Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/5123—Organic compounds, e.g. fats, sugars
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates

Definitions

• BACKGROUND AKT-1 (Archexin), a protein product of akt-1 proto-oncogene, plays a role in cancer progression by promoting cell proliferation and inhibiting apoptosis of cancer cells (see Revathidevi S, et al., Semin Cancer Biol.2019;59:80-91; and Uko NE, et al., Curr Top Med Chem.2020;20(10):883-900).
• Archexin is a fully phosphorothioated 20-mer antisense oligonucleotide, which can specifically bind to AKT-1 mRNA resulting in RNase H-based AKT-1 downregulation. Archexin inhibits AKT-1 mRNA translation and inhibits tumor growth.
• compositions and methods disclosed herein address these and other needs.
• Described herein are pharmaceutical compositions including a lipid nanoparticle encapsulating an active agent.
• the lipid nanoparticle can include 2.5 mol% to 15 mol% one or more cationic lipids; 30 mol% to 50 mol% one or more ionizable lipids; 30 mol% to 65 mol% one or more neutral lipids; and 2.5 mol% to 15 mol% one or more PEGylated lipids.
• the active agent can include RX- 0201, 5′ gctgcatuatctccttggcg 3′, SEQ. ID. NO.1.
• methods for treating cancer, preventing cancer, preventing cancer metastasis, preventing cancer recurrence or preventing angiogenesis can include administering a pharmaceutical composition described herein to a subject in need thereof.
• the method can include (a) combining one or more ethanolic solutions comprising a mixture of lipids with an aqueous solution and acidifying to induce formation of a population of empty lipid nanoparticles; (b) contacting the population of empty lipid nanoparticles with an aqueous solution comprising the active agent, thereby encapsulating the active agent in the population of empty lipid nanoparticles to produce the population of lipid nanoparticles encapsulating the active agent; and (c) subjecting the population of lipid nanoparticles encapsulating the active agent to tangential flow filtration to replace buffer and remove residual ethanol.
• the mixture of lipids comprises 2.5 mol% to 15 mol% one or more cationic lipids; 30 mol% to 50 mol% one or more ionizable lipids; 30 mol% to 65 mol% one or more neutral lipids; and 2.5 mol% to 15 mol% one or more PEGylated lipids.
• Figures 1A-1B show graphs of the effects of WGI-0301 on tumor growth (Fig.1A) and survival (Fig.1B) in Hepa1-6 syngeneic hepatocellular carcinoma model.
• Figures 2A-2B are graphs of branch point number versus concentration (Fig.2A) and capillary length versus concentration (Fig.2B).
• Figures 3A-3C shows images of anti-angiogenesis effect of WGI-0301 alone and in therapy combinations (0.1% DMSO and WGI-0301 (Fig.3A), 2 ⁇ M Sorafenib and WGI-0301 (Fig.3B), and 5 ⁇ M Lenvatinib and WGI-0301 (Fig.3C)).
• Figure 4 shows a flow chart of the manufacturing process of the lipid nanoparticles described herein.
• Figure 5 shows the effects of test articles on mice body weight in Hepa 1-6 model.
• Figure 6 shows the effects of test articles on mice body weight change in Hepa 1-6 model.
• Figure 7 shows the effects of test articles on tumor volume in Hepa 1-6 model.
• Figure 8 shows the survival curves of test articles in Hepa 1-6 model.
• Constant administration means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.
• Systemic administration refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject's body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems.
• beneficial agent and “active agent” are used interchangeably herein to refer to a chemical compound or composition that has a beneficial biological effect.
• beneficial biological effects include both therapeutic effects, i.e., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., prevention of a disorder or other undesirable physiological condition.
• the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like.
• the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
• therapeutic agent or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
• a "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
• “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. “Inactivate”, “inactivating” and “inactivation” means to decrease or eliminate an activity, response, condition, disease, or other biological parameter due to a chemical (covalent bond formation) between the ligand and a its biological target.
• reduce or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the Attorney Docket No.11650-003WO1 standard or relative value to be referred to.
• reduced tumor growth means reducing the rate of growth of a tumor relative to a standard or a control.
• the terms “prevent” or “suppress” can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition.
• a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms.
• the term “preventing” a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and/or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event.
• effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect.
• the amount of beneficial agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount”. However, an appropriate “effective’ amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. Attorney Docket No.11650-003WO1 An “effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response.
• a “therapeutically effective amount” of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result
• a “prophylactically effective amount” of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition.
• Therapeutically effective and prophylactically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.
• terapéuticaally effective amount can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect.
• the precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and/or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
• the term “pharmacologically active” can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
• a “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive” or “negative.” As used herein, by a “subject” is meant an individual.
• oligonucleotide denotes single- or double-stranded nucleotide multimers of from about 2 to up to about 100 nucleotides in length.
• Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPSTM technology.
• double-stranded When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA.
• double-stranded As used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry, Third Ed., (1988), incorporated herein by reference for all purposes.
• nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment Attorney Docket No.11
• sequences are then said to be “substantially identical.”
• This definition also refers to, or may be applied to, the compliment of a test sequence.
• the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
• the preferred algorithms can account for gaps and the like.
• identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length.
• percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
• the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res.25:3389-3402, and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
• This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
• T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol.215:403-410).
• Cumulative scores are calculated using, for Attorney Docket No.11650-003WO1 nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
• M forward score for a pair of matching residues; always >0
• N penalty score for mismatching residues; always ⁇ 0.
• a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• W wordlength
• E expectation
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc.
• nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
• nucleobase refers to the part of a nucleotide that bears the Watson/Crick base- pairing functionality.
• a “sense” oligonucleotide will hybridize with a normal gene sequence, and will not affect the amount of, or properties of, the protein.
• a “nonsense” sequence will not yield a product, or may yield a non-functional product. For example, if a “nonsense” codon or oligomer is inserted into a gene, a truncated, non-functional protein may result.
• An “antisense” oligonucleotide will hybridize with a normal gene, but will yield a protein altered with respect to its Attorney Docket No.11650-003WO1 structure, or amount. It has been found that antisense oligomers, that is antisense compounds that are relatively short, can be easily inserted into cells, where they alter gene function.
• Antisense compounds are commonly used as research reagents for the exploration of gene function because they are able to alter gene expression with extraordinar specificity, and may be used to elucidate the function of particular genes. Antisense compounds can be used, for example, to distinguish between functions of various members of a biological pathway. Antisense oligonucleotides can be used to selectively block disease-causing genes, thereby inhibiting production of disease-associated proteins. Some antisense oligonucleotides have been safely and effectively administered to humans, and numerous clinical trials are presently underway. It is thus possible that oligonucleotides can be used to treat cells, tissues, and animals, especially humans.
• oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.
• Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a nucleic acid target and increased stability in the presence of nucleobases.
• compositions and methods described herein employ oligomeric nucleotide compounds, particularly antisense oligonucleotides, which are targeted to a portion of a nucleic acid encoding Akt-1, and which modulate the expression of Akt-1.
• the oligonucleotide compounds are designed to specifically hybridize with one or more nucleic acids encoding Akt-1.
• To target an antisense compound to a particular gene means to identify the nucleic acid sequence of interest, and select one or more sites within the nucleic acid sequence to be modified.
• nucleic acid encoding Akt-1 encompasses DNA encoding Akt-1, RNA (including pre-mRNA) transcribed from such DNA, and also cDNA derived from such RNA.
• the specific hybridization of an antisense oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid.
• the functions of DNA to be interfered with include replication and transcription.
• RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, Attorney Docket No.11650-003WO1 splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.
• the overall effect of such interference with target nucleic acid function is modulation of the expression, or production of, a protein.
• modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
• to hybridize means to hydrogen bond, which may be via Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
• adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
• “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides.
• oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position.
• the oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
• “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target.
• an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
• An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
• antisense oligonucleotides are a preferred form of antisense compound
• the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below.
• the antisense compounds in accordance with this invention preferably comprise from about 10 to about 30 nucleobases. Particularly preferred are antisense oligonucleotides comprising about 20 nucleobases (i.e. about 20 linked nucleosides).
• a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base.
• Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
• the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar.
• the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred.
• the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
• the normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
• Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
• modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
• Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′–5′ linkages, 2′–5′ linked analogs of these, and those having inverted polarity wherein
• modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
• morpholino linkages formed in part from the sugar portion of a nucleoside
• siloxane backbones sulfide, sulfoxide and sulfone backbones
• formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
• alkene containing backbones sulfamate backbones
• sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH2 component parts.
• both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with new groups.
• the base units are maintained for hybridization with an appropriate nucleic acid target compound.
• One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
• nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
• Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O— N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—].
• oligonucleotides having morpholino backbone structures may also contain one or more substituted sugar moieties.
• Preferred oligonucleotides comprise one of the following 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 may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.
• oligonucleotides comprise one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
• a preferred modification includes 2′-methoxyethoxy (2′- O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486–504) i.e., an alkoxyalkoxy group.
• a further preferred modification includes 2′- dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in examples hereinbelow.
• Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
• Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
• base include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
• Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-Me-C), 5-hydroxymethyl cytosine, 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-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted
• nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the 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 nucleic acid duplex stability by 0.6–1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O- methoxyethyl sugar modifications.
• oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
• moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl- ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety
• the present invention also includes antisense compounds which are chimeric compounds.
• “Chimeric” antisense compounds or “chimeras,” in the Attorney Docket No.11650-003WO1 context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
• oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
• An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
• RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
• RNA target Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.
• Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
• Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
• the antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed.
• the antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.
• the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
• the antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically Attorney Docket No.11650-003WO1 acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
• prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
• prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives.
• pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
• salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.
• acid addition salts formed with inorganic acids for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like
• salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid
• compositions including a lipid nanoparticle encapsulating an active agent.
• the active agent can include RX-0201, 5′ gctgcatgatctccttggcg 3′, SEQ. ID. NO.1.
• RX-0201 is targeted to a site in the coding region of the Akt-1 gene having the following sequence: 5′ cgccaaggagatcatgcagc 3′ at site 1,478 of Akt-1 gene (Genebank # BC000479) (Seq. Id. No. 2).
• the sequence for the backbone of RX-0201 is complementary to this site. Archexin (RX-0201) is described in U.S.
• the compound is an antisense oligonucleotide.
• the antisense oligonucleotide has at least one modified internucleoside linkage that is a phosphorothioate linkage.
• Suitable cationic lipids can include, but are not limited to, DOTMA: [1-(2,3-sioleyloxy)propyl)]- N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DODAP, DOSPA (2,3-dioleyloxy-N-[2-(spermine carboxamido)ethyl]-N,N-dimethyl-1-propanaminium Attorney Docket No.11650-003WO1 trifluoroacetate), DORIE (N-[1-(2,3-dioleyloxypropyl)]-N,N-dimethyl-N-hydroxyethylammonium bromide), DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromid
• DORIE 2,3-dioleyloxy-N-[2-(spermine carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate
• the lipid nanoparticle can include 2.5 mol% to 15 mol% one or more cationic lipids.
• the one or more cationic lipids can be present in the lipid nanoparticle in an amount of at least 2.5 mol%, (e.g., at least 5 mol%, at least 7.5 mol%, at least 10 mol%, or at least 12.5 mol%). In some embodiments, the one or more cationic lipids can be present in the lipid nanoparticle in an amount of 15 mol% or less, (e.g., 12.5 mol% or less, 10 mol% or less, 7.5 mol% or less, or 5 mol% or less). The one or more cationic lipids can be present in the lipid nanoparticle in an amount ranging from any of the minimum values described above to any of the maximum values described above.
• the one or more cationic lipids can be present in the lipid nanoparticle in an amount of from 2.5 mol% to 15 mol% (e.g., from 2.5 mol% to 12.5 mol%, from 2.5 mol% to 10 mol%, from 2.5 mol% to 7.5 mol%, from 2.5 mol% to 5 mol%, from 5 mol% to 15 mol%, from 5 mol% to 12.5 mol%, from 5 mol% to 10 mol%, or from 5 mol% to 7.5 mol%).
• 2.5 mol% to 15 mol% e.g., from 2.5 mol% to 12.5 mol%, from 2.5 mol% to 10 mol%, from 2.5 mol% to 7.5 mol%, from 2.5 mol% to 5 mol%, from 5 mol% to 15 mol%, from 5 mol% to 12.5 mol%, from 5 mol% to 10 mol%, or from 5 mol% to 7.5 mol%.
• Suitable neutral lipids can include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol, or any combination thereof.
• the one or more neutral lipids can include cholesterol, DOPE, DOPC, or a combination thereof.
• the one or more neutral lipids can include (1) cholesterol; and (2) DOPE, DOPC, or a combination thereof.
• the lipid nanoparticle can include 30 mol% to 65 mol% one or more neutral lipids.
• the one or more neutral lipids are present in the lipid nanoparticle in an amount of at least 30 mol%, (e.g., at least 35 mol%, at least 40 mol%, at least 45 mol%, at least 50 mol%, at least 55 mol%, or at least 60 mol%).
• the one or more neutral lipids are present in the lipid nanoparticle in an amount of 65 mol% or less, (e.g., 60 mol% or less, 55 mol% or less, 50 mol% or less, 45 mol% or less, 40 mol% or less, or 35 mol% or less).
• the one or more neutral lipids can be present in the lipid nanoparticle in an amount ranging from any of the minimum values described above to any of the maximum values described above.
• the one or more neutral lipids are present in the lipid nanoparticle in an amount of from 30 mol% to 65 mol%, (e.g., from 35 mol% to 60 mol %, from 40 mol% to 55 mol %, or from 45 mol% to 50 mol %).
• Suitable PEGylated lipids can include, but are not limited to, a PEG-ditetradecylacetamide, a PEG-myristoyl diglyceride, a PEG-diacylglycerol, a PEG dialkyloxypropyl, a PEG-phospholipid, a PEG- ceramide, PEG-DMG, PEG-DSPE, or any combinations thereof.
• the one or more PEGylated lipids comprise 1,2-dimyristoyl-sn-glycerol (DMG-PEG).
• the lipid nanoparticle can include 2.5 mol% to 15 mol% one or more PEGylated lipids.
• the one or more PEGylated lipids can be present in the lipid nanoparticle in an amount of at least 2.5 mol%, (e.g, at least 5 mol%, at least 7.5 mol%, at least 10 mol%, or at least 12.5 mol%).
• the one or more PEGylated lipids can be present in the lipid nanoparticle in an amount of 15 mol% or less, (e.g., 12.5 mol% or less, 10 mol% or less, 7.5 mol% or less, or 5 mol% or less).
• the one or more PEGylated lipids can be present in the lipid nanoparticle in an amount ranging from any of the minimum values described above to any of the maximum values described above.
• the one or more PEGylated lipids can be present in the lipid nanoparticle in an amount of from 2.5 mol% to 15 mol%, (e.g., from 2.5 mol% to 10 mol%, from 2.5 mol% to 7.5 mol%, from 2.5 mol% to 5 mol%, from 5 mol% to 15 mol%, from 5 mol% to 10 mol%, from 5 mol% to 7.5 mol%, from 7.5 mol% to 10 mol%, from 7.5 mol% to 15 mol%, from 7.5 mol% to 12.5 mol%, from 10 mol% to 12.5 mol%, from 10 mol% to 15 mol%, or from 12.5 mol% to 15 mol%).
• 2.5 mol% to 15 mol% e.g., from 2.5 mol% to 10 mol%, from 2.5 mol% to 7.5 mol%, from 2.5 mol% to 5 mol%, from 5 mol% to 15 mol%, from 5 mol% to 10
• Suitable ionizable lipids can include, but are not limited to, N,N-dimethyl-2,3- dioleyloxypropylamine (DODMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8- ⁇ (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino ⁇ octanoate (SM-102), DLin-MC3-DMA, DLin-KC2-DMA, DLinDMA and the like as disclosed in International Publication No.
• DODMA N,N-dimethyl-2,3- dioleyloxypropylamine
• ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(
• the one or more ionizable lipids comprise N,N-dimethyl- 2,3-dioleyloxypropylamine (DODMA).
• the lipid nanoparticle can include 30 mol% to 50 mol% one or more ionizable lipids.
• the one or more ionizable lipids can be present in the lipid nanoparticle in an amount of at least 30 mol%, (e.g., at least 35 mol%, at least 40 mol%, or at least 45 mol%). In some embodiments, the one or more ionizable lipids can be present in the lipid nanoparticle in an amount of 50 mol% or less, (e.g., 45 mol% or less, 40 mol% or less, or 35 mol% or less). The one or more ionizable lipids can be present in the lipid nanoparticle in an amount ranging from any of the minimum values described above to any of the maximum values described above.
• the one or more ionizable lipids can be present in the lipid nanoparticle in an amount of from 30 mol% to 50 mol%, (e.g, from 30 mol% to 45 mol%, from 30 mol% to 40 mol%, from 30 mol% to 35 mol%, from 35 mol% to 50 mol%, from 35 mol% to 45 mol%, from 35 mol% to 40 mol%, from 40 mol% to 50 mol%, from 40 mol% to 45 mol%, or from 45 mol% to 50 mol%).
• the lipid nanoparticle comprises DOTAP, DODMA, DOPC, cholesterol, and DMG-PEG.
• the DOTAP, DODMA, DOPC, cholesterol, and DMG-PEG can be present at a molar ratio of DOTAP:DODMA:DOPC:cholesterol:DMG-PEG of 5:40:25:20:10, 5:40:27.5:20:7.5, or 5:40:30:20:5.
• the DOTAP, DODMA, DOPC, cholesterol, and DMG-PEG can be present at a molar ratio of DOTAP:DODMA:DOPC:cholesterol:DMG-PEG of 5:40:27.5:20:7.5.
• the lipid nanoparticle and the active agent can be present at a weight ratio of lipid nanoparticle to active agent of from 5:1 to 20:1, from 7.5:1 to 15:1, from 7.5:1 to 10:1, from 7.5:1 to 12:1, from 10:1 to 12:1, from 10:1 to 15:1, or from 12:1 to 15:1. In some embodiments, the lipid nanoparticle and the active agent are present at a weight ratio of lipid nanoparticle to active agent of 15:1, 12:1, 10:1, or 7.5:1.
• the population of the lipid nanoparticles can have an average particle size, as determined by dynamic light scattering, of about 55 nm.
• the composition can include a population of the lipid nanoparticles having an average zeta potential of at least -0.6 mV (e.g, at least -0.1 mV, at least 0.5 mV, at least 1 mV, at least 1.5 mV, or at least 2 mV).
• carrier encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
• excipients include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
• General considerations in formulation and/or manufacture can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.
• excipients include, but are not limited to, any non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• materials which can serve as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium Attorney Docket No.11650-003WO1 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;
• the excipients may be chosen based on what the composition is useful for.
• the choice of the excipient will depend on the route of administration, the agent being delivered, time course of delivery of the agent, etc., and can be administered to humans and/or to animals, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), buccally, or as an oral or nasal spray.
• the active compounds disclosed herein are administered topically.
• Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.
• Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross- linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.
• cross-linked poly(vinyl-pyrrolidone) crospovidone
• sodium carboxymethyl starch sodium starch glycolate
• Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
• stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol Attorney Docket No.11650-003WO1 distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
• Cremophor polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
• Exemplary binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g.
• natural and synthetic gums e.g. acacia, sodium alginate, extract of Irish moss, pan
• Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
• Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
• Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.
• EDTA ethylenediaminetetraacetic acid
• salts and hydrates thereof e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like
• citric acid and salts and hydrates thereof e.g., citric acid mono
• Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
• Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
• Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
• Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
• preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.
• the preservative is an anti-oxidant.
• the preservative is a chelating agent.
• buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyr
• Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
• Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasqua
• Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.
• Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
• the liquid composition may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
• the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
• injectable compositions for example, injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation may also be an injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
• acceptable vehicles and solvents for pharmaceutical or cosmetic compositions that may be employed are water, Ringer's solution, U.S.P.
• the particles are suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween 80.
• the injectable composition can be sterilized, for example, by filtration through a bacteria-retaining filter, or by Attorney Docket No.11650-003WO1 incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
• Solid compositions include capsules, tablets, pills, powders, and granules.
• the particles are mixed with at least one excipient and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate,
• the dosage form may also comprise buffering agents.
• Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• Tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
• compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• Compositions for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active compound is admixed with an excipient and any needed preservatives or buffers as may be required.
• the ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.
• Excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.
• Attorney Docket No.11650-003WO1 Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances.
• Sprays can additionally contain customary propellants such as chlorofluorohydro
• Transdermal patches have the added advantage of providing controlled delivery of a compound to the body.
• dosage forms can be made by dissolving or dispensing the nanoparticles in a proper medium.
• Absorption enhancers can also be used to increase the flux of the compound across the skin.
• the rate can be controlled by either providing a rate controlling membrane or by dispersing the particles in a polymer matrix or gel.
• Method of Use Described herein are also methods for treating cancer, preventing cancer, preventing cancer metastasis, preventing cancer recurrence or preventing angiogenesis.
• the methods can include administering to a subject in need thereof the pharmaceutical composition described herein.
• the cancer can be hepatocellular carcinoma.
• compositions as used in the methods described herein can be administered by any suitable method and technique presently or prospectively known to those skilled in the art.
• the active components described herein can be formulated in a physiologically- or pharmaceutically- acceptable form and administered by any suitable route known in the art including, for example, oral and parenteral routes of administering.
• parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection.
• the active agent may be administered by any route.
• the active ingredient is administered via a variety of routes, including oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
• routes including oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, enteral, sublingual; by intratracheal instillation, bronchial instillation
• the most appropriate route of administration will depend upon a variety of factors including the nature of the active ingredient (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.
• Administration of the active components of their compositions can be a single administration, or at continuous and distinct intervals as can be readily determined by a person skilled in the art. In certain embodiments, it may be desirable to provide continuous delivery of one or more compounds to a patient in need thereof. For intravenous or intraarterial routes, this can be Attorney Docket No.11650-003WO1 accomplished using drip systems, such as by intravenous administration.
• the active ingredient may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result.
• the exact amount of the active ingredient will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular active ingredient, its mode of administration, its mode of activity, and the like.
• the active ingredient, whether the active compound itself, or the active compound in combination with an agent, is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the active ingredient will be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
• the exact amount of an active ingredient required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like.
• the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
• Useful dosages of the compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
• the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected.
• the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
• the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
• the dosage can be adjusted by the individual physician Attorney Docket No.11650-003WO1 in the event of any counterindications.
• Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
• the compositions described herein can be administered in conjunction with additional active agent or therapy.
• Active Agents refers to therapeutic agents, diagnostic agents, or prophylactic agents.
• the therapeutic agents can be released from the disclosed compounds, compositions, and systems in a biologically active form.
• therapeutic agents refers to one or more therapeutic agents, active ingredients, or substances that can be used to treat a medical condition.
• Therapeutic agent includes any synthetic or naturally occurring biologically active compound or composition of matter which, when administered to an organism (human or nonhuman animal), induces a desired pharmacologic, immunogenic, and/or physiologic effect by local and/or systemic action.
• the term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like.
• therapeutic agents are described in well-known literature references such as the Merck Index (14th edition), the Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and they include, without limitation, medicaments; vitamins and minerals such as essential amino acids, calcium, iron, potassium, zinc, vitamin B12, and the like; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.
• the term “therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; an antimicrobial agents (including antibiotics, antiviral agents, antiparasitic, and anti-fungal agents), anti-inflammatory agents (including steroids and non-steroidal anti-inflammatory agents), anti-coagulant agents, ophthalmic agents, gastrointestinal drugs, antiplatelet agents, and antiseptic agents, steroidal agent, anti-neoplastic agent, anti-cancer agent, antigen, antibody, birth control agent, progestational agent, anti-cholinergic, nutritional agent, analgesics and analgesic combinations such as acetaminophen, acetylsalicylic acid, and the like; anesthetics such as lidocaine, xylocaine, and the like, anorexics such as dexadrine, phendimetrazine tartrate, and the like; anti-epileptics, local and general anesthetics, hypermal
• the agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas.
• the agent to be delivered may be a mixture of active agents.
• antibiotics include amikacin, amoxicillin, ampicillin, atovaquone, azithromycin, aztreonam, bacitracin, carbenicillin, cefadroxil, cefazolin, cefdinir, cefditoren, cefepime, cefiderocol, cefoperazone, cefotetan, cefoxitin, cefotaxime, cefpodoxime, cefprozil, ceftaroline, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, chloramphenicol, colistimethate, cefuroxime, cephalexin, cephradine, cilastatin, cinoxacin, cip
• antiviral agents include, but are not limited to, abacavir, acyclovir, adefovir, amantadine, amprenavir, atazanavir, balavir, baloxavir marboxil, boceprevir, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docasanol, dolutegravir, doravirine, ecoliever, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, forscarnet, fosnonet, famciclovir, favipravir, fomivirsen, foscavir, ganciclovir, ibacitabine, idoxuridine, indinavir, in
• anticoagulant agents include, but are not limited to, heparin, warfarin, rivaroxaban, dabigatran, apixaban, edoxaban, enoxaparin, and fondaparinux.
• Attorney Docket No.11650-003WO1 Representative examples of antiplatelet agents include, but are not limited to, clopidogrel, ticagrelor, prasugrel, dipyridamole, dipyridamole/aspirin, ticlopidine, and eptifibatide.
• antifungal agents include, but are not limited to, voriconazole, itraconazole, posaconazole, fluconazole, ketoconazole, clotrimazole, isavuconazonium, miconazole, caspofungin, anidulafungin, micafungin, griseofulvin, terbinafine, flucytosine, terbinafine, nystatin, and amphotericin b.
• steroidal anti-inflammatory agents include, but are not limited to, hydrocortisone, dexamethasone, prednisolone, prednisone, triamcinolone, methylprednisolone, budesonide, betamethasone, cortisone, and deflazacort.
• non-steroidal anti-inflammatory drugs include ibuprofen, naproxen, ketoprofen, tolmetin, etodolac, fenoprofen, flurbiprofen, diclofenac, piroxicam, indomethacin, sulindax, meloxicam, nabumetone, oxaprozin, mefenamic acid, and diflunisal.
• active agents include chloroquine, hydrochloroquine, Pyridoxal phosphate, Vitamin D, and Vitamin C.
• anticytokine or immunomodulatory agents include, but are not limited to, tocilizumab, sarilumab, bevacizumab, fingolimod, imiquimod, and eculizumab.
• contraceptives include, but are not limited to, progestins, estrogens, or any combination thereof.
• suitable progestins include, but are not limited to, natural and synthetic compounds having progestational activity, such as, for example, progesterone, chlormadinone acetate, norethindrone, cyproterone acetate, norethindrone acetate, desogestrel, levonorgestrel, drospirenone, trimegestone, norgestrel, norgestimate, norelgestromin, etonogestrel, gestodene, and other natural and/or synthetic gestagens.
• progesterone chlormadinone acetate, norethindrone, cyproterone acetate, norethindrone acetate, desogestrel, levonorgestrel, drospirenone, trimegestone, norgestrel, norgestimate, norelgestromin, etonogestrel, gestodene, and other natural and/or synthetic gestagens.
• suitable estrogens include, but are not limited to, natural and synthetic compounds having estrogenic activity, such as, for example, estradiol (17 ⁇ -estradiol), 17 ⁇ -estradiol, estriol, estrone, and their esters, such as the acetate, sulfate, valerate or benzoate esters of these compounds, including, for example, estradiol 17 ⁇ -cypionate, estradiol 17- propionate, estradiol 3-benzoate, and piperazine estrone sulfate; ethinyl estradiol; conjugated estrogens (natural and synthetic); mestranol; agonistic anti-estrogens; and selective estrogen receptor modulators.
• estradiol 17 ⁇ -estradiol
• 17 ⁇ -estradiol estriol
• estrone and their esters
• esters such as the acetate, sulfate, valerate or benzoate esters of these compounds, including, for example, estradiol 17 ⁇ -cypion
• contraceptives include gonodotropin releasing hormone (GnRh) or anologs thereof such as deslorelin, avorelin, leuprolide, triptorelin, nafarelin, goserelin, buserelin, and fertirelin.
• GnRh gonodotropin releasing hormone
• steroid refers to compounds belonging to or related to the following illustrative families of compounds: corticosteroids, mineralicosteroids, and sex steroids (including, for example, Attorney Docket No.11650-003WO1 potentially androgenic or estrogenic or anti-androgenic and anti- estrogenic molecules).
• the therapeutic agent may comprise a steroid.
• exemplary cancer drugs or anti-cancer agents can include, but are not limited to, antimetabolite anti- cancer agents and antimitotic anti-cancer agents, and combinations thereof.
• antimetabolite and antimitotic anti-cancer agents including single such agents or combinations of such agents, may be employed in the methods and compositions described herein.
• Antimetabolic anti-cancer agents typically structurally resemble natural metabolites, which are involved in normal metabolic processes of cancer cells such as the synthesis of nucleic acids and proteins.
• the antimetabolites differ enough from the natural metabolites such that they interfere with the metabolic processes of cancer cells.
• antimetabolites are mistaken for the metabolites they resemble, and are processed by the cell in a manner analogous to the normal compounds.
• the presence of the “decoy” metabolites prevents the cells from carrying out vital functions and the cells are unable to grow and survive.
• antimetabolites may exert cytotoxic activity by substituting these fraudulent nucleotides into cellular DNA, thereby disrupting cellular division, or by inhibition of critical cellular enzymes, which prevents replication of DNA.
• the antimetabolite anti-cancer agent is a nucleotide or a nucleotide analog.
• the antimetabolite agent may comprise purine (e.g., guanine or adenosine) or analogs thereof, or pyrimidine (cytidine or thymidine) or analogs thereof, with or without an attached sugar moiety.
• Suitable antimetabolite anti-cancer agents for use in the present disclosure may be generally classified according to the metabolic process they affect, and can include, but are not limited to, analogues and derivatives of folic acid, pyrimidines, purines, and cytidine.
• the antimetabolite agent(s) is selected from the group consisting of cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, and combinations thereof.
• the antimetabolite agent is a cytidine analog.
• the cytidine analog may be selected from the group consisting of cytarabine (cytosine arabinodside), azacitidine (5-azacytidine), and salts, analogs, and derivatives thereof.
• the antimetabolite agent is a folic acid analog.
• Folic acid analogs or antifolates generally function by inhibiting dihydrofolate reductase (DHFR), an enzyme involved in the formation of nucleotides; when this enzyme is blocked, nucleotides are not formed, Attorney Docket No.11650-003WO1 disrupting DNA replication and cell division.
• the folic acid analog may be selected from the group consisting of denopterin, methotrexate (amethopterin), pemetrexed, pteropterin, raltitrexed, trimetrexate, and salts, analogs, and derivatives thereof.
• the antimetabolite agent is a purine analog.
• Purine- based antimetabolite agents function by inhibiting DNA synthesis, for example, by interfering with the production of purine containing nucleotides, adenine and guanine which halts DNA synthesis and thereby cell division.
• Purine analogs can also be incorporated into the DNA molecule itself during DNA synthesis, which can interfere with cell division.
• the purine analog may be selected from the group consisting of acyclovir, allopurinol, 2-aminoadenosine, arabinosyl adenine (ara-A), azacitidine, azathiprine, 8-aza-adenosine, 8-fluoro-adenosine, 8-methoxy- adenosine, 8-oxo-adenosine, cladribine, deoxycoformycin, fludarabine, gancylovir, 8-aza-guanosine, 8- fluoro-guanosine, 8- methoxy-guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-beta- L-2-aminofucose, guanosine diphosphate-D-arabinose, guanosine diphosphate-2- fluorofucose, guanosine diphosphate, guanos
• the antimetabolite agent is a pyrimidine analog. Similar to the purine analogs discussed above, pyrimidine-based antimetabolite agents block the synthesis of pyrimidine-containing nucleotides (cytosine and thymine in DNA; cytosine and uracil in RNA). By acting as “decoys,” the pyrimidine-based compounds can prevent the production of nucleotides, and/or can be incorporated into a growing DNA chain and lead to its termination.
• the pyrimidine analog may be selected from the group consisting of ancitabine, azacitidine, 6-azauridine, bromouracil (e.g., 5-bromouracil), capecitabine, carmofur, chlorouracil (e.g.5-chlorouracil), cytarabine (cytosine arabinoside), cytosine, dideoxyuridine, 3′-azido-3′- deoxythymidine, 3′- dideoxycytidin-2′-ene, 3′-deoxy-3′-deoxythymidin-2′-ene, dihydrouracil, doxifluridine, enocitabine, floxuridine, 5-fluorocytosine, 2-fluorodeoxycytidine, 3-fluoro-3′- deoxythymidine, fluorouracil (e.g., 5-fluorouracil (also known as 5-FU), gemcitabine, 5-methylcytosine, 5-fluorouracil (also
• the pyrimidine analog is other than 5- fluorouracil.
• the pyrimidine analog is gemcitabine or a salt thereof.
• the antimetabolite agent is selected from the group consisting of 5- fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof.
• the Attorney Docket No.11650-003WO1 antimetabolite agent is selected from the group consisting of capecitabine, 6- mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof.
• the antimetabolite agent is other than 5-fluorouracil.
• the antimetabolite agent is gemcitabine or a salt or thereof (e.g., gemcitabine HCl (Gemzar®)).
• antimetabolite anti-cancer agents may be selected from, but are not limited to, the group consisting of acanthifolic acid, aminothiadiazole, brequinar sodium, Ciba-Geigy CGP-30694, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, Wellcome EHNA, Merck & Co.
• EX-015 benzrabine, fludarabine phosphate, N-(2′-furanidyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5- FU-fibrinogen, isopropyl pyrrolizine, Lilly LY-188011; Lilly LY-264618, methobenzaprim, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner- Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, tiazofurin, Erbamont TIF, tyrosine kinase inhibitors, Taiho UFT and uricytin, among others.
• the antimitotic agent is a microtubule inhibitor or a microtubule stabilizer.
• microtubule stabilizers such as taxanes and epothilones, bind to the interior surface of the beta-microtubule chain and enhance microtubule assembly by promoting the nucleation and elongation phases of the polymerization reaction and by reducing the critical tubulin subunit concentration required for microtubules to assemble.
• the microtubule stabilizers such as taxanes, decrease the lag time and dramatically shift the dynamic equilibrium between tubulin dimers and microtubule polymers towards polymerization.
• the microtubule stabilizer is a taxane or an epothilone.
• the microtubule inhibitor is a vinca alkaloid.
• the therapeutic agent may comprise a taxane or derivative or analog thereof.
• the taxane may be a naturally derived compound or a related form, or may be a chemically synthesized compound or a derivative thereof, with antineoplastic properties.
• the taxanes are a family of terpenes, including, but not limited to paclitaxel (Taxol®) and docetaxel (Taxotere®), which are derived primarily from the Pacific yew tree, Taxus brevifolia, and which have activity against certain tumors, particularly breast and ovarian tumors.
• the taxane is docetaxel or paclitaxel.
• Paclitaxel is a preferred taxane and is considered an antimitotic agent that promotes the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization.
• Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Pat.
• the taxane may also be a taxane conjugate such as, for example, paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, docetaxel-PEG, docetaxel- dextran, docetaxel- xylose, and the like.
• Other derivatives are mentioned in “Synthesis and Anticancer Activity of Taxol Derivatives,” D. G. I. Scientific et al., Studies in Organic Chemistry, vol.26, entitled “New Trends in Natural Products Chemistry” (1986), Atta-ur-Rabman, P. W. le Quesne, Eds. (Elsevier, Amsterdam 1986), among other references.
• the antimitotic agent can be a microtubule inhibitor; in one preferred aspect, the microtubule inhibitor is a vinca alkaloid.
• the vinca alkaloids are mitotic spindle poisons.
• the vinca alkaloid agents act during mitosis when chromosomes are split and begin to migrate along the tubules of the mitosis spindle towards one of its poles, prior to cell separation. Under the action of these spindle poisons, the spindle becomes disorganized by the dispersion of chromosomes during mitosis, affecting cellular reproduction.
• the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, and salts, analogs, and derivatives thereof.
• the antimitotic agent can also be an epothilone.
• members of the epothilone class of compounds stabilize microtubule function according to mechanisms similar to those of the taxanes.
• Attorney Docket No.11650-003WO1 Epothilones can also cause cell cycle arrest at the G2-M transition phase, leading to cytotoxicity and eventually apoptosis.
• Suitable epithiolones include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, and salts, analogs, and derivatives thereof.
• One particular epothilone analog is an epothilone B analog, ixabepilone (IxempraTM).
• the antimitotic anti-cancer agent is selected from the group consisting of taxanes, epothilones, vinca alkaloids, and salts and combinations thereof.
• the antimitotic agent is a taxane.
• the antimitotic agent is paclitaxel or docetaxel, still more preferably paclitaxel.
• the antimitotic agent is an epothilone (e.g., an epothilone B analog).
• the antimitotic agent is a vinca alkaloid.
• cancer drugs examples include, but are not limited to: thalidomide; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin; anthracenediones such as mitoxantrone; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N- methylhydrazine, MIH); adrenocortical suppressants such as mitotane (o,p′-DDD) and aminoglutethimide; RXR agonists such as bexarotene; and tyrosine kinase inhibitors such as sunitimib, imatinib, axitinib, dasatinib, erlotinib, nilotinib, and pazopanib.
• platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin
• alkylating agents examples include nitrogen mustards such as mechlorethamine, cyclophosphainide, ifosfamide, melphalan sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU) and streptozocin (streptozotocin); DNA synthesis antagonists such as estramustine phosphate; and triazines such as dacarbazine (DTIC, dimethyl-triazenoimidazolecarboxamide) and temozolomide.
• alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphainide, ifosfamide, melphalan sarcolysin) and chlorambuci
• antimetabolites include folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracin (5-fluorouracil, 5-FU, SFU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside) and gemcitabine; purine analogs such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG) and pentostatin (2′-deoxycoformycin, deoxycoformycin), cladribine and fludarabine; and topoisomerase inhibitors such as amsacrine.
• folic acid analogs such as methotrexate (amethopterin)
• pyrimidine analogs such as fluorouracin (5-fluorouracil, 5-FU, SFU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytos
• Examples of natural products include vinca alkaloids such as vinblastine (VLB) and vincristine; taxanes such as paclitaxel, protein bound paclitaxel (Abraxane) and docetaxel (Taxotere); epipodophyllotoxins such as etoposide and teniposide; camptothecins such as topotecan and irinotecan; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, histrelin, bleomycin, mitomycin (mitomycin C), Attorney Docket No.11650-003WO1 idarubicin, epirubicin; enzymes such as L-asparaginase; and biological response modifiers such as interferon alpha and interlelukin 2.
• VLB vinblastine
• vincristine taxanes
• paclitaxel protein bound paclitaxel
• hormones and antagonists include luteinising releasing hormone agonists such as buserelin; adrenocorticosteroids such as prednisone and related preparations; progestins such as hydroxyprogesterone caproate, rnedroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol and related preparations; estrogen antagonists such as tamoxifen and anastrozole; androgens such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs such as leuprolide.
• releasing hormone agonists such as buserelin
• adrenocorticosteroids such as prednisone and related preparations
• progestins such as hydroxyprogesterone caproate, rnedroxyprogesterone a
• the anti-cancer agent may comprise a chemotherapeutic agent.
• Suitable chemotherapeutic agents include, but are not limited to, alkylating agents, antibiotic agents, antimetabolic agents, hormonal agents, plant-derived agents and their synthetic derivatives, anti- angiogenic agents, differentiation inducing agents, cell growth arrest inducing agents, apoptosis inducing agents, cytotoxic agents, agents affecting cell bioenergetics i.e., affecting cellular ATP levels and molecules/activities regulating these levels, biologic agents, e.g., monoclonal antibodies, kinase inhibitors and inhibitors of growth factors and their receptors, gene therapy agents, cell therapy, e.g., stem cells, or any combination thereof.
• alkylating agents include, but are not limited to, alkylating agents, antibiotic agents, antimetabolic agents, hormonal agents, plant-derived agents and their synthetic derivatives, anti- angiogenic agents, differentiation inducing agents, cell growth arrest inducing agents, apoptosis inducing agents, cytotoxic agents, agents affecting cell bioenergeti
• the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, chlorambucil, melphalan, mechlorethamine, ifosfamide, busulfan, lomustine, streptozocin, temozolomide, dacarbazine, cisplatin, carboplatin, oxaliplatin, procarbazine, uramustine, methotrexate, pemetrexed, fludarabine, cytarabine, fluorouracil, floxuridine, gemcitabine, capecitabine, vinblastine, vincristine, vinorelbine, etoposide, paclitaxel, docetaxel, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, mitomycin, hydroxyurea, topotecan, irinotecan, amsacrine, tenipos
• the therapeutic agent may comprise a biologic drug, particularly an antibody.
• the antibody is selected from the group consisting of cetuximab, anti-CD24 antibody, panitumumab and bevacizumab.
• Growth factors useful as therapeutic agents include, but are not limited to, transforming growth factor- ⁇ (“TGF- ⁇ ”), transforming growth factors (“TGF- ⁇ ”), platelet-derived growth factors (“PDGF”), fibroblast growth factors (“FGF”), including FGF acidic isoforms 1 and 2, FGF basic form 2 and FGF 4, 8, 9 Attorney Docket No.11650-003WO1 and 10, nerve growth factors (“NGF”) including NGF 2.5s, NGF 7.0s and beta NGF and neurotrophins, brain derived neurotrophic factor, cartilage derived factor, bone growth factors (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (G-CSF), insulin like growth factor (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factors (TGF), including TGFs alpha, beta,
• Vascular endothelial growth factor (VEGF) inhibitors useful as therapeutic agents include, but are not limited to, sunitinib, pazopanib, sorafenib, tivozanib, cabozantinib, bevacizumab, aflibercept, ranibizumab, dasatinib, and nilotinib.
• Cytokines useful as therapeutic agents include, but are not limited to, cardiotrophin, stromal cell derived factor, macrophage derived chemokine (MDC), melanoma growth stimulatory activity (MGSA), macrophage inflammatory proteins 1 alpha (MIP-1alpha), 2, 3 alpha, 3 beta, 4 and 5, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TNF- ⁇ , and TNF- ⁇ .
• Immunoglobulins useful in the present disclosure include, but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof.
• VEGF vascular endothelial growth factor
• NGFs nerve growth factors
• PDGF-AA vascular endothelial growth factor
• PDGF-BB PDGF-BB
• PDGF-AB vascular endothelial growth factor
• FGFb FGFa
• BGF BGF
• Other molecules useful as therapeutic agents include but are not limited to growth hormones, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor alpha and beta, endostatin, thrombospondin, osteogenic protein-1, bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, osteocalcin, , interferon alpha, interferon alpha A, interferon beta, interferon gamma, interferon 1 alpha, and interleukins 2, 3, 4, 56, 7, 8, 9, 10, 11, 12,13, 15, 16, 17 and 18.
• Diagnostic agents include gases; metals; commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.
• PET positron emissions tomography
• CAT computer assisted tomography
• MRI magnetic resonance imaging
• suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium.
• Examples of materials useful for CAT and x-ray imaging include iodine-based materials.
• Therapeutic and prophylactic agents include, but are not limited to, antibiotics, nutritional supplements, and vaccines.
• Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, cell extracts, and RNA encoding at least one antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic Attorney Docket No.11650-003WO1 fragment capable of inducing an immune response to the antigenic polypeptide).
• Therapeutic and prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc.
• Prophylactic agents can include infection agents such as antigens of such bacterial organisms as Streptococccus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallid
• antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof. Also described herein are methods of inducing cytotoxicity in a cancer cell including contacting the cell with a pharmaceutical composition described herein.
• Also described herein are methods of producing a population of lipid nanoparticles encapsulating an active agent comprising (a) combining one or more ethanolic solutions comprising a mixture of lipids with an aqueous solution and acidifying to induce formation of a population of empty lipid nanoparticles; (b) contacting the population of empty lipid nanoparticles with an aqueous solution comprising the active agent, thereby encapsulating the active agent in the population of empty lipid nanoparticles to produce the population of lipid nanoparticles encapsulating the active agent; and (c) subjecting the population of lipid nanoparticles encapsulating the active agent to tangential flow Attorney Docket No.11650-003WO1 filtration to replace buffer and remove residual ethanol.
• the mixture of lipids or lipid nanoparticle comprises 2.5 mol% to 15 mol% one or more cationic lipids; 30 mol% to 50 mol% one or more ionizable lipids; 30 mol% to 65 mol% one or more neutral lipids; and 2.5 mol% to 15 mol% one or more PEGylated lipids.
• the active agent comprises RX-0201, 5′ gctgcatgatctccttggcg 3′, SEQ. ID. NO.1.
• the RX-0201 is an antisense oligonucleotide.
• the RX-0201 has at least one modified internucleoside linkage that is a phosphorothioate linkage.
• the lipid nanoparticles comprise DOTAP, DODMA, DOPC, cholesterol, and DMG-PEG.
• the DOTAP, DODMA, DOPC, cholesterol, and DMG-PEG in the lipid nanoparticles are present at a molar ratio of DOTAP:DODMA:DOPC:cholesterol:DMG-PEG of 5:40:25:20:10, a molar ratio of DOTAP:DODMA:DOPC:cholesterol:DMG-PEG of 5:40:27.5:20:7.5, or a molar ratio of DOTAP:DODMA:DOPC:cholesterol:DMG-PEG of 5:40:30:20:5.
• the lipid nanoparticle and the active agent are present at a weight ratio of lipid nanoparticle to active agent of from 5:1 to 20:1, from 7.5:1 to 15:1, from 7.5:1 to 10:1, from 7.5:1 to 12:1, from 10:1 to 12:1, from 10:1 to 15:1, or from 12:1 to 15:1.
• the population of the lipid nanoparticles has an average particle size, as determined by dynamic light scattering, of from 50 nm to 80 nm, from 55 nm to 75nm, or from 55 nm to 60 nm.
• the population of the lipid nanoparticles has an average zeta potential of from -06 mV to 2.5 mV. In some embodiments, the population of the lipid nanoparticles has a polydispersity index (PDI) of from 0.15 to 0.5, from 0.15 to 0.4, from 0.15 to 0.3, from 0.15 to 0.2, from 0.2 to 0.3, from 0.2 to 0.5, from 0.2 to 0.4, from 0.2 to 0.3, from 0.3 to 0.4, from 0.3 to 0.5, or from 0.4 to 0.5.
• PDI polydispersity index
• Example 1 The anti-tumor and anti-angiogenesis efficacy of a lipid nanoparticle suspension of an AKT-1 anti-sense oligonucleotide AKT-1 plays a critical role in cancer progression by promoting cell proliferation and inhibiting apoptosis (see Revathidevi S, et al., Semin Cancer Biol.2019;59:80-91; and Uko NE, et al., Curr Top Med Chem.2020;20(10):883-900). Inhibition of AKT has been shown to inhibit tumor growth and angiogenesis (see Nitulescu GM, et al., Int J Oncol.2016;48(3):869-85).
• Archexin is a fully phosphorothioated 20-mer antisense oligonucleotide, which can specifically bind to AKT-1 mRNA Attorney Docket No.11650-003WO1 resulting in RNase H-based AKT-1 downregulation.
• WGI-0301 is a proprietary lipid nanoparticle (LNP) formulation of Archexin designed for enhanced delivery.
• LNP lipid nanoparticle
• a phase I clinical study has recently been initiated for WGI-0301 in solid tumors.
• Anti-tumor efficacy of WGI-0301 was studied in vivo in a Hepa1-6 syngeneic murine tumor model for hepatocellular carcinoma (HCC). The mice were injected i.v.
• WGI-0301 was investigated in vitro using human umbilical vein endothelial cells (HUVEC) (see DeCicco-Skinner KL, et al., J Vis Exp.2014 Sep 1;(91):e51312; and Arnaoutova I, et al., Nat Protoc.2010;5(4):628-35).
• HUVEC human umbilical vein endothelial cells
• Three experimental groups were set up, including WGI-0301(0, 0.2, 2 and 20 ⁇ M), WGI-0301+ 2 ⁇ M Sorafenib, and WGI-0301+ 5 ⁇ M Lenvatinib.
• the cells were plated at 1.5 ⁇ 10 4 per well.
• the cells were treated with 50% Matrigel and then cultured in the 24 well plate in triplicates and were incubated for an additional 6 hours.
• the in vivo study demonstrated anti-tumor efficacy of WGI-0301 in the Hepa1-6 model.
• the tumor growth inhibition % (TGI) for WGI-0301 was 46.16%, and the medium survival time (MST) of the treatment group was 55 days, which was significantly different from that of the control group (37 days).
• WGI-0301 The dose-dependent inhibitory effect of WGI-0301 on angiogenesis was observed both as a monotherapy and in combination with other agents, particularly with 5 ⁇ M Lenvatinib, indicating that angiogenesis inhibition may be a crucial mechanism of its antitumor activity. Furthermore, the combination therapy of WGI-0301 and Lenvatinib demonstrated high effectiveness. The study results showed that WGI-0301 had significant anti-tumor and anti-angiogenic efficacy in the Hepa1-6 syngeneic murine tumor model for HCC. Moreover, combining WGI-0301 with Sorafenib or Lenvatinib resulted in even greater anti-angiogenic activity compared to WGI-0301 monotherapy.
• WGI-0301 as a selective AKT-1 inhibitor.
• Example 2 WGI-0301 lipid nanoparticles Attorney Docket No.11650-003WO1
• the drug substance is the antisense oligonucleotide, Archexin (RX-0201), developed by Rexahn Pharmaceuticals (Rockville, MD).
• the drug product WGI-0301 is manufactured as a lyophilized powder.
• RX-0201 was found to have promising antitumor activity and tolerability.
• RX-0201 also achieved early success in Phase II trials for renal cell carcinoma and pancreatic cancers.
• RX-0201 Due to its poor membrane permeability and in vivo stability, and further demanding administration regimen of 14-day continuous infusion, RX-0201 was limited by the challenges inherent to antisense oligonucleotides.
• Zhejiang Haichang Biotech Co., Ltd. HCBio
• WGI-0301 lipid nanoparticle suspension formulation
• WGI-0301 uses a combination of quaternary and tertiary lipoamines for pH sensitive delivery along with other neutral and stabilizing lipids.
• WGI-0301 is proposed as a promising therapeutic candidate for hepatocellular carcinoma.
• Table 1 Composition of WGI-0301 Amount per Vial Component Components Concentration Function Quality d e e e e e F F F F F F F 1.
• DOTAP as the cationic lipid and the full chemical name is 1,2-dioleoyl-3-trimethylammonium- propane.
• DODMA as the ionizable lipid and the full chemical name is 1,2-dioleyloxy-3-dimethylamino-propane.
• DOPC as the neutral lipid and the full chemical name is 1,2-dioleoyl-sn-glycero-3-phosphocholine. 4.
• DMG-PEG2000 as the PEGylated lipid and the full chemical name is 1,2-Dimyristoyl-rac-glycero-3- methylpolyoxyethylene 2000.
• Attorney Docket No.11650-003WO1 5.
• Sucrose, Ammonium chloride, Acetic acid, Sodium hydroxide and Ethanol will be removed during process preparation.
• LNPs are suspended in a solution of 0.9% sodium chloride. Since aqueous form of RX-0201 cannot be stored stably at 2–8°C, the long-term storage recommendation for WGI-0301 is set at -20°C, with thawing before use.
• WGI-0301 is provided as a 6.573 mg Free Acid of Archexin (which is equivalent to 7.0 mg Archexin/RX-0201) per 7 mL to fulfill clinical need. Based on the established experience of HCBio, different formulations were developed and studied. These formulations were investigated based on the CQAs and efficacies to screen the appropriate formulation and formulation process. Table 2. WGI-0301 Critical Quality Attributes (CQAs) Quality Attributes Target CQA Justification . e . e Attorney Docket No.11650-003WO1 Quality Attributes Target CQA Justification e s, ly s.
• CQAs Critical Quality Attributes
• Permanently ionized lipids (e.g., lipids bearing quaternary ammonium moieties) and conditionally ionizable lipids (e.g., lipids bearing tertiary amine moieties) have been widely used in LNP formulation for the delivery of nucleic acid drugs.
• Quaternary cationic lipids, such as DOTAP-Cl carry a permanent positive charge with the cationic property remained unchanged in different pH conditions.
• the quaternary cationic lipids are widely used in the formulation of LNP delivery systems for gene delivery.
• DODMA ionizable lipids
• DOTAP-Cl quaternary lipoamines
• DODMA conditionally ionizable tertiary lipoamines
• DODMA cationic lipids
• endosomal anionic membrane leading to exocytosis and promoting the escape of oligonucleotide into the cytoplasm.
• commercially available DOTAP-Cl and DODMA were selected as cationic lipids for WGI-0301 product formulation.
• PEGylated lipid and neutral lipids PEGylated lipids are widely used in nanoparticle delivery systems. Cationic lipids interact non- specifically with charged components in the serum through electrostatic interaction and may be recognized and eliminated by the mononuclear phagocytic system easily.
• PEGylated lipids can confer stealth properties to LNPs, reduce non-specific interactions with negatively charged serum components, and suppress the drug uptake by the reticuloendothelial system (RES), thereby prolonging the half-life of the drug in the plasma.
• RES reticuloendothelial system
• PEGylated lipids are able to enhance formulation stability by reducing interaction with nucleases and improving overall colloidal stability.
• the presence of a PEG layer can sterically hinder the interaction between LNPs and cell membranes, result in reduced cell uptake and inhibited interaction of LNPs with the endosomal and lysosomal membranes.
• PEGylated lipids are necessary to improve the formulation stability and circulating time in blood, they need to be rapidly released from LNPs after the nanoparticles reach Attorney Docket No.11650-003WO1 the target organ to increase the cellular internalization and facilitate the endosomal escape. Therefore, the timely release of PEGylated lipids from LNPs is critical. Many studies showed that the duration that PEGylated lipids attached to the LNP were determined by the length of the fatty acid chain of PEGylated lipids. Longer chain PEGylated lipids (e.g. PEG-C20) cannot be easily released from the LNPs due to the stronger intermolecular forces, resulting in LNPs with greater stability and circulation time.
• PEG-C20 Longer chain PEGylated lipids
• shorter chain PEGylated lipids e.g. PEG-C8
• the length of PEGylated lipid chain is independent from the LNP’s ability to accumulate at the tumor site. It is also reported that PEGylated lipids have a strong immune response.
• the clearance rate of LNPs with longer fatty acid chains e.g., PEG-DSPE, PEG-s-DSG
• PEG-s-DMG or PEG-CerC14 was faster compared to the ones with shorter fatty acid chains.
• Formulation 1 DOTAP-Cl, DODMA, DOPC, cholesterol, and DMG-PEG 2000
• Formulation 2 DOTAP-Cl, DODMA, DSPC, cholesterol, and DMG-PEG 2000
• Formulation 3 DOTAP-Cl, DODMA, DOPC, cholesterol, and DSPE-PEG 2000.
• Particle size of API loaded LNPs was used as a formulation screening criterium. The design of the different formulations is shown in the table below. Table 3.
• Formulation Formulation 1 Formulation 2
• Formulation 3 composition Molar acd caton, sucrose as an Osmoarty reguator, NaO as a p reguator, an ydrous et ano as a pd solubilizer, and water for injection as solvent.
• the amount of the aforementioned components is the same in three formulations, so they will not be discussed in this report.
• Table 4 Formulation screening of LNP formulations.
• WGI-0301 is intended to be developed as a potential treatment for hepatocellular carcinoma, so DMG-PEG 2000 was selected as the PEGylated lipid.
• Neutral lipids such as DOPC and DOPE are used as bilayer forming lipids for LNPs due to their low cytotoxicity and immunogenicity.
• the biophysical property of neutral lipids such as DOPC, DOPE, are not related to pH but limits the ability of LNPs to load and deliver negatively charged oligonucleotide drugs. Based on the formulation screening results above, DOPC was chosen as the neutral lipid for our LNP formulation.
• Cholesterol is a neutral lipid used as a regulator of membrane fluidity, which plays a role in lipid self-assembly and stabilization of LNPs.
• DOTAP-Cl and DODMA provide positive charge balance to interact with anionic oligonucleotides.
• DOPC is used as a bilayer forming lipid.
• Cholesterol is a lipid regulator of membrane fluidity, which increases membrane rigidity and provides stabilization.
• DMG-PEG 2000 is selected as a lipid to reduce the off-target uptake and immunogenicity and promote the circulation time.
• Sucrose and sodium Attorney Docket No.11650-003WO1 chloride are osmolarity regulators.
• Ammonium chloride and glacial acetic acid are lipid acidifiers used to protonate DODMA. NaOH is used to adjust pH.
• DOTAP-Cl cationic quaternary lipoamines
• DODMA conditionally ionizable tertiary lipoamines
• DOTAP-Cl also contributes to the structural stability of LNPs under physiological pH conditions: permanently positively charged DOTAP-Cl better interacts with the negative charged oligonucleotides, resulting in nanoparticles with a relatively smaller particle size compared to the nanoparticles only containing tertiary lipoamines.
• DOPC is used as a bilayer forming lipid.
• Cholesterol is a lipid regulator of membrane fluidity, which increases membrane rigidity and provides stabilization.
• DMG-PEG 2000 is selected as a lipid to reduce the off-target uptake and immunogenicity and promote the circulation time.
• Sucrose plays a role as an Osmolarity regulator.
• Ammonium chloride and glacial acetic acid were used as lipid acidifiers to protonate DODMA to interact with the API under low pH conditions. After formation of an electrostatic complex, pH is elevated to physiological pH with NaOH as a pH modifier. The buffer system is then replaced with 0.9% NaCl solution by tangential flow filtration. Determination of lipid molar ratio Several different formulations were screened to determine the optimal ratio of lipid components.
• the particle size and zeta potential of LNPs were examined. The molar ratios of lipid were confirmed by HPLC-ELSD.
• the formulation designs can be found in the table below: Table 5 LNP formulations design Formulation composition Formulation 1 Formulation 2 Formulation 3 Attorney Docket No.11650-003WO1 Note: The formulation also includes the use of ammonium chloride and glacial acetic acid as lipid acidifiers, sucrose as osmolarity regulator, NaOH as pH regulator, anhydrous ethanol, and water for injection as the solvent. The amount of each component is the same in three formulations and they are not discussed in this section of the report.
• WGI- 0301 (12:1) (8mg/kg) treatment group’s MTV was 1027.11 mm 3 , TGI(%) was 50%, and the difference was statistically significant compared with the negative control group (p 0.041).
• the results showed that WGI-0301 (12:1) Attorney Docket No.11650-003WO1 and WGI-0301 (7.5:1) had significant anti-tumor effects on the syngeneic Hepa1-6 model in C57BL/6 mice. Survival Analysis Table 11. Survival Analysis of Each Group.
• WGI-0301 15:1), WGI-0301 (12:1), and WGI- 0301(10:1) are not significantly different than that of RX-0201 control group.
• WGI-0301 (7.5:1) has a significantly higher survival time compared to the RX-0201 control, WGI-0301 (15:1), and WGI-0301 (12:1) groups.
• the lipid:drug ratio selected in this study was 7.5:1.
• Manufacturing process This product is an injectable suspension, the preparation process includes API solution preparation, empty LNPs preparation, drug loading, pH titration, tangential flow filtration, sterile filtration, filling, capping and sealing, packaging, and freezing.
• the ethanol dilution method is used to prepare LNPs.
• Ethanolic lipid solutions containing cationic lipids (DOTAP-Cl, DODMA), bilayer forming lipids (DOPC), PEGylated lipids (DMG-PEG 2000), and cholesterol are combined and acidified to around pH 4.0.
• the empty LNPs and oligonucleotide aqueous solutions are mixed rapidly to prepare the Attorney Docket No.11650-003WO1 LNPs at a 1:1 (v/v) lipid:drug ratio, which also brings the pH value to a physiological range.
• Tangential flow filtration is used to replace buffer and remove ethanol to stabilize the LNP.
• high-throughput and controllable microfluidic technology is used to obtain high-quality, nanosized, stable LNPs.
• the advantage of microfluidic technology in the preparation of LNPs is that the prepared LNPs have higher encapsulation efficiency and smaller particle size, which enables the rapid production of LNPs, from laboratory scale to GMP production scale.
• the microfluidic technology is an in-line, continuous process for the preparation of LNPs.
• One advantage of this technology is that there was no change in formulation composition and proportion, equipment, or process parameters from laboratory scale to lab scale, to pilot scale, and to GMP production scale. In addition, there was no obvious change in the key quality parameters of the drug product, which indicates the stability of the process.
• the manufacturing process of empty LNPs used for nonclinical studies is similar to that of WGI-0301 except using 20% sucrose solution to replace the API solution.
• Figure 4 shows a diagram of the manufacturing process.
• Preparation of 0.9%(w/w) sodium chloride solution weigh 450 g of solidum chloride, dissolve with appropriate amount of water for injection, mix and ultrafiltrate by an ultrafiltration membrane to remove bacterial endotoxin.
• Preparation of 1 M sodium hydroxide (NaOH) solution weigh 20.0 g sodium hydroxide, dissolve with appropriate amount of water for injection.
• Preparation of WGI-0301 lipid nanoparticles Set the water bath temperature of the heated magnetic stirrer (DF-101T; #2) to 40°C. With magnetic stirring, pre-heat 5.25 L RX-0201 solution (in a 5 L screw-top reagent bottle) to 40 °C (acceptable temperature range: 38–42°C). With magnetic stirring, transfer the 5 L screw-top reagent bottle that contains the blank lipid nanoparticle solution from the #3 stirrer (DF-101T; #3) to the #2 stirrer (DF-101T; #2). Set the water bath temperature of the heated magnetic stirrer (DF-101T; #3) to 40°C.
• Pretreat the new hollow fiber column filter rinse with water first, then repeatedly rinse with a 0.2 M NaOH solution for 60 min. After that, rinse with sterile water for injection until the pH of the flushed water becomes neutral.
• the particle size increased after the drug loading and titration steps, especially for the 37.5 mg/mL lipid concentration group, where the particle size increased from 35.87 nm to 65.28 nm, showing its instability.
• the lipid concentration was 75 mg/mL, the final particle size was the smallest and most stable. Therefore, the lipid concentration was selected to be 75 mg/mL.
• a SY03 syringe pump system is used to prepare empty LNPs.
• lipid solution and acidifying solutions pass through a T-connector at a certain flow rate (flow rates are 40 and 160 mL/min, respectively) through a microfluidic channel of a certain size, under laminar flow, the lipid solution and acidifying solution can be fully dispersed rapidly and completely.
• Needle sizes tested include 14G (internal diameter 1.60 mm; length 300 mm), 16G (internal diameter 1.19 mm; length 300 mm), and 18G (internal diameter 0.84 mm; length 300 mm). The particle size distribution was used as the criterion for selection.
• the SY03 syringe pump system is used to prepare empty LNPs.
• the lipid solution and acidifying solution can be fully dispersed rapidly and completely.
• Needle sizes tested include 14G (internal diameter 1.60mm; length 300 mm), 16G (internal diameter 1.19mm; length 300 mm), and 18G (internal diameter 0.84mm; length 300 mm).
• the particle size distribution was used as the criterion for selection. The experimental design and results are shown in the table below. Table 17. Screening of Needle Specifications. Process Batch No.
• the selection of needle diameter was 16G for drug loading via microfluidic method.
• Screening of syringe pump system of empty LNPs and API solutions In the process of drug loading optimization, the SY03 syringe pump system was used to prepare API loaded LNPs.
• the empty LNPs and API solution were combined by a T-connector, at a certain flow rate combination (40 & 160 mL/min) through a microfluidic channel of a certain size under laminar flow, which allowed for rapid and complete mixing.
• a 16G needle was equipped to determine the effect of varying syringe pump speed combination of empty LNPs and API solutions: 160 &160 mL/min, 80 & 80 mL/min, and 40 & 40 mL/min.
• RX-0201 is unstable and impurities may increase. Small quantities of API were dissolved, and the Assay value and impurities were evaluated under different temperature conditions. The experimental design and inspection results are as follows. Table 19. API Stability under Different Temperature. Sample information RX-0201 Related Substances (%) Assay value Attorney Docket No.11650-003WO1 API solution at 25°C for 6 h 7.34 2.864 5.754 1.94 API solution at 25°C for 12 h 7.34 2.844 5.740 1.96 ° . Acid of Archexin, which is the API solution (RX-0201 is soluble in 20% sucrose solution and the dissolution time is very short, so no investigation is necessary).
• the particle size of empty LNPs increased slowly with the incubation time (from 37.23 nm to 46.46 nm), the PDI decreased slowly with the incubation time (from 0.340 to 0.233), and LPC content remained unchanged at 12 h, but increased at 24 h.
• the incubation temperature was 55°C, the particle size of the empty LNPs within 12 h increased slowly corresponding to increased incubation time (from 41.54 nm to 49.75 nm), but suddenly decreased to 41.54 nm at 24 h, indicating that the empty LNPs may be damaged or broken at 24 h.
• Table 23 Screening of TFF Replacement Solution. Batch No. 20051902-3 20051902-4A 20051902-3-4B 6 cycles of TFF using 6 cycles of TFF using FF Attorney Docket No.11650-003WO1 Drug loaded LNPs with physiological pH were buffer exchanged with different buffer systems (0.9% NaCl solution or pH 7.4 PBS solution) using TFF. The results showed that there was no significant difference in the particle size distribution, assay value, encapsulation efficiency, lipids, LPC content, impurities, or residual solvent (ethanol) in different buffer systems. The results of the freeze-thaw experiment showed no significant change in particle size distribution.
• Example 3 In Vivo Efficacy of WGI-0301 in the Liver Cancer Mice Model
• the AKT pathway is an important therapeutic target for cancer drug discovery as it functions as the main point for transducing extracellular and intracellular oncogenic signals. Moreover, alternations of the AKT pathway have been found in a wide range of cancers (Cheng, J.
• Archexin (also named RX-0201) is a fully phosphorothioated 20-mer oligonucleotide complementary to Akt1 mRNA. Archexin is able to specifically target the mRNA sequence of Akt-1 where it causes inhibition of translation and downstream pathway activity of Akt-1 mRNA (Bellacosa, A., et al., Adv Cancer Res, 94, 29-86 (2005), Liang, J., et al., J. M.,Cell Cycle, 2(4), 339-345 (2003), Staal, S. P.
• Akt-1 mRNA levels and AKT-1 protein expression in human tumor cells of brain, breast, cervix, liver, lung, ovary, prostate, and stomach, as well as melanoma were significantly reduced following treatment with Archexin.
• Archexin suppressed cell proliferation of these human cancer cells.
• the growth inhibition by Archexin appeared to be dose-dependent and the concentration causing 50% inhibition (IC50) of cell growth ranged from 2 nM to 50 nM in these human cancer cells.
• Three clinical trials of Archexin were conducted under IND 69763: one Phase I monotherapy trial and two Phase II combination therapy trials.
• WGI-0301 is a lipid nanoparticle formulation of Archexin.
• Nonclinical studies carried out to support the development of WGI-0301 include primary pharmacology studies demonstrating tumor Attorney Docket No.11650-003WO1 inhibition efficacy of WGI-0301 in the liver cancer mice model in vivo, as well as to study the survival prolongation effect.
• WGI-0301 is a reformulated lipid nanoparticle suspension of Archexin. Due to the tissue distribution profile of lipid nanoparticle, liver is the main target of WGI-0301. In the pharmacodynamics study, mice liver cancer models were selected to study the efficacy of WGI-0301. In an in vivo Hepa1-6 liver cancer mice model study (Study No.
• WGI-0301 showed a better tumour inhibition efficacy and significant longer survival time than Archexin.
• the tumour growth inhibition% of WGI-0301 was 46.16%, while that of Archexin at the same dosing regimen was 3.32%, as compared to the vehicle control group.
• the lipid nanoparticle formulation of Archexin, WGI-0301 is expected to achieve promising antitumor and/or survival prolongation effects with a lower dosing frequency and less amount of Archexin.
• Antitumor growth efficacy and survival prolongation of WGI-0301 at a lower dose level, 8 mg/kg (0.65 mg/kg HED), in the liver cancer mice model were demonstrated.
• no test article-related effect on neurobehavioral changes was noted in rats, and no test-article related changes in qualitative and quantitative electrocardiogram evaluation, blood pressure, heart rate, and respiratory parameters, neurological examination parameters were founded.
• the in vitro hERG assay showed little inhibitory effect at concentrations of up to 274.1 ⁇ M Free Acid of Archexin.
• cancer is the first or second leading cause of death before the age of 70 years in 112 of 183 countries and ranks third or fourth in a further 23 countries.
• WHO World Health Organization
• available therapeutic approaches to cancer include surgery, chemotherapy, hormone therapy, immunotherapy, and radiation.
• Each of these therapies has their limitation, such as intolerable toxicities, insufficient efficacy, or not well accessible for the public. Therefore, the need of safe and effective therapies is always increasing.
• Akt protein kinase B or PKB gene family
• Akt-1 protein kinase B gene family
• Akt protein Attorney Docket No.11650-003WO1 products can phosphorylate a range of proteins and thereby control several cellular processes (Staal, 1987) [2] .
• overexpression of constitutively activated Akt-1 gene promotes cellular transformation by two distinct mechanisms. Strong evidence demonstrates that Akt-1 protein plays a very important role in cancer progression process by stimulating cell proliferation and inhibiting apoptosis. Akt-1 appears to promote proliferation under conditions in which cells should normally be growth arrested.
• Akt-1 also prevents apoptosis by inhibiting virtually all cell-death inducing molecules. These mechanisms enable Akt-1 to promote the survival of tumor cells under conditions in which those cells should die.
• overexpression of p-Akt may contribute to the development and progression of malignancies (e.g., prostate (Van de Sande, T et al., 2005) [3] , breast (Stal et al, 2003) [4] , ovarian (Kurose et al., 2001) [5] , endometrial (Uegaki et al., 2005) [6] , squamous cell (Massarelli et al., 2005) [7] , and renal (Rathmell et al., 2005) cancers) [8] and have a negative impact on prognosis.
• malignancies e.g., prostate (Van de Sande, T et al., 2005) [3] , breast (Stal et al, 2003
• Akt-1 High anti-phosphorlation specific Akt immunostaining was significantly associated with poor cancer specific survival rate and metastases in renal cell carcinoma (Horiguchi et al., 2003) [9] . Increases in both cytoplasmic and nuclear p-Akt levels were independent prognostic factors for reduced renal cancer subject survival (Hager et al, 2009) [10] . Successful regulation of Akt-1 activity will be an effective way to control the survival of cancer cells. Therefore, Akt-1 may be an attractive drug target for the treatment of cancer. The potential of antisense oligonucleotides in gene silencing was discovered around 40 years ago, which resulted in increasing interest in chemistry, mechanism of action, and metabolic pathways aspects.
• Archexin is a 20-mer oligonucleotide, the API of WGI-0301, that is complementary to Akt-1 mRNA. Archexin had been tested in several in vitro and in vivo models and the results of these studies have demonstrated that Archexin is a selective and specific antitumor agent. In vitro studies have demonstrated that Archexin specifically inhibits the proliferation of human cancer cells of the brain, breast, cervix, colon, kidney, liver, lung, ovary, pancreas, prostate, skin, and stomach by inhibiting the mRNA and protein expression of Akt.
• WGI-0301 was designed to improve the in vivo delivery of drug substance Archexin and its therapeutic performance. WGI-0301 is formulated as a lipid nanoparticle suspension formulation of Archexin using a combination of quaternary and tertiary lipoamines for pH sensitive delivery along with other neutral and stabilizing lipids. Permanently ionized and conditionally ionizable lipids have been widely used in LNP formulation for the delivery of nucleic acid drugs.
• WGI-0301 product used quaternary cationic lipids, DOTAP-Cl, which carries a permanent positive charge regardless of the pH conditions, to help condense the large nucleic acid drugs into nanosized stable complexes.
• the LNP formulation also includes DODMA as ionizable lipid, which is mostly uncharged under at neutral pH and becomes cationized only under in acidic conditions.
• DODMA When incorporate DODMA into the LNP formulation, after endocytosis of LNPs, DODMA becomes charged and subsequently promotes the escape of LNP-encapsulated nucleic acids gene materials loaded in the LNPs under in the low pH condition, such as of late endosome or lysosome.
• LNP formulation of Archexin can also take advantage of the well documented enhanced permeation and retention (EPR) effect and a cleavable PEG coating which reduces mononuclear phagocyte system (MPS) uptake, and so that Archexin can accumulate preferentially within the tumor site, limiting off-target exposure.
• EPR enhanced permeation and retention
• MPS mononuclear phagocyte system
• WGI-0301 enhanced the efficacy and altered the tissue distribution profile in the liver, making liver cancer a very promising target. Further investigation has been planned after the proposed first in human study. WGI-0301 will be evaluated in patients with advanced solid tumors. Pre-clinical Studies Drug Product WGI-0301 WGI-0301 is a lipid nanoparticle suspension of Archexin.
• WGI-0301 is a lipid nanoparticle preparation of Archexin ® for the treatment of advanced solid tumors. WGI-0301 will be administered weekly by intravenous infusion over 1 hour at a starting dose of 0.1 mg/kg for 4 consecutive weeks (a cycle). Treatment cycles will continue unless disease progression, unacceptable toxicity, or a clinical observation meeting any withdrawal criteria is noted. A subject that has a Dose Limiting Toxicity (DLT) will be removed and other subjects in the same cohort will receive precedent cohort dose. Please refer to Pharmacy Manual for more investigational products handling and administration details.
• DLT Dose Limiting Toxicity
• Method A ‘3+3’ design will be deployed to determine dose limiting toxicities (DLT), maximum tolerated dose (MTD)/ the Recommended Phase 2 dose (RP2D).
• DLT dose limiting toxicities
• MTD maximum tolerated dose
• R2D Recommended Phase 2 dose
• Three to six patients per treatment cohort will be assigned to receive weekly 1-h IV infusion of WGI-0301 for 4 weeks (1 cycle) at a starting dose of 0.1 mg/ kg. All relevant safety data will be reviewed and adjudicated 28 days following the cycle’s start date. All toxicities will be considered related to WGI-0301 if they cannot be definitively explained by underlying disease, intercurrent illness or concomitant medications; Treatment cycles will continue unless disease progression, unacceptable toxicity, or a clinical observation meeting any withdrawal criteria is noted.
• the 3 + 3 design will be conducted as follows.
• both 1.3 and 1.75 mg/kg/weekly dose of WGI-0301 satisfies the requirement of endotoxin under the USP ⁇ 85> recommendation of 5 EU/kg in one hour; the endotoxin level of the drug products for the clinical trials has been retested and the results is ⁇ 2.5 mg/kg.
• the total study duration will be approximately 16 months.
• Subjects who continue to derive clinical benefit from the study treatment in the absence of withdrawal of consent, PD, or unacceptable toxicity may continue the study treatment. Patients may withdraw their consent at any time.
• the Primary Investigator may discontinue therapy at any time for the best interest of subjects. Specific criteria for the discontinuation of a patient’s participation are outlined.
• a DLT is defined as any treatment-emergent adverse event (TEAE) not attributable to disease or disease-related processes that occurs during the DLT evaluation period (Day 1 to Day 28) according to National Cancer Institute Common Terminology Criteria for Adverse Event (NCI-CTCAE) version 5.0.
• TEAE treatment-emergent adverse event
• NCI-CTCAE National Cancer Institute Common Terminology Criteria for Adverse Event
• Study Participants Approximately 24 subjects, depending on the number of cohorts explored. Subjects must meet all the following criteria to participate in this study: Subject with measurable disease based on RECIST 1.1. Advanced, histologically or cytologically confirmed solid tumors who have progressed from current therapy or who have relapsed after prior therapy and are not candidates for potentially curative therapy. Pathologically confirmed solid tumors.
• Adequate renal function [calculated estimate glomerular filtration rate eGFR of ⁇ 50mL/min] using the CKD-EPI Creatinine Equation (2021).
• Adequate hepatic function [total bilirubin ⁇ 1.5 x UNL; AST (aspartate transaminase) or ALT (alanine transaminase) ⁇ 3 x UNL or ⁇ 5 x UNL if due to liver involvement by tumor.
• Concomitant malignancies except carcinoma in situ, basal or squamous cell skin carcinoma; low grade prostate cancer treated with prostatectomy more than 5 years ago; early-stage melanoma treated with complete surgical excision more than 5 years ago; carcinoma in situ of cervix treated with cone procedure more than 8 years ago.
• CNS central nervous system
• CHF New York Heart Association [NYHA] classes II-IV
• serious cardiac arrhythmia requiring treatment has a medical history of symptomatic CHF (New York Heart Association [NYHA] classes II-IV) or serious cardiac arrhythmia requiring treatment.
• Example 5 In Vivo Efficacy Study of Test Articles in the Treatment of Subcutaneous Hepa 1-6 Murine Liver Cancer Model in Female C57BL/6 Mice The objective of this study is to evaluate the in vivo therapeutic efficacy of test articles in the treatment of subcutaneous Hepa 1-6 murine liver cancer model in female C57BL/6 mice.
• Materials Female Mus musculus C57BL/6. The mice were 7-8 weeks old.
• mice were housed at a density of up to 5 mice per cage at a temperature of 20-26 °C, humidity of 40-70%, 12 hours of light and 12 hours dark, on a standard rodent chow diet, irradiated, ad libitum, and 0.2 ⁇ m filtered, reverse osmosis (RO) water, autoclaved. Mice were monitored daily cage side observations, and weekly clinical observations. Hepa 1-6 tumor cells were maintained in vitro with 10% fetal bovine serum in DMEM medium at 37°C in an atmosphere of 5% CO2. The tumor cells in an exponential growth phase were harvested and counted for tumor inoculation.
• RO reverse osmosis
• mice were inoculated subcutaneously at the right front flank region with Hepa 1-6 tumor cells (5 x 106) in 0.1 mL of PBS for tumor development.
• Experimental Treatment for Hepa 1-6 model study is shown in Table 28 below.
• Table 28. Treatment for Hepa 1-6 Model Study.
• Dose Level Dosing Dosing Dosing G Mi T t t /k S l ti V l ROA F & records. Change was under the approval of sponsor.
• Attorney Docket No.11650-003WO1 The randomization started when the mean tumor size reached approximately 81 mm 3 .
• a total of 48 mice were enrolled in the study and allocated into 6 groups shown in Table 4, with 8 mice per group.
• Randomization was performed based on "Matched distribution" method (Study Director TM software, version 3.1.399.19). The date of randomization was denoted as Day O. After tumor inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (body weights were measured twice per week after randomization), eye/hair matting, and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail.
• Dose Items Level Cone Pre aration Ph sical Attorney Docket No.11650-003WO1 Diluted 2ml lmg/mL RX0201(12:l) with 0.5ml Solution RX-0201(12:1) 10% sucrose to make 2.5ml 0.8mg/mL The treatment was initiated on the same day of randomization per study design (Table 4). Dosing of this Hepa 1-6 model study started from Day 0 through Day 21. All mice would be fed with Dietgel Recovery if>l0% mean BWL is observed in the therapeutic groups.
• any animal exhibiting an ulcerated or necrotic tumor was separated immediately and singly housed and monitored daily before the animal was euthanized or until tumor regression was complete.
• Mouse with tumor ulceration of approximately 25% or greater on the surface of the tumor was euthanized.
• Bartlett's test was first used to check the assumption of homogeneity of variance across all groups. When the p-value of Bartlett's test is0.05, one-way ANOVA was ran to test the overall equality of means across all groups.
• mice showed slight or medium body weight loss after the treatment, they could recover gradually during the dosing interval.
• G4-118 mouse was sacrificed and GS- 197 mouse was found dead on Day 9.
• the maximum mean body weight loss in Negative control, Empty LNP, RX-0201 (8 mg/kg), RX-0301(15:1) (8 mg/kg), RX-0301(12:1) (8 mg/kg), RX-0301(10:1) (8 mg/kg) and RX-0301(7.5:1) (8 mg/kg) (dosing started from Day 0) treated groups was respectively none, - 13.91% (on Day 2), none, -13.39% (on Day 2), -16.13% (on Day 3), -13.37% (on Day 2) and-10.71% (on Day 2).
• RX-0301(12:1) administered at 8 mg/kg, QW produced slight anti-tumor efficacy against Hepa 1- 6 model, with a TGI value of 41.87% on Day 30, however no statistically significant difference (P>0.05) was observed compared with control group.
• RX-0301(10:1) administered at 8 mg/kg, QW produced slight anti-tumor efficacy against Hepa 1- 6 model, with a TGI value of 38.09% on Day 30, however no statistically significant difference (P>0.05) was observed compared with control group.
• Survival Analysis was assessed by time to tumor volume reaching 3000 mm3,the data were analyzed using Kaplan-Meier survival curves and shown in Table 30 and Figure 8. Table 30. Survival Analysis of Test Articles in Hepa 1-6 Model. Group Treatment Description MST (days) ILS (%) p value Ne ative control 10 L/ iv 37 The detailed statistical survival analysis for multiple comparisons is shown below: Attorney Docket No.11650-003WO1 In this study, the also investigated.
• mice whose tumor volume exceeded 3000 mm 3 were euthanized.
• RX-0301(7.5:1) showed significant efficacy in prolonging the survival time of Hepa 1-6 tumor-bearing mice while other test articles did not.
• the tolerability and efficacy of test articles in subcutaneous Hepa 1-6 liver cancer model in female C57BL/6 mice were investigated in this study.
• Hepa 1-6 tumor-bearing mice were well tolerated during the 4-week dosing phase with the designed dosing regimens.
• the tumor volume statistical analysis was conducted based on the data on Day 30 when all the animals were alive or did not meet the exclusion criteria of exceeding 3000 mm 3 tumorvolume.
• the TGI3 ⁇ 4 of EmptyLNP, RX-0201 (8 mg/kg), RX-0301(15:1) (8 mg/kg), RX-0301(12:1) (8 mg/kg), and RX-0301(10:1) (8 mg/kg), and RX-0301 (7.5:1) (8 mg/kg) groups were 26.56%, 3.32%, 27.93%, 41.87%, 38.09%, and 46.16% on Day 30 compared to vehicle control, respectively. There were no significant differences between the treated groups and the vehicle control group. The experiment was terminated on Day 56 to study the survival prolongation effects of the test articles.
• RX-0301 (7.5:1) (8 mg/kg) group showed significant efficacy in prolonging the survival time compared to the vehicle control group and RX-020 I-treated group in Hepa 1-6 tumor-bearing mice.
• Example 6 Free acid of archexin: effects on electric current passing through cloned herg potassium channels stably expressed in human embryonic kidney (hek293) cells using manual patch-clamp technique The objective of this study was to evaluate the in vitro concentration-response relationship of the effect of Free Acid of Archexin on the electric current passing through hERG (human ether a-go-go- related gene) potassium channels (a surrogate for IKr, the rapidly activating, delayed rectifier cardiac potassium current) stably expressed in a HEK293 cell line using manual patch clamp technique.
• Free Acid of Archexin is the active pharmaceutical ingredient; the concentration of test article in this report indicates the concentration of Free Acid of Archexin.
• Free Acid of Archexin was soluble in extracellular solution (ECS) at concentrations up to 300 ⁇ M without precipitate by visual check.
• ECS extracellular solution
• the pH value of ECS containing the highest concentration of Free Acid of Archexin was 6.9, which was within the target range of 6.5 to 7.8. No pH adjustment was required in the treatment medium.
• a dose range-finding assay was performed first to support the dose selection for the definitive assay.
• the definitive hERG assay was used to determine the ICso of Free Acid of Archexin.
• ECS was chosen as the solvent based on the solubility of Free Acid of Archexin.
• the measured concentration of the samples for concentration verifications were within 82% to 104% of the nominal values, and the measured concentration of the samples for homogeneity were within 98% to 102% of the nominal values.
• RSD relative standard deviation
• No Free Acid of Archexin was detected in the vehicle/negative control working solution.
• hERG currents were stable for at least 15 min in the negative control.
• Inhibition of positive control, terfenadine at 100 nM, on the hERG current was 78.84%, which was within the historical positive control data range.
• the seal criteria, current amplitude, and leak criteria met the pre-defined ranges. All these data demonstrated the validity of this assay.
• the objective of this study was to evaluate the in vitro concentration-response relationship of the effect of Free Acid of Archexin on the electric current passing through hERG (human ether-a-go-go- related gene) potassium channels (a surrogate for IKr, the rapidly activating, delayed rectifier cardiac potassium current) stably expressed in a HEK293 cell line using manual patch-clamp technique.
• hERG human ether-a-go-go- related gene potassium channels
• IKr the rapidly activating, delayed rectifier cardiac potassium current
• the increased action potential duration causes prolongation of the QT interval in the electrocardiogram that is associated with a dangerous ventricular arrhythmia, torsade de pointes. Therefore, testing the interaction of a compound with the hERG potassium channel in heterologous expression systems is recommended by the International Conference on Harmonisation (ICH) as one of the non-clinical testing methods for assessing the potential of a test compound for prolonging the QT interval.
• ICH International Conference on Harmonisation
• hERG channels were stably expressed in Human Embryonic Kidney (HEK293) cells.
• the Human Embryonic Kidney cell line, HEK293-hERG was employed in this assay.
• HEK293-hERG cells were obtained from Sophion Biosciences BPS Biosciences (San Diego, CA), subcultured and frozen in WuXi AppTec (Suzhou) Co., Ltd.
• the cell stocks were stored in liquid nitrogen. Every batch of the cell stocks was tested and determined to be free from mycoplasma contamination. Cells were not used after the 20th passage.
• Medium and Cell Culture Condition Complete medium was MEM medium, supplemented with 10% fetal bovine serum, 1% non- essential amino acids, 1 mM Na pyruvate, 400 ⁇ g/mL Geneticin@ selective antibiotic (G418), and 1% Penicillin/Streptomycin.
• Recovery medium was MEM medium with 10% fetal bovine serum, 1% non- essential amino acids, and 1 mM Na pyruvate.
• HEK293-hERG cells were cultured in a humidified incubator of5% CO2 (4% to 8%) in air at 37°C ( ⁇ 2°C).
• Attorney Docket No.11650-003WO1 The cells were recovered with recovery medium and subcultured in complete medium. The culture medium was switched to recovery medium in the last subculture before patch-clamp experiment.
• Solubility and pH Tests Test article was soluble in ECS at concentrations up to 300 ⁇ M. The pH of the ECS containing the highest soluble concentration was 6.9 in the pH test, which was within the range of 6.5 to 7.8.
• the hERG current was activated by depolarizing at +60 mV for 850 ms, after which the current was taken back to -50 mV for 1275 ms to remove the inactivation and to observe the deactivating tail current; the peak tail current was measured and collected for data analysis. Finally, the voltage was decreased to the holding potential (-80 mV). This command voltage protocol was repeated every 15 s continuously during the test article application. See Figure 9. During the initial recording period with vehicle control working solution, the peak tail current amplitude was monitored until it was stable for at least 10 sweeps. The average peak tail current Attorney Docket No.11650-003WO1 amplitude of the last 5 sweeps during the monitored period was used as current amplitude for vehicle control working solution (initial current).
• Negative Control Group The negative control was conducted in a separate group of cells (three cells) to evaluate the current stability (run-down or run-up) during at least 15 min recording period. The average peak tail current amplitude of the last five sweeps of every 5 min was used to evaluate current run-down or run- up. All the values were normalized to that of first 5 min and were presented as percentages. Less than 15% current during run-down or run-up measurement period was considered acceptable. Positive Control Terfenadine was used as the positive control article to evaluate the validity of the test system. The final working solution of positive control article at 100 nM was prepared for this study.
• the positive control was tested with 3 cells.
• the inhibition ratio for the positive control should be comparable with the historical positive control data range, to ensure the consistent responsiveness of the test system.
• Quality Control for Manual Patch-clamp Data Acceptance Seal criteria When acquiring whole-cell configuration, a holding potential (e.g., -80 mV) was applied while membrane parameters were collected (Cm, Rm and Rs).
• a "good" whole-cell recording is generally defined as: series resistance (Rs) was less than 10 M ⁇ ; membrane resistance (Rm) was greater than 500 M ⁇ , and membrane capacitance (Cm) was less than 100 pF.
• the stable current amplitude is defined as the CV of peak current amplitudes in 10 continuous sweeps that is less than 10% or less than 30% if the average current amplitude of the 10 sweeps is less than 200 pA. If an average of percent inhibition value is less than 70%, the Standard Deviation (SD) of percent inhibition values from different recorded cells must be less than 15%. If the average of percent inhibition value is over 70%, the SD of percent inhibition values from different recorded cells must be less than 10%. Results Solubility and pH Tests Test article was soluble in ECS at concentrations up to 300 ⁇ M, no precipitate was observed at any concentration by visual check.
• the pH value for the ECS containing 300 ⁇ M of test article was 6.9 in the pH test, which was within the acceptable range of 6.5 to 7.8. Therefore, no pH adjustment was required in the treatment medium.
• hERG current inhibition The results of hERG current inhibition are provided in Table 31. Mean current inhibitions of 5.99%, 6.63%, and 11.69% were observed at 3, 30, and 300 ⁇ M, respectively.
• the hERG current inhibition data for the negative control are presented in Table 32; the hERG current inhibition data for the positive control are presented in Table 33.
• the concentration-response curve of Free Acid of Archexin is shown in Figure 10; and the hERG current inhibition data for Free Acid of Archexin are presented in Table 34.
• Table 32. hERG Current Stability of Negative Control in the Definitive hERG Assay.
• Table 33 Terfenadine: hERG Current Inhibition in the Definitive hERG Assay.
• Attorney Docket No.11650-003WO1 Negative Control The negative control was tested in a separate group of cells (three cells) to evaluate the current stability (run-down or run-up) during at least 15 min recording period. The results indicated that no significant current run-down or run-up was observed during the recording period.
• HCC Hepatocellular carcinoma
• HCC liver cancer
• HCC is a highly vascular tumor in which angiogenesis plays a major role in tumor growth and metastasis [4].
• the proposed clinical use of WGI-0301 is to treat advanced HCC as a second line treatment for patients who failed frontline immunotherapies (IOs) in combination with Tyrosine Kinase Inhibitor (TKI), Attorney Docket No.11650-003WO1 Sorafenib. Immunotherapy or immunotherapy combinations with better efficacy data has gradually taken over the crown of first line Advanced HCC setting in recent years.
• Atezolizumab withBevacizumab (Atezo-Bev) is currently the first choice first-line treatment, as it confers a superior survival benefit compared to Sorafenib [5] .
• Atezo-Bev is currently the first choice first-line treatment, as it confers a superior survival benefit compared to Sorafenib [5] .
• Sorafenib is an important therapeutic advance as a single agent in advanced HCC. Sorafenib has a well-studied safety profile and excellent patient access with many years of clinical use. It is the first systemic treatment that has shown overall survival (OS) benefit over placebo in Attorney Docket No.11650-003WO1 HCC patients validated through randomized controlled trial [6]. For more than a decade, it was used in the treatment of advanced HCC globally. However, the combination of Atezo-Bev or Durvalumab- Tremelimumab exhibited superior OS outcomes over Sorafenib [5,7].
• Sorafenib becomes an alternative option of first- line therapy for patients with contraindication or limited access to Atezo-Bev or Durvalumab-Tremelimumab, or empirical use as second-line therapy after IO treatment.
• Sorafenib can potentially provide moderate survival benefits over placebo in patients with advanced HCC after disease progression on Atezo-Bev [8].
• Sorafenib-combinations regimens as second-line therapy in patients who progress on first-line IO therapy.
• the Combination of WGI-0301 and Sorafenib has shown potential synergistic anti-tumor activity in HCC.
• WGI-0301 has the potential to enhance Sorafenib’s anti-tumor effect through various mechanisms of action (MoA). Synergistic effects have been observed with the co- administration of WGI-0301 and Sorafenib in preclinical models, resulting in enhanced inhibition of angiogenesis and augmentation of tumor growth suppression. This combination therapy has garnered attention due to the absence of significant cytochrome P450 enzyme interaction by WGI-0301, suggesting a reduced likelihood of pharmacokinetic drug-drug interactions with concurrent Sorafenib treatment.
• Resistance is a complex phenomenon involving multiple mechanisms, including activation of signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR).
• Sorafenib can activate the PI3K/AKT pathway, and the potential compensation mechanism presented by the PI3K/AKT pathway can cause Sorafenib resistance in HCC patients [9].
• Current preclinical and clinical evidence suggests that inhibitors of the PI3K/AKT/mTOR pathway could have utility in combination with other anticancer therapies to circumvent resistance by cancer cells [10] .
• Dual Attorney Docket No.11650-003WO1 blockade with an AKT-1 inhibitor might prevent or delay the development of resistance.
• Stage 1 a Dose Escalation Stage
• Stage 2 advanced HCC patients who have received up to one line of immunotherapy
• Stage 1 will use “Traditional 3 + 3” study design to explore the MTD / RP2D of WGI-0301 when combined with Sorafenib, and to evaluate the PK, PD, safety, tolerability and preliminary efficacy of the combination therapy for advanced HCC.
• the starting dose of WGI-0301 is 0.6 mg / kg / week with 400 mg Sorafenib PO, BID.
• Stage 1 three dose levels of WGI-0301, 0.6, 1.0 and 1.3 mg / kg / week, combined with Sorafenib are planned. Once MTD / RP2D of WGI-0301 is determined from Stage 1 by Safety Monitoring Committee (SMC), Stage 2 will start once. In Stage 2, patients will be treated with combination of different WGI-0301 dose levels and standard dose Sorafenib or standard dose Sorafenib alone in a 2:2:1 ratio to evaluate the safety and efficacy in patients with advanced HCC who have previously received up to one line of PD-1/PD- L1 immune checkpoint inhibitor or combination in the first line setting. Stage 1 and Stage 2 will be conducted in both the US and China.
• SMC Safety Monitoring Committee
• the US sites will start Stage 1 once approved by FDA regardless of China segment status. Study will be conducted in a competing enrollment manner when more than 1 site is activated. Kinds of Clinical Trials to be Conducted in the First Year Following submission An open-label phase 2 study of WGI-0301 plus Sorafenib in patients with advanced HCC as second line therapy will be conducted in the U.S. and China.
• the US sites will start Stage 1 once approved by FDA regardless of China segment status. Study will be conducted in a competing enrollment manner when more than 1 site is activated. Stage 2 will start once the MTD / RP2D of WGI- 0301 in combination with Sorafenib is determined from Stage 1 by SMC.
• LNP infusion product For events related to WGI-0301, class adverse events of LNP infusion product that may occur during or shortly after the administration of an LNP-containing therapy based on severity of symptoms, absence of respiratory function loss, respond to treatment (steroids and antihistamines) was lean towards.
• the exact cause of infusion reactions to LNP products is not always clear, but it is thought that they may result from the body’s immune response to the LNP itself or the payload delivered by the LNP.
• the components of the LNP formulation such as the lipid mixture, may also play a role in these reactions.
• prophylaxis regimen was proposed containing corticosteroids, antihistamines, with or without nonsteroidal anti- inflammatory drugs, which has been utilizing during phase I study recommended by SMC committee.
• a serum tryptase test will also be implemented to help excluding anaphylaxis.
• the site should remain alert on differentiating anaphylaxis and infusion reactions with emergency medications and equipment on hand.
• Liver Toxicity For liver toxicity, clinically significant liver function tests (LFTs) elevation has not been captured during Phase I study as of 30Oct2023.
• WGl-0301 is a proprietary lipid nanoparticle formulation (QTsome TM ) of Archexin designed to enhance delivery.
• QTsome TM lipid nanoparticle formulation
• a phase I clinical study of WGl-0301 as monotherapy in patients with advanced solid tumors is currently ongoing in the U.S.
• WGl- 0301 combined with TKls may augment the therapeutic response to TKls by enhancing angiogenic blockade, inhibiting AKT-1 , and overcome resistance.
• WGI-0301 An efficacy study of WGI-0301 was conducted in combination with Lenvatinib, sorafenib, or cabozantinib in the treatment of human hepatocellular cancer Hep3B-luciferase orthotopic model in the female Balb/c nude mice.
• Sixty-four female Balb/c nude mice were randomly divided into groups ⁇ 8 mice in each group), including a vehicle control.
• the mice were given WGI-0301 or Lenvatinib, sorafenib, or cabozantinib or WGI-0301 in combination with lenvatinib/sorafenib/cabozantinib for 28 days.
• WGI-0301 was administered at a dose of 8 mg/kg once a week intravenously for 4 times, and lenvatinib/sorafenib/cabozantinib were given by oral at doses of 10 mg/kg, 20 mg/kg, and 20 mg/kg daily respectively for 28 days.
• Body weight was monitored twice a week.
• Whole body fluorescent Attorney Docket No.11650-003WO1 imaging was performed twice a week, and tumor load was measured using bioluminescence. Treatment was suspended from day 29 to day 70 for survival observation after drug discontinuation. Table 35. Groups and Treatment Information.
• Dosing Dosing G roup N a Treatment b Dose Survival ( mg/kg) Volume Route Schedule d O e ( ⁇ L/g) c bservation was formulated in cremophor EL: (95%) ethanol 1:1 (v/v) and WGI-0301 was formulated in saline. Vehicle in group 1 was saline. c. Dosing volume: adjust dosing volume based on body weight 10 ⁇ L/g. d. Grouping was performed on PG-D0, treatment was started from PG-D1. Dosing schedule of Lenvatinib, Sorafenib and Cabozantinib were QD, dosing schedule of Vehicle and WGI-0301 were QW.
• mice were weighed and intraperitoneally administered luciferin at a dose of 150 mg/kg. Ten minutes after the luciferin injection, the animals were pre-anesthetized with the mixture gas of oxygen and isoflurane. When the animals were in a complete anesthetic state, the mice were moved into the imaging chamber for bioluminescence measurements with an IVIS (Lumina III) imaging system. The major endpoint was to see if the tumor bioluminescence growth can be delayed, decreased, or vanished, and another major endpoint was to observe the survival period of the mice after treatment, such as the death of a single mouse or the mouse reaching the requirement of euthanasia.
• IVIS Lumina III
• Tumor bioluminescence metastasis can be prevented or the mice can be cured.
• the body weight was measured twice weekly.
• TGI (%) [1-(T i -T 0 )/ (C 1- C 0 )] ⁇ 100;
• T i is the average tumor bioluminescence value of a treatment group on a given day,
• T 0 is the average tumor bioluminescence value of the treatment group on the first day of treatment,
• Ci is the average tumor bioluminescence value of the vehicle control group on the same day with Ti, and
• C0 is the average tumor bioluminescence value of the vehicle group on the first day of treatment.
• Mean survival time (Days) of each group was calculated based on the survival time of the animals within, increased life span was analyzed according to the mean survival time in treatment groups and vehicle group.
• the fluorescence signal intensity of the combination group (56.74*10 8 , 22.28*10 8 , 40.40*10 8 photons/s) was slightly lower than that of the lenvatinib/sorafenib/cabozantinib group (81.97*10 8 , 73.95*10 8 , 86.96*10 8 photons/s).
• the median survival time (MST) for the combination group was 55.5, 58.0, and 53.5 days, which was slightly higher than the lenvatinib/sorafenib/cabozantinib group (46.0, 55.5, 51.5 days).
• mice #8-3 in group 4 (Sorafenib, 20 mg/kg, p.o., QD x 4W) exhibited body weight loss over 15% on PG-D24, it was provided with diet gel from PG-D25 to PG-D27 to maintain its bodyweight.
• the mouse #1-1 in group 1 (Vehicle, i.v., QW x 4W) was found died at PG-D27, according to the veterinary autopsy, the cause of death was likely related to deteriorating health status caused by huge tumor.
• 3 mice showed enlarged abdomen at 17 days after administration, and the liver protruded in situ.
• Bioluminescence value (x10 8 photon/s) a Group Treatment 0 b 3 7 10 14 17 21 24 28 c Vehi 0.39 ⁇ 0.87 ⁇ 4.35 ⁇ 9.46 ⁇ 30.48 ⁇ 59.39 ⁇ 115.07 ⁇ 166.36 ⁇ 144.20 1 cle, i .v., QW x 4W 0.05 0.15 0.93 2.11 5.31 8.87 17.15 27.90 ⁇ 26.27 WGI-0301, 2 8 mg/kg, i.v., 0.39 ⁇ 0.80 ⁇ 3.71 ⁇ 8.34 ⁇ 22.80 ⁇ 52.63 ⁇ 101.94 ⁇ 161.59 ⁇ 153.21 QW x 4W 0.05 0.11 0.87 2.33 2.81 10.39 15.50 29.49 ⁇ 22.29 Lenvatinib, 0.39 ⁇ 0.70 ⁇ 3.94 ⁇ 9.59 ⁇ 23.98 ⁇ 32.18 ⁇ 48.30 ⁇ 9 81.97 ⁇ 1 110.41 3 10 mg/kg p.o., 0.06 0.11 0.84 0.99 3.21 4.56 .96
• Tumor Growth Inhibition Analysis Analysis (Calculated based on the bioluminescence data obtained on PG-D24).
• Bioluminescence value p c Group Treatment (x10 8 photon/s) T/C b (%) TGI b (%) value a WGI-0301 + Sorafenib, 8 mg/kg + 20 mg/kg, 7 i.v., + p.o., (QW + QD) x 22.28 ⁇ 9.27 12.75 86.81 * 4W WGI-0301 + Cabozantinib, 8 8 mg/kg + 20 mg/kg, 40.40 ⁇ 9.27 23.52 75.89 * i.v., + p.o., (QW + QD) x 4W a. Data is shown as Mean ⁇ SEM.
• TGI Tumor Growth Inhibition
• T/C Antitumor activity
• c A one-way ANOVA with SPSS was performed to compare the bioluminescence value among vehicle group and treatment groups. ns: no significance, * indicates p ⁇ 0.05.
• Table 41 Tumor Growth Inhibition Analysis (Calculated based on the bioluminescence data obtained on PG-D24).
• Independent-Samples T Test with SPSS was performed to compare the bioluminescence value between group 3 and group 6. ns: no significance.
• b. Independent-Samples T Test using SPSS was performed to compare the bioluminescence value between group 4 and group 7. ns: no significance.
• c. Independent-Samples T Test using SPSS was performed to compare the bioluminescence value between group 5 and group 8. ns: no significance.
• Survival Animals with deteriorating health status due to excessive tumor size or severe bodyweight loss were euthanized according to the IACUC protocol. The survival time of the animals was observed for 42 days. The mean survival time of animals in vehicle group was 37.63 days.
• the mean survival time of the animals from Group 2 (WGI-0301, 8 mg/kg), Group 3 (Lenvatinib, 10 mg/kg), Group 4 (Sorafenib, 20 mg/kg), Group 5 (Cabozantinib, 20 mg/kg), Group 6 (WGI-0301+Lenvatinib, 8+10 mg/kg), Group 7 (WGI- 0301+Sorafenib, 8+20 mg/kg) and Group 8 (WGI-0301+Cabozantinib, 8+20 mg/kg) were 35.88, 47.00, 53.25, 52.25, 53.00, 58.88 and 53.75 days respectively.
• the mean survival rates of each treatment group were 95.35%, 124.90%, 141.51%, 138.85%, 140.85%, 156.47% and 142.84% respectively.
• the median survival time of vehicle group was 35 days.
• the Kaplan-Meier survival curves for animals in each group is shown in Figure 15, 16 and 17.
• the result of survival time analysis is shown in Table 42.
• Table 42. Survival Time analysis. Mean a mean survival Treatment rates (%) b Median c p Attorney Docket No.11650-003WO1 Vehicle, i .v., QW x 4W 37.63 ⁇ 3.74 -- 35.00 -- Cabozantinib, 2 0 mg/kg p.o., QD x 4W 52.25 ⁇ 3.03 138.85 51.50 ns WGI-0301 + Lenvatinib 8 mg/kg + 10 mg/kg, 53.00 ⁇ 1.72 140.85 55.50 ** i.v., + p.o., (QW + QD) x4W WGI-0301 + Sorafenib 8 mg/kg + 20 mg/kg, 58.88 ⁇ 3.49 156.47 58.00 ** i.v., + p.o., (QW
• mice #1-1 in the vehicle group was found dead on the 27th day of administration. After veterinary autopsy evaluation, it may be related to the decline in physical fitness caused by tumor overload. The mice treated with Lenvatinib, 10 mg/kg and Sorafenib, 20 mg/kg (monotherapy or combination) showed slight bodyweight loss during treatment.
• Endpoint data based on mortality, clinical symptoms, and body weight showed no substantial increase in toxicity in mice treated with the combination of WGI-0301 + Lenvatinib, 8+10 mg/kg, WGI-0301 + Sorafenib, 8+20 mg/kg and WGI-0301 + Cabozantinib, 8+20 mg/kg compared to the Lenvatinib / Sorafenib / Cabozantinib monotherapy group.
• Both T/C and TGI were the indicators of antitumor effectiveness, the data on PG-D24 are shown in Table 40 and Table 41.
• the survival time of the animals was observed for 42 days after 28-days treatment completion.
• the animals were died or euthanized in accordance to the IACUC protocol due to obvious weight loss or deterioration of health status.
• the mean and median survival time of animals in vehicle group was 37.63 days and 35 days, respectively.
• Sorafenib 20 mg/kg, WGI-0301+Lenvatinib, 8+10 mg/kg, WGI-0301+Sorafenib, 8+20 mg/kg and WGI-0301+Cabozantinib, 8+20 mg/kg could significantly prolong the survival time of mice bearing the Hep3B-luc orthotopic tumor model. Compared with the monotherapy, the combined treatment showed a little more survival time of mice.
• mice treated with WGI-0301 combined with Sorafenib, 8+20 mg/kg, and WGI- 0301 combined with Cabozantinib, 8+20 mg/kg showed a significant anti-tumor effect on Hep3B-luc orthotopic tumor-bearing mice, and can prolong the survival time of mice.
• Sorafenib, 20 mg/kg and WGI-0301+Lenvatinib, 8+10 mg/kg showed minor tumor inhibitory effects and can prolong the survival time of mice as well.
• compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited.
• a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.
• the term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms.

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