WO2013062982A1

WO2013062982A1 - Poly(lysine) homopolymers for the delivery of oligonucleotides

Info

Publication number: WO2013062982A1
Application number: PCT/US2012/061510
Authority: WO
Inventors: Stephanie E. Barrett; J. Michael Williams; Robert A. Kowtoniuk
Original assignee: Merck Sharp & Dohme Corp.
Priority date: 2011-10-27
Filing date: 2012-10-24
Publication date: 2013-05-02

Abstract

The present invention provides poly(lysine) polymers, polyconjugates, compositions and methods for the delivery of oligonucleotides for therapeutic purposes.

Description

TITLE OF THE INVENTION

POLY(LYSINE) HOMOPOLYMERS FOR THE DELIVERY OF OLIGONUCLEOTIDES

BACKGROUND OF THE INVENTION

Oligonucleotides conjugated to polymers are known. Further, the delivery of oligonucleotides conjugated to polymers (polyconjugates) for therapeutic purposes is also known. See WO2000/34343; WO2008/022309; and Rozema et al. PNAS (2008) 104, 32: 12982-12987.

Poly(amide) polymers are known. Tokunaga et al., (2003) J. Pharm. Sci.

Technol., Jpn., 63, 71-78; Plank et al., (1999) Human Gene Therapy, 10, 319-332; De Paula et al., (2007) RNA 13, 431-456; Duksin et al. (1970) P.N.A.S. 67, 185-192; Bichowsky- Slomnicki et al. (1956) Archives of Biochemistry and Biophysics 65, 400-413; Duksin et al. (1975) FEBS Letters 60, 21-25; Yang et al. (1998) J. Am. Chem. Soc. 120, 10646-10652;

Miyata et al. (2008) J. Am. Chem. Soc. 130, 16287-16294; Sato et al. (2010) Biol. Pharm. Bull. 33(7), 1246-1249); WO2008/070141 and US2009/0232762.

Herein, we disclose and describe novel poly(lysine) homopolymers and polyconjugates useful for the delivery of oligonucleotides for therapeutic purposes.

SUMMARY OF THE INVENTION

The present invention provides poly(lysine) homopolymers, polyconjugates, compositions and methods for the delivery of oligonucleotides for therapeutic purposes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Analytical results from polyconjugates prepared from poly(lyisne) homopolymers (Method 2).

FIG. 2. RBC hemolysis data of an exemplery poly(lysine) homopolymer (Method 2).

FIG. 3. Mouse in vitro bDNA data of masked polyconjugates prepared from poly(lysine) homopolymers (Method 2).

FIG. 4. Rat in vivo data of masked polyconjugates from polyconjugates prepared from poly(lysine) homopolymers (Method 1).

FIG. 5. Rat in vivo data of masked polyconjugates from polyconjugates prepared from poly(lysine) homopolymers (Method 2).

FIG. 6. Rat in vivo ALT data of masked polyconjugates from poly(L-lysine) homopolymers.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment of the instant invention is a polymer of Formula Z:

z wherein:

x is 50 to 2000;

R is independently selected from an end group;

Ri is butyl amine;

R_a is independently hydrogen, methyl, ethyl, propyl or butyl optionally substituted with N¾ and OH; and

Rb is independently hydrogen, methyl, ethyl, propyl or butyl optionally substituted with N¾ and OH;

or a stereoisomer thereof.

In another embodiment of the instant invention is a polymer of Formula Z: wherein:

x is 50 to 1000

R is independently selected from an end group;

Ri is butyl amine;

Ra is hydrogen; and

Rb is hydrogen;

or a stereoisomer thereof.

x is 100 to 1000;

R is independently selected from an end group;

Ri is butyl amine;

Ra is hydrogen; and

Rb is hydrogen;

or a stereoisomer thereof.

In another embodiment of the instant invention is a polyconjugate of Formula I:

I wherein:

x is 50 to 2000;

R is independently selected from an end group;

R_\ is independently selected from butyl amine, butyl amine with a linker- oligonucleotide, butyl amine with a linker-targeting ligand and butyl amine with a linker-PEG;

Ra is independently hydrogen, methyl, ethyl, propyl or butyl optionally substituted with N¾ and OH; and

Rb is independently hydrogen, methyl, ethyl, propyl or butyl optionally substituted with NH₂ and OH;

or a stereoisomer thereof.

In another embodiment of the instant invention is a polyconjugate of Formula I: wherein:

x is 50 to 1000;

R is independently selected from an end group;

R_\ is independently selected from from butyl amine, butyl amine with a linker- oligonucleotide, butyl amine with a linker-targeting ligand and butyl amine with a linker-PEG;

R_a is hydrogen; and

Rb is hydrogen;

or a stereoisomer thereof.

x is 100 to 1000;

R is independently selected from an end group;

R_a is hydrogen; and

R¾ is hydrogen;

or a stereoisomer thereof.

In another embodiment of the instant invention, the polyconjugate is:

CDM-PEG

n_aVg=10

oligonucleotide

CDM-NAG wherein

R is independently selected from n-butyl amine or rnPEG-amine (where the PEG molecular weight can range from 500 g/mol to 12,000 g/mol), a hydrogen, hydroxyl, and carboxylate and; x is 50-2000;

or a stereoisomer thereof.

In another embodiment of the instant invention is a method of treating a disease in a patient by administering a polyconjugate composition of the instant invention.

DEFINITIONS

"Disease" means a disorder or incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors; illness; sickness; ailment. An example of a disease is cancer.

"Linker" means a chemical moiety that physically conjugates a specified group with the polymer of Formula Z. An example of a linker is the chemical moiety which is made by the conjugation of a derivative of 4-succinimidyloxycarbonyl-methyl-(2- pyridyldithio)toluene (SMPT) and a derivative of N-Succininn^yl-S-acetylthioacetate (SATA).

"Linker-oligonucleotide" means a chemical moiety that physically conjugates the oligonucleotide with the polymer of Formula Z.

"Linker-targeting ligand" means a chemical moiety that physically conjugates the targeting ligand with the polymer of Formula Z.

"Linker-PEG" means a chemical moiety that physically conjugates the PEG with the polymer of Formula Z.

"Oligonucleotide" means deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinations of DNA, RNA and other natural and synthetic nucleotides, including protein nucleic acid (PNA). DNA maybe in form of cDNA, in vitro polymerized DNA, plasmid DNA, parts of a plasmid DNA, genetic material derived from a virus, linear DNA, vectors (PI, PAC, BAC, YAC, and artificial chromosomes), expression vectors, expression cassettes, chimeric sequences, recombinant DNA, chromosomal DNA, anti-sense DNA, or derivatives of these groups. RNA may be in the form of messengerRNA (mRNA), in vitro polymerized RNA, recombinant RNA, transfer RNA (tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), chimeric sequences, anti-sense RNA, interfering RNA, small interfering RNA

(siRNA), microRNA (miRNA), ribozymes, external guide sequences, small non-messenger RNAs (snmRNA), untranslatedRNA (utRNA), snoRNAs (24-mers, modified snmRNA that act by an anti-sense mechanism), tiny non-coding RNAs (tncRNAs), small hairpin RNA (shRNA), or derivatives of these groups. In addition, DNA and RNA may be single, double, triple, or quadruple stranded. Double, triple, and quadruple stranded polynucleotide may contain both RNA and DNA or other combinations of natural and/or synthetic nucleic acids.

Oligonucleotides can be chemically modified. The use of chemically modified oligonucleotides can improve various properties of the oligonucleotides including, but not limited to: resistance to nuclease degradation in vivo, cellular uptake, activity, and sequence-specific hybridization. Non-limiting examples of such chemical modifications include: phosphorothioate

internucleotide linkages, LNA, 2'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation. These chemical modifications, when used in various oligonucleotide constructs, are shown to preserve oligonucleotide activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Chemically modified siRNA can also minimize the possibility of activating interferon activity in humans. See WO2008/022309 for a more detailed description of oligonucleotides.

"Patient" means a mammal, typically a human, in need of treatment for a disease.

"PDF (or polydispersity index) is defined as the distribution of molecular weights in a particular polymer sample. It can be calculated from the weight average molecular weight divided by the number average molecular weight (PDI = M_w/M_n). The PDI is always greater than or equal to 1.

"Polymer" means a molecule built up by repetitive smaller units called monomers. A polymer can be linear, branched, network, star, comb, or ladder type.

"Targeting ligand", also referred to as "targeting agent", means an agent that can deliver a polymer or polyconjugate to target cells or tissues, or specific cells types. Targeting ligands enhance the association of molecules with a target cell. Thus, targeting ligands can enhance the pharmacokinetic or biodistribution properties of a polyconjugate to which they are attached to improve cellular distribution and cellular uptake of the conjugate. See

WO2008/022309 for a more detailed description of targeting ligands.

In an embodiment of Formula Z or I, x is 2 to 5000.

In another embodiment of Formula Z or I, x is 50 to 2000.

In another embodiment of Formula Z or I, x is 50 to 1000.

In another embodiment of Formula Z or I, x is 100 to 1000.

In another embodiment of Formula Z or I, x is 200 to 800.

In another embodiment of Formula Z or I, x is 300 to 600.

In an embodiment, R is an end group independently selected from R'R"N and R'O where R' and R" are independently hydrogen, alkyl (C_1-18) or substituted alkyl, or aryl or substitued aryl, or heterocyclyl or substituted heterocyclyl, or a PEG.

In an embodiment, R is an end group independently selected from an amine, alcohol, water, hydrogen, alkali halide, alkoxide, or a hydroxide.

In an embodiment, R is an end group independently selected from an amine.

In an embodiment, R is an end group independently selected from a monoamine, a diamine, a bisamine, a monoprotected diamine, and a dendrimer having multiple amines as end groups.

In an embodiment, R is an end group independently selected from, n-butyl amine, mPEG 2K amine, mPEG 5K amine, mPEG 12K amine, QO'-bis(2- aminoethyl)polyethylene glycol, ethylene diamine, 1,6-hexanediamine, 2-(2-aminoethoxy)ethyl 2-(acetylamino)-2-deoxy-P-D-galactopyranoside, N-Boc-ethylenediamine, L-aspartic acid β- benzyl ester, and poly(amido amine) (PAMAM) dendimers with surface amino groups.

In an embodiment, R is an end group independently selected from, triemylamine, n-butyl amine and mPEG 2K amine.

In an embodiment, R is an end group independently selected from hydroxy, carboxylate and hydrogen.

In an embodiment, R is an end group which is n-butyl amine.

In another embodiment, Ri is independently selected from butyl amine, linker- oligonucleotide, linker-targeting ligand and linker-PEG.

In an embodiment, Ra and R_b are independently hydrogen, methyl, ethyl, butyl or butyl optionally substituted with N¾ and OH. In another embodiment, R_a and ¾ are hydrogen.

In an embodiment, a linker-oligonucleotide is selected from both degradable and non-degradable moieties (included but not limited to the moiety that is formed when SMPT reacts with a SATA-modified oligonucleotide).

In an embodiment, a linker-targeting ligand is selected from compounds with affinity to cell surface molecules, cell receptor ligands, and antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules.

In another embodiment, a targeting ligand is selected from carbohydrates, glycans, saccharides (including, but not limited to: galactose, galactose derivatives, mannose, and mannose derivatives), vitamins, folate, biotin, antibodies, aptamers, and peptides

(including, but not limited to: RGD-containing peptides, insulin, EGF, and transferrin).

In another embodiment, a targeting ligand is selected from N- acetylgalactosamine (NAG), mannose and glucose.

In another embodiment, a targeting ligand is selected from N- acetylgalactosamine (NAG).

In an embodiment, an oligonucleotide is selected from siRNA, miRNA and antisense. In another embodiment, an oligonucleotide is an siRNA.

FORMULATION

The polyconjugates of Formula I are formed by covalently linking the oligonucleotide to the polymer. Conjugation of the oligonucleotide to the polymer can be performed in the presence of excess polymer. Because the oligonucleotide and the polymer may be of opposite charge during conjugation, the presence of excess polymer can reduce or eliminate aggregation of the polyconjugate. Excess polymer can be removed from the polyconjugate prior to administration of the polyconjugate to a patient. Alternatively, excess polymer can be co-administered with the polyconjugate to the patient.

Similarly, the polymer can be conjugated to a masking agent in the presence of an excess of polymer or masking agent. Because the oligonucleotide and the polymer may be of opposite charge during conjugation, the presence of excess polymer can reduce or eliminate aggregation of the polyconjugate. Excess polymer can be removed from the polyconjugate prior to administration of the polyconjugate to a patient. Alternatively, excess polymer can be coadministered with the polyconjugate to the patient. The polymer can be modified prior to or subsequent to conjugation of the oligonucleotide to the polymer.

Parenteral routes of administration include intravascular (intravenous, interarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal, intrathecal, subdural, epidural, and intralymphatic injections that use a syringe and a needle or catheter. Intravascular herein means within a tubular structure called a vessel that is connected to a tissue or organ within the body. Within the cavity of the tubular structure, a bodily fluid flows to or from the body part. Examples of bodily fluid include blood, cerebrospinal fluid (CSF), lymphatic fluid, or bile. Examples of vessels include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, bile ducts, and ducts of the salivary or other exocrine glands. The intravascular route includes delivery through the blood vessels such as an artery or a vein. The blood circulatory system provides systemic spread of the pharmaceutical. An adniinistration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes. Intraparenchymal includes direct injection into a tissue such as liver, lung, heart, muscle (skeletal muscle or diaphragm), spleen, pancreas, brain (including intraventricular), spinal cord, ganglion, lymph nodes, adipose tissues, thyroid tissue, adrenal glands, kidneys, prostate, and tumors. Transdermal routes of administration have been affected by patches and iontophoresis. Other epithelial routes include oral, nasal, respiratory, rectum, and vaginal routes of administration.

The polyconjugates can be injected in a pharmaceutically acceptable carrier solution. Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view. The phrase pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a patient. Preferably, as used herein, the term pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

UTILITY

The polyconjugates of Formula I may be used for research purposes or to produce a change in a cell that can be therapeutic. The use of polyconjugates for therapeutic purposes is known. See WO2000/34343; WO2008/022309; and Rozema et al. PNAS (2008) 104, 32: 12982-12987.

EXAMPLES

Examples and schemes provided are intended to assist in a further understanding of the invention. Particular materials employed, species and conditions are intended to be further illustrative of the invention and not limitative of the reasonable scope thereof.

Polymers can be prepared following several different mechanisms (see Deming, Journal of Polymer Science: Part A: Polymer Chemistry (2000) 38, 3011-3018). The first uses a nucleophile to initiate the polymerization by ring-opening the N-carboxyanhydride and is called the "normal amine" mechanism.

The second, the "activated monomer" mechanism also uses the N- carboxyanhydride monomer but is initiated by the deprotonation of the monomer with a base. This NCA anion then becomes the nucleophile that initiates the polymerization.

MONOMER SYNTHESIS

Boc-L-lysine N-carboxyanhydride (NCA (Scheme 1 :

To a slurry of boc-L-lysine (10 g, 40.6 mmol) in 600 mL of THF under nitrogen was charged triphosgene (4.82 g, 16.24 mmol) in THF (420 mL). The reaction was heated at 50- 55°C for 1 h then cooled to ambient temperature. The remaining solid was removed by filtration washing with THF (100 mL). The filtrate was concentrated by vacuum distillation to give a white solid. The white solid was dissolved in EtOAc (100 mL) and DCM (100 mL) was added. A solid precipitate was observed and filtered. The filtrate was evaporated to dryness. The solid was dissolved in THF (100 mL) and switched to cyclopentylmethyl ether (CPME).

The resulting slurry was cooled to ambient temperature and stirred under nitrogen overnight. The solid was isolated by filtration washing with 70 mL of CPME and vacuum dried to give 8.56 g of white crystalline product. Solid was collected and stored at -20°C in a sealed bottle. Ή NMR (500 MHz, DMSO-i e ): δ 9.06 (s, 1 H); 6.78 (s, 1 H); 4.44 (dd, J = 7.0 Hz, 1 H); 2.90 (dd, J = 6.50 Hz, 2 H); 1.74-1.68 (m, 1 H); 1.66-1.63 (m, 1 H); 1.37 (s, 9H); 1.33-1.29 (m, 2 H).

POLYMER SYNTHESIS

Polv(Boc-L-Lysine) - Polvmeization Method 1 (Scheme 3

Boc-L- lysine-N-carboxyanhydride (500 mg) was placed in an oven dried 40 mL vial and was purged with an atmosphere of nitrogen. Anhydrous dimethylacetamide (DMA, 5.6 mL, water content = 90 ug/mL) was added to give a clear solution. n-Butylamine (4.03 mg) was added. The solution was stirred at room temperature under vacuum overnight. The polymer was precipitated by adding the reaction mixture into 500 mL of water with vigorous stirring. The solid was isolated by filtration washing with 500 mL of water. The collected precipitate was frozen and placed on a lyophilizer for 48 hours. Gel-permeation chromatography (GPC) analysis of the protected polymer, giving a polymer with a M_n=l 8,400 g/mol and a PDI of 1.2. Molecular weight and molecular weight distributions were estimated using a (GPC) (Waters Alliance 2695 Separations Module) system equipped with a TOSOH TSKgel Alpha 3000 column and a Waters 2414 refractive index detector. The columns were eluted with

dimethylformamide (DMF) containing lithium chloride (10 mM) (0.5 mL/min) at 40 °C. The molecular weights and molecular weight distributions of poly(lysine) polymers were compared to poly(styrene) standards (Sigma-Aldrich).

Deprotection of the amines was carried out (see below for procedure).

Deprotection:

The protected polymer was dissolved in dichloromethane (35 mg/mL polymer in DCM). The resulting hazy solution was stirred at room temperature under nitrogen, and trifluoroacetic acid (1 :1 DCM:TFA by volume) was added . The solution became clear immediately and was stirred for 20 minutes. The deprotected polymer was obtained after the solvent and volatile byproducts were removed by vacuum.

Shown below for a polymer prepared from Method 1 :

Poly(Boc-L-Lvsine) - Polymerization Method 2 (Scheme 4)

L-Boc-lysine-N-carboxyanhydride (500 mg) was placed in an oven dried 500 mL round-bottom flask and was purged with nitrogen. Anhydrous THF (27 mL, water content = 44 ug/mL) was added to give a clear solution. Triethylamine (37 mg) was added in one portion and the reaction was aged at room temperature under nitrogen for 15 h. The polymer was precipitated by adding the reaction mixture into 400 mL of cold water with vigorous stirring, isolated by filtration, and rinsed with an additional 500 mL of water then dried under vacuum. A white solid was obtained (412 mg). Gel-permeation chromatography (GPC) analysis of the protected polymer, giving a polymer with a M_n=l 94,000 g/mol and a PDI of 1.1. Molecular weight and molecular weight distributions were estimated using a (GPC) (Waters Alliance 2695 Separations Module) system equipped with a TOSOH TSKgel Alpha 3000 column and a Waters 2414 refractive index detector. The columns were eluted with

^!H spectra were recorded on Varian spectrometer operating at 500 MHz with a relaxation delay of 0.5 s. 1H NMR spectra were in full accordance with the expected structures. All NMR spectra were taken in deuterated DMSO. In the example below, 10 μΐ, of MeOD was also added to the polymer solution in d-DMSO.

1H of polymer prepared from method 1 :

wherein x is 50-400.

9 8 7 6 5 4 3 2 l pp«

Molecular weight and molecular weight distributions were estimated using a gel-permeation chromatography (GPC) (Waters Alliance 2695 Separations Module) system equipped with a TOSOH TSKgel Alpha 3000 column and a Waters 2414 refractive index detector. The columns were eluted with dimethylformamide (DMF) containing hthium chloride (10 mM) (0.5 mL/min) at 40 °C. The molecular weights and molecular weight distributions of poly(lysine) polymers were compared to poly(styrene) standards (Sigma-Aldrich).

A typical GPC trace is shown below;

Exemplary polymers of the instant invention made by the Schemes above include:

wherein

x is 2 to 5000; and

R is independently selected from n-butyl amine or mPEG-amine (where the PEG molecular weight can range from 500 g/mol to 12,000 g/mol), a hydrogen, hydroxyl, and carboxylate; or a stereoisomer thereof.

Polymer prepared by method 1

wherein

R is hydrogen; and

x is 50-400.

Polymer prepared by method 2

wherein,

R is hydroxyl;

R' is hydrogen; and

x is 150 to 2500.

CONJUGATION

The polymers comprising Formula Z and the specific examples shown above were synthesized for use in the following conjugation steps to ultimately create the polyconjugates of the instant invention. The polymers comprising Formula Z and the specific examples disclosed are useful in the preparation of polyconjugates of Formula I which are, in turn, useful for the delivery of oligonucleotides, specifically the delivery of siRNA. Other methods for the synthesis of polyconjugates are described in WO2008/022309. Polymer 1 (Scheme 5)

Step 1 : Activation of polymer Polymer (362 mg) was dissolved in 6.75 mL of 5 mM TAPS, pH=9. The solution was mixed until the polymer was completely dissolved and 1149 μί, of a solution of SMPT in DMSO (lmg/100 μΚ) was added (corresponding to 1.5 wt% with respect to the polymer weight).

Step 2: Activation of oligonucleotide

Oligonucleotide (lg, 0.0714 mmol) was dissolved in 0.1M sodium bicarbonate buffer (20 ml, 50 mg/mL) in a vial with magnetic stir bar and cooled to 0-5 °C in an ice water bath. In a separate vial, SATA (83 mg, 0.357 mmol, 5 equivalents) was dissolved in 0.78 mL of DMSO. The SATA solution was added over lmin and the clear, colorless reaction mixture was stirred at 0-5 °C. After 2h, the reaction mixture was sampled and analyzed by UPLC or HPLC for completion of the reaction. Additional SATA can be added to effect complete conversion of the oligonucleotide (<5% remaining unreacted). The reaction mixture was purified by tangential flow filtration (TFF) using water (~ 2L). The retentate was lyophilized to give a white solid. The recovery was ~ 95% and the purity was greater than 70% by UPLC. Step 3: Polymer-oligonucleotide conjugation

The activated polymer was diluted with 100 mM TRIS, 5% glucose, buffer at pH=9 resulting in a final polymer concentration of ~ 2.7 mg/mL. About 34 mg of

oligonucleotide was added to the acvitated polymer solution and allowed to react at room temperature for one hour until the final masking step. In situ, the primary amine on the polymer is assumed to deprotect the SATA modified siRNA to produce the free thiol siRNA, which can then react with the SMPT-modified polymer.

Step 4: Masking of the polymer conjugate

In a separate vial, 897 mg of carboxydimethylmaleic anhydride-N- acetylgalactosamine (CDM-NAG) and 445 mg of carboxydimethylmaleic anhydride poly(ethylene glycol (CDM-PEG) were weighed out. The siRNA-polymer conjugate solution was then transferred into the vial containing CDM-NAG and CDM-PEG and the resulting solution was stirred at room temperature for 10 minutes. The pH of the polyconjugate solution was 8.5).

Step 5: Purification of the polymer conjugate (optional)

Tangential flow filtration (TFF) process was used to purify polymer conjugate formulations of un-incorporated components and to exchange buffer to a pharmaceutically acceptable formulation vehicle. The TFF filter material was made of either modified polyethersulfone (PES) or regenerated cellulose. The selection of molecular weight cutoff for these membranes was done with efficiency of purification and retention of polymer conjugate in mind. The processing parameters, including but not limited to feed pressure, retentate pressure, crossflow rate and filtrate flux, were set to allow reproducibility from batch to batch and linear scaling of the process. Using the difiltration mode of TFF, the reaction impurities were filtered out into the permeate and the buffer for the retained polymer conjugate is exchanged. After TFF, the final product was concentrated to 0.4-2.0 mg/mL of siRNA and sterile filtered using a 0.2um PES syringe filter and stored at -20 °C until use.

- 18-

- 19-

wherein x is 50-2000.

Within the schemes and examples provided, polymers with randomly oriented repeating units are denoted by round brackets with a forward slash between repeating units. For example, a random copolymer of monomer A and monomer B will be represented by the formula . In contrast, a block copolymer having m repeating umts of monomer

A and n repeating units of monomer B will be represented by the following formula

POLYCONJUGATE (Scheme 6)

Exemplary polyconjugates of the instant invention made by the Scheme above include:

-21 -

-22-

wherein,

R is an end group independently selected from a hydrogen, hydroxyl, and carboxylate; and x is 50 to 2000.

EXAMPLE 1

TFA Analysis:

TFA concentration in polymer samples was determined by reversed-phase HPLC using a Waters Atlantis T3 column and mobile phases of 0.025% phosphoric acid in water and THF. Polymer samples were dissolved in water to an approximate concentration of 2-3 mg/mL prior to analysis. siRNA Conjugation Efficiency:

Free RNA duplex as well as free RNA duplex-dimer was determined by aqueous SEC using a GE Heathsciences Superdex 75HR 10/300 column. The mobile phase was composed of 1 OOmM Tris with 2M NaCl, pH 8.4. Total RNA (both free and bound) was determined by using Inductively Coupled Plasma (ICP) spectroscopy. Since the RNA is the only phosphorus containing species in the formulations, determining the total phosphorus content can be used to directly determine the total RNA concentration. Once the free RNA (duplex and duplex-dimer) and total RNA is determined, the amount of RNA conjugated to the polymer can be calculated (i.e. conjugation efficiency).

Example SEC chromatogram of a masked polymer conjugate:

Example SEC chromatogram of a masked polymer conjugate with siRNA dimer present: ree CDM

Masking Efficiency:

Total concentrations of CDM-NAG and CDM-PEG were determined using reverse-phase HPLC with mobile phases of 0.1% TFA in water and 0.1% TFA in acetonitrile. Rapid demasking of the polymer after injection onto the column allows quantitation of CDMs with the polymer removed using a CI 8 guard column to prevent chromatographic interference. Free (i.e. unbound) CDM-NAG and CDM-PEG is analyzed by first filtering through a 10K centrifuge filter followed by analysis using the same reverse-phase HPLC method. Masking Efficiency can be calculated by first calculating the bound RNA, CDM-NAG and CDM-PEG. The polymer molecular weight in combination with the total amines available for conjugation is then used with the bound ligands to calculate masking efficiency. Example chromatogram of CDM-NAG and CDM-PEG:

As shown in Figure 1, the siRNA conjugation efficiency is >90%, and the masking efficiency is ~60%.

Polymer Assay:

Quantitation of poly-L-lysine homopolymers was accomplished by derivitazation of the primary amines with TNBS (trinitrobenzene sulfonic acid) and

comparison to a polymer standard. Sample, water and 0. IN HC1 were first combined and mixed well to ensure demasking of the amines. A 0.01% TNBS solution prepared using DMSO and sodium borate was then mixed with the sample and the final solution dispensed to a 96 well plate. A similarly prepared polymer standard covering a concentration range of 0 - 20ug/mL was also dispensed into the 96 well plate. The standard used must match the polymer used in the formulation for accurate quantitation.

EXAMPLE 2

RBC Hemolysis Assay:

Human blood was collected in 10ml EDTA Vacutainer tubes. A small aliquot was assessed for evidence of hemolysis by centrifugation at 15000 RCF for 2 min and non- hemolyzed samples were carried forward into the assay. Red blood cells (RBCs) were washed three times in either 150mM NaCl/20mM MES, pH 5.4, or 150mM NaCl/20mM HEPES, pH 7.5 by centrifuging at 1700 x g for 3 min and resuspending in the same buffer to yield the initial volume. RBCs were then diluted in appropriate pH buffer to yield 10 cells in suspension. A lOx stock concentration of the polymer was prepared and a 10 point, 2-fold dilution was performed in appropriate pH buffers. The diluted test agents were added to the RBCs in appropriate pH buffers in Costar 3368 flat-bottom 96 well plates. Solutions were mixed 6 to 8 times and the microtiter plate was covered with a low evaporation lid and incubated in a 37°C warm room or incubator for 30 minutes to induce hemolysis. The plate was then centrifuged at 1700 x g for 5 min and 150 μΐ supernatants were transferred to a Costar 3632 clear bottom 96 well plate. Hemoglobin absorbance was read at 541nM using a Tecan Satire plate reader and percent hemolysis was calculated assuming 100% lysis to be measured by the hemoglobin released by RBCs in 1% Triton X-100.

As shown in Figure 2, the data demonstrate that the polymers are non lytic at either pH tested.

EXAMPLE 3

HepG2 gene silencing and toxicity data:

HepG2 cells were plated in 96-well microtiter plates at 6000 cells/well and incubated overnight at 37 °C to allow cell adherence. lOx stock of PCs (polyconjugates) were prepared in media and 20μ1 lOx PC was added to 180 μΐ media already in wells resulting in lx final treatment and a 300-0 nM 10-point half log titration, based on siRNA concentration. Cells were incubated with PCs in 37 degrees C0₂ incubator for 24 -72h. MTS Toxicity Assay was performed on 24h - 72h treated cells and cytotoxicity was assessed by CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega #G3581, Madison, WI). 40μ1 MTS Solution was added, incubated in 37 degrees C0 incubator 1 hour, absorbance at 490nm was read on Tecan Satire. Cells were then washed 3x in PBS and 150μ1Λνε11 bDNA DLM Lysis Buffer (Panomics "Quantigene" 1.0 bDNA kit #QG0002, Fremont, CA) was added. Plate was then incubated at 37 degrees for 30 min. Lysates were removed and frozen at -70 degrees C overnight. The next day, all cell lysates were thawed at RT and 20μ1 of each lysate was removed and used for determination of total protein using Micro BCA Protein Assay kit (Pierce #23235, through Thermo Scientific, Rockford, IL). Absorbance was measured on Tecan Satire: Wavelength = 562nM, Plate = Costar96ft, Number of Reads = 100, Time between Reads = 5. 50μ1 each lysate was also used to determine mRNA expression levels in cells treated with SSB siRNA.

Apolipoprotein B (ApoB) mRNA knockdown was determined using Quantigene

1.0 bDNA Assay (Panomics # QG0002 Lot # 51CW36, Fremont, CA), a kit designed to quantitate RNA using a set of target-specific oligonucleotide probes.

Oligonucleotide synthesis is well known in the art. (See US patent applications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US

2009/0285881 and PCT patent applications: WO 2009/086558, WO2009/127060,

WO2009/132131, WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 and WO2010/054406). The siRNAs disclosed and utilized in the Examples were synthesized via standard solid phase procedures.

ScilO ApoB siRNA was utilized in the experiments.

ScilO ApoB siRNA

5'-iB-CUUUA4CA4UUCCUGA4^UTsT-iB-3' (SEQ ID NO.: 1) 3'-UsUG^4U^/GUUA4GG^CUsUsUsA-5' (SEQ ID NO.:2)

U - Ribose iB - Inverted deoxy abasic

AGU- 2' Fluoro

T - 2^* Deoxy

CU - 2* OCH₃

s - phophorothioate linkage

Low Hex 9 siR A was utilized in the experiments as a control siRNA.

Low Hex 9 siRNA

5^,-amil-iB-C^ AG ί/GGACA Gi7CGAί7ATsT-iB-3^, (SEQ ID NO.: 3)

3'-UsUGA^CGACCC/GC/GCAGCUAU-5' (SEQ ID NO.: 4)

amil - amino linker

iB - Inverted deoxy abasic

Ct/- 2*-Fluoro (F)

AGT - 2^*-Deoxy

UGA - 2'-Methoxy (OMe)

AU - Ribose

s - phosphorothioate linkage

Day 1

Make diluted lysis mixture (DLM) by mixing 1 volume of lysis mixture with 2 volumes of Nuclease Free water (Ambion cat # AM9930). Aspirate (PBS) from plate. Add 150μ1 DLM to each well and mix. (Include Column 1 as Buffer Alone Background). Incubate at 37°C for 30 minutes. (After heating, Lysates can be placed in the -70°C freezer until analysis is performed. If lysates are frozen, thaw at Room Temperature and incubate at 37°C for 30 minutes and mix well before adding the samples to the capture plate.) Bring all reagents to Room Temperature before use, including the capture plates. Dilute CE, LE and BL probe set components: Ο.ΐμΐ/well each into DLM. Add (100-X) μΐ diluted probe set/well. Add ( X ) μΐ cell lysate/well. Cover with foil plate sealer. Incubate at 53 °C for 16-20 hrs. Note: If assay contains multiple plates, perform steps 7, 8, 9 on 2-3 plates at a time and place at 53 °C before going on to next 2-3 plates.

Day 2

Bring Amplifier, Label Probe and Substrate to Room Temperature. Vortex and briefly centrifuge the tubes of Amplifier and Label Probe to bring the contents to the bottom of the tube. Prepare Wash buffer: add 3ml Component 1 and 5ml Component 2 to 1L distilled water. (Wash Buffer is stable at Room Temperature for up to 6 months)

Prepare as needed: Amplifier Working solution, Label Probe Working Solution, and Substrate Working Solution:

Amplifier Working Solution - 1 : 1000 dilution into Amplifier/Label Probe diluent. Label Probe Working solution - 1:1000 dilution into Amplifier/Label Probe diluent.

Substrate Working Solution - 1 :333 dilution of 10% Lithium Lauryl Sulfate Substrate into

Substrate Solution (protect from light).

Add 200μ1 /well of wash buffer to overnight hybridization mixture. Repeat washes 3x with 300μ1 of Wash Buffer. *Do not let the capture plates stand dry for longer than

5 minutes. Add ΙΟΟμΙ/well of Amplifier Working Solution. Seal plate with clear seal and incubate at 53°C for 30 minutes. Wash plate 3x with 300μ1 of Wash Buffer. Add ΙΟΟμΙ/well of

Label Probe Working Solution. Seal plate with clear seal and incubate at 53°C for 30 minutes.

Wash plate 3x with 300μ1 of Wash Buffer. Add ΙΟΟμΙ/well Substrate Working Solution. Seal plate with foil seal and incubate at 53°C for 15 minutes. Let plate stand at Room Temperature for 10 minutes. Read in luminometer with integration time set to 0.2 seconds. bDNA data was normalized to protein and graphed using GraphPad Prism® Program using non-linear regression curve fit analysis.

As shown in Figure 3, the data demonstrate an IC50 of >300 nM for the polyconjugate prepared from a poly(lysine) homopolymer with an MTS IC50 of >300 nM.

In Vivo Evaluation of Efficacy

CD1 mice were tail vein injected with the siRNA containing polymer conjugates at a dose of 3, and 6 mg/kg. In the case of rat studies, Sprague-Dawley rats were used. Rats were dosed at 3, 6, 9, and 12 mg/kg.

Five days post dose, mice were sacrificed and liver tissue samples were immediately preserved in RNALater (Ambion). Preserved liver tissue was homogenized and total RNA isolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNA isolation kit following the manufacturer's instructions. Liver ApoB mRNA levels were determined by quantitative RT-PCR. Message was amplified from purified RNA utilizing primers against the mouse ApoB mRNA (Applied Biosystems Cat. No. Mm01545156_ml). The PGR reaction was run on an ABI 7500 instrument with a 96- well Fast Block. The ApoB mRNA level is normalized to the housekeeping PPIB mRNA and GAPDH. PPIB and GAPDH mRNA levels were determined by RT-PCR using a commercial probe set (Applied Biosytems Cat. No.

Mm00478295_ml and Mm4352339E_ml). Results are expressed as a ratio of ApoB mRNA/ PPIB / GAPDH mRNA. All mRNA data is expressed relative to the vehicle control.

Alanine aminotransferanse (ALT) was measured using the AD VIA Chemistry Systems Alanine Aminotransferase (ALT) method, 03815151, Rev. A., according to the following reference, Clinical and Laboratory Standards Institute. Laboratory Documents:

Development and Control; Approved Guideline - Fifth Edition. CLSI document GP2-A5

[ISBN 1-56238-600-X]. Clinical and Loboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania, 19807-1898 USA, 2006.

As shown in Figures 4 and 5, the data demonstrate that the polyconjugates of the instant invention can deliver siRNA to hepatocytes in rat. In Figure 5, a polyconjugate prepared from a poly(L-lysine) (85,000 g/mol) showed ~80% knockdown of ApoB with a 3 mg/kg dose in rat (at a 5 day timepoint), with no increase in liver or kidney toxicity markers.

As shown in Figure 6, polyconjugates prepared with poly(L-lysine) (85,000 g/mol) show no increase in liver or kidney toxicity markers (up to 12 mpk at a 48 hour timepoint).

Claims

WHAT IS CLAIMED IS:

1. A polymer comprising Formula Z:

Z wherein: x is 50 to 2000;

R is independently selected from an end group; Ri is butyl amine;

R_a is independently hydrogen, methyl, ethyl, propyl or butyl optionally substituted with NH₂ and OH; and

R_b is independently hydrogen, methyl, ethyl, propyl or butyl optionally substituted with N¾ and OH; or stereoisomer thereof.

2. A polyconjugate of Formula I:

I wherein: x is 50 to 2000;

R is independently selected from an end group;

Ri is independently selected from butyl amine, butyl amine with a linker-oligonucleotide, butyl amine with a linker-targeting ligand and butyl amine with a linker-PEG;

Rb is independently hydrogen, methyl, ethyl, propyl or butyl optionally substituted with N¾ and OH; or stereoisomer thereof.

3. A polyconjugate composition comprising the polymer of Formula Z of Claim 1 and a linker-oligonucleotide.

4. The polyconjugate composition of Claim 3 further comprising a linker-

PEG.

5. The polyconjugate composion of Claim 4 further comprising a linker- targeting ligand.