffer at a pH greater than the pKa of the lipid used.

Optionally, lipid vesicles (i.e., lipid particles) can be formed by hydration in an aqueous buffer and sized using any of the methods described above, followed by addition of nucleic acid. As mentioned above, the pH of the aqueous buffer should be below the pKa of the amino lipid. A solution of nucleic acid is then added to these sized, preformed vesicles. To encapsulate nucleic acids in such "pre-formed" vesicles, the mixture should comprise an alcohol, such as ethanol. In the case of ethanol, it should be present at a concentration of about 20% (w/w) to about 45% (w/w). In addition, depending on the lipid vesicle composition and the nature of the nucleic acid, it may be necessary to heat the mixture of preformed vesicles and nucleic acid in an aqueous buffer-ethanol mixture to a temperature of about 25 ℃ to about 50 ℃. It is clear to the skilled person that optimizing the encapsulation process to obtain the desired level of nucleic acid in the lipid vesicle will require control of variables such as ethanol concentration and temperature. Examples of suitable conditions for encapsulating nucleic acids are provided in the examples. Once the nucleic acid is encapsulated within the preformed vesicle, the external pH can be raised to at least partially neutralize the surface charge. The unencapsulated and surface-adsorbed nucleic acids can then be removed as described above.

Application method

The lipid particles of the invention can be used to deliver therapeutic agents to cells in vitro or in vivo. In a specific embodiment, the therapeutic agent is a nucleic acid, which is delivered to a cell using the nucleic acid-lipid particle of the invention. The following description of various methods of using the lipid particles and related pharmaceutical compositions of the present invention is illustrated by the description in relation to nucleic acid-lipid particles, with the understanding that these methods and compositions can be readily used to deliver any therapeutic agent for the treatment of any disease or condition that would benefit from such treatment.

In certain embodiments, the invention provides methods for introducing nucleic acids into cells. Preferred nucleic acids for introduction into cells are siRNA, immuno-stimulatory oligonucleotides, plasmids, antisense and ribozymes. These methods can be performed by contacting the particles or compositions of the invention with the cells for a period of time sufficient for intracellular delivery to occur.

The compositions of the invention can be adsorbed on virtually any cell type. Once adsorbed, the nucleic acid-lipid particle can be endocytosed by a portion of the cell, exchange lipids with the cell membrane, or fuse with the cell. Transfer or binding of the nucleic acid portion of the complex can occur via any of these pathways. Without limiting the scope of the invention, it is believed that in the case where the particles are taken up by the cell by endocytosis, the particles then interact with the endosomal membrane, resulting in destabilization of the endosomal membrane, perhaps by forming a non-bilayer phase, resulting in entry of the encapsulated nucleic acid into the cytoplasm. Similarly, in the case of direct fusion of the particle and cytoplasmic membrane, when fusion occurs, the liposomal membrane is integrated into the cellular membrane and the liposomal contents are bound to the intracellular fluid. Contact between the cell and the lipid-nucleic acid composition (when performed in vitro) will occur in a biologically compatible medium. The concentration of the composition may vary depending on the particular application, but is typically from about 1. mu. mol to about 10 mmol. In certain embodiments, the treatment of the cells with the lipid-nucleic acid composition is typically performed at physiological temperature (about 37 ℃) for about 1 to 24 hours, preferably about 2 to 8 hours. For in vitro applications, the nucleic acid may be delivered to any cell, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type, grown in culture. In a preferred embodiment, the cell is an animal cell, more preferably a mammalian cell, most preferably a human cell.

In one set of embodiments, the lipid-nucleic acid particle suspension is added to cells that are 60-80% confluent and have a cell density of about 10³To about 10⁵Individual cell/mL, preferably about 2X 10⁴Individual cells/mL. The concentration of the suspension added to the cells is preferably about 0.01 to 20. mu.g/mL, more preferably about 1. mu.g/mL.

Typical applications include providing intracellular delivery of siRNA using well known methods to inhibit or silence a particular cellular target. In addition, applications include the delivery of DNA or mRNA sequences encoding therapeutically useful polypeptides. In this manner, treatment for genetic diseases is provided by providing an insufficient or absent gene product (i.e., for Duchenne muscular dystrophy, see Kunkel, et al, Brit. Med. Bull.45 (3): 630-643(1989), for cystic fibrosis, see Goodfellow, Nature 341: 102-103 (1989)). Other uses of the compositions of the invention include the introduction of antisense oligonucleotides into cells (see Bennett, et al, mol. pharm. 41: 1023-1033 (1992)).

Alternatively, the compositions of the invention may also be used to deliver nucleic acids to cells in vivo by methods known to those skilled in the art. For the use of the present invention for the delivery of DNA or mRNA sequences, Science 261 of Zhu et al, which is incorporated herein by reference: 209-211(1993) describe the intravenous delivery of Cytomegalovirus (CMV) -Chloramphenicol Acetyltransferase (CAT) expression plasmids using DOTMA-DOPE complexes. Hyde et al Nature 362, incorporated herein by reference: 250-256(1993) describes the use of liposomes to deliver cystic fibrosis transmembrane conductance regulator (CFTR) genes to the airway epithelium and alveoli of the lung in mice. Am.j.med.sci.298 of Brigham et al, incorporated herein by reference: 278-281(1989) describe the in vivo transfection of mouse lungs with a functional prokaryotic gene encoding the intracellular enzyme Chloramphenicol Acetyltransferase (CAT). Thus, the compositions of the invention are useful for treating infectious diseases.

For in vivo administration, the pharmaceutical composition is preferably administered parenterally, i.e., intra-articularly, intravenously, intra-abdominally, subcutaneously, or intramuscularly. In a specific embodiment, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection. See, as an example, Stadler et al, U.S. patent No.5,286,634, which is incorporated herein by reference. Intracellular nucleic acid delivery is also described in Straubringer et al Methods in enzymology, Academic Press, New York.101: 512-527 (1983); mannino, et al, Biotechniques 6: 682-690 (1988); nicolau, et al, crit.rev.ther.drug carrier system.6: 239-271(1989), and Behr, acc, chem, res.26: 274-278 (1993). Other methods of administering lipid-based therapeutics are described, for example, in Rahman et al, U.S. patent No.3,993,754; sears, U.S. patent No.4,145,410; papahadjopoulos et al, U.S. Pat. No.4,235,871; schneider, U.S. patent No.4,224,179; lenk et al, U.S. Pat. Nos. 4,522,803; and Fountain et al, U.S. patent No.4,588,578.

In other methods, the pharmaceutical formulation may be contacted with the target tissue by applying the formulation directly to the tissue. The application may be by a local, "open" or "closed" procedure. By "topical" is meant the direct application of the pharmaceutical formulation to the tissue exposed to the environment, e.g., skin, oropharynx, external auditory meatus, etc. An "open" procedure is to cut the patient's skin and directly visualize the underlying tissue to which the pharmaceutical formulation is applied. This is typically accomplished by a surgical procedure, such as a thoracotomy to the lungs, an abdominal laparotomy to the abdominal viscera, or other direct surgery to access the target tissue. A "closed" procedure is an invasive procedure in which the internal target tissue is not directly visualized, but is reached via an insertion instrument through a small wound in the skin. For example, the formulation may be administered to the peritoneum by needle lavage. Similarly, the pharmaceutical formulation may be administered to the meninges or spinal cord by infusion during lumbar puncture, and the patient then positioned as is commonly done for spinal anesthesia or spinal panoxanide imaging. Alternatively, the formulation may be administered by an endoscopic device.

The lipid-nucleic acid composition may also be administered as an aerosol that is inhaled into the lung (see Brigham, et al, am. J. Sci.298 (4): 278-281(1989)) or by direct injection into the lesion (Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)).

The methods of the invention may also be carried out in a variety of hosts. Preferred hosts include mammalian species, such as humans, non-human primates, dogs, cats, cows, horses, sheep, and the like.

The dosage of the lipid-therapeutic agent particles of the present invention will depend on the ratio of therapeutic agent to lipid and the opinion of the attending physician based on age, weight and condition of the patient.

In one embodiment, the invention provides a method of modulating the expression of a target polynucleotide or polypeptide. These methods generally comprise contacting a cell with a lipid particle of the invention that binds to a nucleic acid capable of modulating expression of a target polynucleotide or polypeptide. As used herein, the term "modulate" means to alter the expression of a target polynucleotide or polypeptide. In various embodiments, modulation may refer to increasing or strengthening, or it may refer to decreasing or decreasing. Methods for determining the expression level of a target polynucleotide or polypeptide are known and available in the art and include, for example, methods using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical techniques. In particular embodiments, the expression level of the target polynucleotide or polypeptide is increased or decreased by at least 10%, 20%, 30%, 40%, 50% or greater than 50% as compared to an appropriate control value. For example, if increased polypeptide expression is desired, the nucleic acid can be an expression vector comprising a polynucleotide encoding the desired polypeptide. On the other hand, if it is desired to reduce expression of a polynucleotide or polypeptide, the nucleic acid may be, for example, an antisense oligonucleotide, siRNA or microrna comprising a polynucleotide sequence that specifically hybridizes to a polynucleotide encoding the target polypeptide, thereby interfering with expression of the target polynucleotide or polypeptide. Alternatively, the nucleic acid may be a plasmid expressing such an antisense oligonucleotide, siRNA or microrna.

In one embodiment, the invention provides a method of modulating the expression of a polypeptide in a cell comprising providing to the cell a lipid particle consisting of or consisting essentially of a cationic lipid of formula a, a neutral lipid, a sterol, a PEG or PEG-modified lipid, e.g., at a molar ratio of about 35-65% of the cationic lipid of formula a, 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid, wherein the lipid particle is associated with a nucleic acid capable of modulating the expression of the polypeptide. In specific embodiments, the lipid molar ratio is about 60/7.5/31/1.5 or 57.5/7.5/31.5/3.5 (mol% lipid a/DSPC/cholesterol/PEG-DMG). In another set of embodiments, the neutral lipids in these compositions are replaced by DPPC (dipalmitoyl lecithin), POPC, DOPE, or SM.

In a specific embodiment, the therapeutic agent is selected from the group consisting of an siRNA, a microrna, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, microrna, or antisense oligonucleotide, and wherein the siRNA, microrna, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide or its complement such that expression of the polypeptide is reduced.

In other embodiments, the nucleic acid is a plasmid encoding a polypeptide or a functional variant or fragment thereof such that expression of the polypeptide or functional variant or fragment thereof is increased.

In related embodiments, the invention provides a method for treating a disease or disorder characterized by overexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the invention, wherein the therapeutic agent is selected from the group consisting of an siRNA, a microrna, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, microrna, or antisense oligonucleotide, and wherein the siRNA, microrna, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide, or the complement thereof.

In one embodiment, the pharmaceutical composition comprises a lipid particle consisting of or consisting essentially of lipid A, DSPC, cholesterol and PEG-DMG, PEG-C-DOMG, or PEG-DMA, such as a cationic lipid of formula a at a molar ratio of about 35-65%, 3-12% neutral lipid, 15-45% sterol, and 0.5-10% PEG or PEG-modified lipid PEG-DMG, PEG-C-DOMG, or PEG-DMA, wherein the lipid particle is conjugated to a therapeutic nucleic acid. In specific embodiments, the lipid molar ratio is about 60/7.5/31/1.5 or 57.5/7.5/31.5/3.5 (mol% lipid a/DSPC/cholesterol/PEG-DMG). In another set of embodiments, the neutral lipids in these compositions are replaced by DPPC, POPC, DOPE, or SM.

In another related embodiment, the invention includes a method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject comprising providing to the subject a pharmaceutical composition of the invention, wherein the therapeutic agent is a plasmid encoding the polypeptide or a functional variant or fragment thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Examples

Example 1: dsRNA synthesis

Sources of reagents

When the present invention does not specifically address the source of the reagents, such reagents may be obtained from any molecular biology reagent supplier at the quality/purity standards used in molecular biology.

SiRNA synthesis

For screening of dsRNA, an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass as a solid support (CPG,prooligo Biochemie GmbH, Hamburg, Germany) single stranded RNA was prepared on a 1. mu. mol scale by solid phase synthesis. RNA and RNA containing 2 '-O-methyl nucleotides were generated by solid phase synthesis using the corresponding phosphoramidite and 2' -O-methyl phosphoramidite (Prologo Biochemie GmbH, Hamburg, Germany), respectively. John Wiley using, for example, Current protocols in nucleic acid chemistry, Beaucage, S.L, et al (Edrs.), John Wiley&Standard nucleoside phosphoramidite chemistry described in Sons, Inc., New York, NY, USA binds these building blocks at selected sites within the oligoribonucleotide chain sequence. Phosphorothioate linkages were introduced by replacing the iodine oxidizer solution with a solution of Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). In addition, auxiliary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were performed according to the specified procedure. The yields and concentrations were determined by UV absorption of solutions of the corresponding RNAs at a wavelength of 260nm using a spectrophotometer (DU 640B, Beckman Coulter GmbH, Unterschlei. beta. heim, Germany). Double-stranded RNA was produced by mixing equimolar solutions of the complementary strands in annealing buffer (20mM sodium phosphate, pH 6.8; 100mM sodium chloride), heating in a water bath at 85-90 ℃ for 3 minutes and cooling to room temperature over 3-4 hours. The annealed RNA solution was stored at-20 ℃ until use.

dsRNA targeting Eg5 gene

Initial screening set

siRNA design was performed to identify sirnas targeting Eg5 (also known as KIF11, HSKP, KNSL1, and TRIP 5). Using the human mRNA sequence of Eg5, RefSeq ID No.: NM _004523.

siRNA duplexes were designed to cross-react with human and mouse Eg 5. Twenty-four duplexes were synthesized for screening. (Table 1 a). A second screening group was defined with 266 sirnas targeting human Eg5 and its rhesus ortholog (table 2 a). The expanded screening group was selected with 328 sirnas targeting human Eg5 without hitting any Eg5mRNA from other species (table 3 a).

The sequences of human and partial rhesus Eg5mRNA were downloaded from the NCBI nucleotide database, the human sequence further used as a reference sequence (human Eg 5: NM — 004523.2, 4908bp, rhesus Eg 5: XM — 001087644.1, 878bp (5' part of human Eg5 only).

For the tables: key words: a, G, C, U-ribonucleotide: t-deoxythymidine: u, c-2' -O-methyl nucleotides: s-phosphorothioate linkages.

TABLE 1A. sequence of Eg5/KSP dsRNA duplexes

TABLE 1b analysis of Eg5/KSP ds duplexes

TABLE 2a sequence of Eg5/KSP dsRNA duplexes

TABLE 2b analysis of Eg5/KSP dsRNA duplexes

TABLE 3 sequence and analysis of Eg5/KSP dsRNA duplexes

DsRNA targeting VEGF gene

Four hundred target sequences were identified within exons 1-5 of the VEGF-A121 mRNA sequence. The reference transcript is: NM _ 003376.

1 augaacuuuc ugcugucuug ggugcauugg agccuugccu ugcugcucua ccuccaccau

61 gccaaguggu cccaggcugc acccauggca gaaggaggag ggcagaauca ucacgaagug

121 gugaaguuca uggaugucua ucagcgcagc uacugccauc caaucgagac ccugguggac

181 aucuuccagg aguacccuga ugagaucgag uacaucuuca agccauccug ugugccccug

241 augcgaugcg ggggcugcug caaugacgag ggccuggagu gugugcccac ugaggagucc

301 aacaucacca ugcagauuau gcggaucaaa ccucaccaag gccagcacau aggagagaug

361 agcuuccuac agcacaacaa augugaaugc agaccaaaga aagauagagc aagacaagaa

421 aaaugugaca agccgaggcg guga(SEQ ID NO：1539)

Table 4a includes the identified target sequences. Corresponding sirnas targeting these sequences were bioinformatically screened.

To ensure that this sequence is specific for the VEGF sequence and not for sequences from any other gene, the nuclear target sequence was checked against sequences in Genbank using the BLAST search engine provided by NCBI. Use of the BLAST algorithm is described in Altschul et al, j.mol.biol.215: 403, 1990; and Altschul and Gish, meth.enzymol.266: 460, 1996.

siRNAs were also prioritized according to their ability to cross-react with monkey, rat and human VEGF sequences.

Out of these 400 potential target sequences, 80 were selected for analysis by experimental screening to identify a small number of lead candidates. A total of 114 siRNA molecules were designed for these 80 target sequences 114 (Table 4 b).

Target sequences in VEGF-121

Table 4 b: VEGF-targeting duplexes

Chain: s is sense, AS is antisense

Example 2: in vitro screening of Eg5 siRNA via cell proliferation

Since silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, et al [2002] Biotechniques 33: 1244-8), cell viability assays were used for siRNA activity screening. HeLa cells (HeLa cells) (14000 per well [ selection 1 and 3] or 10000 per well [ selection 2]) were seeded in 96-well plates and simultaneously transfected with ipofectamine 2000(Invitrogen) with a final siRNA concentration of 30nML in the wells, 50nM for the first selection and 25nM for the second selection. Duplex subsets were tested at 25nM in the third screen (table 5).

Seventy-two hours after transfection, WST-1 reagent (Roche) was added to the medium, followed by measurement of absorbance at 450nm, and cell proliferation was tested. The absorbance value of control (non-transfected) cells was considered 100%, and the absorbance of siRNA from transfected wells was compared to the control value. Six of the three screens were run per experiment. Subsets of sirnas were then tested at a range of siRNA concentrations. The experiments were performed in HeLa cells (14000 wells per well; the procedure was as above, Table 5).

Table 5: 25n ofEffect of M duplexes targeting Eg5 on cell viability

The 9 siRNA duplexes showing the greatest growth inhibition in table 5 were retested in HeLa cells at a range of siRNA concentrations. The siRNA concentrations tested were 100nM, 33.3nM, 11.1nM, 3.70nM, 1.23nM, 0.41nM, 0.14nM and 0.046 nM. The assay was repeated six times and the calculation resulted in fifty percent inhibition of cell proliferation (IC)₅₀) Each siRNA concentration of (a). For each duplex, the dose-response assay was performed two to four times. Average IC₅₀The values (nM) are given in Table 6.

Table 6: IC50 of siRNA: cell proliferation in HeLa cells

Double chain body	Average IC50
		AL-DP-6226	15.5
AL-DP-6229	3.4
		AL-DP-6231	4.2
AL-DP-6232	17.5
		AL-DP-6239	4.4
AL-DP-6242	5.2
		AL-DP-6243	2.6
AL-DP-6244	8.3
		AL-DP-6248	1.9

Example 3: in vitro screening of Eg5 siRNA via mRNA inhibition

Shortly before transfection, HeLa S3 (ATCC-Number: CCL-2.2, LCGPromochem GmbH, Wesel, Germany) cells were plated at 1.5X10 ⁴Cells/well were seeded in 75. mu.l growth medium (Ham's F12, 10% fetal bovine serum, 100u penicillin/100. mu.g/ml streptomycin, all from Bookrom AG, Berlin, Germany) in 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany). The transfection was performed in quadruplicate. In each well 0.5. mu.l Lipofectamine2000(Invitrogen GmbH, Karlsruhe, Germany) and 12. mu.l Opti-MEM (Invitrogen) were mixed and incubated for 15min at room temperature. For the case where the siRNA concentration in the transfection volume was 50nM for 100. mu.l, 1. mu.l of 5. mu.M siRNA and 11.5. mu.l of Opti-MEM were mixed per well, combined with the Lipofectamine2000-Opti-MEM mixture and incubated at room temperature for an additional 15 minutes. The siRNA-Lipofectamine 2000-complex was added completely to the cells, which were incubated at 37 ℃ in a humid incubator (Heroes GmbH, Ha)nau) 5% CO₂And (4) performing medium incubation for 24 h. Single dose screening was performed at 50nM and 25nM, respectively.

Cells were harvested by applying 50 μ l of lysis mix (contents of QuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well containing 100 μ l of growth medium and lysed at 53 ℃ for 30 min. Then, 50 μ l were incubated with probe sets specific for human Eg5 and human GAPDH and manipulated according to the supplier's protocol for QuantiGene. Finally, chemiluminescence was measured as RLU (relative Light units) in Victor2-Light (Perkin Elmer, wissbaden, Germany), and the values obtained for the hEg5 probe set were normalized to the corresponding GAPDH values for each well. The values obtained for siRNA against Eg5 correlated with the values obtained for non-specific siRNA (against HCV) set at 100% (tables 1b, 2b and 3 b).

The effective siRNA resulting from the screening was further characterized using a dose response curve. Transfection of dose response curves was performed at the following concentrations: 100nM, 16.7nM, 2.8nM, 0.46nM, 77picoM, 12.8picoM, 2.1picoM, 0.35picoM, 59.5fM, 9.9fM and mock (no siRNA) and diluted with Opti-MEM to a final concentration of 12.5. mu.l according to the protocol described above. Data analysis was performed using Microsoft Excel embedding software XL-fit 4.2(IDBS, Guildford, Surrey, UK) and using dose response model 205 (tables 1b, 2b and 3 b).

The lead siRNA AD12115 was additionally analyzed by applying WST-proliferation assay from Roche (as described above).

The 34 duplex subsets from table 2 that showed the greatest activity were tested by transfection in HeLa cells at final concentrations of 100nM to 10 fM. The transfection was performed in quadruplicate. Two dose-response experiments were performed for each duplex. The concentrations at which each duplex reduced KSP mRNA by 20% (IC20), 50% (IC50) and 80% (IC80) were calculated (Table 7).

Table 7: dose responsive mRNA inhibition of Eg5/KSP duplexes in HeLa cellsConcentrations given in pM

(ND-not measured)

Example 4: single dose bolus injection of siRNA formulated in LNP01 into young rats Silencing of liver Eg5/KSP

Eg5/KSP expression was detectable in the liver of growing rats from birth until about 23 days of age. Target silencing of Eg5/KSP siRNA formulated in young rats was evaluated using duplex AD-6248.

Assayed KSP duplexes

Method of producing a composite material

And (4) animal administration. Male, young Sprague-Dawley rats (19 days old) were administered a single dose of liposome ("LNP 01") formulated siRNA via tail vein injection. Ten animals in each group received a 10 milligram dose per kilogram body weight (mg/kg) of AD6248 or non-specific siRNA. Dosage level means the amount of siRNA duplex administered in the formulation. The third group received phosphate buffered saline. Animals were sacrificed two days after siRNA administration. The livers were excised, snap frozen in liquid nitrogen and ground to powder.

And (3) mRNA measurement. The level of Eg5/KSP mRNA from the livers of all treatment groups was determined. Each liver powder sample (approximately ten mg) was homogenized in tissue lysis buffer containing proteinase K. The Eg5/KSP and GAPDH mRNA levels were determined for each sample using the Quantigene branched DNA assay (GenoSpectra) and the assay was repeated three times. The mean Eg5/KSP values for each sample were normalized to the mean GAPDH value. Group means were determined for each experiment and normalized to the PBS group.

And (5) carrying out statistical analysis. Significance was determined by ANOVA followed by Tukey post hoc tests.

Results

Summary of data

The mean (. + -. SD) of the Eg5/KSP mRNA is given. Statistical significance (p-value) was shown for the comparative PBS group (ns, not significant [ p > 0.05 ]).

TABLE 8, experiment 1

KSP/GAPDH P values

PBS 1.0±0.47

AD6248 10mg/kg 0.47±0.12 ＜0.001

Nonspecific 10mg/kg 1.0. + -. 0.26 ns

A statistically significant reduction in liver Eg5/KSP mRNA was obtained after administration of formulation AD6248 at a dose of 10 mg/kg.

Example 5: silencing of rat liver VEGF following intravenous infusion of LNP01 formulated VSP Transforming

A "lipid-like" formulation containing an equimolar mixture of two sirnas was administered to rats. As used herein, VSP means a composition containing two siRNAs, one siRNA directed against Eg5/KSP and the other siRNA directed against VEGF. In this experiment, duplex AD3133 against VEGF and AD12115 against Eg5/KSP were used. Because Eg5/KSP expression was barely detectable in adult rat liver, VEGF levels were only measured after siRNA treatment.

Administered siRNA duplexes (VSP)

Key words: a, G, C, U-ribonucleotides; c, u-2' -O-Me nucleotides; s-phosphorothioate. The unmodified form of each strand and the target of each siRNA are as follows:

Method of producing a composite material

And (4) animal administration. Lipid-like ("LNP 01") formulated siRNA was administered to adult, female Sprague-Dawley rats by infusion into the femoral vein over a two hour period. Four animals in each group received the formulated siRNA at doses of 5, 10 and 15 milligrams per kilogram of body weight (mg/kg). The dose level refers to the total amount of siRNA duplex administered in the formulation. The fourth group received phosphate buffered saline. Animals were sacrificed 72 hours after the siRNA infusion was completed. The livers were excised, snap frozen in liquid nitrogen and ground to powder.

Preparation method

The lipid ND 98.4 HCl (MW 1487) (formula 1 above), cholesterol (Sigma-Aldrich) and PEG-ceramide C16(Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; cholesterol, 25mg/ml, PEG-ceramide C16, 100 mg/ml. Stock solutions of ND98, cholesterol, and PEG-ceramide C16 were then mixed in a molar ratio of 42: 48: 10. The mixed lipid solution was mixed with aqueous siRNA (in sodium acetate at pH 5) such that the final ethanol concentration was about 35-45% and the final sodium acetate concentration was about 100-300 mM. Once mixed, lipid-siRNA nanoparticles spontaneously formed. Depending on the desired particle size distribution, the resulting nanoparticle mixture is in some cases extruded through a polycarbonate membrane (100nm cut-off) using a thermal barrier Extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step is omitted. Removal of ethanol and concurrent buffer exchange can be accomplished by dialysis or tangential flow filtration. The buffer was replaced with Phosphate Buffered Saline (PBS) at pH 7.2.

Characterization of the formulations

Formulations prepared by standard methods or by extrusion-free methods can be characterized in a similar manner. The formulation was first characterized by visual inspection. They should be white colored translucent solutions free of aggregates or precipitates. The particle size and particle size distribution of the lipid-nanoparticles were determined by dynamic light scattering using a Malvern zetasizer Nano ZS (Malvern, USA). The size of the particles should be about 20-300nm, ideally 40-100 nm. The particle size distribution should be monomodal. The total siRNA concentration in the formulation was evaluated using a dye exclusion assay, as well as the capture fraction. The formulated siRNA samples were incubated with the RNA-binding dye Ribogreen (molecular probe) in the presence or absence of formulation-disrupting surfactant 0.5% Triton-X100. The signal from the surfactant-containing sample can be compared to a standard curve to determine total siRNA in the formulation. The capture fraction was determined by subtracting the "free" siRNA content (determined from the signal in the absence of surfactant) from the total siRNA content. The percentage of captured siRNA is typically > 85%. For a SNALP formulation, the particle size is at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 110nm, and at least 120 nm. Preferred ranges are from about at least 50nm to about at least 110nm, preferably from about at least 60nm to about at least 100nm, most preferably from about at least 80nm to about at least 90 nm. In one example, each particle size comprises an Eg5dsRNA and a VEGF dsRNA in a ratio of at least about 1: 1.

And (3) mRNA measurement. Each liver powder sample (approximately ten mg) was homogenized in tissue lysis buffer containing proteinase K. The levels of VEGF and GAPDH mRNA were determined for each sample using Quantigene branched DNA assay (GenoSpectra) in triplicate. The VEGF mean for each sample was normalized to the mean GAPDH value. Group means for each experiment were determined and normalized to the PBS group.

Protein assay

Each liver powder sample (approximately 60 mg) was homogenized in 1ml of RIPA buffer. The total protein concentration was determined using the MicroBCA protein assay kit (Pierce). VEGF protein levels were determined by VEGF ELISA assay (R & D system) using total protein samples from each animal. Group means for each experiment were determined and normalized to the PBS group.

And (5) carrying out statistical analysis. Significance was determined by ANOVA followed by Tukey post hoc tests.

Results

Summary of data

Mean values (± standard deviation) of mRNA (VEGF/GAPDH) and protein (rel. VEGF) are shown for each treatment group. Statistical significance (p-value) is shown for each experimental control PBS group.

TABLE 9

Statistically significant reductions in liver VEGF mRNA and protein were measured in all three siRNA dose levels.

Example 6: evaluation of VSP SNALP in mouse model of human liver tumor

These studies utilized VSP siRNA mixtures comprising dsRNA targeting KSP/Eg5 and dsRNA targeting VEGF. As used herein, VSP means a composition containing two siRNAs, one siRNA directed against Eg5/KSP and the other siRNA directed against VEGF. In this experiment, duplexes AD3133 (for VEGF) and AD12115 (for Eg5/KSP) were used. The siRNA mixture was formulated into SNALP as follows.

20-25 mice were used for the maximum study scale. To test the efficacy of the siRNA SNALP cocktail in treating liver cancer, 1x10^6 tumor cells were injected directly into the left lobe of the test mouse. The incision was closed by suturing and the mice were allowed to recover for 2-5 hours. Mice fully recovered within 48-72 hours. SNALP siRNA treatment was started 8-11 days after tumor inoculation.

The SNALP formulation used was (i) VSP (KSP + VEGF siRNA mixture (1: 1 molar ratio)); (ii) KSP (KSP + Luc siRNA mixture); and (iii) VEGF (VEGF + LucsiRNA mixture). All formulations contained equal amounts (mg) of each active siRNA. All mice received a total siRNA/lipid dose and each mixture was formulated as 1: 57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC and 34.3% cholesterol), 6: 1 lipid: drug using the original citrate buffer conditions.

Human Hep3B study a: antitumor Activity of VSP-SNALP

Human hepatoma Hep3B tumors were formed in scid/beige mice by intrahepatic inoculation. Group a (n-6) animals were dosed with PBS; group B (n-6) animals were administered VSP SNALP; group C (n ═ 5) animals were dosed with KSP/Luc SNALP; group D (n-5) animals were administered VEGF/Luc SNALP.

SNALP treatment was started eight days after tumor inoculation. SNALP were administered at 3mg/kg total siRNA twice weekly (monday and thursday) for a total of six doses (18 mg/kg siRNA accumulated). The final dose was administered on day 25, and the endpoint was on day 27.

By (a) body weight; (b) liver weight; (c) visual inspection and photography on day 27; (d) human specific mRNA analysis; and (e) determining blood alpha-fetoprotein levels on day 27 to determine tumor burden.

Table 10 below illustrates the results of visual scoring of the tumor burden measured in the inoculated (left) lobe. Scoring: "-", no visible tumor; the injection site had obvious tumor tissue; "+ +" ═ discrete tumor nodules protruding from liver lobes; "+ + + +" -large tumors protruding from bilateral lobes; "+ ++", a large tumor, multiple nodules, spread over the lobes of the liver.

Watch 10

The percentage of liver weight relative to body weight is shown in figure 1. Fig. 2A, 2B, 2C, and 2D show the effect of PBS, VSP, KSP, and VEGF on body weight of mice with human hepatoma Hep3B tumor.

The following conclusions were drawn from this study: (1) VSP SNALP showed potential anti-tumor effects in the Hep3B 1H model; (2) the antitumor activity of VSP mixtures appears to be very relevant to the KSP component; (3) anti-KSP activity was confirmed by single dose histological analysis; and (4) vegfssirna showed no detectable effect on the inhibition of tumor growth in this model.

Human Hep3B study B: extended survival following treatment with VSP

In a second Hep3B study, human hepatoma Hep3B tumors were formed by intrahepatic inoculation into scid/beige mice. Lymphocytes and natural killer cells of these mice are deficient, which is the minimal range of immune-mediated antitumor effects. Group a (n ═ 6) mice were untreated; group B (n ═ 6) mice were dosed with luciferase (luc)1955 SNALP (Lot No. app10-02); VSP SNALP (Lot No. app10-01) was administered to group C (n ═ 7) mice. The SNALP is 1: 57cDMASNALP, and 6: 1 lipid: drug.

SNALP treatment was started eight days after tumor inoculation. SNALP were administered at 3mg/kg siRNA twice weekly (monday and thursday) for a total of six doses (18 mg/kg siRNA accumulated). The final dose was administered on day 25 and the end point of the study was on day 27.

By (1) body weight; (2) visual inspection and photography on day 27; (3) human specific mRNA analysis; and (4) determining blood alpha-fetoprotein levels on day 27 to determine tumor burden.

Figure 3 shows body weights measured daily (days 8, 11, 14, 18, 21 and 25) and on the day of sacrifice.

TABLE 11

Scoring: "-", no visible tumor; the injection site had obvious tumor tissue; "+ +" ═ discrete tumor nodules protruding from liver lobes; "+ + + +" -large tumors protruding from bilateral lobes; "+ ++", a large tumor, multiple nodules, spread over the lobes of the liver.

The correlation between body weight and tumor burden is shown in figures 4, 5 and 6. Figure 4 shows the percentage of body weight of untreated mice over 27 days. Fig. 5 shows the percentage of body weight over 27 days in 1955 Luc SNALP treated mice. Figure 6 shows the percentage of body weight of VSP SNALP treated mice over a 27 day period.

Administration of a single dose of VSP SNALP (2mg/kg) to Hep3B mice resulted in the formation of mitotic spindles in liver tissue samples as detected by histological staining.

Tumor burden was quantified by quantitative RT-PCR (pRT-PCR) (Taqman). Human GAPDH was normalized to mouse GAPDH by species-specific Taqman assay. Figure 7A shows the tumor scores shown by visual inspection in the above table in relation to GADPH levels.

Serum ELISA was performed to determine the alpha-fetoprotein (AFP) secreted by the tumor. As described below, if AFP levels decrease after treatment, the tumor does not grow. Figure 7B shows that treatment with VSP reduced AFP levels in some animals compared to treatment with controls.

Human HepB3 study C：

In a third study, human HCC cells (HepB3) were injected directly into the liver of SCID/beige mice and treatment was initiated 20 days later. Group a animals were given PBS; group B animals were administered 4mg/kg Luc-1955 SNALP; group C animals were administered 4mg/kg SNALP-VSP; group D animals were administered 2mg/kg SNALP-VSP; group E animals were given 1mg/kg SNALP-VSP. A single dose intravenous (iv) treatment was performed and the mice were sacrificed 24 hours later.

Tumor burden and target silencing were determined by qRT-PCR (Taqman). Tumor scores were also determined visually as described above and the results are shown in the table below. hGAPDH levels as shown in figure 8 correlate with the macroscopic tumor scores shown in the table below.

TABLE 12

Scoring: "-" — "visible tumor/some small tumors; "+ +" ═ discrete tumor nodules protruding from liver lobes; "+ + + +", large tumors protruding from bilateral lobes.

Human (tumor-derived) KSP silencing was determined by Taqman analysis and the results are shown in FIG. 9. hKSP expression was normalized to hGAPDH. About 80% tumor KSP silencing was observed with 4mg/kg SNALP-VSP, with a significant efficacy at 1 mg/kg. Figure 9 is a clear bar graph showing results from small (low GAPDH) tumors.

Human (tumor-derived) VEGF silencing was determined by Taqman analysis and the results are shown in fig. 10. hVEGF expression was normalized to hGAPDH. About 60% tumor VEGF silencing was observed with the 4mg/kg SNALP-VSP, with a significant efficacy at 1 mg/kg. Figure 10 is a clear bar graph showing results from small (low GAPDH) tumors.

Mouse (liver-derived) VEGF silencing was determined by Taqman analysis and the results are shown in fig. 11A. mVEGF expression was normalized to hGAPDH. About 50% liver VEGF silencing was observed with the 4mg/kg SNALP-VSP, with a significant efficacy at 1 mg/kg.

Human HepB3 study D: contribution of each dsRNA to tumor growth

In a fourth study, human HCC cells (HepB3) were injected directly into the liver of SCID/beige mice and treatment was started 8 days later. This treatment was performed with intravenous (iv) bolus injections twice weekly for a total of six doses. The final dose was administered on day 25, and the endpoint was on day 27.

Tumor burden was determined by total histology, human specific mRNA analysis (hGAPDH qPCR) and blood alpha-fetoprotein levels (serum AFP determined by ELISA).

In study 1, group A was treated with PBS, group B was treated with SNALP-KSP + Luc (3mg/kg), group C was treated with SNALP-VEGF + Luc (3mg/kg), and group D was treated with SNALP-VSP (3 mg/kg).

In study 2, group A was treated with PBS, group B was treated with SNALP-KSP + Luc (1mg/kg), and group C was treated with ALN-VSP02(1 mg/kg).

GAPDH mRNA levels and serum AFP levels both showed decreases after treatment with SNALP-VSP (as shown in fig. 11B).

Histological study：

Human hepatoma Hep3B tumors were formed by intrahepatic inoculation in mice. SNALP treatment started 20 days after tumor inoculation. Tumor bearing mice (three per group) were treated Intravenously (IV) with a single dose of either (i) VSP SNALP or (ii) control (Luc) SNALP (2mg/kg total siRNA).

Liver/tumor samples were collected 24 hours after a single SNALP administration for routine H & E histology.

Large tumor nodules (5-10mm) were visually evident upon autopsy.

Effect of SNALP-VSP in Hep3B mice：

Treatment with SNALP-VSP (a mixture of KSP dsRNA and VEGF dsRNA) reduced tumor burden and expression of KSP and VEGF from tumor sources. A decrease in GAPDH mRNA levels (a measure of tumor burden) was also observed following SNALP-VSP dsRNA administration (shown in fig. 12A, 12B, and 12C). The reduction in tumor burden seen visually after SNALP-VSP administration was also evident.

A single IV bolus SNALP-VSP also resulted in mitotic spindle formation, which was clearly detected in Hep3B mouse liver tissue samples. This result indicates cell cycle arrest.

Example 7: survival of SNALP-VSP animals versus SNALP-Luc treated animals Rate of change

To determine the effect of siRNA SNALP on survival in cancer subjects, tumors were formed by intrahepatic inoculation in mice and mice were treated with SNALP-siRNA. These studies used a mixture of VSP siRNA containing dsRNA targeting KSP/Eg5 and VEGF. The control was dsRNA targeting Luc. The siRNA mixture is formulated into SNALP.

Tumor cells (human hepatoma Hep3B, 1x10^6) were injected directly into the left lobe of scid/beige mice. Lymphocytes and Natural Killer (NK) cells of these mice are deficient, which is the minimal range of immune-mediated anti-tumor effects. The incision was closed by suturing and the mice were allowed to recover for 2-5 hours. Mice fully recovered within 48-72 hours.

All mice received a total siRNA/lipid intravenous (iv) dose and each mixture was formulated as 1: 57cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC and 34.3% cholesterol), 6: 1 lipid: drug using the original citrate buffer conditions.

siRNA-SNALP treatment was started on the days shown below (18 or 26 days) after tumor inoculation. The siRNA-SNALP was administered twice weekly at a dose of 4mg/kg after 18 or 26 days for three weeks. Survival was monitored and animals were sacrificed based on human surrogate endpoints (e.g., animal body weight, abdominal distension/discoloration, and overall health).

Survival data from treatments starting 18 days after tumor inoculation are summarized in table 13, table 14 and fig. 13A.

TABLE 13 Kaplan-Meier (survival) data (% survival)

Sky	SNALP-Luc	SNALP-VSP
			18	100％	100％
22	100％	100％
			25	100％	100％
27	100％	100％
			28	100％	100％
28	86％	100％
			29	86％	100％
32	86％	100％
			33	86％	100％
33	43％	100％
			35	43％	100％
36	43％	100％
			36	29％	100％
38	29％	100％
			38	14％	100％
38	14％	88％
			40	14％	88％
43	14％	88％
			45	14％	88％
49	14％	88％
			51	14％	88％
51	14％	50％
			53	14％	50％
53	14％	25％
			55	14％	25％
57	14％	25％
			57	0％	0％

TABLE 14 days of survival for each animal

FIG. 13A shows the average survival rates of SNALP-VSP and SNALP-Luc treated animals relative to days post tumor inoculation. The mean survival of SNALP-VSP animals was extended by approximately 15 days compared to SNALP-Luc treated animals.

TABLE 15 serum alpha-fetoprotein (AFP) concentration (concentration expressed in. mu.g/ml) of each animal before and at the end of treatment

Tumor burden was monitored during the experiment with serum AFP levels. Alpha-fetoprotein (AFP) is the major plasma protein produced by the yolk sac and liver during fetal life. This protein is considered to be the fetal counterpart of serum albumin, and the human AFP and albumin genes are present in tandem on chromosome 4 in the same transcriptional orientation. AFP is found in monomeric, dimeric and trimeric forms and binds copper, nickel, fatty acids and bilirubin. AFP levels gradually decrease after birth, reaching adult levels in 8-12 months. Normal adult AFP levels are low, but detectable. AFP has no known function in normal adults, and AFP expression in adults is often associated with a proportion of tumours such as hepatomas and teratomas. AFP is a tumor marker for monitoring testicular cancer, ovarian cancer, and malignant teratomas. The major tumors that secrete AFP include endodermal sinoma (yolk sac carcinoma), neuroblastoma, hepatoblastoma, and hepatocellular carcinoma. In patients with tumors that secrete AFP, serum levels of AFP are often correlated with tumor size. Serum levels can be used to assess response to treatment. Typically, if AFP levels decrease after treatment, the tumor does not grow. The possibility that a temporary increase in AFP immediately after chemotherapy indicates that it is not tumor growth, but that it is shrinking (and releasing AFP due to tumor cell death). Resection is usually accompanied by a decrease in serum levels. As shown in fig. 14, tumor burden was significantly reduced in SNALP-VSP treated animals.

Experiments were repeated with SNALP-siRNA treatment on days 26, 29, 32, 35, 39 and 42 post-implantation. Data are shown in fig. 13B. The mean survival of SNALP-VSP animals was extended by about 15 days, and SNALP-Luc treated animals were extended by about 19 days, or 38%.

Example 8: induction of monosomes in established tumors

Inhibition of KSP in dividing cells results in the formation of single stars, which can be readily observed in tissue sections. To determine whether monosomic formation occurred in SNALP-VSP treated tumors, 2mg/kg SNALP-VSP was administered to tumor-bearing animals via tail vein injection (three weeks after Hep3B cell implantation). Control animals received 2mg/kg SNALP-Luc. And each mixture was formulated using the original citrate buffer conditions into 1: 57cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC and 34.3% cholesterol), 6: 1 lipid: drug.

Animals were sacrificed twenty-four hours later and tumor-bearing liver lobes were processed for histological analysis. A typical image of the H & E stained tissue section is shown in fig. 15. Extensive monarchid formation was evident in SNALP-VSP treated (a) but not SNALP-Luc treated (B) tumors. In the latter, normal mitotic images are evident. Monosomic production is characteristic of KSP inhibition and also provides evidence that SNALP-VSP has significant activity in established liver tumors.

Example 9: preparation method and product specification of ALN-VSP02(SNALP-VSP)

The ALN-VSP02 product contained the drug substance ALN-VSPDS01 at 2mg/mL formulated as a sterile lipid particle formulation (referred to as SNALP) for IV administration via infusion. The drug substance ALN-VSPLDS 01 consists of two siRNAs (ALN-12115 targeting KSP and ALN-3133 targeting VEGF) in equimolar ratios. The drug product was packaged in a 10mL glass vial with a fill volume of 5 mL.

The drug substance may be formulated, for example, with the cationic lipids XTC, ALNY-100, and MC3 into other nucleic acid-lipid particle formulations described herein.

The present invention uses the following terms:

^*optional names AD-12115, AD 12115;^**optional names AD-3133 and AD3133

9.1 preparation of the drug substance ALN-VSPLDS 01

Two siRNA components, ALN-12115 and ALN-3133, of the drug substance ALN-VSPDS01 were chemically synthesized using commercially available synthesis equipment and starting materials. The preparation method comprises synthesis of two single-stranded oligonucleotides for each duplex (19562 sense and 19563 antisense to ALN 12115 and 3981 sense and 3982 antisense to ALN 3133) by conventional solid phase oligonucleotide synthesis using phosphoramidite chemistry and 5 ' O Dimethoxytriphenylmethyl (DMT) protecting groups with either a tert-butyldimethylsilyl group (TBDMS) for the 2 ' hydroxyl group or a 2 ' hydroxyl group replaced by a 2 ' methoxy (' OMe). The oligonucleotide chains are assembled on a solid support such as controlled pore glass or polystyrene by the phosphoramidite method. The cycle consists of 5' deprotection, coupling, oxidation and capping reactions. Each coupling reaction is carried out using 5 (ethylthiol) 1 Htetrazole reagent to activate the appropriately protected ribose, 2 'OMe or deoxyribonucleoside amide, followed by coupling to the free 5' hydroxyl group of the support-immobilized protected nucleoside or oligonucleotide. After an appropriate number of cycles, the final 5' protecting group is removed by acid treatment. The crude oligonucleotide is cleaved from the solid support by treatment with aqueous methylamine solution and concomitant removal of the cyanoethyl protecting group and the nucleobase protecting group. The 2' O TBDMS group is then cleaved using a reagent comprising hydrogen fluoride to produce a crude oligoribonucleotide, which is purified using strong anion exchange High Performance Liquid Chromatography (HPLC) followed by desalting using ultrafiltration. The purified single strands were analyzed to confirm the correct molecular weight, molecular sequence, impurity profile and oligonucleotide content, and then annealed into duplexes. The annealed duplex intermediates ALN 12115 and ALN 3133 were lyophilized and stored at 20 ℃ or the solutions were mixed in a 1: 1 molar ratio and lyophilized to yield the drug substance ALN VSPLDS 01. If the duplex intermediates are stored as dry powders, they are redissolved in water prior to mixing. The mixing process was monitored by HPLC to achieve equimolar ratio.

Example specifications are shown in table 16a.

TABLE 16a example Specification of ALN-VSPLDS 01

The stability test results of ALN-VSPDS01 drug substance up to 12 months are shown in table 16 b. Assays were selected to assess physical properties (appearance, pH, water content), purity (by SEC and denaturing anion exchange chromatography) and efficacy (by denaturing anion exchange chromatography) [ AX-HPLC ].

Table 16 b: stability of drug substances

9.2 preparation of pharmaceutical product ALN-VSP02

ALN VSP02 was a sterile formulation of two sirnas (at a 1: 1 molar ratio) and a lipid excipient in an isotonic buffer. The lipid excipient binds to both sirnas, protecting them from degradation in the circulatory system, and facilitating their delivery to the target tissue. The specific lipid excipients and the quantitative ratios of each (shown in table 17) were selected by a repeated series of experiments comparing the physicochemical properties, stability, pharmacodynamics, pharmacokinetics, toxicity and product manufacturability of a large number of different formulations. The excipient DLinDMA is a titratable amino lipid that is positively charged at low pH, such as those found in mammalian cellular endosomes, but which is relatively uncharged at the more neutral pH of whole blood. This feature promotes efficient encapsulation of negatively charged siRNA at low pH, preventing empty particle formation, but allows for conditioning (reduction) of particle charge by replacing formulation buffer with a more neutral storage buffer prior to use. Cholesterol and the neutral lipid DPPC were introduced to provide physicochemical stability of the particles. The polyethylene glycol lipid conjugate PEG2000C DMA contributes to drug product stability and provides optimized cycle times for the proposed use. The ALN VSP02 lipid particles had an average diameter of about 80-90nm, with low polydispersity values. At neutral pH, the particles are essentially uncharged, with zeta potential values of less than 6 mV. There was no evidence of empty (unsupported) particles based on this preparation method.

Table 17: quantitative composition of ALN-VSP02

^*The 1: 1 molar ratio of the two siRNAs in the drug product was maintained throughout the particle size distribution of the drug product particles.

The solution of lipids (in ethanol) and ALN VSPDS01 drug substance (in aqueous buffer) was mixed and diluted to form a colloidal dispersion of siRNA lipid particles with an average particle size of about 80-90 nm. The dispersion was then filtered through a 0.45/0.2 μm filter, concentrated and diafiltered by tangential flow filtration. After in-process testing and adjustment of the concentration to 2.0mg/mL, the product is sterilized by filtration, filled into glass vials under aseptic conditions, stoppered, capped and placed at 5. + -. 3 ℃. Ethanol and all aqueous buffer components are usp grade; all water used was usp sterile water for injection grade. ALN-VSP 02.

Similar methods are used to formulate other lipid formulations of ALN-VSPDS01, for example, those containing the cationic lipids XTC, ALNY-100, and MC 3.

Example 10: in vitro efficacy of ALN-VSP02 on human cancer cell lines

The efficacy of ALN-VSP02 treatment on human cancer cell lines was determined after treatment by measuring KSP mRNA, VEGF mRNA and cell viability. IC50(nM) values for KSP and VEGF were determined for each cell line.

Table 19: cell lines

Cells were seeded in complete medium in 96-well plates on the first day to reach 70% density on the next day. The following day, the medium was changed to Opti-MEM reduced serum medium (InVitrogen Cat N: 11058-. After 6 hours the medium was changed to complete medium. Three plates were inoculated in duplicate for each cell line in each experiment.

ALN-VSP02 was formulated as described in table 17.

Cells were harvested 24 hours after transfection. Determining KSP levels using bDNA; VEGF mRNA levels were determined using a human TaqMan assay.

Viability was determined at 48 and/or 72 hours using Cell Titer Blue reagent (Promega Cat N: G8080) according to manufacturer recommendations.

As shown in table 20, VSP02 at nM concentrations was effective in reducing KSP and VEGF expression in a variety of human cell lines. Viability of the treated cells was not

Table 20: results

Example 11: VSP SNALP vs sorafenib in established Hep3B intrahepatic tumors Antitumor efficacy of Nini

The antitumor effect of multiple doses of VSP SNALP vs sorafenib in scid/beige mice bearing established Hep3B intrahepatic tumors was studied. Sorafenib is a small molecule inhibitor of protein kinases approved for the treatment of hepatocellular carcinoma (HCC).

Tumors were formed by inoculation in the liver of scid/beige mice as described herein. Treatment was started 11 days after inoculation. Mice were treated with sorafenib and control siRNA-SNALP, sorafenib and vspasi-SNALP or VSP siRNA-SNALP alone. Control mice were treated with buffer only (DMSO instead of sorafenib, PBS instead of siRNA-SNALP). Sorafenib was administered parenterally from monday to friday for three weeks at a dose of 15mg/kg body weight for a total of 15 injections. Sorafenib was administered a minimum of 1 hour after SNALP injection. siRNA-SNALPS was administered intravenously at 3mg/kg via the lateral tail vein on days 1, 4, 7, 10, 14, and 17 according to the newly recorded body weight (10ml/kg) for three weeks (6 doses total).

Each siRNA-SNALP was formulated as 1: 57cDMASNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC and 34.3% cholesterol), 6: 1 lipid: drug using the original citrate buffer conditions.

Mice were sacrificed based on assessment of tumor burden, including gradual weight loss and clinical signs (including symptoms, abdominal distension/discoloration, and mobility).

The percent survival data is shown in figure 16. The co-administration of VSP siRNA-SNALP and sorafenib increased the survival rate compared to sorafenib or VSP siRNA-SNALP administered alone. VSP siRNA-SNALP increased survival rates compared to sorafenib.

Example 12: VSP in vitro efficacy using variants of AD-12115 and AD-3133

Two sets of duplexes targeting Eg5/KSP and VEGF were designed and synthesized. Each set included duplexes extending (tilling) 10 nucleotides in each direction at the target site of either of AD-12115 and AD-3133.

The target, sense and antisense strand sequences for each duplex are shown in the table below.

Inhibition of expression by each duplex was determined using the assay described herein. Duplexes, e.g., Eg5/KSP dsRNA and VEGF dsRNA combinations, are administered alone and/or in combination. In some embodiments, the dsRNA is administered as a nucleic acid lipid particle (e.g., a SNALP formulation as described herein).

Table 21: dsRNA sequences (extensions) targeting VEGF and Eg5/KSP

Example 13: VEGF-targeting dsRNA with single blunt end

A set of dsRNA duplexes targeting VEGF was designed and synthesized. Each set includes duplexes that extend 10 nucleotides in each direction of the target site of AD-3133. Each duplex includes a 2 base overhang at the end corresponding to the 3 'end of the antisense strand and no overhang, e.g., blunt end, at the end corresponding to the 5' end of the antisense strand.

The individual strand sequences of these duplexes are shown in the table below.

Inhibition of expression by each duplex was determined using the assay described herein. The VEGF duplexes are administered alone and/or in combination with Eg5/KSP dsRNA (e.g., AD-12115). In some embodiments, the dsRNA is administered as a nucleic acid lipid particle (e.g., a SNALP formulation as described herein).

Table 22: target sequences for VEGF-targeting blunt-ended dsRNA

Table 23: strand sequence of blunt-ended dsRNA targeting VEGF

Example 14: dsRNA oligonucleosidesAcid synthesis

Synthesis of

All oligonucleotides were synthesized on an AKTAoligopilot synthesizer. A commercially available controlled pore glass solid support (dT-CPG,prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5 ' -O-dimethoxytrityl N6-benzoyl-2 ' -tert-butyldimethylsilyl-adenosine-3 ' -O-N, N ' -diisopropyl-2-nitriloethylphosphonamide, 5 ' -O-dimethoxytrityl-N4-acetyl-2 ' -tert-butyldimethylsilyl-cytidine-3 ' O-N, N ' -diisopropyl-2-nitriloethylphosphonamide, 5 ' -O-dimethoxytrityl-N2-isobutyl-2 ' -tert-butyldimethylsilyl-guanosine-3 ' -O-N, N ' -diisopropyl-2-nitriloethylphosphonamide and 5 ' -O-dimethoxytrityl Benzyl-2 ' -tert-butyldimethylsilyl-uridine-3 ' -O-N, N ' -diisopropyl-2-nitriloethylphosphatimide (Pierce Nucleic Acids Technologies) was used to synthesize the oligonucleotides. 2 ' -F phosphoramidite, 5 ' -O-dimethoxytrityl-N4-acetyl-2 ' -fluoro-cytidine-3 ' -O-N, N ' -diisopropyl-2-carbonitrile ethyl phosphoramidite and 5 ' -O-dimethoxytrityl-2 ' -fluoro-uridine-3 ' -O-N, N ' -diisopropyl-2-cyanoethyl phosphoramidite were purchased from Promega. All phosphoramidites were used as 0.2M acetonitrile (CH3CN) solution, except for guanosine, which was used as 0.2M 10% THF/ANC (v/v) solution. The coupling/recirculation time used was 16 minutes. The activator is 5-ethylmercaptotetrazole (0.75M, American International Chemicals); for PO oxidation, iodine/water/pyridine was used, for PS-oxidation, PADS (2%) in 2, 6-lutidine/ACN (1: 1v/v) was used.

3' -ligand conjugated chains were synthesized with solid supports containing the corresponding ligands. The introduction of the cholesterol unit into the sequence is carried out, for example, by hydroxyprolinol-cholesterol phosphoramidite. Binding cholesterol to trans-4-hydroxyprolinol via a 6-aminocaproate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5' -terminal Cy-3 and Cy-5.5 (fluorophore) -labeled siRNAs were synthesized from the corresponding Quasar-570(Cy-3) phosphoramidites purchased from Biosearch Technologies. Ligand conjugates that bind to the 5' -end and or internal position are obtained by using appropriately protected ligand-phosphoramidite building blocks. A0.1M solution of phosphoramidite in anhydrous CH3CN was extendedly coupled to the solid-support bound oligonucleotide in the presence of 5- (ethylmercapto) -1H-tetrazole activator for 15 min. Oxidation of internucleotide phosphites to phosphates was performed using standard iodine-water as reported in (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10: 87: 3) (10 min oxidation latency for conjugated oligonucleotides). The phosphorothioates are incorporated by oxidation of phosphites to phosphorothioates using sulfur transfer agents such as DDTT (available from AM Chemicals), PADS, and or Beaucage reagents. Cholesterol phosphoramidite was synthesized internally and used at a concentration of 0.1M in dichloromethane. The coupling time for the cholesterol phosphoramidite was 16 minutes.

Deprotection I (nucleobase deprotection)

After completion of the synthesis, the support was transferred to a 100mL glass Vial (VWR). The oligonucleotides were cleaved from the support while deprotecting the base and phosphate groups with 80mL of a mixture of ethanolamines [ ammonia: ethanol (3: 1) ] at 55 ℃ for 6.5 h. The bottle was cooled briefly on ice and the ethanolamino mixture was then filtered into a new 250-mL bottle. CPG was washed with 2X40mL parts of ethanol/water (1: 1 v/v). The mixture volume was then reduced to about 30mL by a rotary evaporator (roto-vap). The mixture was then frozen on dry ice and dried in vacuo using a vacuum centrifuge concentrator (speed vac).

Deprotection II (removal of 2' -TBDMS group)

The dried residue was resuspended in 26mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3: 4: 6) and heated at 60 ℃ for 90 minutes to remove the tert-butyldimethylsilyl group at the 2' -position (TBDMS). The reaction was then quenched with 50mL of 20mM sodium acetate and the pH adjusted to 6.5. The oligonucleotides were stored in a refrigerator until purified.

Analysis of

The oligonucleotides are analyzed by High Performance Liquid Chromatography (HPLC) and then purified, the choice of buffer and column depending on the sequence and or nature of the conjugated ligand.

HPLC purification

The ligand-conjugated oligonucleotides were purified by reverse phase preparative HPLC. Unconjugated oligonucleotides were purified by anion exchange HPLC on an internally packed TSK gel column. The buffer was 10% CH₃20mM sodium phosphate (pH 8.5) (buffer A) in CN and 10% CH₃CN, 20mM sodium phosphate (pH 8.5) in 1M NaBr (buffer B). Fractions containing full-length oligonucleotides were pooled. Desalted and lyophilized. Approximately 0.15OD of the desalted oligonucleotide was diluted to 150. mu.L in water and then pipetted into special vials for CGE and LC/MS analysis. The compounds were then analyzed by LC-ESMS and CGE.

SiRNA preparation

To prepare siRNA, equimolar amounts of sense and antisense strands were heated at 95 ℃ in 1xPBS for 5min and slowly cooled to room temperature. The integrity of the duplex was confirmed by HPLC analysis. The AD-3133 and AD-12115 described in the present invention were synthesized.

Example 15: synthesis of conjugated lipids

PEG-lipids were synthesized using the following method, e.g. mPEG2000-1, 2-di-O-alkyl-sn 3-carbamoylglyceride (PEG-DMG):

mPEG2000-1, 2-di-O-alkyl-sn 3-carbamoylglyceride

Preparation of compound 4 a: will be provided with1, 2-di-O-tetradecyl-sn-glyceride 1a (30g, 61.80mmol) and N, N' -succinimidyl carbonate (DSC, 23.76g, 1.5eq) were added to dichloromethane (DCM, 500mL) and stirred in a mixture of ice and water. Triethylamine (25.30mL, 3eq) was added to the stirred solution, and the reaction mixture was subsequently stirred at ambient temperature overnight. The progress of the reaction was monitored by TLC. The reaction mixture was diluted with DCM (400mL), water (2X500mL), NaHCO ₃The organic layer was washed with aqueous solution (500mL) followed by standard procedures. The obtained residue was dried at ambient temperature under high vacuum overnight. After drying, the crude carbonate 2a thus obtained was dissolved in dichloromethane (500mL) and stirred in an ice bath. Adding mPEG into the stirring solution under the argon atmosphere₂₀₀₀-NH₂(3, 103.00g, 47.20mmol, available from NOF Corporation, Japan) and anhydrous pyridine (80mL, excess). In some embodiments, x in methoxy- (PEG) x-amine is 45-49, preferably 47-49, more preferably 49. The reaction mixture was then stirred at ambient temperature overnight. The solvent and volatiles were removed in vacuo and the residue was dissolved in DCM (200mL) and loaded onto a silica gel column packed with ethyl acetate. The column was eluted first with ethyl acetate followed by a gradient of 5-10% methanol in dichloromethane to give the desired PEG-lipid 4a as a white solid (105.30g, 83%).¹H NMR(CDCl₃，400MHz)＝5.20-5.12(m，1H)，4.18-4.01(m，2H)，3.80-3.70(m，2H)，3.70-3.20(m，-O-CH₂-CH₂-O-，PEG-CH₂) 2.10-2.01(m, 2H), 1.70-1.60(m, 2H), 1.56-1.45(m, 4H), 1.31-1.15(m, 48H), 0.84(t, J ═ 6.5Hz, 6H). MS range measured value 2660-.

4b preparation: 1, 2-di-O-hexadecyl-sn-glyceride 1b (1.00g, 1.848mmol) and DSC (0.710g, 1.5eq) were added together in dichloromethane (20mL) and cooled to 0 ℃ in an ice water mixture. To this was added triethylamine (1.00mL, 3eq) and stirred overnight. The reaction was followed by TLC, dilution with DCM, water (2X), NaHCO ₃The solution was washed and dried over sodium sulfate. The solvent was removed under reduced pressure and the residue 2b was placed under high vacuum overnight. This compound was used in the next reaction without additional purification. MPE reaction under argon atmosphereG₂₀₀₀-NH₂3(1.50g, 0.687mmol, available from NOF Corporation, Japan) and Compound 2b from the previous reaction (0.702g, 1.5eq) were dissolved in dichloromethane (20 mL). The reaction was cooled to 0 ℃. To this was added pyridine (1mL, excess) and stirred overnight. The reaction was monitored by TLC. The solvents and volatiles were removed in vacuo and the residue was purified by chromatography (eluting first with ethyl acetate and then with a gradient of 5-10% MeOH in DCM) to give the desired compound 4b as a white solid (1.46g, 76%).¹H NMR(CDCl₃，400MHz)＝5.17(t，J＝5.5Hz，1H)，4.13(dd，J＝4.00Hz，11.00Hz，1H)，4.05(dd，J＝5.00Hz，11.00Hz，1H)，3.82-3.75(m，2H)，3.70-3.20(m，-O-CH₂-CH₂-O-，PEG-CH₂) 2.05-1.90(m, 2H), 1.80-1.70(m, 2H), 1.61-1.45(m, 6H), 1.35-1.17(m, 56H), 0.85(t, J ═ 6.5Hz, 6H). MS range found: 2716-2892.

4c preparation: 1, 2-di-O-octadecyl-sn-glyceride 1c (4.00g, 6.70mmol) and DSC (2.58g, 1.5eq) were added together in dichloromethane (60mL) and cooled to 0 ℃ in a mixture of ice and water. To this was added triethylamine (2.75mL, 3eq) and stirred overnight. The reaction was followed by TLC, dilution with DCM, water (2X), NaHCO ₃The solution was washed and dried over sodium sulfate. The solvent was removed under reduced pressure and the residue 2b was placed under high vacuum overnight. This compound was used in the next reaction without additional purification. Under argon atmosphere, MPEG₂₀₀₀-NH₂3(1.50g, 0.687mmol, available from NOF Corporation, Japan) and compound 2c (0.760, 1.5eq) from the previous reaction were dissolved in dichloromethane (20 mL). The reaction was cooled to 0 ℃. To this was added pyridine (1mL, excess) and stirred overnight. The reaction was monitored by TLC. The solvents and volatiles were removed in vacuo and the residue was purified by chromatography (eluting first with ethyl acetate and then with a gradient of 5-10% MeOH in DCM) to give the desired compound 4c as a white solid (0.92g, 48%).¹H NMR(CDCl₃，400MHz)＝5.22-5.15(m，1H)，4.16(dd，J＝4.00Hz，11.00Hz，1H)，4.06(dd，J＝5.00Hz，11.00Hz，1H)，3.81-3.75(m，2H)，3.70-3.20(m，-O-CH₂-CH₂-O-，PEG-CH₂) 1.80-1.70(m, 2H), 1.60-1.48(m, 4H), 1.31-1.15(m, 64H), 0.85(t, J ═ 6.5Hz, 6H). MS range found: 2774-2948.

Example 16: general scheme for extrusion Process

Lipids (e.g., lipid A, DSPC, cholesterol, DMG-PEG) were dissolved in ethanol and mixed according to the desired molar ratio. Liposomes were formed by ethanol injection, in which the mixed lipids were added to sodium acetate buffer at pH 5.2. This resulted in the spontaneous formation of liposomes in 35% ethanol. The liposomes were extruded through a 0.08 μm polycarbonate membrane at least 2 times. A stock solution of siRNA was prepared in sodium acetate and 35% ethanol was added to the liposomes for loading. The siRNA-liposome solution was incubated at 37 ℃ for 30min, followed by dilution. Ethanol was removed and replaced with PBS buffer by dialysis or tangential flow filtration.

Example 17: general scheme for in-line mixing process

Preparation of individual and separate stock solutions: one comprising a lipid and the other comprising an siRNA. Lipid stocks, for example, comprising lipid A, DSPC, cholesterol, and PEG lipids, were prepared by dissolution in 90% ethanol. The remaining 10% was low pH citrate buffer. The concentration of the lipid stock solution was 4 mg/mL. The citrate buffer may have a pH of 3-5 depending on the type of fusogenic lipid used. siRNA was also dissolved in citrate buffer at a concentration of 4 mg/mL. For small scale, 5mL of each stock solution was prepared.

The stock solution is completely transparent and the lipids must be completely dissolved before mixing with siRNA. The stock solution can thus be heated to completely dissolve the lipids. The siRNA used in this method may be an unmodified oligonucleotide or a modified oligonucleotide, and may be conjugated to a lipophilic moiety such as cholesterol.

Mixing by pumping each solution into the T-junctionAnd combining the separate stock solutions. A double-headed Watson-Marlow pump was used to control the start and stop of both streams simultaneously. The 1.6mm polypropylene tube was further reduced to a 0.8mm tube to increase the linear flow rate. Polypropylene lines (ID ═ 0.8mm) were attached to either side of the T-junction. The linear edge of the polypropylene T was 1.6mm, and the resulting volume was 4.1mm ³. Each large end (1.6mm) of the polypropylene line was placed in a test tube containing a stock solution of dissolved lipids or dissolved siRNA. After the single pipe is placed at the T-junction, the combined flow will flow out there. The tubing was then extended into a container containing 2 volumes of PBS. PBS was rapidly stirred. The flow rate of the pump was set at 300rpm or 110 mL/min. Ethanol was removed and replaced with PBS by dialysis. The lipid preparation is then concentrated to the appropriate use concentration by centrifugation or diafiltration.

FIG. 17 shows a schematic of an in-line mixing process.

Example 18: characterization of Hep3B tumors in mouse liver by LNP-08 formulated VSP SiRNA silencing

VSP (VEGF and KSP) silencing was performed in orthotopic (intrahepatic) Hep3B tumors following intravenous administration of siRNA formulated in XTC-containing nucleic acid-lipid particles, such as LNP-08.

By mixing 1X10⁶Hep3B cells transplanted into the right flank of 8-week old female Fox scid/beige mice formed tumors. Cells were genetically engineered to stably express firefly luciferase. Tumor burden was monitored weekly by in vivo biophotonic imaging using the IVIS system (Caliper, Inc.). Approximately 4 weeks after tumor implantation, the tumor-bearing animal group received intravenous (tail vein) injections of the following test articles:

LNP08-1955 is a lipid nanoparticle formulated with siRNA AD-1955 (targeting firefly luciferase) containing XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%) and PEG-cDMG (1.5 mol%), N: P ratio about 3.0.

LNP08-VSP is a lipid nanoparticle formulated with siRNAs AD-12115 (targeting KSP) and AD-3133 (targeting VEGF) at a 1: 1 molar ratio, containing XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%) and PEG-cDMG (1.5 mol%), N: P ratio of about 3.0.

One day after treatment, animals were sacrificed and tumor bearing liver lobes were collected for analysis. Total RNA was extracted, and then cDNA was synthesized by random primer. Using human-specific custom TaqmanThe test (Applied Biosystems, Inc.) determined human KSP and human VEGF levels normalized to human GAPDH. Group means were calculated and normalized to the LNP08-1955 treatment group.

As shown in FIG. 18, treatment with LNP08-VSP (group 2) resulted in a tumor KSP mRNA reduction of more than 60%, e.g., 68% (p < 0.001), and a VEGFmRNA reduction of at least 40% (p < 0.05) compared to LNP08-1955 treatment (group 1).

Example 19: LIPID FATIONS OF LNP-011 and LNP-012 IN THE MOULD HEP3b TUMOR MODEL IN MOSES Evaluation of agents

The effect of various VSP preparations on KSP 3B tumor expression in mouse liver was compared. 1X10^6Hep3B-Luc cells suspended in 0.025cc PBS were injected into thirty-five female Fox Scid beige mice via direct intrahepatic surgery. Tumor growth was monitored by Xenogen via Luc readings.

Mice received a single bolus (4mg/kg) of one of the following formulations: SNALP-1955 (luciferase control); ALN-VSP 02; SNALP-T-VSP (with C-18PEG) -VSP; LNP-11-VSP and LNP-12 VSP. Animals were sacrificed 24 hours after dosing and TaqMan methods were used to determine tumor-specific KSP and VEGF inhibition.

The results are shown in FIG. 21. SNAPL-T-VSP, LNP-11-VSP and LNP-12VSP show increased inhibition of KSP expression compared to ALN-VSP 02.

Example 20: LNP-08+/-C18 lipid formulation in mouse Hep3b tumor model Evaluation of

The following VSP formulations were tested for their effect in the HEP3B tumor model. One of the following formulations was injected into (intrahepatic) tumor-bearing mice, prepared according to the above method and administered as a single bolus IV:

ALN-VSP02 formulation was as described in example 9.

LNP08-Luc is a lipid nanoparticle formulated with siRNA AD-1955 (targeting firefly luciferase) containing XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%) and PEG-cDMG (1.5 mol%), with a N: P ratio of about 3.0.

LNP08-VSP is a lipid nanoparticle formulated with siRNA AD-12115 (targeting KSP) and AD-3133 (targeting VEGF) at a 1: 1 molar ratio, containing XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%) and PEG-cDMG (1.5 mol%), with a N: P ratio of about 3.0.

LNP08-C18-VSP is a lipid nanoparticle formulated with siRNA AD-12115 (targeting KSP) and AD-3133 (targeting VEGF) at a 1: 1 molar ratio, containing XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%) and PEG-cDSG (1.5 mol%), N: P ratio of about 3.0.

FIG. 19 illustrates the chemical structures of PEG-DSG and PEG-C-DSA. PEG-DSG is polyethylene glycol distyryl glycerol, wherein PEG is C18-PEG or PEG-C18, and the average molecular weight of PEG is 2000 Da.

Twenty-four hours after treatment, animals were sacrificed and tumors were collected for analysis. Total RNA was extracted from the tumor, and cDNA was synthesized from random primers. Using human-specific custom TaqmanThe test (Applied Biosystems, Inc.) determined human KSP and human VEGF levels normalized to human GAPDH.

The results are shown graphically in fig. 22, which shows KSP and VEGF silencing comparable to silencing by ALN-VSP 02.

Example 21: role of ApoE in cellular uptake of liposomes in HeLa cells

LNP formulated dsRNA was prepared by the addition of recombinant human ApoE. The resulting LNP-ApoE formulated dsRNA was tested in HeLa cells for its effect on dsRNA uptake by the cells. Compositions and methods of using ApoE and ionizable lipids are described in International patent application No. PCT/US10/22614, which is incorporated herein by reference in its entirety.

Experimental methods

HeLa cells were seeded overnight at 6000 cells per well in 96-well plates (Grenier). Three different liposomal formulations of Alexa-fluor 647-labeled GFP siRNA: 1) LNP01, 2) SNALP, 3) LNP05, diluted to a final concentration of 50nM in one of the three media conditions. The media conditions tested were OptiMem, DMEM with 10% FBS or DMEM with 10% FBS plus 10ug/mL human recombinant ApoE (Fitzgerald industries). The indicated liposomes in culture medium or in culture medium pre-complexed with ApoE for 10 minutes were added to the cells for 4, 6 or 24 hours. Each experimental condition was performed in triplicate. After adding HeLa cells in the plates at the indicated time points, the cells were fixed in 4% paraformaldehyde for 15 minutes, and then stained for nuclei and cytoplasm with DAPI and Syto dyes. Images were obtained using an Opera rotating disk automated confocal system from Perkin Elmer. Quantification of Alexa Fluor 647siRNA uptake was performed using Acapella software. Four different parameters were quantified: 1) number of cells, 2) number of siRNA positive spots per region, 3) number of siRNA positive spots per cell, and 4) integrated spot signal or average number of siRNA spots per cell multiplied by average spot intensity. The average spot signal is thus a rough estimate of the total number of siRNA levels per cell.

In addition, 4 different LNP-ApoE formulated dsrnas (SNALP (DLinDMa), XTC, MC3, ALNY-100) were tested in the following cell lines and the effect on dsRNA uptake by the cells was determined:

a375 (melanoma), B16F10 (melanoma), BT-474 (breast cancer), GTL-16 (gastric cancer), Hct116 (colon cancer), Hep3B (liver cancer), HepG2 (liver cancer), HeLa (cervical cancer), HUH 7 (liver cancer), MCF7 (breast cancer), Mel-285 (uveal melanoma), NCI-H1975 (lung cancer), OMM-1.3 (uveal melanoma), PC3 (prostate cancer), SKOV-3 (ovarian cancer), U87 (glioblastoma).

Example 22: k of KSP siRNA in the presence of ApoE _d

The effect of ApoE on the Kd (affinity) of KSP-targeting siRNA formulated with LNP-08 was evaluated in a variety of cell lines. Sirnas formulated with C18PEG were used with LNP08 and LNP 08. The siRNA duplex targeting KSP is AL-DP-6248.

The following cell lines were used.

Cell lines	Cell species	Species (II)
			HeLa	Adenocarcinoma of cervix	Human being
HCT116	Cancer of colon	Human being
			A375	Melanoma (MEA)	Human being
MCF7	Adenocarcinoma of breast	Human being
			B16F10	Melanoma (MEA)	Mouse
Hep3b	Liver cancer	Human being
			HUH 7	Liver cancer	Human being
HepG2	Liver cancer	Human being
			Skov 3	Ovarian cancer	Human being
U87	Glioblastoma	Human being
			PC3	Prostate cancer	Human being

The first day, cells were seeded at 20000 cells/well in 96-well plates. The following day, the formulated siRNA was incubated with serum-containing medium +/-ApoE for 15-30 minutes at 37 ℃. The medium was removed from the cells and the pre-warmed complexes were plated on the cells at 100 uL/well with a siRNA concentration of 20 nM. ApoE concentrations were titrated at 1.0, 3.0, 9.0 and 20.0. mu.g/ml. Cells were incubated with the formulated duplexes for 24 hours. On the third day, cells were lysed and prepared for bDNA analysis and kD calculation.

The presence of Apo E improved kD in a number of cell lines including HCT-116, HeLa, A375 and B16F10 (data not shown).

Example 23: IC of KSP siRNA in the presence of ApoE ₅₀

Evaluation of IC of ApoE on KSP-targeting siRNA formulated with LNP-08 in various cell lines₅₀(efficacy). Sirnas formulated with C18PEG were used with LNP08 and LNP 08. The siRNA duplex targeting KSP is AL-DP-6248.

On day 0, cells were seeded at 15000-20000 cells/well in 96-well plates. The first day, serum-containing medium, formulated duplex and +/-3ug/ml ApoE were incubated for 15-30 minutes at 37 ℃. Serial dilutions of siRNA were used in the range of 0.01nM to 1.0. mu.M. The medium was removed from the cells and the pre-warmed complex was plated on the cells at 100 uL/well. Cells were incubated with siRNA for 24 hours. On the next day, cells were lysed and prepared for bDNA analysis as described herein. KSP mRNA levels were determined using Quantigene 1.0 to determine KSP levels and compared to GAPDH. The negative control was siRNA targeting luciferase, AD-1955.

The results are shown in the following table. The siRNA formulated with LNP-08 was active in all cell lines. In some cell lines, the addition of ApoE improves the efficacy of siRNA treatment, e.g., lower IC₅₀As confirmed.

Example 24: inhibition of Eg5/KSP and VEGF expression in humans

Treating a human subject with a pharmaceutical composition, e.g., a nucleic acid-lipid particle comprising a dsRNA targeting the Eg5/KSP gene and a dsRNA targeting the VEGF gene, to inhibit expression of the Eg5/KSP and VEGF genes in the nucleic acid-lipid particle. For example, the nucleic acid-lipid particle comprises XTC, MC3, or ALNY-100.

Selecting or identifying a subject in need of treatment. The subject may be in need of cancer treatment, such as liver cancer.

At time zero, an appropriate first dose of the composition is administered subcutaneously to the subject. The compositions are formulated as described herein. After a period of time, the subject's condition is assessed, for example, by measuring tumor growth, measuring serum AFP levels, and the like. The assay may be accompanied by assaying Eg5/KSP and/or VEGF expression in the subject, and/or assaying Eg5/KSP and/or VEGF mRNA for successful siRNA-targeted products. Other relevant criteria were also determined. The number and intensity of doses are adjusted according to the needs of the subject.

After treatment, the condition of the subject is compared to the condition present prior to treatment, or to the condition of a subject having a similar condition but not treated.

Those skilled in the art are familiar with methods and compositions other than those specifically set forth in the invention, which will allow them to practice the invention within the full scope of the appended claims.

Claims (17)

1. A composition comprising a nucleic acid lipid particle comprising a first double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a human kinesin family member 11(Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of a human VEGF in the cell, wherein:

the nucleic acid lipid particle comprises a lipid preparation comprising 45-65 mol% of a cationic lipid, 5 mol% to 10 mol% of a non-cationic lipid, 25-40 mol% of a sterol, and 0.5-5 mol% of PEG or PEG-modified lipid, wherein the cationic lipid comprises MC3(4- (dimethylamino) butanoic acid (6Z,9Z,28Z,31Z) -thirty-seven carbon-6, 9,28, 31-tetraen-19-yl ester),

the first dsRNA consists of a first sense strand consisting of SEQ ID No. 1534 (5'-UCGAGAAUCUAAACUAACUTT-3') and a first antisense strand consisting of SEQ ID No. 1535 (5'-AGUUAGUUUAGAUUCCUGATT-3'); and is

The second dsRNA consists of a second sense strand consisting of SEQ ID NO:1536 (5'-GCACAUAGGAGAGAUGAGCUU-3') and a second antisense strand consisting of SEQ ID NO:1537 (5'-AAGCUCAUCUCUCCUAUGUGCUG-3').

2. The composition of claim 1, wherein the cationic lipid comprises MC3 and the lipid formulation is selected from the group consisting of:

3. the composition of claim 1, wherein each strand is modified as described below to include a 2' -O-methyl ribonucleotide represented by the lower case "c" or "u" and a phosphorothioate represented by the lower case "s":

the first dsRNA consists of a sense strand consisting of SEQ ID NO. 1240(5 ' -ucGAAucuAAAcuAAcuTST-3 ') and an antisense strand consisting of SEQ ID NO. 1241(5 ' -AGUuAGUUUuAGAUUCGATST);

the second dsRNA consists of the sense strand consisting of SEQ ID NO:1242(5 '-GcAuAGGAUGAUGAGCAUUUU-3') and the antisense strand consisting of SEQ ID NO:1243(5 '-AAGCUcAUCUCUCUCUCCUAUGUGCusG-3').

4. The composition of claim 1 or 2, wherein the first and second dsRNA comprise at least one modified nucleotide.

5. The composition of claim 4, wherein the modified nucleotide is selected from the group consisting of: 2 '-O-methyl modified nucleotides, nucleotides having a 5' -phosphorothioate group, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid didecylamide group.

6. The composition of claim 4, wherein the modified nucleotide is selected from the group consisting of: 2' -deoxy-2 ' -fluoro modified nucleotides, 2' -deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base containing nucleotides.

7. The composition of claim 1 or 2, wherein the first and second dsRNA each comprise at least one 2 '-O-methyl modified ribonucleotide and at least one nucleotide having a 5' -phosphorothioate group.

8. The composition of claim 1 or 2, wherein the first and second dsRNA are present in an equimolar ratio.

9. The composition of claim 1 or 2, further comprising sorafenib.

10. The composition of claim 1 or 2, further comprising a lipoprotein.

11. The composition of claim 1 or 2, further comprising apolipoprotein e (apoe).

12. Use of a composition according to any one of claims 1-11 in the manufacture of a medicament for inhibiting the expression of Eg5/KSP and VEGF in a cell.

13. Use of a composition according to any one of claims 1 to 11 in the manufacture of a medicament for preventing tumor growth, reducing tumor growth, or prolonging the survival of a mammal in need of treatment for cancer.

14. The use of claim 13, wherein the mammal has liver cancer.

15. The use of claim 13 or 14, wherein the mammal is a human suffering from liver cancer.

16. Use of the composition of any one of claims 1-11 in the manufacture of a medicament for reducing tumor growth in a mammal in need of treatment for cancer, the medicament reducing tumor growth by at least 20%.

17. The use of claim 16, wherein the agent reduces KSP expression by at least 60%.