HK1220380B - Compositions and methods for inhibiting expression of eg5 and vegf genes - Google Patents

Description

Compositions and methods for inhibiting Eg5 and VEGF gene expression

The present application is a divisional application of invention patent application No. 200980115656.5, filed on March 5, 2009, and entitled “Compositions and methods for inhibiting Eg5 and VEGF gene expression”.

Related applications

This application claims priority to U.S. Provisional Application No. 61/034,019, filed March 5, 2008, U.S. Provisional Application No. 61/083,367, filed July 24, 2008, U.S. Provisional Application No. 61/086,381, filed August 5, 2008, U.S. Provisional Application No. 61/112,079, filed November 6, 2008, and U.S. Provisional Application No. 61/150,664, filed February 6, 2009, which are hereby incorporated by reference in their entireties.

Field of the Invention

The present invention relates to compositions containing double-stranded ribonucleic acid (dsRNA), and their use in mediating RNA interference to inhibit the expression of a gene combination (e.g., the genes for Eg5 and vascular endothelial growth factor (VEGF) formulated in SNALP), and the use of the compositions in treating pathological processes mediated by the expression of Eg5 and VEGF (e.g., cancer).

Background of the Invention

The maintenance of cell populations in an organism is controlled by the cellular processes of cell division and programmed cell death. In 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 interrupted. For example, cancer cells may lose control of the cell division cycle (checkpoint control) through overexpression of positive regulatory factors or loss of negative regulatory factors (possibly due to mutations).

Alternatively, cancer cells may lose the ability to undergo programmed cell death through overexpression of negative regulatory factors.Therefore, there is a need to develop new chemotherapeutic drugs that will restore checkpoint control and programmed cell death processes in cancerous cells.

One approach to treating human cancers is to target proteins that are critical for cell cycle progression. Certain prerequisite events must occur in order for the cell cycle to progress from one phase to the next. There are checkpoints in the cell cycle that enforce the correct sequence of events and phases. One such checkpoint is the spindle checkpoint, which occurs during the metaphase stage of mitosis. Small molecules targeting proteins with important functions in mitosis can trigger the spindle checkpoint, arresting cells in mitosis. Among the small molecules that arrest cells in mitosis, those that have clinically demonstrated anti-tumor activity also induce apoptosis, a morphological change associated with programmed cell death. Therefore, effective chemotherapeutic agents for cancer treatment could be those that induce checkpoint control and programmed cell death. Unfortunately, few compounds are available to control these processes in cells. Most known compounds that cause mitotic arrest and apoptosis act as tubulin-binding agents. These compounds alter the dynamic instability of microtubules and indirectly alter the function/structure of the mitotic spindle, thereby arresting mitosis. Because most of these compounds specifically target tubulin, a component of all microtubules, they may also affect one or more of the many normal cellular processes in which microtubules play a role. Therefore, there is also a need for agents that more specifically target proteins associated with proliferating cells.

Eg5 is one of several kinesin-like motor proteins localized to the mitotic spindle and known to be essential for the formation and/or function of the bipolar mitotic spindle. Recently, small molecules that interfere with the polarity of the mitotic spindle have been reported (Mayer, T.U. et al., 1999. Science 286(5441)971-4, incorporated herein by reference). More specifically, these small molecules induce the formation of an abnormal mitotic spindle, in which a monoastral array of microtubules emanates from the central centrosome pair, allowing chromosomes to attach to the distal ends of the microtubules. These small molecules were named "monastrol" based on their monoastral arrangement. This monoastral arrangement has previously been observed in mitotic cells immunodepleted of the Eg5 motor protein. This distinctive monoastral arrangement facilitated the identification of monastrol as a potential Eg5 inhibitor. Indeed, monastrol has been further shown to inhibit microtubule motility driven by the Eg5 motor in in vitro assays. The Eg5 inhibitor monastrol has no significant effect on the related kinesin motors or motors responsible for Golgi movement in cells. Cells that exhibit monostellate formation by immunodepletion of Eg5 or monastrol inhibition of Eg5 are arrested in the M phase of the cell cycle. However, the mitotic arrest induced by immunodepletion or inhibition of Eg5 is transient (Kapoor, T.M., 2000. J Cell Biol 150(5)975-80). Both the monostellate formation in mitosis and the cell cycle arrest induced by monastrol are reversible. Cells recover to form normal bipolar mitotic spindles, complete mitosis and progress through the cell cycle and undergo normal cell proliferation. These data suggest that Eg5 inhibitors that induce transient mitotic arrest may be ineffective in the treatment of cancer cell proliferation. Nevertheless, the discovery that monastrol causes mitotic arrest has aroused people's interest and thus requires further research and identification of compounds that can be used to modulate the Eg5 motor protein in a manner that is effective for the treatment of human cancer. There is also a need to study the combination of these compounds with other antitumor drugs.

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

Recently, double-stranded RNA molecules (dsRNA) have been shown to inhibit gene expression using a highly conserved regulatory mechanism called RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of dsRNAs of at least 25 nucleotides in length to inhibit gene expression in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), fruit flies (see, e.g., Yang, D. et al., Curr. Biol. (2000) 10: 1191-1200), and mammals (see WO 00/44895, Limmer; and DE 10100586.5, Kreutzer et al.). This natural mechanism has currently become the focus of new therapeutic agents for treating conditions caused by abnormal or undesirable regulation of genes.

SUMMARY OF THE INVENTION

The present invention discloses a composition comprising two double-stranded ribonucleic acids (dsRNAs) for inhibiting the expression of the human kinesin family member 11 (Eg5/KSP) and human VEGF genes in cells. The dsRNAs are formulated into stable nucleic acid lipid particles (SNALPs). Also disclosed are methods for using the composition to reduce the expression of Eg5/KSP and/or VEGF in cells, as well as methods for using the composition to treat diseases (such as liver cancer).

Thus, the present invention discloses a composition having a first double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the human kinesin family member 11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting the expression of human VEGF in a cell, wherein the first and second dsRNAs are both formulated into stable nucleic acid lipid particles (SNALPs); the first dsRNA consists of a first sense strand and a first antisense strand, and the first sense strand has a first sequence, and the first antisense strand has a second sequence that is complementary to at least 15 consecutive nucleotides of SEQ ID NO: 1311 (5'-UCGAGAAUCUAAACUAACU-3'), wherein the first sequence is complementary to the second sequence, and wherein the first dsRNA is 15 to 30 base pairs in length; and the second dsRNA consists of a second sense strand and a second antisense strand, the second sense strand has a third sequence, and the second antisense strand has a second sequence that is complementary to at least 15 consecutive nucleotides of SEQ ID NO: 1311 (5'-UCGAGAAUCUAAACUAACU-3'). A fourth sequence that is complementary to at least 15 consecutive nucleotides of NO:1538 (5’-GCACAUAGGAGAGAUGAGCUU-3’), wherein the third sequence is complementary to the fourth sequence, and wherein each chain is 15 to 30 base pairs in length.

In some embodiments, the first antisense strand has a second sequence complementary to SEQ ID NO: 1311 (5'-UCGAGAAUCUAAACUAACU-3') and the second antisense strand has a fourth sequence complementary to SEQ ID NO: 1538 (5'-GCACAUAGGAGAGAUGAGCUU-3'). In other embodiments, the first dsRNA consists of a sense strand consisting of SEQ ID NO: 1534 (5'-UCGAGAAUCUAAACUAACUTT-3') and an antisense strand consisting of SEQ ID NO: 1535 (5'-AGUUAGUUUAGAUUCUCGATT-3'), and the second dsRNA consists of a sense strand consisting of SEQ ID NO: 1536 (5'-GCACAUAGGAGAGAUGAGCUU-3') and an antisense strand consisting of SEQ ID NO: 1537 (5'-AAGCUCAUCUCUCCUAUGUGCUG-3'). In a further embodiment, each strand is modified as follows to include a 2'-O-methyl ribonucleotide represented by a lowercase "c" or "u" and a phosphorothioate represented by a lowercase "s": the first dsRNA consists of a sense strand consisting of SEQ ID NO: 1240 (5'-ucGAGAAucuAAAcuAAcuTsT-3') and an antisense strand consisting of SEQ ID NO: 1241 (5'-AGUuAGUUUuAGAUUCUCGATsT); the second dsRNA consists of a sense strand consisting of SEQ ID NO: 1242 (5'-GcAcAuAGGAGAGAuGAGCUsU-3') and an antisense strand consisting of SEQ ID NO: 1243 (5'-AAGCUcAUCUCUCCuAuGuGCusG-3').

In some embodiments, the first dsRNA comprises two overhangs, and the second dsRNA comprises an overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

The first and second dsRNA can have at least one modified nucleotide.For example, each dsRNA can have at least one nucleotide selected from following modification: 2'-O-methyl modified nucleotide, nucleotide with 5'-thiophosphate group and the terminal nucleotide connected with cholesterol derivative or dodecanoic acid bisdecylamide (bisdecylamide) group.The nucleotide of modification can be selected from: 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide (locked nucleotide), abasic nucleotide (abasic nucleotide), 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide (morpholino nucleotide), phosphoramidate (phosphoramidate) and nucleotide with non-natural base.In some embodiments, the first and second dsRNA respectively comprise at least one 2'-O-methyl modified nucleotide and at least one nucleotide with 5'-thiophosphate group.

Each strand of each dsRNA can be, for example, 19-23 bases in length, or alternatively 21-23 bases in length. In one embodiment, each strand of the first dsRNA is 21 bases in length, and 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 some embodiments, the first and second dsRNA are present in an equimolar ratio.

As described herein, dsRNA is formulated into SNALPS. In some embodiments, the SNALP formulation includes DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA. For example, SNALP can have ingredients in the ratios listed in Table 17.

The compositions of the present invention can be used to reduce the expression of Eg5 and/or VEGF. In some embodiments, the compositions of the present invention, upon contact with cells expressing Eg5, inhibit the expression of Eg5 by at least 40%, 50%, 60%, 70%, 80%, or at least 90%. In other embodiments, the compositions of the present invention, upon contact with cells expressing VEGF, inhibit the expression of VEGF by at least 40%, 50%, 60%, 70%, 80%, or at least 90%. Administration of the composition to cells can simultaneously inhibit the expression of both Eg5 and VEGF in the cells. The composition of claims 1-17, wherein the composition is administered at a nM concentration.

Administration of the compositions of the present invention to cells can result in, for example, an increase in mono-aster formation in the cells. Administration of the compositions to mammals can result in at least one effect selected from the group consisting of: preventing tumor growth, reducing tumor growth, or prolonging survival in the mammal. This effect can be measured 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. Compositions that exhibit these effects when administered at nM concentrations are included.

In a further embodiment, the composition of the present invention comprises Sorafenib.

The present invention also includes methods of using the compositions of the present invention. One embodiment is a method of inhibiting the expression of Eg5/KSP and VEGF in a cell by administering any of the compositions of the present invention to the cell. Other embodiments are methods of administering the compositions to a mammal to prevent tumor growth, reduce tumor growth, or prolong survival in a mammal in need of cancer treatment. In some embodiments, the mammal has liver cancer, for example, the mammal is a human with liver cancer. The method may include the further step of administering sorafenib.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing liver weight as a percentage of body weight after administration of SNALP-siRNA in a Hep3B mouse model.

2A-2D are graphs showing the effects of SNALP-siRNA on body weight in the Hep3B mouse model.

FIG3 is a graph showing the effect of SNALP-siRNA on body weight in the Hep3B mouse model.

Figure 4 is a graph showing body weights of untreated control animals.

FIG5 is a graph showing the effect of control luciferase-SNALP siRNA on body weight in the Hep3B mouse model.

FIG6 is a graph showing the effect of VSP-SNALP siRNA on body weight in the Hep3B mouse model.

FIG7A is a graph showing the effect of SNALP-siRNA on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG7B is a graph showing the effect of SNALP-siRNA on serum AFP levels measured by serum ELISA in the Hep3B mouse model.

FIG8 is a graph showing the effect of SNALP-siRNA on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG9 is a graph showing the effect of SNALP-siRNA on human KSP levels normalized to human GAPDH levels in the Hep3B mouse model.

FIG10 is a graph showing the effect of SNALP-siRNA on human VEGF levels normalized to human GAPDH levels in the Hep3B mouse model.

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

FIG11B is a set of graphs showing the effects of SNALP-siRNA on human GAPDH levels and serum AFP levels in the Hep3B mouse model.

12A-12C are graphs showing the effects of SNALP-siRNA on tumor KSP, VEGF, and GAPDH levels in the Hep3B mouse model.

Figures 13A and 13B are graphs showing the effect of SNALP-siRNA on survival in mice bearing liver tumors. Treatment was initiated 18 days (Figure 13A) and 26 days (Figure 13B) after tumor cell inoculation.

FIG14 is a graph showing the effect of SNALP-siRNA on serum alpha-fetoprotein (AFP) levels.

Figures 15A and 15B are images of H&E-stained sections from tumor-bearing animals (3 weeks after Hep3B cell implantation) administered 2 mg/kg of SNALP-VSP (A) or 2 mg/kg of SNALP-Luc (B). Twenty-four hours later, the tumor-bearing liver lobes were processed for histological analysis. Arrows indicate single astrocytes.

FIG16 is a flow chart showing the preparation process of ALN-VSPDS01.

FIG17 is a cryogenic transmission electron microscopy (cryo-TEM) image of ALN-VSP02.

FIG18 is a flow chart showing the preparation process of ALN-VSP02.

FIG19 shows the treatment regimen of a transgenic mouse model of MYC-oncogene driven de novo liver cancer using siRNA targeting KSP.

FIG20 is a graph showing the effect of administration of SNALP-formulated siRNA and sorafenib on survival rate.

Detailed Description of the Invention

The present invention provides compositions and methods for inhibiting the expression of the Eg5 and VEGF genes in cells or mammals using dsRNA. The dsRNA is preferably encapsulated in a stable nucleic acid microparticle (SNALP). The present invention also provides compositions and methods for treating pathological conditions and diseases (such as liver cancer) caused by the expression of the Eg5 and VEGF genes in mammals. The dsRNA directs the 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 the Eg5 gene and the VEGF gene, respectively, as well as compositions and methods for treating diseases and conditions caused by the expression of these genes, such as cancer. The pharmaceutical compositions of aspects of the present invention include dsRNA having an antisense strand comprising a complementary region less than 30 nucleotides in length (generally 19-24 nucleotides in length) that is substantially complementary to at least a portion of the RNA transcript of the Eg5 gene, together with a pharmaceutically acceptable carrier. The compositions of aspects of the present invention also include dsRNA having an antisense strand having a complementary region less than 30 nucleotides in length, generally 19-24 nucleotides in length, that is substantially complementary to at least a portion of the RNA transcript of the VEGF gene.

Therefore, certain aspects of the present invention provide pharmaceutical compositions containing Eg5 and VEGF dsRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit the expression of Eg5 and VEGF genes, respectively, and methods of using the pharmaceutical combinations to treat diseases caused by the expression of Eg5 and VEGF genes.

I. Definition

For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. If there is an apparent discrepancy between the usage of a term in other parts of the specification and the definition provided in this section, the definition in this section shall prevail.

"G," "C," "A," and "U" generally represent nucleotides comprising guanine, cytosine, adenine, and uracil as bases, respectively. "T" and "dT" are used interchangeably herein and refer to deoxyribonucleic acids in which the nucleoside base is thymine, such as deoxyribothymine. However, it is understood that the term "ribonucleotide" or "nucleotide" may also refer to modified nucleotides (as described in further detail below) or alternative replacement moieties. It is well known to those skilled in the art that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide having such a replacement moiety. For example, but not limitation, a nucleotide comprising inosine as its base can base pair with a nucleotide comprising adenine, cytosine, or uracil. Thus, a nucleotide comprising uracil, guanine, or adenine may be replaced in the nucleotide sequences of the present invention by a nucleotide comprising, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form G-U wobble base pairing with the target mRNA. Sequences containing such replacement moieties are embodiments of the present invention.

As used herein, "Eg5" refers to human kinesin family member 11, also known as KIF11, Eg5, HKSP, KSP, KNSL1, or TRIP5. The Eg5 sequence can be found as NCBI GeneID: 3832, HGNC ID: HGNC: 6388, and RefSeq ID: NM_004523. The terms "Eg5," "KSP," and "Eg5/KSP" can be used interchangeably.

As used herein, VEGF, also known as vascular permeability factor, is an angiogenic growth factor. VEGF is a homodimeric 45kDa glycoprotein that exists in at least three different isoforms. VEGF isoforms are expressed in endothelial cells. The VEGF gene comprises 8 exons expressing 189 amino acid protein isoforms. 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 comprise 145 amino acids and 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 involved in endothelial cell differentiation and angiogenesis. A third receptor, VEGFR-3, is involved in lymphogenesis.

Various isoforms have different biological activities and clinical significance. For example, VEGF145 induces angiogenesis, and similar to VEGF189 (but different from VEGF165), VEGF145 effectively binds to the extracellular matrix by a mechanism that is independent of heparan sulfate associated with the extracellular matrix. VEGF shows activity as an endothelial cell mitogen and chemoattractant in vitro, and induces vascular permeability and angiogenesis in vivo. VEGF is secreted by various cancer cell types and promotes tumor growth by inducing the occurrence of adipose duct systems associated with tumors. The inhibition of VEGF function has been shown to limit the growth of primary experimental tumors and the occurrence of metastasis in immunocompromised mice. Various dsRNAs directed to VEGF are described in pending U.S. patent applications 11/078,073 and 11/340,080, which are fully introduced herein by reference.

As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Eg5/KSP and/or VEGF gene, including mRNA that is an RNA processing product of the primary transcript.

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

As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence refers to 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 conditions understood by the skilled artisan. These conditions can be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours, followed by washing. Other conditions may apply, such as physiologically relevant conditions that may be encountered in an organism. A skilled artisan will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences based on the ultimate application of the hybridizing nucleotides.

In some embodiments, the present invention provides the oligonucleotides or polynucleotides containing the first nucleotide sequence and the oligonucleotides or polynucleotides containing the second nucleotide sequence base pairing on the total length of the first and second nucleotide sequences.Herein, this sequence can be referred to as "complete complementarity" to each other.However, when the first sequence is referred to as "substantially complementary" with the second sequence herein, the two sequences can be complete complementarity, or they can form one or more but usually no more than 4,3 or 2 mismatched base pairs when hybridizing, and simultaneously retain the ability to hybridize under the most relevant conditions with their final application.However, in the case where two oligonucleotides are designed to form one or more single-stranded overhangs when hybridizing, this overhang should not be considered as mispairing when determining complementarity.For example, according to the purpose of the present invention, the dsRNA (wherein the longer oligonucleotide comprises the sequence of 21 nucleotides that are completely complementary to the shorter oligonucleotide) comprising an oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length can also be referred to as "complete complementarity."

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

As understood from the context of their use, the terms "complementary," "fully complementary," and "substantially complementary" herein may refer to base matching between the sense and antisense strands of a dsRNA or between the antisense strand of a dsRNA and a target sequence.

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 (including the 5'UTR, open reading frame (ORF), or 3'UTR) of an mRNA of interest (e.g., an mRNA encoding Eg5/KSP and/or VEGF). For example, a polynucleotide sequence is complementary to at least a portion of an Eg5 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Eg5.

As used herein, the term "double-stranded RNA" or "dsRNA" refers to a duplex structure having two antiparallel and substantially complementary nucleic acid chains as defined above. The two chains forming the duplex structure can be different parts of a larger RNA molecule, or they can be independent RNA molecules. When the two chains are part of a larger molecule and therefore connected by the uninterrupted nucleotide chain between the 3' end of a chain forming the duplex structure and the 5' end of the corresponding another chain, the connecting RNA chain is referred to as a "hairpin loop." When the two chains are covalently linked by other means other than the uninterrupted nucleotide chain between the 3' end of a chain forming the duplex structure and the 5' end of the corresponding another chain, this connection structure is referred to as a "linker." The RNA chain can have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides of the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA can comprise one or more nucleotide overhangs. Generally, the majority of the nucleotides in each strand are ribonucleotides, but as described in detail herein, each strand or both strands may also contain at least one non-ribonucleotide, for example, a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, "dsRNA" may include chemical modifications to ribonucleotides, including substantial modifications of multiple nucleotides and including all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, "dsRNA" includes any such modifications (as used in siRNA-type molecules).

As used herein, "nucleotide overhang" refers to unpaired nucleotides 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. "Blunt" or "blunt end" means that there are no unpaired nucleotides at that end of the dsRNA, i.e., there are no nucleotide overhangs. A "blunt-ended" dsRNA is a dsRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. In some embodiments, a dsRNA can have an overhang at one end of the duplex and a blunt end at the other end.

The term "antisense strand" refers to a strand of a dsRNA comprising a region that is substantially complementary to a target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence as defined herein (e.g., a target sequence). When the complementary region is not fully complementary to the target sequence, mispairing may occur in the interior or terminal regions of the molecule. The most tolerable mispairing is typically in the terminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides at the 5' and/or 3' ends.

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

As used herein, the term "SNALP" refers to a stable nucleic acid lipid particle. SNALP represents a lipid vesicle enclosing a low aqueous interior containing a nucleic acid (eg, an iRNA agent or a plasmid from which the iRNA agent is transcribed).

When "introduced into a cell" is used to describe a dsRNA, it means facilitating uptake or absorption into the cell, as understood by those skilled in the art. The absorption or uptake of the dsRNA can occur by unassisted diffusive or active cellular processes or by auxiliary reagents or devices. The meaning of this term is not limited to cells in vitro; dsRNA can also be "introduced into a cell," where the cell is part of a living organism. In this case, introduction into a cell includes delivery to the organism. For example, for in vivo delivery, the dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection.

When the terms "silencing" and "preventing expression," "downregulating expression," "inhibiting expression," and the like are used to describe the Eg5 and/or VEGF genes, they refer herein to at least partial inhibition of Eg5 gene expression, as indicated by a decrease in the amount of Eg5 mRNA and/or VEGF mRNA that can be isolated from a first cell or cell population (in which the Eg5 and/or VEGF gene is transcribed and has been treated such that expression of the Eg5 and/or VEGF gene is inhibited) compared to a second cell or cell population (substantially equivalent to the first cell or cell population but not treated) (control cells). The degree of inhibition is generally expressed using the following formula:

Alternatively, the degree of inhibition can be expressed as a reduction in a parameter functionally associated with the expression of the Eg5 and/or VEGF genes, for example, the amount of protein encoded by the Eg5 and/or VEGF genes produced by cells, or the number of cells exhibiting a certain phenotype (e.g., apoptosis). In theory, target gene silencing can be measured in any cell expressing the target, either constitutively or through genetic engineering and by any suitable detection method. However, when a reference is needed to determine whether a given dsRNA inhibits the expression of the Eg5 gene to a specific extent and is therefore included in the present invention, the analysis provided in the following examples will serve as such a reference.

For example, in some cases, the 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 administering a double-stranded oligonucleotide of the invention. In some embodiments, the 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 values for inhibition of expression using various Eg5 and/or VEGF dsRNA molecules at various concentrations.

As used herein in the context of Eg5 expression (or VEGF expression), the terms "treat," "treatment," and the like refer to the alleviation or alleviation of pathological processes mediated by Eg5 and/or VEGF expression. In the context of the present invention, as long as any of the other conditions listed below (other than those mediated by Eg5 and/or VEGF expression) are involved, the terms "treat," "treatment," and the like refer to the alleviation or alleviation of at least one symptom associated with the condition, or the slowing or reversing of the progression of the condition, such as the slowing or reversing of the progression of liver cancer.

As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that produces a therapeutic benefit in the treatment, prevention, or management of a pathological process mediated by Eg5 and/or VEGF expression, or a symptom manifested by a pathological process mediated by Eg5 and/or VEGF expression. The specific amount that is therapeutically effective can be readily determined by a medical professional of ordinary skill and can 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 the 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 pharmaceutically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically effective amount," "therapeutically effective amount," or simply "effective amount" refers to an amount of RNA effective to produce the desired pharmacological, therapeutic, or preventive 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%, then the therapeutically effective amount of the drug for the treatment of that disease or condition is the amount required to achieve at least a 25% reduction in that parameter.

The term "pharmaceutically acceptable carrier" refers to a carrier for administering therapeutic agents. As described in detail below, such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. This term particularly excludes cell culture media. For orally administered medicines, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, adhesives, lubricants, sweeteners, aromatics, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Adhesives can include starch and gelatin, and lubricants (if present) are typically magnesium stearate, stearic acid, or talc. If desired, tablets can be coated with materials 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 has been introduced a vector from which a dsRNA molecule can be expressed.

II. Double-stranded RNA (dsRNA)

As described in more detail below, the present invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of Eg5 and/or VEGF genes in cells or mammals, wherein the dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of an mRNA formed during the expression of the Eg5 and/or VEGF genes, and wherein the complementary region is less than 30 nucleotides in length, typically 19-24 nucleotides in length, and wherein the dsRNA inhibits the expression of the Eg5 and/or VEGF genes when contacted with a cell expressing the Eg5 and/or VEGF genes.

As discussed further below, dsRNA can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc.

dsRNA comprises two chains that are fully complementary to each other to hybridize and form a duplex structure. One chain of the dsRNA (the antisense strand) comprises a complementary region that is substantially complementary and generally fully complementary to a target sequence derived from an mRNA sequence formed during the expression of the Eg5/KSP and/or VEGF gene, and the other chain (the sense strand) comprises a region complementary to the antisense strand, such that when combined under appropriate conditions, the two chains hybridize and form a duplex structure. In general, the length of the duplex structure is 15 to 30, more generally 18 to 25, but more generally 19 to 24, and most generally 19 to 21 base pairs. In other embodiments, the length of the duplex structure is 25-30 base pairs.

In one embodiment, the duplex length is 19 base pairs. In another embodiment, the duplex length is 21 base pairs. When two different siRNAs are used in combination, the duplex lengths can be the same or different. For example, a composition can include a first dsRNA targeting Eg5 with a duplex length of 19 base pairs and a second dsRNA targeting VEGF with a duplex length of 21 base pairs.

Similarly, the region of complementarity to the target sequence is 15 to 30, more typically 18 to 25, but more typically 19 to 24, and most typically 19 to 21 nucleotides in length. In other embodiments, the complementary region is 25-30 nucleotides in length.

In one embodiment, the complementary region is 19 nucleotides in length. In another embodiment, the complementary region is 21 nucleotides in length. When two different siRNAs are used in combination, the complementary regions can be the same or different in length. For example, a composition can include a first dsRNA targeting Eg5 having a complementary region of 19 nucleotides and a second dsRNA targeting VEGF having a complementary region of 21 nucleotides.

The length of each strand of the dsRNA of the present invention is generally 15 to 30, or 18 to 25, or 18, 19, 20, 21, 22, 23 or 24 nucleotides. In other embodiments, the length of each strand is 25-30 base pairs. The strands of the duplex can have the same or different lengths. When two different siRNAs are used in combination, the lengths of the strands of each siRNA can be the same or different. For example, a composition can include a dsRNA targeting Eg5 with a sense strand of 21 nucleotides and an antisense strand of 21 nucleotides and a second dsRNA targeting VEGF with a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides.

The dsRNA of the present 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, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides at each of the 3' and 5' ends that exceeds the sense strand. In a further embodiment, the sense strand of the dsRNA has an overhang of 1-10 nucleotides at each of the 3' and 5' ends that exceeds the antisense strand.

dsRNA with at least one nucleotide overhang has unexpectedly excellent inhibitory properties compared with their blunt-ended counterparts. In some embodiments, the presence of only a nucleotide overhang can enhance the interference activity of dsRNA without affecting its overall stability. It has been shown that dsRNA with only one overhang is particularly stable and effective in vivo and in various cells, cell culture media, blood and serum. Usually, the single-stranded overhang is located at the 3' end of the antisense strand or, alternatively, at the 3' end of the sense strand. dsRNA can also have a blunt end, usually at the 5' end of the antisense strand. This dsRNA can have improved stability and inhibitory activity, and therefore allows low-dose administration, i.e., less than 5mg/kg recipient body weight every day. Usually, the antisense strand of dsRNA has a nucleotide overhang at the 3' end, and the 5' end is a blunt end. In another embodiment, one or more nucleotides in the overhang are replaced with nucleoside thiophosphates.

As described in more detail herein, the compositions of the present invention include a first dsRNA targeted to Eg5 and a second dsRNA targeted to VEGF. The first and second dsRNAs can have the same overhang configuration, e.g., the number of nucleotide overhangs on each strand, or the dsRNAs can have different configurations. In one embodiment, the first dsRNA targeted to Eg5 includes a 2-nucleotide overhang at the 3' end of each strand, and the second dsRNA targeted to VEGF includes 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 present invention is the human Eg5 gene. In one embodiment, the antisense strand of the dsRNA targeting Eg5 comprises at least 15 consecutive nucleotides of one of the antisense sequences in Tables 1-3. In a specific embodiment, the first sequence of the dsRNA is selected from one of the sense strands in Tables 1-3, and the second sequence is selected from the antisense sequences in Tables 1-3. The target sequence and the flanking Eg5 sequence can be used to easily determine alternative antisense substances targeted elsewhere in the target sequence provided in Tables 1-3. In some embodiments, the dsRNA targeting Eg5 comprises 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 the mRNA sequence produced during expression of the Eg5 gene. Therefore, the dsRNA comprises 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 where a second dsRNA targeted to VEGF is used, such agents are exemplified in the Examples, Tables 4a and 4b, and in co-pending U.S. patent applications 11/078,073 and 11/340,080, which are incorporated herein by reference. In one embodiment, the dsRNA targeted to VEGF has an antisense strand that is complementary to at least 15 consecutive nucleotides of a VEGF target sequence as described in Table 4a. In other embodiments, the dsRNA targeted to VEGF comprises one of the antisense sequences of Table 4b or one of the sense sequences of Table 4b, or comprises one of the duplexes (sense and antisense strands) of Table 4b.

It is well known to those skilled in the art that dsRNA comprising a duplex structure of 20 to 23 (but particularly 21) base pairs has been found to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888). However, others have found that longer or shorter dsRNA is also effective. In the above-mentioned embodiment, according to the properties of the oligonucleotide sequences provided in Tables 1-3, the dsRNA of the present invention may comprise a chain having at least one minimum length of 21nt. It is reasonably expected that a shorter dsRNA comprising a sequence in Tables 1-3, which is only deducted from a few nucleotides at one or both ends, may be equally effective compared to the above-mentioned dsRNA. Thus, the present invention contemplates partial sequences comprising at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a sequence from Tables 1-3, and wherein the ability to inhibit Eg5 gene expression in a FACS assay described below differs by no more than 5%, 10%, 15%, 20%, 25%, or 30% compared to a dsRNA comprising the full sequence. Further dsRNAs that cleave within the target sequences provided in Tables 1-3 can be readily prepared using the Eg5 sequence and the provided target sequences. Additional dsRNAs targeting VEGF can be designed in a similar manner using the sequences disclosed in Tables 4a and 4b, the Examples, and co-pending U.S. patent applications Ser. Nos. 11/078,073 and 11/340,080, which are incorporated herein by reference.

In addition, the RNAi agent provided in Tables 1-3 identifies sites in the Eg5 mRNA that are susceptible to RNAi-based cutting. Therefore, the present invention also includes RNAi agents, such as dsRNA, that are targeted within the sequence of a target of a reagent of the present invention. As used herein, if a second RNAi agent shears the messenger RNA at any position in the mRNA that is complementary to the antisense strand of the first RNAi agent, the second RNAi agent is said to be targeted within the sequence of the first RNAi agent. Such a second agent is generally composed of at least 15 consecutive nucleotides from a sequence provided in Tables 1-3 coupled to another nucleotide sequence taken from the region adjacent to the selected sequence in the Eg5 gene. For example, the last 15 nucleotides from SEQ ID NO: 1 are combined with the next 6 nucleotides from the target Eg5 gene to produce a single-stranded agent of 21 nucleotides based on one of the sequences provided in Tables 1-3. Additional RNAi agents, such as dsRNAs, targeted to VEGF can be designed in a similar manner using the sequences disclosed in Tables 4a and 4b, the Examples, and co-pending US patent applications 11/078,073 and 11/340,080, which are incorporated herein by reference.

The dsRNA of the present invention may comprise one or more mispairings with the target sequence. In a preferred embodiment, the dsRNA of the present invention comprises no more than 3 mispairings. If the antisense strand of the dsRNA comprises a mispairing with the target sequence, the region of the mispairing is preferably not located at the center of the complementary region. If the dsRNA antisense strand comprises a mispairing with the target sequence, the mispairing is preferably limited to 5 nucleotides from either end, for example, 5, 4, 3, 2 or 1 nucleotides from the 5' or 3' ends of the complementary region. For example, for a 23-nucleotide dsRNA chain complementary to the region of the Eg5 gene, the dsRNA does not contain any mispairing in the middle 13 nucleotides generally. The method of the present invention can be used to determine whether the dsRNA comprising a mispairing with the target sequence effectively inhibits the expression of the Eg5 gene. It is important to consider the effect of the dsRNA with a mispairing in inhibiting the expression of the Eg5 gene, particularly if the specific complementary region in the Eg5 gene is known to have a polymorphic sequence variation in a population.

Modification

In another embodiment again, dsRNA is chemically modified to enhance stability. Nucleic acid of the present invention can be synthesized and/or modified by the method that this area has established, such as " Current protocols in nucleic acid chemistry ", Beaucage, S.L. et al. (editor), John Wiley＆Sons, Inc., New York, NY, those methods described in USA, the document is incorporated into this paper by reference. The specific example of the preferred dsRNA compound useful in the present invention includes the backbone containing modification or the dsRNA that does not have natural internucleoside connection. As defined in this specification, the dsRNA with modified backbone includes those dsRNA that retain phosphorus atom in the backbone and those dsRNA that do not have phosphorus atom in the backbone. For the purpose of this specification and as sometimes indicated in this area, the modified dsRNA that does not contain phosphorus atom in the internucleoside backbone can also be considered as oligonucleoside (oligonucleoside).

Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphates (including 3'-alkylene phosphates and chiral phosphates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thioalkylphosphoesters, thioalkylphosphotriesters, and boranophosphates with normal 3'-5' bonds, their 2'-5' bond analogs, and those with reversed polarity (wherein adjacent nucleoside units are modified from 3'-5' to 5'-3' bonds or from 2'-5' to 5'-2' bonds). Various salts, mixed salts, and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patents 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399, 676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,316, 5,550,111, 5,563,253, 5,571,799, 5,587,3615,625,050, each of which is incorporated herein by reference.

Preferred modified dsRNA backbones that do not contain phosphorus atoms have backbones formed from short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatom and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatom or heterocyclic nucleoside linkages. These include those with morpholine linkages (formed in part by the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimine and methylenehydrazine backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones with mixed N, O, S and _CH2 components.

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

In other preferred dsRNA mimics, the sugar and internucleoside connections (i.e., backbone) of the nucleotide unit are replaced by new groups. The base unit is kept to hybridize with an appropriate nucleic acid target compound. A type of such oligomeric compound (a dsRNA mimic having excellent hybridization properties) is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of dsRNA is replaced by an amide-containing backbone, especially an aminoethylaminoacetic acid backbone. The nucleoside bases are retained and directly or indirectly bound to the nitrogen atoms of the backbone amide portion. Representative U.S. patents for teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 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 embodiments of the present invention are dsRNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones, particularly the _--CH2 -NH-- _CH2-- , --CH2 _-- N(CH3)--O-- _CH2-- [referred to as a methylene(methylimine) or MMI backbone], --CH2--O--N( _CH3 )--CH2--, _--CH2 -- _N ( _CH3 )--N( _CH3 )--CH2--, and --N( _CH3 )-- _CH2 -- _CH2-- [wherein the natural phosphodiester backbone is represented as --O--P--O-- _CH2-- ] of _the aforementioned U.S. Patent _{No. 5,489,677} , and the amide backbone of the aforementioned U.S. Patent _No. 5,602,240. Also preferred are dsRNAs containing a morpholine backbone structure of the aforementioned U.S. Patent No. 5,034,506.

Modified dsRNAs can also contain one or more substituted sugar moieties. Preferred dsRNAs contain one of the following groups at the 2' position: 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 groups _can be substituted or unsubstituted _C1 to _C10 alkyl or _C2 to _C10 alkenyl and alkynyl groups. Particularly preferred are O[( _CH2 ) _nO ] _mCH3 , _O ( _CH2 ) _nOCH3 , O( _CH2 ) _nNH2 , O( _CH2 ) _{nCH3 ,} O( _CH2 ) _{nONH2 ,} and O( _CH2 ) _nON [( _CH2 ) _nCH3 )] ₂ , _{wherein n} and m are 1 to about 10. Other preferred dsRNAs comprise one of the following groups at the 2' position: _C1 to _C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, _SCH3 , _OCN , Cl, Br, CN, CF3, _OCF3 , _SOCH3 , SO2CH3, _ONO2 , _NO2 , _N3 , _NH2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group that _improves the pharmacokinetic properties of the dsRNA _or group that improves the pharmacodynamic properties of the dsRNA, and other substituents with similar properties. Preferred modifications include 2'-methoxyethoxy (2'-O--CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxy-alkoxy group. Further preferred modifications include 2'-dimethylaminooxyethoxy, i.e., an O(CH ₂ ) ₂ ON(CH ₃ ) ₂ group, also known as 2'-DMAOE, as described in the examples below herein, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH ₂ --O--CH ₂ --N(CH ₂ ) ₂ , as described in the examples below herein.

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

dsRNA can also include modifications or substitutions of nucleoside bases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleoside bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleoside bases include other synthetic and natural nucleoside bases 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, cytos ... uracil, thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 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 as well as 3-deazaguanine and 3-deazaadenine. Other nucleoside bases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, J.L., ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pp. 289-302, Crooke, S.T. Lebleu, B., ed., CRC Press, 1993. Certain of these nucleoside bases are particularly advantageous for increasing the binding affinity of the oligomeric compounds of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 degrees Celsius (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are currently the preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases and other modified nucleobases include, but are not limited to, the above-mentioned U.S. Patent 3,687,808, as well as U.S. Patents 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; and 5,681,941, each of which is incorporated herein by reference, and U.S. Patent 5,750,692, which is also incorporated herein by reference.

Coupling

Another modification of the dsRNA of the invention involves chemically linking to the dsRNA one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the dsRNA. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994 41053-1060), thioethers (such as beryl-S-tritylthiol) (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain (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 di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphate) (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & 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. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Patents 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,1 38,045, 5,414,077, 5,486,603, 5,512,439, 5,578,718, 5,608,046, 4,587,044, 4,605,735, 4,667,025, 4,762,779, 4,789,737, 4,824,941, 4,835,263, 4,876,335, 4,904,582, 4,958,013 、5,082,830、5,112,963、5,214,136、5,082,830、5,112,963、5,214,136、5,245,022、5,254,469、5,258,506、5,262,536、5,272,250、5,292,873、5,317,098、5,371,241、5,391,723、5,416 ,203, 5,451,463, 5,510,475, 5,512,667, 5,514,785, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928 and 5,688,941, each of which is incorporated herein by reference.

It is not necessary to modify all positions in a given compound in unison, and in fact more than one of the aforementioned modifications can be added to a single compound or even a single nucleoside within a dsRNA. The present invention also includes dsRNA complexes that are chimeric complexes. In the context of the present invention, "chimeric" dsRNA compounds or "chimeras" are dsRNA complexes containing two or more chemically distinct regions, particularly dsRNAs, each of which is composed of at least one monomeric unit (i.e., a nucleotide in the case of a dsRNA complex). These dsRNAs typically include at least one region in which the dsRNA is modified to impart improved nuclease degradation resistance, improved cellular uptake, and/or improved binding affinity to the target nucleic acid. Other regions of the dsRNA can be used as substrates for enzymes that can shear RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that shears the RNA chain of an RNA:DNA duplex. Therefore, activation of RNase H causes shearing of the RNA target, thereby greatly enhancing the efficiency of dsRNA in inhibiting gene expression. Thus, comparable results can generally be achieved with shorter dsRNAs when using chimeric dsRNAs compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if desired, by relevant nucleic acid hybridization techniques known in the art.

In some cases, dsRNA can be modified with non-ligand groups. Many non-ligand molecules have been conjugated to dsRNA to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4:1053), thioethers (e.g., hexyl-S-tritylthiol) (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), aliphatic chains (e.g., dodecanediol or undecyl residues) (Saison-Behmoaras et al., EMBO J, 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids (such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphate) (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantane acetic acid (Manoharan et al., Tetrahedron The conjugates may be prepared by combining a dsRNA with a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), an octadecylamine or hexylamino-carbonyl hydroxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such dsRNA conjugates are listed above. A typical conjugation protocol involves synthesizing a dsRNA with an aminolinker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using a suitable coupling or activating reagent. The conjugation reaction can be performed with the dsRNA still bound to the solid support or after shearing the dsRNA in the liquid phase. Purification of the dsRNA conjugate by HPLC generally provides a pure conjugate.

In some cases, the ligand can be multifunctional and/or the dsRNA can be coupled to more than one ligand. For example, a dsRNA can be coupled to one ligand for enhancing uptake and a second ligand for enhancing release.

Vector-encoded RNAi agent

In another aspect of the invention, dsRNA molecules specific for Eg5 and VEGF expressed by transcriptional units are inserted into DNA or RNA vectors (see, e.g., Couture, A et al., TIG. (1996), 12:5-10; Skillern, A et al., International PCT Publication WO 00/22113, Conrad, International PCT Publication WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors that can be incorporated and inherited as transgenes integrated into the host genome. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The independent chain of dsRNA can be transcribed by the promoter on two independent expression vectors, and co-transfected into target cell.Selectively, each independent chain of dsRNA can be transcribed by the promoter all being positioned at on the same expression plasmid.In a preferred embodiment, dsRNA is expressed as the inverted repeat sequence that is combined by connecting polynucleotide sequence, so that dsRNA has stem and loop structure.

Recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expression viral vectors can be based on adeno-associated virus (for review, see Muzyczka et al., Curr. Topics Micro. Immunol. (1992) 158: 97-129), adenovirus (see, for example, 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 alphavirus and other species known in the art, but are not limited thereto. Retroviruses have been used to introduce various genes into many different cell types in vitro and/or in vivo, including epithelial cells (e.g., see, Eglitis et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. 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 87:6141-6145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Patent 4,868,116; U.S. Patent 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into a suitable 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 wide variety of cells and tissues in susceptible hosts (e.g., rats, hamsters, dogs, and chimpanzees) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also 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 can be used, such as vectors derived from adenovirus (AV), adeno-associated virus (AAV), retroviruses (such as lentivirus (LV), rhabdovirus, murine leukemia virus), herpes virus, etc. The tropism of the viral vector can be appropriately modified by standardizing the vector with envelope proteins or other surface antigens from other viruses or by substituting different viral capsid proteins.

For example, the lentiviral vectors of the present invention can be quasi-typed with surface proteins from vesicular stomatitis virus (VSV), rabies virus, Ebola virus, Mokola virus, etc. The AAV vectors of the present 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. This serotype 2 capsid gene in an AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. The technology for constructing AAV vectors expressing different capsid protein serotypes is the technology of the art, see, for example, Rabinowitz J E et al. (2002), J Virol 76:791-801, which is incorporated herein by reference in its entirety.

The selection of recombinant viral vectors suitable for use in the present invention, methods for inserting nucleic acid sequences for expressing dsRNA into vectors, and methods for delivering viral vectors to cells of interest are all within the skill of the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis MA (1988), Biotechniques 6: 608-614; Miller AD (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson DA et al., Nat. Genet. 33: 401-406, the entire disclosures of which are incorporated herein by reference.

Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the present invention is expressed as two independent complementary single-stranded RNA molecules by a recombinant AAV vector having, for example, the U6 or H1 RNA promoter or the cytomegalovirus (CMV) promoter.

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

Suitable AAV vectors for expressing the dsRNA of the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R 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. Patent 5,252,479; U.S. Patent 5,139,941; International Patent Application WO 94/13788; and International Patent Application WO 93/24641, the entire disclosures of which are incorporated herein by reference.

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

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

In general, the recombinant vector that can express dsRNA molecule is delivered as described below, and remains in target cell.Selectively, the viral vector that transient expression of dsRNA molecule is provided can be used.If desired, this type of vector can be repeatedly administered.Once expressed, dsRNA combines target RNA and regulates its function or expression.The delivery of dsRNA expression vector can be systemic, as by intravenous or intramuscular administration, by to the administration of the target cell that then reintroduces in the patient from patient's transplantation, or by any other mode that allows to introduce the target cell of hope.

The DNA plasmid for dsRNA expression is usually transfected into the target cell as a complex with a cationic lipid carrier (such as Oligofectamine) or a non-cationic lipid-based carrier (such as Transit-TKO ^™ ). The present invention also contemplates multiple lipid transfections for dsRNA-mediated knockdown (knockdown) targeting different regions of a single EG5 gene (or VEGF gene) or a plurality of Eg5 genes (or VEGF genes) over a period of one week or longer. Various known methods can be used to monitor the successful introduction of the vector of the present invention into the host cell. For example, transient transfection can be indicated by a reporter molecule (such as a fluorescent marker, such as green fluorescent protein (GFP)). Markers that provide resistance (such as hygromycin B resistance) to specific environmental factors (such as antibiotics and drugs) to the transfected cells can be used to ensure stable transfection of isolated cells.

The specific dsRNA molecules of Eg5 and VEGF can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to subjects by, for example, intravenous injection, local administration (see U.S. Patent No. 5,328,470) or by stereotactic injection (see, for example, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a sustained-release matrix in which the gene therapy vector is embedded. Alternatively, when a complete gene delivery vector can be produced intactly from recombinant cells (such as retroviral vectors), the pharmaceutical preparation can include one or more cells that produce a gene delivery system.

Pharmaceutical compositions containing dsRNA

In one embodiment, the present invention provides pharmaceutical compositions comprising a dsRNA as described herein and a pharmaceutically acceptable carrier, and methods of administering the pharmaceutical compositions. Pharmaceutical compositions comprising dsRNA are used to treat diseases or conditions associated with the expression or activity of 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.

dose

Pharmaceutical compositions described herein are administered in a dosage sufficient to inhibit EG5/KSP and/or VEGF gene expression. Typically, the appropriate dosage of dsRNA is 0.01 to 200.0 milligrams per kilogram of recipient body weight per day, generally 1 to 50 milligrams per kilogram of recipient body weight per day. For example, dsRNA can be administered in a dosage of 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg or 50 mg/kg.

The pharmaceutical composition can be administered once daily, or the dsRNA can be administered as 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 long-lasting, allowing subsequent doses to be administered at intervals of no more than 7 days, or at intervals of no more than 1, 2, 3, or 4 weeks.

In some embodiments, dsRNA is administered by continuous infusion or delivery through a controlled release formulation. In this case, the dsRNA contained in each subdose must be correspondingly smaller to reach a total daily dose. Dosage unit can also be compounded to deliver within a few days, as used in a few days to provide a conventional sustained-release formulation of the dsRNA of sustained release. Sustained-release formulations are well known in the art and are particularly useful for delivering substances to specific sites, for example, can be used together with reagents of the present invention. In this embodiment, dosage unit comprises corresponding multiple daily doses.

Skilled artisans 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 treatment, the subject's general health and/or age, and other existing diseases. In addition, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. As described elsewhere herein, the effective amount and in vivo half-life of individual dsRNAs encompassed by the present invention can be estimated using conventional methods or based on in vivo testing using appropriate animal models.

Advances in mouse genetics have yielded numerous mouse models for the study of various human diseases, such as pathological processes mediated by expression of EG5/KSP and/or VEGF. Such models are useful for in vivo testing of dsRNA and for determining therapeutically effective amounts. Suitable mouse models are, for example, mice harboring 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 by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture experiments and animal studies can be used to formulate dosage ranges for humans. The dosage of the compositions described herein is generally within a circulating concentration range that includes the ED50 with little or no toxicity. The dosage may vary depending on the dosage form used and the route of administration used. For any compound used in the methods described herein, a therapeutically effective dose can be preliminarily assessed from cell culture experiments. Dosages can be formulated in animal models to achieve a circulating plasma concentration range (e.g., a decreased concentration of the polypeptide) of the compound or, where appropriate, the polypeptide product of the target sequence, that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined by cell culture. This information can be used to more accurately determine useful dosages in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to administration as described above, the dsRNA described herein can be administered in combination with other known drugs that effectively treat the pathological processes mediated by target gene expression. In any case, the physician in charge can adjust the amount and timing of dsRNA administration based on the results observed using standard measures of efficacy known in the art or as described herein.

Application

The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical, pulmonary, for example, by inhalation or insufflation of a powder or aerosol, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, and subcutaneous, oral or parenteral, for example, subcutaneous administration.

Typically, when treating a mammal suffering from hyperlipidemia, the dsRNA molecule is administered systemically by parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or perfusion; or intracranial, such as intraparenchymal, intrathecal or intracavitary administration. For example, coupled or uncoupled dsRNA or dsRNA formulated with or without liposomes can be administered intravenously to the patient. For this, the dsRNA molecule can be formulated into a composition, for example, a sterile and non-sterile aqueous solution, a non-aqueous solution in a general solvent (such as alcohols) or a solution in a liquid or solid oil matrix. Such solutions can also include buffers, diluents and other suitable additives. For parenteral, intrathecal or intracavitary administration, the dsRNA molecule can be formulated into a composition such as a sterile aqueous solution, which can also include buffers, diluents and other suitable additives (for example, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers). Preparations are described in more detail herein.

The dsRNA can be delivered in a manner that targets a specific tissue, such as the liver (eg, hepatocytes of the liver).

preparation

The pharmaceutical formulations of the present invention can be prepared in unit dosage form conveniently according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of combining the active ingredient with a pharmaceutically acceptable carrier or excipient. In general, the formulations are prepared by uniformly and intimately combining the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated into any number of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft capsules, suppositories, and enemas. The compositions of the present invention can also be prepared as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension, including, but not limited to, sodium carboxymethylcellulose, sorbitol, and/or dextran. The suspension can also contain a stabilizer.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be prepared from a variety of ingredients, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. One aspect of the present invention is a formulation that targets the liver for the treatment of liver disorders, such as hyperlipidemia.

In addition, dsRNAs targeting EG5/KSP and/or VEGF genes can be formulated into compositions containing dsRNAs mixed, encapsulated, coupled, or otherwise combined with other molecules, molecular structures, or nucleic acid mixtures. For example, a composition containing one or more dsRNA agents targeting EG5/KSP and/or VEGF genes can include other therapeutic agents, such as other cancer therapeutics or one or more dsRNA compounds targeting genes other than EG5/KSP and/or VEGF.

Oral, parenteral, topical, and biologic agents

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, cachets, tablets or minitablets. Thickeners, fragrances, diluents, emulsifiers, dispersion aids or adhesives may be required. In some embodiments, oral formulations are formulations in which the dsRNA described herein is 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), bile acid, dehydrocholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, sad, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glyceryl monooleate (monoolein), dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptane-2-one, acylcarnitines, acylcholines or monoglycerides, diglycerides or pharmaceutically acceptable salts thereof (such as sodium salt). In some embodiments, a combination of penetration enhancers can be used, such as a combination of fatty acids/salts and bile acids/salts. An exemplary combination is the sodium salt of lauric acid, sad and UDCA. Other penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-octadecyl ether. The dsRNA described in the present invention can be delivered orally in the form of particles comprising spray-dried particles or compounded to form micron or nanoparticles. dsRNA complexing agents include polyamino acids, polyimines, polyacrylates, polyalkyl acrylates, polyoxethanes, polyalkyl cyanoacrylates, cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch, polyalkyl cyanoacrylates, DEAE-derivatized polyimines, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyanoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAB-albumin and DEAE-dextran, polymethacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in US Patent 6,887,906, US Patent Publication 20030027780, and US Patent 6,747,014, each of which is incorporated herein by reference.

Compositions and dosage forms for parenteral, intraparenchymal (into the brain), intrathecal, intracavitary, 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 preparations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners, etc. may also be necessary or desirable. Suitable topical formulations include preparations in which the dsRNA described herein is mixed with a topical delivery agent (such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants). Suitable lipids and liposomes include neutral (such as dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine), anionic (such as dimyristoylphosphatidylglycerol DMPG) and cationic (such as dioleoyltetramethylaminopropane DOTAP and dioleoylphosphatidylethanolamine DOTMA). The dsRNA described herein can be encapsulated in liposomes or can form complexes therewith, especially with cationic liposomes. Alternatively, dsRNA can be complexed with lipids, especially with cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, sad, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glyceryl monooleate, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptane-2-ketone, acylcarnitines, acylcholine or C _1-10 alkyl esters (such as isopropyl myristate IPM), monoglyceride, diglyceride or their pharmaceutically acceptable salts. Topical preparations are described in detail in United States Patent (USP) 6,747,014, which is incorporated herein by reference in its entirety. In addition, dsRNA molecules can be used to mammals using the biological or abiotic methods described in, for example, United States Patent (USP) 6,271,359. Non-biological delivery can be achieved by a variety of methods including, but not limited to, the following: (1) loading liposomes with dsRNA nucleic acid molecules provided herein, and (2) complexing the dsRNA molecules with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. Liposomes can be composed of cationic and neutral lipids commonly used for in vitro transfection of cells. Cationic lipids can be complexed (e.g., electrically bound) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, but are not limited to, lipofectin, lipofectamine, lipofectace, and DOTAP. Methods for forming liposomes are well known in the art. For example, liposome compositions can be formed from phosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylglycerol, or dioleoylphosphatidylethanolamine. Many lipophilic substances are commercially available, including Lipofectin ^™ (Invitrogen/Life Technologies, Carlsbad, Calif.) and Effectene ^™ (Qiagen, Valencia, Calif.). In addition, systemic delivery methods can be optimized using commercially available cationic lipids (such as DDAB or DOTAP), each of which can be mixed with a neutral lipid (such as DOPE or cholesterol). In some cases, liposomes as described in Templeton et al. (Nature Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations (such as polyethyleneimine) can be used to achieve in vivo and ex vivo delivery (Boletta et al., J. Am Soc. Nephrol. 7: 1728 (1996)). Further information on liposomes for delivery of nucleic acids can be found in U.S. Patent 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 the whole bag of tricks including but not limited to using viral vectors.For example, viral vectors (such as adenovirus and herpes virus vectors) can be used for delivering dsRNA molecules to hepatocytes.Standard molecular biology techniques can be used for introducing one or more dsRNA provided by the invention into one of the many different viral vectors for nucleic acid delivery to cells developed before.The viral vectors obtained can be used for example for infecting one or more dsRNA delivered to cells.

Identification of prepared dsRNA

Preparations prepared by online mixing or extrusion-free method can be identified in a similar manner. For example, preparations are usually identified by visual inspection. They should be whitish, translucent solutions that do not contain aggregates or sediments. Particle size and particle size distribution can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particle size should be approximately 20-300nm, such as 40-100nm. Particle size distribution should be unimodal. Dye exclusion analysis is used to assess the total siRNA concentration and entrapped fraction in the preparation. Samples of formulated siRNA can be incubated with an RNA-binding dye such as Ribogreen (Molecular Probes) in the presence or absence of a formulation-disrupting surfactant (e.g., 0.5% Triton-X100). The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant relative to a standard curve. The fraction captured is determined by subtracting the "free" siRNA content (as measured by the signal in the absence of the surfactant) from the total siRNA content. The percentage of captured siRNA is typically >85%. For SNALP formulations, the microparticle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. Suitable ranges are typically from about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Liposomal preparations

In addition to microemulsions, many organized surfactant structures that have been studied and used for pharmaceutical preparations are also available. These structures include unilamellar, micelle, bilayer and vesicle. Vesicle (such as liposome) has attracted widespread attention due to the persistence of its specificity and the effect it provides in drug delivery. As used in the present invention, the term "liposome" refers to the vesicle that is composed of the amphiphilic lipids that are arranged into one or more spherical bilayers.

Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic substance and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with cell walls. Non-cationic liposomes, while not as effectively fused with cell walls, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of an appropriate transdermal gradient. Therefore, it is ideal to use liposomes that are highly deformable and able to pass through such micropores.

Other advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; and liposomes can protect drugs encapsulated in their lumen from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

Liposomes can be used to transfer and deliver active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissue, they begin to fuse with the cell membrane. As the liposome and cell fuse, the contents of the liposome are released into the cell where the active ingredient can act.

Liposomal formulations have been the focus of intensive research as a mode of delivery for a variety of drugs. Growing evidence indicates that liposomes offer several advantages over other dosage forms for topical administration. These advantages include reduced side effects associated with high systemic absorption of the administered drug, improved accumulation of the administered drug at the intended target, and the ability to administer a wide range of hydrophilic and hydrophobic drugs into the skin.

Several reports have detailed the ability of liposomes to deliver substances (including large molecular weight DNA) into the skin. Compounds including analgesics, antibodies, hormones, and large molecular weight DNA have been administered to the skin. Most applications result in targeting the upper epidermis.

Liposomes are divided into two major categories. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized into the endosome. Due to the acidic pH in the endosome, the liposomes rupture, thereby releasing their contents into the cytoplasm (Wang et al., Biochem.Biophys.Res.Commun., 1987, 147, 980-985).

pH-sensitive or negatively charged liposomes capture DNA rather than compounding with it. Because DNA and lipids have similar charges, they repel rather than form complexes. However, some DNA is captured in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. The expression of exogenous genes was detected in target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

An important type of liposome composition includes phospholipids other than naturally derived phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy PC and egg yolk PC. Another type is formed from a mixture of phospholipids and/or phosphatidylcholine and/or cholesterol.

Several studies have evaluated the topical delivery of liposome pharmaceutical preparations to skin. The liposome comprising interferon is applied to guinea pig skin and causes alleviating skin herpes ulcers, and delivering interferon by other means (for example, as solution or emulsion) is invalid (Weiner et al., Journal of Drug Targeting, 1992,2,405-410). In addition, another study has tested the interferon used as a part for liposome preparation relative to the efficiency of utilizing aqueous system to apply interferon, and draws a conclusion: liposome preparation is better than aqueous mode of application (du Plessis et al., Antiviral Research, 1992,18,259-265).

Nonionic liposome systems, particularly systems containing nonionic surfactants and cholesterol, have also been tested to determine their use in drug delivery to the skin. Nonionic liposome formulations containing Novasome ^™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome ^™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin A into the dermis of mouse skin. The results showed that this nonionic liposome system was effective in promoting the deposition of cyclosporin A into different skin layers (Hu et al. STP Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include "sterically stabilized" liposomes, a term used herein to refer to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in an increased circulation life compared to liposomes without such specialized lipids. Examples of sterically stabilized liposomes are liposomes in which a portion of the vesicle-forming lipid portion of the liposomes (A) comprises one or more glycolipids (e.g., monosialoganglioside G _M1 ), or (B) is derived from one or more hydrophilic polymers (e.g., 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, sphingomyelins, or PEG-derived lipids, the increased circulation half-life of these sterically stabilized liposomes is due to reduced uptake in cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G _M1 , galactocerebroside sulfate, and phosphatidylinositol to increase the blood half-life of liposomes. Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949) described these findings in detail. U.S. Patent No. 4,537,025 and WO 55/04924 to Allen et al. disclose liposomes comprising (1) sphingomyelin and (2) ganglioside G _M1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

Many liposomes comprising 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) described liposomes comprising the nonionic detergent 2C _1215G , which contained a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) showed that hydrophilic coatings of polystyrene particles with polyglycols resulted in a significant increase in blood half-life. Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899) described synthetic phospholipids modified by conjugation to carboxyl groups of polyalkylene glycols such as PEG. Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating a significant increase in the blood circulation half-life of liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extend this discovery to other PEG-derived phospholipids, such as DSPE-PEG, which is formed by a combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes with covalently bound PEG moieties on their outer surface are described in Fisher's European Patent EP 0445 131 B1 and WO 90/04384. Woodle et al. (U.S. Patents 5,013,556 and 5,356,633) and Martin et al. (U.S. Patent 5,213,804 and European Patent EP 0496 813 B1) describe liposome compositions comprising 1-20 mole percent of PE derived from PEG and methods of use thereof. Liposomes containing a variety of other lipid-polymer conjugates are described in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and WO 94/20073 (Zalipsky et al.). Liposomes containing PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe liposomes containing PEG, which may be further derivatized with functional moieties on their surface.

Numerous liposomes containing nucleic acids are known in the art. WO 96/40062 to Thierry 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 claims that the contents of such liposomes can include dsRNA. U.S. Patent No. 5,665,710 to Rahman et al. describes a specific method for encapsulating oligodeoxynucleotides into liposomes. WO 97/04787 to Love et al. discloses liposomes containing dsRNA targeted to the raf gene.

Transfersomes are another type of liposome and are highly deformable lipid aggregates (which are attractive candidates for drug delivery vehicles). Transfersomes can be described as lipid droplets that are highly deformable to the point that they can easily penetrate pores smaller than the droplets. Transfersomes can adapt to the environment in which they are used, such as their self-optimization (adapting to the shape of the pores in the skin), self-repairing, and generally do not need to be fragmented to reach their target and are generally self-loading. In order to prepare transfersomes, it is possible to add a surface boundary activator, generally a surfactant, to a standard liposome composition. Transfersomes have been used to deliver serum albumin to the skin. The delivery of serum albumin mediated by transfersomes 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 way to classify and grade the properties of many different types of surfactants, both natural and synthetic, is to use the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also referred to as the "head") provides the most useful way to classify the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceuticals and cosmetics and can be used over a wide range of pH values. Generally, their HLB values vary 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 fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers) are also included in this category. Polyoxyethylene surfactants are the most commonly used members of the nonionic surfactant class.

If the surfactant molecule has a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates (such as soaps), acyl lactylates, acyl amide compounds of amino acids, sulfates (such as alkyl sulfates and ethoxylated alkyl sulfates), sulfonates (such as alkylbenzenesulfonates, acyl isethionates, acyl taurates and sulfosuccinates), and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

If the surfactant molecule has a positive charge when dissolved or dispersed in water, the surfactant is classified as a cationic surfactant. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

If the surfactant molecule can carry either a positive or negative charge, the surfactant is classified as an amphoteric surfactant. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamide compounds, N-alkyl betaines, and phospholipids.

The use of surfactants in pharmaceutical products, formulations, and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

SNALP

In one embodiment, the dsRNA described in the present invention is fully encapsulated in a lipid formulation to form SPLP, pSPLP, SNALP or other nucleic acid-lipid microparticles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid microparticles, including SPLP. As used herein, the term "SNLP" refers to nucleic acid-lipid microparticles comprising plasmid DNA encapsulated in lipid vesicles. SNALP and SPLP generally comprise cationic lipids, non-cationic lipids and lipids that prevent microparticles (e.g., PEG-lipid conjugates) from assembling. SNALP and SPLP are particularly advantageous for systemic application because they exhibit prolonged circulation life after intravenous (i.v.) injection and assemble at distal sites (e.g., sites physically separated from the site of administration). SPLP includes "pSPLP," which includes the coagulant-nucleic acid complex of the encapsulation shown in PCT Publication WO 00/03683. The microparticles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. In addition, when nucleic acids are present in the nucleic acid-lipid microparticles of the present invention, the nucleic acids are resistant to degradation by nucleases in aqueous solution. For example, U.S. Patents 5,976,567, 5,981,501, 6,534,484, 6,586,410, 6,815,432 and PCT Publication WO 96/40964 disclose nucleic acid-lipid microparticles and methods for their preparation.

In one embodiment, the ratio of lipid to drug (mass/mass ratio) (e.g., lipid to dsRNA ratio) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dioleyloxy Oxy-N,N-dimethylaminopropane (DLinDMA), l,2-Dilinoleyoxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleyl 1,2-Dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-Dilinoleyloxy- 3-(N-methylpiperazine)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, or mixtures thereof. The cationic lipid may comprise about 20 mol% to about 50 mol%, or about 40 mol%, of the total lipid present in the microparticle.

In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61/107,998, filed October 23, 2008, which is incorporated herein by reference.

In one embodiment, the liposome-siRNA microparticles comprise 40% 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% cholesterol: 10% PEG-C-DOMG (molar percentages), with a particle size of 63.0±20 nm and an siRNA/lipid ratio of 0.027.

The non-cationic lipid can be an anionic lipid or a neutral lipid, including but not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or a mixture thereof. If cholesterol is included, the non-cationic lipid may comprise from about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the microparticle.

The coupled lipid that inhibits microparticle aggregation can be, for example, polyethylene glycol (PEG) -lipid, including but not limited to PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer) or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci ₂ ), PEG-dimyristyloxypropyl (Ci ₄ ), PEG-dipalmityloxypropyl (Ci ₆ ) or PEG-distearyloxypropyl (Ci ₈ ). The coupled lipid that prevents microparticle aggregation can be about 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the microparticle.

In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, eg, from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98·4HCl (molecular weight 1487) (Formula 1), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar LIpids) can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01 particles). The following stock solutions of each in ethanol can be prepared: ND98, 133 mg/ml; cholesterol, 25 mg/ml; and PEG-ceramide C16, 100 mg/ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions can then be combined in a molar ratio, for example, of 42:48:10. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate at pH 5) to a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. The lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, for example, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., a 100 nm cutoff) using a thermobarrel extruder (e.g., a Lipex extruder (Northern Lipids, Inc)). In some cases, the extrusion step can be omitted. For example, ethanol removal and simultaneous buffer exchange can be achieved by dialysis or tangential flow filtration. The buffer can be exchanged with, for example, phosphate buffered saline (PBS) having a pH of about 7, such as about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

For example, LNP01 formulations are described in International Application Publication No. WO 2008/042973, which is incorporated herein by reference.

emulsion

The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are generally heterogeneous systems in which one liquid is dispersed in another liquid in the form of small droplets, usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are typically two-phase systems containing two immiscible liquid phases that are intimately mixed and dispersed with each other. Typically, emulsions can be of the water-in-oil (w/o) or oil-in-water (o/w) variety. When the aqueous phase is finely divided and dispersed as tiny droplets into 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 divided and dispersed as tiny droplets into a bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o/w) emulsion. In addition to the dispersed phase and the active drug (which may be present as a solution in the aqueous phase, the oil phase, or as a separate phase on its own), an emulsion may also contain other components. If desired, pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion. Pharmaceutical emulsions may also be multiple emulsions consisting of more than two phases, as in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often offer certain advantages over simple two-phase emulsions. Multiple emulsions in which individual oil droplets of an o/w emulsion also contain small water droplets form a w/o/w emulsion. Similarly, a system in which small oil droplets are encapsulated within small water droplets stabilized in an oil continuous phase constitutes an o/w/o emulsion.

Emulsion is characterized in that has less or does not have thermodynamic stability.Usually, the dispersed phase or discontinuous phase of emulsion are well dispersed in external phase or continuous phase and keep this form by the viscosity of emulsifier or preparation.Any phase of emulsion can be semisolid or solid, as in the case of emulsion type ointment base or cream.The mode of other stable emulsions needs to use the emulsifier that can be incorporated into any phase of emulsion.Emulsifier can be broadly divided into four types: synthetic surfactant, naturally occurring emulsifier, absorption matrix and well-dispersed solid (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (editor), 1988, Marcel Dekker, Inc., New York, N.Y., the 1st volume, the 199th page).

Synthetic surfactants, also known as surface-active agents, have found widespread use in emulsion formulations and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, page 199). Surfactants are generally amphoteric and comprise a hydrophilic portion and a hydrophobic portion. The ratio of the hydrophilic and hydrophobic properties of a surfactant is called the hydrophilic/hydrophobic balance (HLB), which is an important tool for classifying and selecting surfactants during formulation. Surfactants can be divided into different types depending on the nature of the hydrophilic group: nonionic, anionic, cationic and zwitterionic (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and gum arabic. Absorbent matrices have hydrophilic properties so they can absorb water to form w/o emulsions while still maintaining their semisolid consistency, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as excellent emulsifiers, especially in combination with surfactants and in viscous products. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or tristearin.

In emulsion dosage forms, a variety of non-emulsifying substances are also included, which affect the properties of the emulsion. These non-emulsifying substances include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, wetting agents, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 199).

Hydrophilic colloids 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 gum), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomers, cellulose ethers, and carboxyvinyl polymers). These substances disperse or swell in water to form colloidal solutions that stabilize emulsions by forming a strong interfacial film around the dispersed phase droplets and by increasing the viscosity of the external phase.

In some embodiments, the emulsion preparation comprises a plurality of preservatives, such as carbohydrates, proteins, sterols and phosphatides, which can easily support microbial growth. These preparations contain preservatives usually. The commonly used preservatives included in the emulsion preparations include methyl parahydroxybenzoate, propyl parahydroxybenzoate, quaternary ammonium salts, benzalkonium chloride, parahydroxybenzoate and boric acid. Antioxidants are usually added to the emulsion preparations to prevent the deterioration of the preparation. Antioxidants used can be free radical scavengers such as tocopherol, alkyl gallates, butylated hydroxyanisole, butylated 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 by dermal, oral, and parenteral routes and their methods of production have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 199). Emulsion formulations for oral delivery have been widely used because of their simplicity of formulation and their efficacy in terms of absorption and bioavailability (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins and high-fat nutritional products are among the substances commonly administered orally as o/w emulsions.

In one embodiment of the invention, the composition of dsRNA and nucleic acid is prepared as a microemulsion. A microemulsion can be defined as a system of water, oil, and an amphiphilic substance that is a single, optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 245). Typically, a microemulsion is a system prepared by first dispersing an oil into an aqueous surfactant solution and then adding a sufficient fourth component (usually a medium-chain alcohol) to form a transparent system. Therefore, microemulsions are also described as thermodynamically stable, isotropic, clear dispersions of two immiscible liquids stabilized by an interfacial film of surfactant molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Typically, microemulsions are prepared by a combination of 3 to 5 components (including oil, water, surfactant, cosurfactant, and electrolyte). Whether a microemulsion is water-in-oil (w/o) or oil-in-water (o/w) depends on the properties of the oil and surfactant used and the structure and geometric packing 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).

Phenomenological methods using phase diagrams have been extensively studied and have resulted in knowledge about how to formulate microemulsions that is readily understood by those skilled in the art (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 335). An advantage of microemulsions over conventional emulsions is the ability to solubilize water-insoluble drugs into a simultaneously formed thermodynamically stable droplet formulation.

The surfactant used in the preparation of microemulsions includes, but is not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglyceryl fatty acids, monolauric acid tetraglyceride (ML310), monooleic acid tetraglyceride (MO310), monooleic acid hexaglyceride (PO310), pentaoleic acid hexaglyceride (PO500), monocapric acid decaglyceride (MCA750), monooleic acid decaglyceride (MO750), sesquioleic acid decaglyceride (SO750), decaglyceride decaglyceride (DAO750). The cosurfactant is typically a short-chain alcohol (such as ethanol, 1-propanol, and 1-butanol) that improves interfacial fluidity by penetrating into the surfactant film and thereby generating a disordered film due to the free space generated between the surfactant molecules. However, microemulsions can be prepared without cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. Typically, the aqueous phase can be (but not limited to) water, aqueous solutions of drugs, glycerol, PEG300, PEG400, polyglycerol, propylene glycol and ethylene glycol derivatives. The oil phase can include, but is not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di- and tri-glycerides, polyoxyethylated glycerol fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oils.

Microemulsions are particularly interesting from the perspective of drug solubility and enhanced drug absorption. The use of lipid-based microemulsions (both o/w and w/o) has been proposed to enhance 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: increased drug solubility, protection of the drug from enzymatic hydrolysis, enhanced drug absorption possibly due to changes in membrane fluidity and permeability caused by surfactants, simple preparation, easier oral administration than solid dosage forms, improved clinical efficacy and reduced toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Typically, microemulsions can form spontaneously when their components are mixed together at ambient temperature. This may be particularly advantageous when formulating heat-labile drugs, peptides, or dsRNAs. In cosmetic and pharmaceutical applications, microemulsions are effectively used for transdermal delivery of active ingredients. We anticipate that the microemulsion compositions and formulations of the present invention will be beneficial in enhancing systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract and in enhancing local cellular uptake of dsRNAs and nucleic acids.

The microemulsions of the present invention may also include other ingredients and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the performance of the formulation and enhance the absorption of the dsRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be divided into one of five major categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92). Each type has been discussed above.

penetration enhancers

In one embodiment, the present invention uses various penetration enhancers to achieve efficient delivery of nucleic acids, particularly dsRNA, to animal skin. Most drugs exist in solution in ionic and nonionic forms. However, generally only fat-soluble or lipophilic drugs can easily cross cell membranes. It has been found that even non-lipophilic drugs can cross cell membranes if the cell membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across the cell membrane, penetration enhancers can also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five major categories, namely 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 the above-mentioned penetration enhancers is described in detail below.

Surfactants: In the context of the present invention, a surfactant (or "surface active agent") is a chemical entity that, when dissolved in an aqueous solution, reduces the surface tension of the solution or the interfacial tension between an aqueous solution and another liquid, with the result that absorption of the dsRNA through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92), and perfluorochemical emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives used as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, their _C1-10 alkyl esters (such as methyl, isopropyl and t-butyl esters) and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological effects of bile salts include promoting the dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38: 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 act as penetration enhancers. The term "bile salts" therefore includes any naturally occurring component of bile and any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycocholic acid (sodium glycocholate), 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 glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th ed., Gennaro ed., Mack Publishing Co., Easton, Pa., 1990, pp. 782-783; Muranishi, 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 agents: Chelating agents, as used in the present invention, can be defined as compounds that remove metal ions from solution by forming complexes with them, resulting in enhanced absorption of dsRNA through the mucosa. With regard to their use as penetration enhancers in the present invention, since most identified DNA nucleases require divalent metal ions for catalytic reactions and are therefore inhibited by chelating agents, chelating agents also have the added advantage of being DNase inhibitors (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 homovanillate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that do not exhibit significant chelating or surfactant activity but can still enhance the absorption of dsRNA through the digestive tract mucosa (Muranishi, Critical 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., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92), and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance the uptake of dsRNA at the cellular level can also be added to the pharmaceutical compositions and other compositions of the present invention. For example, it is known that cationic lipids such as lipofectin (Junichi et al., U.S. Patent No. 5,705,188), cationic glycerol derivatives and polycationic molecules such as polylysine (Lollo et al., PCT application WO 97/30731) also enhance the cellular uptake of dsRNA.

Other agents may also be used to enhance penetration of administered nucleic acids, including glycols (such as ethylene glycol and propylene glycol), pyrroles (such as 2-pyrrole), azones, and terpenes (such as limonene and menthone).

carrier

The dsRNA of the present invention can be formulated into 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 pharmaceutically inert medium. Pharmaceutically acceptable carriers can be liquid or solid and can be selected to provide the desired volume, consistency, and other relevant transport and chemical properties, taking into account the intended mode of administration. Typical pharmaceutically acceptable carriers include, for example, but are not limited to: water, saline solutions, binders (e.g., polyvinyl pyrrolidone or hydroxypropyl methylcellulose), 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 into the formulation. As used herein, a "carrier compound" or "carrier" may refer to a nucleic acid or its analog that is inert (i.e., not biologically active per se) but is recognized as a nucleic acid by an in vivo process that reduces the bioavailability of biologically active nucleic acids (e.g., by degrading the biologically active nucleic acid or promoting its elimination from the circulatory system). Co-administration of a nucleic acid and a carrier compound (typically in excess of the latter) can result in a significant reduction in the amount of nucleic acid recovered from the liver, kidneys, or other organs outside the circulation, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, when co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'-isothiocyanato-stilbene-2,2-disulfonic acid, the recovery of partially phosphorothioated 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, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert medium for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected to provide a desired volume, consistency, etc. when combined with the nucleic acid and other components of a particular pharmaceutical composition, taking into account the intended mode of administration. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose, etc.), fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, or calcium hydrogen phosphate, etc.), lubricants (such as magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (such as starch, sodium starch glycolate, etc.), and wetting agents (such as sodium lauryl sulfate, etc.).

Pharmaceutically acceptable organic or inorganic excipients that do not react adversely with nucleic acids and are suitable for parenteral administration can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, and the like.

The preparation that is used for local administration of nucleic acid can include the aseptic or non-sterile aqueous solution, non-aqueous solution in common solvent (such as alcohols), or the solution of nucleic acid in liquid or solid oil matrix.This solution can also comprise buffer agent, diluent and other suitable additives.Can use pharmaceutically acceptable organic or inorganic excipient that does not have adverse reaction with nucleic acid, is suitable for non-parenteral administration.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

Other ingredients

Composition of the present invention can comprise other auxiliary components commonly used in pharmaceutical compositions with use level well known in the art in addition.Therefore, for example composition can comprise pharmaceutically active substances such as antipruritic, astringent, local anesthetic or anti-inflammatory agent that are additional, compatible, or can comprise other materials that can be used for the various dosage forms of physical preparation composition of the present invention, such as dye, aromatic, preservative, antioxidant, sunscreen, thickener and stabilizer.Yet, when adding this type of material, they should not excessively interfere with the biological activity of the composition of the present invention.Said preparation can be sterilized, and if necessary, can be mixed with auxiliary agents such as lubricant, preservative, stabilizer, wetting agent, emulsifying agent, salt, buffer, coloring matter, flavoring and/or aromatic substance that are used to regulate osmotic pressure, and they do not have harmful interaction with the nucleic acid in the preparation.

Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Combination therapy

On the one hand, the composition of the present invention can be used for combined therapy.Term " combined therapy " includes administration further with other biologically active ingredients (for example, but not limited to, second and different antitumor agent) and non-drug therapy (for example, but not limited to surgery or radiotherapy) in combination with the object compound.For example, the compound of the present invention can be used in combination with other pharmaceutically active compounds, preferably compounds that can improve the effect of the compound of the present invention.The compound of the present invention can be administered simultaneously with other drug treatments (as a single preparation or a separate preparation) or sequentially.Generally, combined therapy includes administering two or more drugs in 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 that modulate protein kinases involved in various disease states. Examples of such kinases can 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 kinases (MAPKs), meiosis-specific kinases (MEKs), RAFs, and aurora kinases. Examples of receptor kinase families include epidermal growth factor receptors (EGFRs) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2, ErbB3, ErbB4, Xmrk, DER, Let23), fibroblast growth factor (FGF) receptors (e.g., FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R), hepatocyte growth/discrete factor receptors (HGFRs) (e.g., MET, RON, SEA, SEX), insulin receptors The non-receptor tyrosine kinase family includes but is not limited to BCR-ABL (e.g., p43 abl , ARG), BTK (e.g., ITK/EMT, TEC), CSK, FAK, ^FPS , JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compound can be administered in combination with one or more agents that modulate non-kinase biological targets or processes. These targets include histone deacetylases (HDACs), DNA methyltransferases (DNMTs), heat shock proteins (e.g., HSP90), and proteosomes.

In one embodiment, the subject compound can be combined with an anti-tumor drug (e.g., small molecules, monoclonal antibodies, antisense RNA, and fusion proteins) that inhibits one or more biological targets, such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar, Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737, and AEE788. Such combinations may achieve enhanced therapeutic efficacy compared to that achieved by either agent alone and may prevent or delay the emergence of drug-resistant mutations.

In certain preferred embodiments, the compounds of the present invention are administered in combination with chemotherapeutic agents. Chemotherapeutic agents include a wide range of therapeutic treatments in the field of tumors. These agents are administered at different stages of the disease for the purpose of shrinking tumors, eliminating residual cancer cells remaining after surgery, inducing symptom regression, maintaining symptom regression, and/or alleviating symptoms associated with cancer or its treatment. Examples of such agents include, but are not limited to, alkylating agents such as mustard gas derivatives (nitrogen mustard, cyclophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimine (thiotepa, hexamethylmelanine), alkyl sulfonates (busulfan), hydrazines and triazines (altretamine, procarbazine, dacarbazine, and temozolomide), nitrosoureas (carmustine, lomustine, and streptozotocin), ifosfamide, and metal salts (carmustine). Plant alkaloids, such as podophyllotoxins (etoposide and tenisopide), taxanes (paclitaxel and docetaxel), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine), and camptothecan analogs (irinotecan and topotecan); antitumor antibiotics, such as chromomycins (dactinomycin and plicamycin); amycin), anthracyclines (doxorubicin, daunorubicin, epirubicin, mitoxantrone, valrubicin, and idarubicin), and various antibiotics, such as mitomycin, dactinomycin, and bleomycin; antimetabolites, such as folic acid antagonists (methotrexate, pemetrexed, raltitrexed, aminopterin), pyrimidine antagonists (5-fluorouracil, floxuridine, cytarabine, 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, etoposide phosphate, teniposide); monoclonal antibodies (alemtuzumab, gemtuzumab, ozogamicin); The invention also includes but is not limited to ozogamicin, rituximab, trastuzumab, ibritumomab tioxetan, cetuximab, panitumumab, tositumomab, bevacizumab; and various anti-tumor drugs, such as ribonucleotide reductase inhibitors (hydroxyurea); adrenocortical steroid inhibitors (mitotane); enzymes (asparaginase and pegaspargase); anti-microtubule drugs (estramustine); and retinoids (bexarotene, isotretinoin, tretinoin (ATRA)). In certain preferred embodiments, the compounds of the present invention are administered in combination with chemotherapeutic protectants. Chemoprotectants act to protect the body or reduce the side effects of chemotherapy. Examples of such agents include, but are not limited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compound is administered in conjunction with radiation therapy. Radiation is typically delivered internally (implanted with radioactive material near the cancer site) or externally using a machine that uses photons (X-rays or gamma rays) or particle radiation. In cases where the combined treatment also includes radiation therapy, radiation therapy can be administered at any appropriate time, as long as the combined effects of the therapeutic agent and the radiation therapy achieve a beneficial effect. For example, in appropriate cases, a beneficial effect can still be achieved when radiation therapy is temporarily separated from the administration of the therapeutic agent (perhaps for days or even weeks).

It will be appreciated that the compounds of the present invention can be used in combination with immunotherapeutic agents. One form of immunotherapy is to generate an active systemic tumor-specific immune response of host origin by administering a vaccine composition at a location distant from the tumor. Various different types of vaccines have been proposed, including isolated tumor antigen vaccines and anti-idiotypic vaccines. Another approach is to utilize tumor cells from the subject to be treated or derivatives of such cells (Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121: 487). In U.S. Patent No. 5,484,596, Hanna Jr. et al. claim a method for treating resectable cancer to prevent tumor recurrence or metastasis: comprising surgical removal of the tumor, dispersing the cells with collagenase, irradiating the cells, and immunizing the patient with at least three consecutive doses of approximately 10 ⁷ cells.

It will be appreciated that the compounds of the present invention may advantageously be used in combination with one or more adjunctive therapeutic agents. Examples of suitable adjunctive therapeutic agents include steroids, such as corticosteroids (amcinolone acetonide, betamethasone, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol 17-propionate, cortisone, deflazacort, desoximetasone, diflumethasone valerate, dexamethasone, dexamethasone sodium phosphate, desonide furoate, fluocinolone acetonide, fluocinolone acetonide, halcinonide, hydrocortisone, hydrocortisone butyrate, hydrocortisone sodium succinate, hydrocortisone valerate, methylprednisolone, mometasone, prednicarbate, prednisolone, triamcinolone, propionate, and halobetasol propionate). Propionate); 5HTi agonists, such as triptans (e.g., sumatriptan or naratriptan); adenosine Al agonists; EP ligands; NMDA modulators, such as glycine antagonists; sodium channel blockers (e.g., lamotrigine); substance P antagonists (e.g., NKi antagonists); cannabinoids; acetaminophen or phenacetin; 5-lipoxygenase inhibitors; leukotriene receptor antagonists; DMARDs (e.g., methotrexate); gabapentin and related compounds; tricyclic antidepressants (e.g., amitriptyline), neuron-stabilizing antiepileptic drugs; monoaminergic uptake inhibitors (e.g., venlafaxine); matrix metalloproteinase inhibitors; nitric oxide synthase (NOS) inhibitors, such as iNOS or nNOS inhibitors; inhibitors of tumor necrosis factor alpha release or activity; antibodies treatment, such as monoclonal antibody therapy; antiviral agents, such as nucleoside inhibitors (such as lamivudine) or immune system modulators (such as interferon); opioid analgesics; local anesthetics; stimulants, including caffeine; H2-antagonists (such as ranitidine); proton pump inhibitors (such as omeprazole); antacids (such as aluminum hydroxide or magnesium hydroxide); antiflatulents (such as simethicone), decongestants (such as phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, naphthazoline, bupropion, propylhexedrine, or levomethadrine); cough suppressants (such as codeine, hydrocodone, carmiphene, toxoplasm, or dextromethorphan); diuretics; or sedating and non-sedating antihistamines.

The compounds of the present invention can be co-administered with siRNAs targeting other genes. For example, the compounds of the present invention can be co-administered with siRNAs targeting the c-Myc gene. For example, AD-12115 can be co-administered with c-Myc siRNAs. Examples of siRNAs targeting c-Myc are disclosed in U.S. patent application Ser. No. 12/373,039, which is incorporated herein by reference.

Methods for treating diseases caused by Eg5 and VEGF gene expression

The present invention particularly relates to the use of a composition comprising at least two dsRNAs (one targeted to the Eg5 gene and one targeted to the VEGF gene) for treating cancer (e.g., liver cancer), for example, to inhibit tumor growth and tumor metastasis. For example, the composition (e.g., pharmaceutical composition) can be used to treat solid tumors such as intrahepatic tumors, such as intrahepatic tumors that may occur in liver cancer. Compositions comprising a dsRNA targeted to Eg5 and a dsRNA targeted to VEGF can also be used to treat 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, Wilms' tumor, multiple myeloma, as well as for the treatment of skin cancers such as melanoma, and for the treatment of lymphomas and blood cancers. The present invention also relates to the use of a composition comprising Eg5 dsRNA and VEGF dsRNA for inhibiting the accumulation of ascites and pleural effusions in various 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, Wilms' tumor, multiple myeloma, skin cancer, melanoma, lymphoma, and blood cancer. Due to the inhibitory effect on the expression of Eg5 and VEGF, the composition of the present invention or the pharmaceutical composition prepared therefrom can improve the quality of life.

In one embodiment, a patient is treated for a tumor associated with the expression of AFP or a tumor that secretes AFP, such as a hepatoma or teratoma. In certain embodiments, the patient is treated for a malignant teratoma, an endodermal sinus tumor (yolk sac carcinoma), a neuroblastoma, a hepatoblastoma, a hepatocellular carcinoma, a testicular cancer, or an ovarian cancer.

The present invention also relates to the use of dsRNA or pharmaceutical compositions thereof for treating cancer or preventing tumor metastasis, for example in combination with other drugs and/or other treatment methods (e.g., in combination with known drugs and/or known treatment methods, such as those currently used to treat cancer and/or to prevent tumor metastasis). Preferably, the dsRNA is combined with radiation therapy and chemotherapeutic agents such as cisplatin, cyclophosphamide, 5-fluorouracil, doxorubicin, daunorubicin, or tamoxifen.

The present invention can also be implemented by including a specific RNAi agent in combination with another anti-tumor chemotherapeutic agent (such as any conventional chemotherapeutic agent). The combination of a specific binding agent with such other agents can enhance the chemotherapeutic regimen. A skilled artisan will readily appreciate the numerous chemotherapeutic regimens that can be incorporated into the methods of the present invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, and natural substances. For example, the compounds of the present invention can be administered together with antibiotics (such as doxorubicin) and other anthracycline analogs, nitrogen mustards (such as cyclophosphamide), pyrimidine analogs (such as 5-fluorouracil), cisplatin, hydroxyurea, paclitaxel, and its natural and synthetic derivatives. As another example, in cases where the tumor includes mixed tumors of gonadotropin-dependent and non-gonadotropin-dependent cells (such as breast adenocarcinoma), the compounds can be administered in combination with leuprorelin or goserelin (synthetic peptide analogs of LH-RH). Other anti-tumor regimens include the use of a tetracycline compound in combination with another treatment modality (such as surgery, radiation, etc.) (also referred to herein as "adjuvant anti-tumor modality"). Therefore, the method of the present invention can be used together with such conventional therapies to have the effects of reducing side effects and enhancing therapeutic effects.

Method for inhibiting the expression of EG5 gene and VEGF gene

In yet another aspect, the present invention provides a method for inhibiting the expression of Eg5 and VEGF genes in a mammal, comprising administering the composition described herein to the mammal, thereby silencing the expression of the Eg5 and VEGF target genes.

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

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or identical to the methods and materials described herein can be used in the implementation or inspection of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. In addition, materials, methods and embodiments are merely illustrative and are not intended to limit the scope of the present invention.

Example

Example 1. dsRNA Synthesis

Reagent Source

Unless the source of a reagent is specifically mentioned herein, such reagent can be obtained from any molecular biology reagent supplier with quality/purity standards suitable for molecular biology applications.

siRNA synthesis

To screen dsRNA, single-stranded RNA was generated by solid phase synthesis using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled micropore glass (Proligo Biochemie GmbH, Hamburg, Germany) as a solid phase carrier at a scale of 1 micromole. RNA and RNA containing 2'-O-methyl nucleotides were generated by solid phase synthesis using the corresponding phosphoramidite and 2'-O-methyl phosphoramidite (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were introduced at selected sites within the oligonucleotide chain sequence using standard nucleoside phosphoramidite chemical reactions (such as those described in current protocols in nucleic acid chemistry (Beaucage, S.L. et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA). Phosphorothioate linkages were introduced by replacing the iodine oxidant solution with a solution of Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further auxiliary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of crude oligoribonucleotides by anion exchange HPLC were performed according to known methods. Yield and concentration were determined by UV absorption of each RNA solution at a wavelength of 260 nm using a spectrophotometer (DU 640B, Beckman Coulter GmbH, Unterschleißheim, Germany). Double-stranded RNA was generated by mixing equimolar complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heating in a water bath at 85-90° C. for 3 minutes and cooling to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.

Conjugate

Below is a prophetic description of the synthesis of siRNA conjugated to 3'-cholesterol (referred to herein as -Chol-3') using a suitably modified solid support for RNA synthesis. The modified solid support was prepared as follows:

2-Azetidine-1,4-dicarboxylic acid diethyl ester AA

A 4.7 M aqueous sodium hydroxide solution (50 mL) was added to a stirred, ice-cold solution of glycine ethyl ester hydrochloride (32.19 g, 0.23 mol) in water (50 mL). Ethyl acrylate (23.1 g, 0.23 mol) was then added, and the mixture was stirred at room temperature until the reaction was complete as determined by TLC. After 19 hours, the solution was separated with dichloromethane (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was distilled to yield AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimide (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0°C. Azetidine-1,4-dicarboxylic acid diethyl ester (5 g, 24.6 mol) and dimethylaminopyridine (0.305 g, 2.5 mmol) were then added. The solution was warmed to room temperature and further stirred for 6 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layers were dried over sodium sulfate and concentrated to give a crude product, which was purified by column chromatography (50% EtOAC/hexanes) to give 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC

Ethyl 3-{ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]-amino}-propionate AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0°C. The solution was stirred for 1 hour. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by converting it to its hydrochloride salt.

3-({6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD

The hydrochloride salt of ethyl 3-[(6-amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionate AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0°C on ice. Diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added to the suspension. Cholesteryl chloroformate (6.675 g, 14.8 mmol) was added to the resulting solution. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE

Potassium tert-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0°C on ice and 5 g (6.6 mmol) of diester AD was slowly added over 20 minutes while stirring. The temperature was maintained below 5°C during the addition. Stirring was continued at 0°C for 30 minutes and 1 mL of glacial acetic acid was added, followed immediately by a solution of 4 g _{of NaH₂PO₄} · _H₂O in 40 mL of water. The resulting mixture was extracted twice with 100 mL of dichloromethane each, and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0°C, and extracted with three 50 mL portions of cold carbonate buffer at pH 9.5. The aqueous extracts were adjusted to pH 3 with phosphoric acid and extracted with five 40 mL portions of chloroform, which were combined, dried, and evaporated to dryness. The residue was purified by column chromatography using 25% ethyl acetate/hexanes to give 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester AF

To a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL) was added methanol (2 mL) dropwise over 1 hour. Stirring was continued at reflux for 1 hour. After cooling to room temperature, 1N HCl (12.5 mL) was added, and the mixture was extracted with ethyl acetate (3 x 40 mL). The combined ethyl acetate layers were dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product, which was purified by column chromatography (10% MeOH/ _CHCl₃ ) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester AG

Diol AF (1.25 g, 1.994 mmol) was dried by vacuum evaporation with pyridine (2 x 5 mL). Anhydrous pyridine (10 mL) and 4,4'-dimethoxytrityl chloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was allowed to proceed overnight at room temperature. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and dichloromethane (50 mL) was added to the residue. The organic layer was washed with 1 M aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. Residual pyridine was removed by evaporation with toluene. The crude product was purified by column chromatography (2% MeOH/chloroform, Rf = 0.5 in 5% MeOH/CHCl ₃ ) (1.75 g, 95%).

Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried overnight at 40°C under vacuum. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added, and the solution was stirred at room temperature under an argon atmosphere for 16 hours. The mixture was then diluted with dichloromethane (40 mL) and washed with ice-cold aqueous citric acid (5 wt%, 30 mL) and water (2 x 20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as is in the next step.

Cholesterol-derived CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To this solution were added DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL) and 2,2'-disulfide-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) successively. To the resulting solution was added triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 mL). The color of the reaction mixture turned bright orange. The solution was briefly stirred using a manual shaker (5 minutes). Long-chain alkylamine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was stirred for 2 hours. The CPG was filtered through a sintered funnel and washed successively with acetonitrile, dichloromethane, and ether. Unreacted amino groups were masked using acetic anhydride/pyridine.The CPG loading was determined by UV measurement (37 mM/g).

Synthesis of siRNAs bearing a 5'-12-dodecanoic acid bisdecylamide group (referred to herein as "5'-C32-") or a 5'-cholesteryl derivative group (referred to herein as "5'-Chol-") was performed as described in WO 2004/065601, except that for the cholesteryl derivative, the oxidation step was performed using Beaucage reagent to introduce a phosphorothioate linkage at the 5' end of the nucleic acid oligomer.

dsRNA targeting the Eg5 gene

Initial screening group

siRNA design was performed to identify siRNAs targeting Eg5 (also known as KIF11, HSKP, KNSL1, and TRIP5). The human mRNA sequence of Eg5, RefSeq ID: NM_004523, was used.

siRNA duplexes were designed that cross-reacted with both human and mouse Eg5. Twenty-four duplexes were synthesized for screening (Table 1a). A second screening panel was defined using 266 siRNAs targeting human EG5 and its rhesus macaque ortholog (Table 2a). An expanded screening panel was selected using 328 siRNAs targeting human EG5, without necessarily hitting any Eg5 mRNA from other species (Table 3a).

The sequences of human and partial rhesus monkey Eg5 mRNA were downloaded from the NCBI nucleotide database, and the human sequence was further used as a reference sequence (human EG5: NM_004523.2, 4908 bp; and rhesus monkey EG5: XM_001087644.1, 878 bp, only the 5′ part of human EG5).

For the table: Legend: A, G, C, U - ribonucleotides: T - deoxythymidine: U, C - 2'-O-methyl nucleotides: S - phosphorothioate bonds.

Table 1a. Sequences of Eg5/KSP dsRNA duplexes

Table 1b. Analysis of Eg5/KSP ds duplex

Table 2a. Sequences 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 the VEGF gene

400 target sequences were identified in exons 1-5 of the VEGF-A121 mRNA sequence. The reference transcript is: NM_003376.

Table 4a includes the identified target sequences. The corresponding siRNAs targeting these sequences were subjected to bioinformatic screening.

To ensure that the sequence was specific for the VEGF sequence and not specific for sequences from any other gene, the target sequence was checked against sequences in GenBank using the BLAST search engine provided by NCBI. The 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 ranked for their ability to cross-react with monkey, rat and human VEGF sequences.

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

Table 4a. Target sequences in VEGF-121

Table 4b: Duplexes targeting VEGF

Chain: S = sense, AS = antisense

Example 2. In vitro screening of Eg5 siRNA by cell proliferation

Since silencing of Eg5 has been shown to cause mitotic arrest (Weil, D et al. [2002] Biotechniques 33:1244-8), cell survival assays were used to screen for siRNA activity. HeLa cells (14,000 per well [screens 1 and 3] or 10,000 per well [screen 2]) were seeded in 96-well plates and transfected simultaneously with Lipofectamine 2000 (Invitrogen) at a final siRNA concentration of 30 nM per well and a final concentration of 50 nM (first screen) and 25 nM (second screen). A subset of duplexes was tested in the third screen at a concentration of 25 nM (see Table 5).

72 hours after transfection, cell proliferation was assayed by adding WST-1 reagent (Roche) to the culture medium and measuring absorbance at 450 nm. The absorbance value of control (non-transfected) cells was considered 100%, and the absorbance of siRNA-transfected wells was compared to the control value. Sextuplicates were performed for each of the three screens. A subset of siRNAs was further tested at a range of siRNA concentrations. Assays were performed in HeLa cells (14,000 per well; same protocol as above, Table 5).

Table 5: Effects of 25 nM duplexes targeting Eg5 on cell viability

The nine siRNA duplexes that showed the greatest growth inhibition in Table 5 were retested in HeLa cells at a range of concentrations. The siRNA concentrations tested were 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.14 nM, and 0.046 nM. The assay was performed in sextuplicate, and the concentration of each siRNA that resulted in 50% inhibition of cell proliferation (IC ₅₀ ) was calculated. For each duplex, this dose-response analysis was performed at two to four times the dose. Table 6 gives the average IC ₅₀ values (nM).

Table 6: _IC50 of siRNA: Cell proliferation in HeLa cells

duplex 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 by mRNA suppression

Directly before transfection, HeLa S3 cells (ATCC No. CCL-2.2, LCG Promochem GmbH, Wesel, Germany) were seeded at 1.5 × ¹⁰ cells/well in 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75 μl of culture medium (Ham's F12, 10% fetal bovine serum, 100 μg/ml penicillin/100 μg/ml streptomycin, all from Bookroom AG, Berlin, Germany). Transfections were performed in quadruplicate. For each well, 0.5 μl of Lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany) was mixed with 12 μl of Opti-MEM (Invitrogen) and incubated at room temperature for 15 minutes. For the siRNA concentration of 50nM in 100 microlitre transfection volumes, the siRNA of 5 μM of 1 μl is mixed with the Opti-MEM of every hole 11.5 μl, merged with the Lipofectamine2000-Opti-MEM mixture, and again incubated at room temperature 15 minutes. siRNA-Lipofectamine2000-complex is applied to cell fully (each 25 μl in every hole), and cell is incubated 24 hours under 37 ℃ and 5% carbon dioxide in moist incubator (Heroes GmbH, Hanau). Carry out a single dose screening with 50nM and 25nM respectively.

Cells were harvested by applying 50 μl of a lysis mixture (from the contents of the QuantiGene bDNA-test kit of Genospectra, Fremont, USA) to each well containing 100 μl of culture medium and lysed at 53°C for 30 minutes. Subsequently, 50 μl of the listed items were incubated with probe sets specific for human Eg5 and human GAPDH and performed according to the protocol of the QuantiGene manufacturer. Finally, chemiluminescence was measured in RLU (relative light units) using Victor2-Light (Perkin Elmer, Wiesbaden, Germany), and the values obtained for each well using the hEg5 probe set were normalized relative to the GAPDH values in Beijing. The values obtained using siRNA directed to Eg5 were correlated with the values obtained using nonspecific siRNA (directed to HCV) (which were set to 100%) (Tables 1b, 2b, and 3b).

The effective siRNA obtained by screening was further characterized by dose-response curves. Dose-response curves were transfected at the following concentrations: 100 nM, 16.7 nM, 2.8 nM, 0.46 nM, 77 pM, 12.8 pM, 2.1 pM, 0.35 pM, 59.5 fM, 9.9 fM and mock (no siRNA) and diluted to a final concentration of 12.5 microliters with Opti-MEM according to the above protocol. Data were analyzed using Microsoft Excel with XL-fit 4.2 (IDBS, Guildford, Surrey, UK) and dose-response model number 205 (Tables 1b, 2b, and 3b).

Lead siRNA AD12115 was further analyzed by applying the WST-proliferation assay from Roche (as described previously).

A subset of the 34 duplexes shown in Table 2 that exhibited maximal activity was analyzed by transfection in HeLa cells at final concentrations ranging from 100 nM to 10 fM. Transfections were performed in quadruplicate. For each duplex, two dose-response analyses were performed. For each duplex, the concentrations that produced a 20% (IC ₂₀ ), 50% (IC ₅₀ ), and 80% (IC ₈₀ ) reduction in KSP mRNA were calculated (Table 7).

Table 7: Dose-response mRNA inhibition of Eg5/KSP duplexes in HeLa cells

Concentration given in pM

(ND - not determined)

Example 4. Silencing of Hepatic Eg5/KSP in Pups Following Single Bolus Administration of LNP01-Formulated siRNA

Eg5/KSP expression can be detected in the liver of growing rats from birth until approximately 23 days of age. Target silencing of formulated Eg5/KSP siRNA was assessed in pups using duplex AD-6248.

Tested KSP duplexes

duplex

method

Animal Dosing. Male juvenile Sprague-Dawley rats (19 days old) were administered a single dose of lipidoid ("LNP01") formulated siRNA via tail vein injection. Groups of 10 animals received 10 mg/kg (mg/kg) body weight of AD6248 or nonspecific siRNA. The dose level refers to the amount of siRNA duplex administered in the formulation. A third group received phosphate-buffered saline. Two days after siRNA administration, the animals were sacrificed. The livers were excised, quickly frozen in liquid nitrogen, and pulverized into a powder.

Measurement of mRNA. The levels of Eg5/KSP mRNA in the livers of all treatment groups were determined. Each liver powder sample (approximately 10 mg) was homogenized in tissue lysis buffer containing proteinase K. For each sample, the levels of Eg5/KSP and GAPDH mRNA were measured in triplicate using Quantigene branched DNA analysis (GenoSpectra). For each sample, the mean value of Eg5/KSP was normalized to the mean value of GAPDH. The group mean values were determined and normalized to the PBS group for each experiment.

Statistical Analysis: Significance was determined by ANOVA followed by Tukey post-hoc test.

result

Data Summary

The mean values (± standard deviation) of Eg5/KSP mRNA are shown. Statistical significance (p value) relative to the PBS group is shown (ns, not significant [p>0.05]).

Table 8. Experiment 1

A statistically significant reduction in hepatic Eg5/KSP mRNA was obtained following treatment with a 10 mg/kg dose of formulated AD6248.

Example 5. Silencing of rat liver VEGF after intravenous infusion of LNP01 formulated VSP

Rats were administered a "lipidoid" formulation containing an equimolar mixture of two siRNAs. As used herein, VSP refers to a composition containing two siRNAs, one directed against Eg5/KSP and one against VEGF. For this experiment, the duplexes AD3133, directed against VEGF, and AD12115, directed against Eg5/KSP, were used. Because Eg5/KSP expression is nearly undetectable in adult rat liver, only VEGF levels were measured after siRNA treatment.

Administered siRNA duplex (VSP)

Examples: A,G,C,U-ribonucleotide; u,c-2’-O-methylribonucleotide: s-phosphorothioate.

The unmodified form of each chain and the target of each siRNA are as follows

method

Animal Dosing. Adult male Sprague-Dawley rats were administered a lipidoid ("LNP01") formulated siRNA via a two-hour infusion into the femoral vein. Groups of four animals received doses of 5, 10, and 15 mg/kg (mg/kg) of body weight of the formulated siRNA. 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 completion of the siRNA infusion. The liver was excised, flash-frozen in liquid nitrogen, and pulverized into a powder.

Preparation process

Lipidoid ND98·4HCl (molecular weight 1487) (Formula 1 above), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of ND98, 133 mg/ml; cholesterol, 25 mg/ml; and PEG-ceramide C16, 100 mg/ml, were prepared in ethanol. The ND98, cholesterol, and PEG-ceramide C16 stock solutions were then mixed at a molar ratio of 42:48:10. The combined lipid solution was rapidly mixed with aqueous siRNA (in sodium acetate at pH 5) to a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of 100-300 mM. Lipid-siRNA nanoparticles typically formed spontaneously upon mixing. Depending on the desired particle size distribution, in some cases, the resulting nanoparticle mixture was extruded through a polycarbonate membrane (100 nm cutoff) using a hot barrel extruder (Lipex extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted. Ethanol removal and simultaneous buffer exchange were achieved by dialysis or tangential flow filtration. The buffer was exchanged with phosphate buffered saline (PBS) at pH 7.2.

Identification of preparations

Formulations prepared by standard methods or without extrusion can be identified in a similar manner. The formulations are first identified by visual inspection. They should be whitish, translucent solutions free of aggregates or sediment. The particle size and size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). The particle size should be approximately 20-300 nm, ideally 40-100 nm. The particle size distribution should be unimodal. The total siRNA concentration and capture fraction in the formulation are assessed using a dye exclusion method. Samples of the formulated siRNA are incubated with the RNA-binding dye Ribogreen (Molecular Probe) in the presence or absence of a formulation-destroying surfactant (0.5% Triton-X100). The total siRNA in the formulation is determined relative to the standard curve by the signal from the surfactant-containing sample. The capture fraction is determined by subtracting the "free" siRNA content (as measured by the signal in the absence of the surfactant) from the total siRNA content. The percentage of captured siRNA is typically >85%. For SNALP formulations, the microparticle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. A preferred range is from about at least 50 nm to about at least 110 nm, preferably from about at least 60 nm to about at least 100 nm, and most preferably from about at least 80 nm to about at least 90 nm. In one example, each microparticle size comprises at least about a 1:1 ratio of Eg5 dsRNA to VEGF dsRNA.

Measurement of mRNA. Each liver powder sample (approximately 10 mg) was homogenized in tissue lysis buffer containing proteinase K. For each sample, the levels of VEGF and GAPDH mRNA were measured in triplicate using Quantigene branched DNA analysis (GenoSpectra). For each sample, the mean value of VEGF was normalized to the mean value of GAPDH. The group mean was determined and normalized to the PBS group for each experiment.

Protein measurement. Each liver powder sample (approximately 60 mg) was homogenized in 1 ml of RIPA buffer. Total protein concentration was determined using a Micro BCA protein assay kit (Pierce). Total protein samples from each animal were used to determine VEGF protein levels using a VEGF ELISA assay (R&D Systems). Group means were determined and normalized to the PBS group for each experiment.

Statistical Analysis: Significance was determined by ANOVA followed by Tukey post-hoc test.

result

Data Summary

Mean values (± standard deviation) for mRNA (VEGF/GAPDH) and protein (rel.VEGF) are given for each treatment group. For each experiment, statistical significance (p value) relative to the PBS group is shown.

Table 9

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

Example 6. Evaluation of VSP SNALP in a mouse model of human liver tumors

These studies utilized a VSP siRNA cocktail containing a dsRNA targeting KSP/Eg5 and a dsRNA targeting VEGF. As used herein, VSP refers to a composition having two siRNAs, one directed against Eg5/KSP and one against VEGF. For this experiment, duplexes AD3133 (directed against VEGF) and AD12115 (directed against Eg5/KSP) were used. The siRNA cocktail was prepared in SNALP.

The largest study used 20-25 mice. To test the efficacy of the siRNA-SNALP mixture in treating liver cancer, ^1x10⁶ tumor cells were injected directly into the left hepatic lobe of the test mice. The incision was closed with sutures, and the mice were allowed to recover for 2-5 hours. Mice fully recovered within 48-72 hours. Treatment with SNALP siRNA began 8-11 days after tumor inoculation.

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

Human Hep3B Study A: Antitumor Activity of VSP-SNALP

Human hepatoma cell Hep3B tumors were established in scid/beige mice by intrahepatic inoculation. Group A (n=6) animals were administered PBS; Group B (n=6) animals were administered VSP SNALP; Group C (n=5) animals were administered KSP/Luc SNALP; and Group D (n=5) animals were administered VEGF/Luc SNALP.

SNALP treatment was initiated 8 days after tumor inoculation. SNALP was administered twice weekly (Monday and Thursday) at 3 mg/kg of total siRNA for a total of 6 doses (18 mg/kg of siRNA cumulatively). The final dose was administered on day 25, and the endpoint was day 27.

Tumor burden was assessed by (a) body weight; (b) liver weight; (c) visual inspection and photography at day 27; (d) human-specific mRNA analysis; and (e) blood alpha-fetoprotein levels at day 27.

Table 10 below shows the results of visual scoring of tumor burden measured in the inoculated (left) liver lobe. Scoring: "-" = no visible tumor; "+" = evidence of tumor tissue at the injection site; "++" = discrete tumor nodules protruding from the liver lobe; "+++" = large tumor protruding from both sides of the liver lobe; "++++" = large tumor, multiple nodules throughout the liver lobe.

Table 10

Liver weight as a percentage of body weight is shown in FIG1 .

Body weights are shown in Figures 2A-2D.

The following conclusions were drawn from this study: (1) VSP SNALP exhibited potent antitumor effects in the Hep3B 1H model; (2) the antitumor activity of the VSP mixture appeared to be largely related to the KSP component; (3) anti-KSP activity was confirmed by single-dose histological analysis; and (4) VEGF siRNA showed no measurable effect on tumor growth inhibition in this model.

Study B of human Hep3B: Treatment with VSP prolongs survival

In the second Hep3B study, human hepatocellular carcinoma Hep3B tumors were established by intrahepatic inoculation of scid/beige mice. These mice lack lymphocytes and natural killer (NK) cells, which is the minimum range for immune-mediated anti-tumor effects. Group A (n = 6) mice were untreated; Group B (n = 6) mice were administered luciferase (Luc) 1955 SNALP (lot number AP10-02); and Group C (n = 7) mice were administered VSP SNALP (lot number AP10-01). The SNALP was a 1:57 ratio of cDMA SNALP and a 6:1 ratio of lipid:drug.

SNALP treatment was initiated 8 days after tumor inoculation. SNALP was administered twice weekly (Monday and Thursday) at 3 mg/kg of siRNA for a total of 6 doses (18 mg/kg of siRNA cumulatively). The final dose was administered on day 25, and the study endpoint was day 27.

Tumor burden was measured by (1) body weight; (2) visual inspection + photography on day 27; (3) human-specific mRNA analysis; and (4) blood alpha-fetoprotein measured on day 27.

Body weights were measured on the days of dosing (Days 8, 11, 14, 18, 21, and 25) and on the day of sacrifice ( FIG3 ).

Table 11.

Scoring: "-" = no visible tumor; "+" = evidence of tumor tissue at the injection site; "++" = discrete tumor nodules protruding from the liver lobe; "+++" = large tumor protruding from both sides of the liver lobe; "++++" = large tumor, multiple nodules throughout the liver lobe.

The correlation between body weight and tumor burden is shown in Figures 4, 5, and 6.

Administration of a single dose of VSP SNALP (2 mg/kg) to Hep3B mice also resulted in the formation of mitotic spindles in liver tissue samples examined 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 analysis. Tumor scores as shown by visual observation in the table above correlated with GAPDH levels (Figure 7A).

Serum ELISA was performed to measure alpha-fetoprotein (AFP) secreted by the tumor. As described below, if AFP levels decreased after treatment, the tumor did not grow. VSP treatment reduced AFP levels in some animals compared to control treatment (Figure 7B).

Human HepB3 Study C:

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

Tumor burden and target silencing were detected by qRT-PCR (Taqman). Tumor scores were also measured visually as described above, and the results are shown in the table below. As shown in Figure 8, hGAPDH levels were correlated with tumor scores observed visually as shown in the table below.

Table 12.

Scoring: "+" = variable tumor gain/some small tumors, "++" = discrete tumor nodules protruding from the liver lobe; "+++" = large tumor protruding from both sides of the liver lobe.

Human (tumor-derived) KSP silencing was detected by TaqMan analysis, and the results are shown in Figure 10. hKSP expression was normalized to hGAPDH. Approximately 80% tumor KSP silencing was observed at 4 mg/kg of SNALP-VSP, and efficacy was evident at 1 mg/kg. The clear bars in Figure 9 represent results for small (low GAPDH) tumors.

Human (tumor-derived) VEGF silencing was detected by TaqMan analysis, and the results are shown in Figure 10. hVEGF expression was normalized to hGAPDH. Approximately 60% tumor VEGF silencing was observed at 4 mg/kg of SNALP-VSP, and efficacy was evident at 1 mg/kg. The clear bars in Figure 10 represent results for small (low GAPDH) tumors.

Mouse (liver-derived) VEGF silencing was detected by TaqMan analysis, and the results are shown in Figure 11A. mVEGF expression was normalized to hGAPDH. Approximately 50% liver VEGF silencing was observed at 4 mg/kg of SNALP-VSP, and efficacy was evident at 1 mg/kg.

Human HepB3 Study D: Effects of Various dsRNAs on Tumor Growth

In the fourth study, human HCC cells (HepB3) were injected directly into the liver of SCID/beige mice and treatment began 8 days later. Treatment was given by intravenous (iv) bolus twice a week for a total of 6 doses. The final dose was administered on day 25, and the endpoint was day 27.

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

In Study 1, Group A was treated with PBS, Group B was treated with SNALP-KSP+Luc (3 mg/kg), Group C was treated with SNALP-VEGF+Luc (3 mg/kg), and Group D was treated with ALN-VSP02 (3 mg/kg).

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

After treatment with ALN-VSP02, both GAPDH mRNA levels and serum AFP levels showed a decrease ( FIG. 11B ).

Histological studies:

Human Hep3B hepatocellular carcinoma (H3B) tumors were established in mice by intrahepatic inoculation. SNALP treatment was initiated 20 days after tumor inoculation. Tumor-bearing mice (three per group) were treated with either (i) VSP SNALP or (ii) control (Luc) SNALP at a single intravenous (IV) dose of 2 mg/kg total siRNA.

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

At autopsy, large macroscopic tumor nodules (5-10 mm) were evident.

Effects of ALN-VSP in Hep3B mice:

Treatment with ALN-VSP (a mixture of KSP dsRNA and VEGF dsRNA) reduced tumor burden and expression of tumor-derived KSP and VEGF. GAPDH mRNA levels, a measure of tumor burden, were also observed to decrease after administration of ALN-VSP dsRNA (see Figures 12A-12C). Visually observed reductions in tumor burden were also evident after administration of ALN-VSP.

A single intravenous injection of ALN-VSP also resulted in the formation of mitotic spindles, which were clearly detectable in liver tissue specimens of Hep3B mice, indicating cell cycle arrest.

Example 7. Survival of SNALP-VSP animals relative to SNALP-Luc treated animals

To test the effect of siRNA SNALP on the survival of cancer subjects, tumors were established in mice by intrahepatic inoculation and the mice were treated with SNALP-siRNA. These studies utilized a VSP siRNA cocktail containing dsRNA targeting KSP/Eg5 and VEGF. The control was a dsRNA targeting Luc. The siRNA cocktail was formulated in SNALP.

Tumor cells (1x10 ⁶ human hepatocarcinoma Hep3B) were injected directly into the left hepatic lobe of scid/beige mice. These mice lack lymphocytes and natural killer (NK) cells, which minimizes immune-mediated anti-tumor effects. The incision was closed with sutures, 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 at a 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA, 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug ratio using native citrate buffer conditions.

After tumor inoculation, siRNA-SNALP treatment was started on the days indicated below (18 or 26 days). After 18 or 26 days at a 4 mg/kg dose, siRNA-SNALP was administered twice a week for 3 weeks. Survival was monitored and animals were euthanized based on humane surrogate endpoints (e.g., weight, abdominal distension/discoloration, and overall health of the animal).

Survival data for treatment initiated 18 days after tumor observation are summarized in Tables 13, 14, and Figure 13A.

Table 13. Kaplan-Meier (Survival) Data (% Survival)

Table 14. Survival days for each animal

animal Treatment group Survival 1 SNALP-Luc 28 sky 2 SNALP-Luc 33 sky 3 SNALP-Luc 33 sky 4 SNALP-Luc 33 sky 5 SNALP-Luc 36 sky 6 SNALP-Luc 38 sky 7 SNALP-Luc 57 sky 8 SNALP-VSP 38 sky 9 SNALP-VSP 51 sky 10 SNALP-VSP 51 sky 11 SNALP-VSP 51 sky 12 SNALP-VSP 53 sky 13 SNALP-VSP 53 sky 14 SNALP-VSP 57 sky 15 SNALP-VSP 57 sky

Figure 13A shows the mean survival of SNALP-VSP animals and SNALP-Luc treated animals relative to the number of days after tumor inoculation. The mean survival time of SNALP-VSP animals was extended by approximately 15 days relative to that of SNALP-Luc treated animals.

Table 15. Serum alpha-fetoprotein (AFP) concentrations before and at the end of treatment for each animal (concentrations in μ g/ml)

During the experiment, serum AFP levels were used to monitor tumor burden. Alpha-fetoprotein (AFP) is the major plasma protein produced by the yolk sac and liver during fetal development. This protein is considered the fetal counterpart of serum albumin, and the human AFP and albumin genes are located in a cluster on chromosome 4 with the same transcriptional orientation. AFP is found in monomeric, dimer, and trimer forms and binds copper, nickel, fatty acids, and bilirubin. AFP levels gradually decrease after birth, reaching adult levels by 8-12 months of age. AFP levels in normal adults are low but detectable. AFP has no known function in normal adults, and AFP expression in adults is often associated with a subset of tumors, such as hepatocellular carcinoma and teratomas. AFP is a tumor marker used to monitor testicular cancer, ovarian cancer, and malignant teratomas. Primary tumors that secrete AFP include endodermal sinus tumors (yolk sac carcinomas), neuroblastomas, hepatoblastomas, and hepatocellular carcinomas. In patients with AFP-secreting tumors, serum AFP levels often correlate with tumor size. Serum levels can be used to assess response to treatment. Typically, if AFP levels drop after treatment, the tumor is not growing. A temporary increase in AFP immediately after chemotherapy may indicate that the tumor is not growing but rather shrinking (and releasing AFP as tumor cells die). Resection is often associated with a drop in serum levels. As shown in Figure 14, tumor burden was significantly reduced in animals treated with SNALP-VSP.

The SNALP-siRNA treatment experiment was repeated at 26, 29, 32, 35, 39, and 42 days post-implantation. The data are shown in Figure 13B. The mean survival of SNALP-VSP animals was extended by approximately 15 days compared to SNALP-Luc treated animals, which were approximately 19 days or 38%.

Example 8. Induction of Monoastrocytes in Established Tumors

Inhibition of KSP in dividing cells leads to the formation of single astral bodies that can be easily observed in tissue sections. To determine whether single astral body formation occurs in tumors treated with SNALP-VSP, 2 mg/kg of SNALP-VSP was administered via tail vein injection to tumor-bearing animals (3 weeks after Hep3B cell implantation). Control animals received 2 mg/kg of SNALP-Luc. After 24 hours, the animals were sacrificed and the tumor-bearing liver lobes were processed for histological analysis. Typical images of H&E stained tissue sections are shown in Figure 15. Extensive single astral body formation is obvious in tumors treated with ALN VSP02 (A), but not in tumors treated with SNALP-Luc (B). In the latter, normal mitotic figures are obvious. The generation of single astral bodies is a unique feature of KSP inhibition and provides further evidence that SNALP-VSP has significant activity in established liver tumors.

Example 9. Manufacturing Method and Product Specifications of ALN-VSP02 (SNALP-VSP)

The ALN-VSP02 product contains 2 mg/mL of the drug substance ALN-VSPDS01 (referred to as SNALP) formulated as a sterile lipid microparticle formulation for IV administration by infusion. The drug substance ALN-VSPDS01 consists of two siRNAs (ALN-12115 targeting KSP and ALN-3133 targeting VEGF) in an equimolar ratio. The drug product is packaged in 10 mL glass vials with a 5 mL fill volume.

The following terms are used in this article:

*Also known as AD-12115, AD12115; **Also known as AD-3133, AD3133

9.1 Preparation of Drug Substance ALN-VSPDS01

The two siRNA components of the drug substances ALN-VSPDS01, ALN-12115, and ALN-3133 were chemically synthesized using commercially available synthesizers and raw materials. The manufacturing method involves synthesizing two single-stranded oligonucleotides of each duplex (the A19562 sense and A19563 antisense strands of ALN 12115 and the A3981 sense and A3982 antisense strands of ALN 3133) using conventional solid-phase oligonucleotide synthesis using phosphoramidite chemistry and a 5'-O-dimethoxytriphenylmethyl (DMT) protecting group with the 2' hydroxyl group protected with tert-butyldimethylsilyl (TBDMS) or substituted with a 2' methoxy group (2'OMe). Assembly of the oligonucleotide chains is performed by the phosphoramidite method on a solid support (such as controlled micropore glass or polystyrene). The cycle consists of 5' deprotection, coupling, oxidation, and capping. Each coupling reaction is carried out by following steps: use 5 (ethylthio) 1H tetrazolium reagent to make the ribose, 2 ' OMe or deoxyribonucleoside amidite (amidite) activation of suitable protection, then the free 5 ' hydroxyl of the protection nucleoside or oligonucleotide that coupling carrier is fixed.After the circulation of appropriate number of times, remove last 5 ' protecting group by acid treatment.Handle by methylamine aqueous solution and excise thick oligonucleotide from solid support, remove cyanoethyl protecting group and core base protecting group simultaneously.Then use the reagent cutting 2 ' O TBDMS group containing hydrogen fluoride, to produce thick oligoribonucleotide, it uses strong anion exchange high performance liquid chromatography (HPLC) then to use ultrafiltration desalination and purification.The single strand of purification is analyzed to confirm correct molecular weight, molecular sequence, impurity characteristics and oligonucleotide content before annealing into duplex. Annealed double-stranded intermediates ALN 12115 and ALN 3133 were lyophilized and stored at 20°C or mixed in a 1:1 molar ratio, and the solution lyophilized to produce the drug substance ALN VSPDS01. If the double-stranded intermediates were stored as dry powders, they were reconstituted in water before mixing. The mixing process was monitored by HPLC to achieve an equimolar ratio.

The manufacturing method flow chart is shown in FIG16 .

Example specifications are shown in Table 16a.

Table 16c shows the results of stability testing of the ALN-VSPDS01 drug substance for up to 12 months. Test methods were selected to assess physical properties (appearance, pH, moisture), purity (by SEC and denaturing anion exchange chromatography), and potency (by denaturing anion exchange chromatography [AX-HPLC]).

Table 16a. Example specifications for the ALN-VSPDS01

Table 16b. Stability of Drug Substance

9.2 Preparation of the Drug Product ALN-VSP02 (SNALP-VSP)

ALN VSP02 is a sterile formulation of two siRNAs (in a 1:1 molar ratio) and a lipid excipient in an isotonic buffer. The lipid excipient binds to the two siRNAs, protecting them from degradation in the circulatory system and facilitating their delivery to target tissues. Specific lipid excipients and the quantitative ratios of each lipid excipient (as shown in Table 17) were selected by comparing the physicochemical properties, stability, pharmacodynamics, pharmacokinetics, toxicity, and product manufacturability of various formulations through repeated series of experiments. The excipient DLinDMA is a titratable amino lipid that is positively charged at low pH values (such as those found in the endosomes of mammalian cells) but relatively uncharged at the more neutral pH of whole blood. This feature facilitates efficient encapsulation of negatively charged siRNAs at low pH values, thereby preventing the formation of empty microparticles, while still allowing the charge of the microparticles to be adjusted (reduced) by replacing the formulation buffer with a more neutral storage buffer before use. To provide physicochemical stability to the microparticles, cholesterol and the neutral lipid DPPC were incorporated. The polyethylene glycol lipid conjugate PEG2000C DMA contributes to drug product stability and provides optimal circulation time for the intended application. ALN VSP02 lipid microparticles have an average diameter of approximately 80-90 nm, characterized by low polydispersity. Representative cryogenic transmission electron microscopy (cryoTEM) images are shown in Figure 17. At neutral pH, the microparticles are essentially uncharged, with zeta potential values below 6 mV. Based on the manufacturing process, there are no significant empty (unloaded) microparticles.

Table 17: Quantitative composition of ALN-VSP02

*Maintain a 1:1 molar ratio of the two siRNAs in the drug product throughout the entire size distribution of the drug product particles.

A solution of lipids (in ethanol) and a solution of the ALN VSPDS01 drug substance (in an aqueous buffer) were mixed and diluted to form a colloidal dispersion of siRNA-lipid microparticles with an average particle size of approximately 80-90 nm. This dispersion was then filtered through a 0.45/0.2 micron filter, concentrated, and diafiltered by tangential flow filtration. After in-line testing and concentration adjustment to 2.0 mg/mL, the product was sterile filtered, aseptically filled into glass vials, stoppered, capped, and stored at 5±3°C. Ethanol and all aqueous buffer components were USP grade; all water used was USP sterile water for injection. A representative ALN-VSP02 process is shown in the flow chart in Figure 18.

Table 18a: Example ALN-VSP02 specifications

9.4 Container/closure system

ALN VSP02 drug product was packaged in 10 mL glass vials with a 5 mL fill volume. The container closure system consisted of a USP/EP Type I borosilicate glass vial, a polytetrafluoroethylene-faced butyl rubber stopper, and an aluminum flip-off cap. The drug product was stored at 5 ± 3°C.

9.5 Stability of the drug ALN-VSP02

Stability data (25°C/60% relative humidity) are found in Tables 18b and 18c.

Table 18b: Stability of example ALN-VSP02 under storage conditions

Table 18c: Example ALN-VSP02 stability at 25°C/ambient humidity

Example 10. In vitro efficacy of ALN-VSP02 in human cancer cell lines

The efficacy of ALN-VSP02 treatment in human cancer cell lines was determined by measuring KSP mRNA, VEGF mRNA and cell viability after treatment. _IC50 (nM) values for KSP and VEGF were determined in each cell line.

Table 19: Cell lines

On day 1, cells were seeded in complete medium in 96-well plates to reach 70% confluency by day 2. On day 2, the medium was replaced with Opti-MEM Reduced Serum Medium (Invitrogen Cat N: 11058-021) and cells were transfected with ALN-VSP02 or the control SNALP-Luc at concentrations ranging from 1.8 μM to 10 pM. Six hours later, the medium was changed to complete medium. Three replicates were cultured for each experiment and cell line.

Cells were harvested 24 hours after transfection. KSP levels were measured using bDNA and VEGF mRNA levels were measured using human TaqMan assay.

Viability was measured at 48 and/or 72 hours using CellTiter Blue reagent (Promega Cat N: G8080) according to the manufacturer's instructions.

As shown in Table 20, nM concentrations of VSP02 effectively reduced the expression of KSP and VEGF in various human cell lines.

Table 20: Results

Example 11. Antitumor efficacy of VSP SNALP relative to sorafenib in established Hep3B intrahepatic tumors

The antitumor effects of multiple doses of VSP SNALP compared with sorafenib, a small molecule protein kinase inhibitor approved for the treatment of hepatocellular carcinoma (HCC), were investigated in scid/beige mice bearing established Hep3B intrahepatic tumors.

As described herein, tumors were established in scid/beige mice by intrahepatic inoculation. Treatment was started 11 days after inoculation. Mice were treated with the following: sorafenib and control siRNA-SNALP, sorafenib and VSP siRNA-SNALP, or only VSP siRNA-SNALP. Control mice were treated with buffer (DMSO for sorafenib and PBS for siRNA-SNALP). Sorafenib was administered intraperitoneally from Monday to Friday for 3 weeks, with a total of 15 injections at 15 mg/kg body weight. Sorafenib was administered at least 1 hour after the injection of SNALP. Based on the most recently recorded body weight, siRNA-SNALPS was administered intravenously through the lateral tail vein at a dose of 3 mg/kg (10 ml/kg) on days 1, 4, 7, 10, 14, and 17 for 3 weeks (6 doses in total).

Mice were euthanized based on evaluation of tumor burden including progressive weight loss and clinical symptoms including status, abdominal distension/discoloration, and activity.

The survival percentage data are shown in Figure 20. Co-administration of VSP siRNA-SNALP with sorafenib increased the survival ratio compared to administration of sorafenib or VSP siRNA-SNALP alone. VSP siRNA-SNALP increased the survival ratio compared to sorafenib.

Example 12. In vitro efficacy of VSP using AD-12115 and AD-3133 variants

Two sets of duplexes targeting Eg5/KSP and VEGF were designed and synthesized. Each set consisted of duplexes tiling 10 nucleotides in each direction of the target sites of AD-12115 and AD-3133.

The sequences of the target, sense, and antisense strands of each duplex are listed in the table below.

Each duplex is tested for expression inhibition using the assays described herein. The duplexes are administered alone and/or in combination, for example, Eg5/KSP dsRNA in combination with VEGF dsRNA. In some embodiments, the dsRNA is administered in a SNALP formulation as described herein.

Table 21: Sequences of dsRNAs targeting VEGF and Eg5/KSP (stacked)

Example 13. VEGF-targeting dsRNA with a single blunt end

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

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

Each duplex is tested for inhibition of expression using the assays described herein. VEGF duplexes are administered alone and/or in combination with Eg5/KSP dsRNA (e.g., AD-12115). In some embodiments, the dsRNA is administered in a SNALP formulation as described herein.

Table 22: Target sequences of blunt-ended dsRNAs targeting VEGF

Table 23: Strand sequences of blunt-ended dsRNAs targeting VEGF

Example 14. Inhibition of Eg5/KSP and VEGF expression in humans

A human subject is treated with a pharmaceutical composition having a SNALP-formulated dsRNA targeted to the Eg5/KSP gene and a SNALP-formulated dsRNA targeted to the VEGF gene, such as ALNVSP02, to inhibit the expression of the Eg5/KSP and VEGF genes.

Select or identify a subject in need of treatment. The subject may be in need of treatment for cancer, such as liver cancer.

At time zero, a suitable first dose of the composition is administered subcutaneously to the subject. The composition is formulated as described herein. After a period of time, the subject's status is assessed, for example, by measuring tumor growth, serum AFP levels, etc. This measurement can be accompanied by measurement of Eg5/KSP and/or VEGF expression in the subject, and/or the product of successful siRNA targeting of the Eg5/KSP and/or VEGF genes. Other relevant indicators can also be measured. The amount and intensity of the dose are adjusted according to the subject's needs.

After treatment, the subject's status is compared to the status that existed before treatment, or relative to the status of untreated subjects with a similar condition.

Those skilled in the art will be familiar with methods and compositions other than those explicitly set forth in this disclosure enabling them to practice the invention within the full scope of the appended claims.

Claims (16)

1. A composition comprising a first double-stranded RNA that inhibits the expression of the Eg5/KSP gene in cells and a second double-stranded RNA that inhibits the expression of human VEGF in cells, wherein: Both the first and second double-stranded ribonucleic acids are formulated into stable nucleic acid lipid microparticles containing DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA, comprising the following proportions: 1.7-2.3 mg/mL for the first and second double-stranded ribonucleic acids, 5.6-10.3 mg/mL for DLinDMA, 2.1-3.9 mg/mL for cholesterol, 0.8-1.5 mg/mL for DPPC, and 0.6-1.1 mg/mL for PEG2000-C-DMA; The first double-stranded ribonucleic acid comprises a first sense strand and a first antisense strand, wherein the first sense strand includes the first sequence of SEQ ID NO:1240 and the first antisense strand includes the second sequence of SEQ ID NO:1241; and The second double-stranded ribonucleic acid is composed of a second sense strand and a second antisense strand, the second sense strand including the third sequence of SEQ ID NO:1242 and the second antisense strand including the fourth sequence of SEQ ID NO:1243, wherein the first and second double-stranded ribonucleic acids are present in an equimolar ratio, wherein the composition inhibits the expression of Eg5 by at least 40% upon contact with cells expressing Eg5 and wherein the composition inhibits the expression of VEGF by at least 40% upon contact with cells expressing VEGF. 2. The composition according to claim 1, wherein the first double-stranded ribonucleic acid comprises a sense strand of SEQ ID NO: 1240 and an antisense strand of SEQ ID NO: 1241; The second double-stranded ribonucleic acid consists of a sense strand composed of SEQ ID NO:1242 and an antisense strand composed of SEQ ID NO:1243. 3. The composition according to claim 1 or 2, comprising the following proportions of the ingredients: 4. The composition according to claim 1 or 2, wherein the composition inhibits the expression of Eg5 by at least 50%, 60%, 70%, 80%, or at least 90% upon contact with cells expressing Eg5. 5. The composition according to claim 1 or 2, wherein the composition inhibits VEGF expression by at least 50%, 60%, 70%, 80%, or at least 90% upon contact with cells expressing VEGF. 6. The composition according to claim 1 or 2, wherein the composition is applied at an nM concentration. 7. The composition according to claim 1 or 2, wherein applying the composition to cells will increase the formation of singlets in the cells. 8. The composition according to claim 1 or 2, wherein administration of the composition to a mammal results in an effect selected from the group consisting of: preventing tumor growth, reducing tumor growth, or prolonging survival time in the mammal. 9. The composition of claim 8, wherein the effect is measured using at least one analysis selected from the following: body weight measurement, organ weight measurement, visual examination, mRNA analysis, serum AFP analysis, and survival monitoring. 10. The composition according to claim 1 or 2, further comprising sorafenib. 11. The composition according to claim 1 or 2, wherein the first double-stranded ribonucleic acid comprises two protruding ends, and the second double-stranded ribonucleic acid comprises a protruding end at the 3' end of the antisense strand and a flat end at the 5' end of the antisense strand. 12. A method for inhibiting the expression of Eg5/KSP and VEGF in cells in vitro, comprising administering to cells the composition of any one of claims 1-11. 13. Use of the composition of any one of claims 1-11 in the preparation of a medicament for use in mammals requiring treatment of cancer to prevent tumor growth, reduce tumor growth, or prolong survival. 14. The use according to claim 13, wherein the mammal suffers from liver cancer. 15. The use according to claim 13, wherein the mammal is a human suffering from liver cancer. 16. The use according to claim 13 or 14 further includes the administration of sorafenib.