In vivo polynucleotide delivery conjugates having enzyme sensitive linkages.

POLYNUCLEOTIDE SUPPLY CONJUGATES '¡WTVTVO

THEY HAVE SENSITIVE COUPLINGS · Λ ΒΝΠΙΐ »

Background of the Invention

The supply of polynucleotide and other substantially cell membrane impermeable compounds into a living cell is highly restricted by the complex membrane system of the cell. Drugs used in antisense, RNAi, and gene therapies are relatively large and frequently highly negatively charged hydrophilic polymers. Both physical characteristics severely restrict its direct infusion through the cell membrane. For this reason the main barrier to polynucleotide delivery is delivery of the polynucleotide across a cell membrane to the cytoplasm of the cell or nucleus.

One means that has been used to deliver small nucleic acid in vivo has been to couple the nucleic acid to either a small target molecule or a lipid or sterol. Although some delivery and activity have been observed with these conjugates, the dose of nucleic acid required with these methods is prohibitively large.

Numerous transfection reagents have also been developed that obtain a reasonably efficient delivery of polynucleotides to cells in vitro however,

REF. 264411 in vitro delivery of polynucleotides using the same transfection reagents is complicated and rendered ineffective through in vivo toxicity, adverse serum interactions, and poor targeting. Transfection reagents that work well in vitro, cationic polymers and lipids, typically form large cationic electrostatic particles and destabilize cell membranes. The positive charge of in vitro transfection reagents facilitates association with nucleic acid through charge-charge (electrostatic) interactions thereby creating the nucleic acid / transfection reagent complex. The positive charge is also beneficial for the non-specific binding of the vehicle to the cell and for the fusion, destabilization and alteration of the membrane. The destabilization of membranes facilitates delivery of substantially cell membrane impermeable polynucleotides across a cell membrane. Although these properties facilitate nucleic acid transfer in vitro, they cause toxicity and ineffective targeting in vivo. The cationic charge results in interaction with serum components, which causes destabilization of the polynucleotide-transfection reagent interaction, poor bioavailability, and poor targeting. The membrane activity of the transfection reagents,

ΙΜΡΙ6

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

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which can be effective in vitro, generally leads to in vivo toxicity.

For in vivo delivery, the carrier (the associated nucleic acid and delivery agent) must be small, less than 100 nm in diameter and preferably less than 50 nm. Even minor complexes, less than 20 nm or less than 10 nm would be more useful. Delivery vehicles larger than 100 nm have very little access to cells other than blood vessel cells in vivo. Complexes formed by electrostatic interactions tend to aggregate or separate when exposed to concentrations of physiological salt or serum components. Furthermore, the cationic load on delivery vehicles in vivo leads to adverse serum interactions and consequently poor bioavailability. Interestingly, the high negative charge can also inhibit target-directed delivery in vivo through interference with interactions necessary for targeting, ie, the binding of target-directed ligands to cellular receptors. In this way, near-neutral vehicles are desired for in vivo delivery and steering. Without careful regulation, membrane disrupting or destabilizing activities are toxic when used in vivo. In the balance between the toxicity of the vehicle and the delivery of the nucleic acid

WICKED ^ ^iwmia moníoai »are obtained much more easily in vitro than in vivo.

Rozema and others, in Publication -'dé * 'Pá'tente de EU

A. 20080152661, provide means for reversibly regulating the membrane-altering activity of a membrane-active polyamine using disubstituted maleic anhydride modification. Maleamate couplings, formed by the reaction of a maleic anhydride with an amine, are pH labile in a pH range suitable for in vivo delivery. This procedure allowed the use of membrane-active polymers for in vivo or nucleic acid delivery. Modified membrane-active polymers having dipeptide-amidobenzyl-carbamate linkages are now provided. Dipeptide-amidobenzyl-carbamate couplings are reversible and physiologically sensitive. Unlike the pH-labile maleamate couplings of disubstituted maleic anhydride modification, coupling of the polymer modifying agents described herein generates enzymatically targetable couplings that are acceptable in vivo circulation.

Brief Description of the Invention

In a preferred embodiment a masking agent is described to reversibly modify and inhibit the membrane activity of an active polyamine in the antipathic membrane comprising: a spherical stabilizer ⁵ WICKED

MEXICAN INSTITUTE

Dt INDUSTRIAL PROPERTY ------- or a targeted ligand coupled to a dipeptidoamidobenzyl carbonate, referred to herein as dipeptide masking agents or protease cleavage masking agents. Dipeptide masking agents have the general form:

R-AlA.²-amidobenzyl carbonate.

where R is a spherical stabilizer, or a targeted ligand, A¹ is an amino acid, and A² it is an amino acid. The carbonate reaction of the masking agent! with a polymeric amine produces a carbonate coupling. The masking agent is stable even when the dipeptide is cleaved in vivo through an endogenous protease, thereby separating the spherical stabilizer or the targeted ligand from the polyamine. After enzymatic separation after the dipeptide (between A² and amidobenzyl), amidobenzyl carbamate undergoes spontaneous reconfiguration resulting in regeneration of a polymer amine. Preferably R is neutral. More preferably, R is unloaded. A preferred spherical stabilizer is polyethylene glycol (PEG). A targeting ligand can be selected from the list comprising haptens such as digoxigenin, vitamin such as biotin, antibody, monoclonal antibody, and cell surface receptor ligand. A targeted ligand can bind to

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OF THE FKLIHEDAU. -,,,. . INDUSTRIAL dipeptide through a linker such as a PEG linker.

A preferred cell surface receptor ligand ..... is an asialoglycoprotein receptor ligand (ASGPr). A preferred ASGPR ligand is one N-acetylgalactosamine (NAG). A preferred dipeptide consists of a hydrophobic amino acid linked to a hydrophilic uncharged amino acid through an amide bond. A preferred amidobenzyl group is a p-amidobenzyl group. A preferred carbonate is a reactive activated amine carbonate.

In a preferred embodiment, the invention provides a composition for delivering an RNA interference polynucleotide (iRNA) to a cell in vivo comprising: a masked antipathetic membrane active polyamine (delivery polymer) wherein the polyamine is masked through the reversible modification with the dipeptide masking agents described herein and an iRNA polynucleotide. The delivery polymer can be covalently linked to the iRNA polynucleotide. A preferred bond for covalent coupling of the delivery polymer to the iRNA polynucleotide is a physiologically labile bond. In one embodiment, this bond is orthogonal to the bond of the dipeptide masking agent. Alternatively, the delivery polymer is not covalently linked to the iRNA polynucleotide and the iRNA polynucleotide is covalently linked to a targeted molecule.

<img file="MX347298B_D0002.tif" />

In a preferred embodiment, it is described¹ A composition comprising: an antipathic membrane active polyamine covalently linked to: a) a plurality of targeted ligands or spherical stabilizers through non-coupling dipeptide-amidobenzylcarbonate bonds; and b) one or more polynucleotides through one or more reversible couplings. In one embodiment, the dipeptide-amidobenzyl carbamate coupling is orthogonal to the reversible covalent polynucleotide bond. The polynucleotide-polymer conjugate is administered to a mammal in a pharmaceutically acceptable carrier or diluent.

In a preferred embodiment, a composition is disclosed comprising: a) an antipathic membrane active polyamine covalently linked to a plurality of targeted ligands or spherical stabilizers via dipeptide-amidobenzylcarbamate linkages; and b) an RNAi polynucleotide covalently linked to a targeted group selected from the list of: a hydrophobic group having 20 or more carbon atoms and a galactose aggregate. In this embodiment, the iRNA polynucleotide is not covalently bound to the modified antipathic membrane-active polyamine. Modified polyamine and target group-RNAi polynucleotide conjugate are synthesized separately and can be supplied in separate packages^-© in single container. The modified polyamine and the target group-iRNA polynucleotide conjugate are administered together or separately to a mammal in pharmaceutically acceptable carriers or diluents.

A preferred dipeptide masking agent comprises a reactive dipeptide-p-amidobenzyl amine carbonate cleavable protease (peptidase) derivative. The protease removable masking agents of the invention utilize a dipeptide linked to a moiety of activated amidobenzyl carbonate. A spherical stabilizer or targeted ligand is coupled to the amino terminus of a dipeptide. The activated amidobenzyl carbonate portion attaches to the carboxy-terminus of the dipeptide. Removable protease linkers suitable for use with the invention have the general structure:

<img file="MX347298B_D0003.tif" />

wherein R4 comprises a spherical stabilizer or targeted ligand, R3 comprises a reactive amine carbonate portion, and R1 and R2 are amino acid side chains. A preferred activated carbonate is paranitrophenol. However, other reactive amine carbonates

<img file="MX347298B_D0004.tif" />

known in the art can easily be substituted for para-nitrophenol. The reaction of the activated carbonate with the amine connects the targeted ligand or spherical stabilizer with the membrane-active polyamine through a cleavable dipeptide coupling of amidobenzyl carbamate peptidase. Separation of the enzyme from the dipeptide, between the amino acid and the amidobenzyl group removes R4 from the polymer and activates an elimination reaction that results in regeneration of the amine from the polymer.

The dipeptide masking agents of the invention are useful for reversible modification / inhibition of active polyamines in the antipathic membrane. A covalent bond is created through the reaction of the activated carbonate of the dipeptide masking agent with a polymeric amine, particularly a primary amine group. Therefore, provided herein is a conjugate comprising a dipeptide-amidobenzylcarbonate masking agent described herein and an antipathic membrane-active polyamine:

R1 HO

<img file="MX347298B_D0005.tif" />

polyamine

The compounds according to the present invention can generally be obtained using known methods.

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by one skilled in the art of organic or medicinal chemistry. Additional objects, features, and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying figures.

Brief Description of Figures

Figure 1. Illustration showing the structure of the dipeptide masking agent where:

R1 and R2 are R groups of amino acids,

R4 is a targeted ligand of a steric stabilizer,

X is -NH-, -O-, or -CH2-,

Y is -NH- or -OR5 is in position 2, 4, or 6 and is -CH2-OC (0) -0-Z where Z is carbonate, and

R6 is independently hydrogen, alkyl, or halide at each of the 2, 3, 4, 5, or 6 positions except for the position occupied by R5.

Figure 2. Illustration showing the structure of the dipeptide masking agent linked to a polyamine where: R1 and R2 are R groups of the amino acids, R4 is a targeted ligand of a steric stabilizer, X is -NH-, -0 -, or -CH₂-, and Y is -NH- or -0-.

Figures 3A-3J. Illustration showing the structures of various dipeptide masking agents.

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Figure 4. Graph illustrating the circulation times of polymers modified with a dipeptide masking agent against two different maleic anhydride-based masking agents.

Detailed description of the invention

Masking agents useful for the reversible modification and inhibition of active polyamines in the amphipathic membrane and delivery polymers formed by modifying the polyamine through dipeptide masking agents are described. The cleavable peptidase bonds are stable to hydrolysis in the absence of the enzyme, electrically neutral, and provide extended DPC stability in storage and circulation in vivo. The improved (longer) half-life in circulation facilitates the widening of the window of opportunity for accumulation as measured by the ligand in tissues, such as tumor tissue. Delivery polymers are particularly useful for in vivo delivery of iRNA polynucleotide. In vivo delivery of iRNA polynucleotides is useful for the therapeutic inhibition (destruction) of gene expression.

Dipeptide masking agents have the general form:

RA ^ -amidobenzyl-carbonate.

where R is a spherical stabilizer or a target-directed ligand, A¹ is an amino acid, A² is an amino acid,

<img file="MX347298B_D0007.tif" />

activated. R is preferably uncharged — Reaction of T carbonate of the masking agent with a polymeric amine produces a carbamate coupling. The masking agent is stable until the dipeptide is removed in vivo via an endogenous protease, thereby separating the spherical stabilizer or the targeted ligand from the polyamine. After enzymatic separation after the dipeptide (between A² and amidobenzyl), amidobenzyl carbamate undergoes spontaneous reconfiguration resulting in regeneration of the polymeric amine. A preferred spherical stabilizer is polyethylene glycol (PEG). A preferred target ligand for delivery to the liver is the ASGPr ligand. A preferred ASGPr ligand is an N-acetylgalactosamine (NAG). A preferred amidobenzyl group is a p-amidobenzyl group.

The dipeptides of the dipeptide masking agents, represented as A^XTO² (or AA), are amino acid dimers connected through amide bonds. Amino acids, including the amino acids oi and β that are well known in biology and chemistry and are molecules that contain an amine group, a carboxylic acid group, and a side chain that varies between different amino acids. A preferred amino acid is an α-amino acid having the generic formula H2NCHRCOOH, where R is an organic substituent.

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A preferred alpha amino acid is a naturally occurring uncharged amino acid. In preferred dipeptide, Al is a hydrophobic amino acid and A2 is an uncharged hydrophilic amino acid. A preferred hydrophobic amino acid is phenylalanine valine, isoleucine, leucine, alanine, or tryptophan. A preferred uncharged hydrophilic amino acid is asparagine, glutamine, or citrulline. A more preferred hydrophobic amino acid is phenylalanine or valine. A more preferred uncharged hydrophilic amino acid is citrulline. Although dipeptides are preferred, it is possible to insert additional amino acids between A¹ and R. It is possible to use a single amino acid instead of a dipeptide by removing amino acid A¹. Any natural amino acid used in the present invention is referred to herein by its common abbreviations. Although charged amino acids can be used, it is preferred that the masking agent is uncharged.

In a preferred embodiment, an antipathic membrane active polyamine is reversibly modified through reaction with a dipeptideamidobenzyl carbonate masking agent of the invention to produce an inactive membrane delivery polymer. Dipeptide masking agents can protect the polymer from nonspecific interactions, increase circulation time, enhance specific interactions, inhibit toxicity, or alter polymer charge,

<img file="MX347298B_D0008.tif" />

Polymers reversibly

INSTITUTO MEXICANO DE LA PROPIEDAIi INDUSTRIAL masked! * Invention comprise the structure:

R1 h or H

<img file="MX347298B_D0009.tif" />

polyamine where:

X is -NH-, -O-, or -CH₂Y is -NH- or -OR1 is preferably

- (CH₂) kf enyl (k is 1, 2, 3, 4, 5, 6k = 1 phenylalanine),

-CH- (CH₃)₂ (valine),

-CH2-CH- (CH₃) 2 (leucine),

-CH (CH₃) -CH₂-CH₃ (isoleucine),

-CH₃ (to the girl),

- (CH₂)₂-COOH (glutamic acid),

<img file="MX347298B_D0010.tif" />

R2 is preferably hydrogen (glycine)

- (CH₂) ₃-NH-C (O) -NH₂ (citrulline),

<img file="MX347298B_D0011.tif" />

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- (CH₂) 4-N- (CH₃) 2 (lysine (CH₃) ₂) , ^{, ND} ~ (CH2) k -C (0) -NH₂; (k is 1, 2, 3, 4; -97 bFT

- CH₂-C (0) -NH₂ (asparagine),

- (CH₂) ₂-C (0) -NH₂ (glutamine),

- CH₂-C (0) -NR ^ -R² (aspartic acid amide),

- (CH₂) 2-C (0) -NR ^² (glutamic acid amide),

- CH₂-C (O) -0R¹ (aspartic acid ester), or

- (CH₂) ₂-C (0) -0R¹ (glutamic acid ester),

R¹ and R² are alkyl groups

R4 comprises a polyethylene glycol or a targeted ligand; and polyamine is an antipathic membrane active polyamine.

Although the above structure indicates a single polymer-bound dipeptide masking agent, in the practice of the invention, 50% to 90% or more of the polymeric amines are modified through the dipeptide masking agents.

In a preferred embodiment, the reversibly masked polymer of the invention comprises the structure:

<img file="MX347298B_D0012.tif" />

wherein Rl, R2, R4 and the polyamine are as described above.

<img file="MX347298B_D0013.tif" />

The reversibly masked polymers of the invention are formed through the reaction of dipeptide masking agents of the invention with amines in the polymer. The dipeptide masking agents of the invention have the structure:

<img file="MX347298B_D0014.tif" />

where:

X, Y, Rl, R2, and R4 are as described above

R5 is in position 2, 4, or 6 and is -CH₂-OC (0) -OZ where Z is

-Haluro,

<img file="MX347298B_D0015.tif" />

<img file="MX347298B_D0016.tif" />

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OF INDUSTRIAL PROPERTY

R6 is independently hydrogen, alkyl, - (CH2) n-CH3 (where n = 0-4), - (CH2) - (CH3) 2, or halide at each of the 2, 3, 4, 5 positions, 06 except for the position occupied by R5

In a preferred embodiment, X is -NH-, Y is -NH-, R4 is uncharged, R5 is at the 4-position, and R6 is hydrogen as shown by:

<img file="MX347298B_D0017.tif" />

In another embodiment, R4 is:

R- (O-CH2-CH2) sO-Yl-, where

R is hydrogen, methyl, or ethyl; ys is = to an integer from 1 to 150, and Y1 is a linker selected from the list comprising:

-O-Y2-NH-C (0) - (CH2) ₂-C (0) -, where Y2 is

- (CH₂)₃-

-C (0) -N- (CH2-CH2-O) P-CH2-CH2- (p is an integer from 1 to 20), and -O-.

A targeted ligand can be selected from the list comprising hapten, vitamin, antibody, monoclonal antibody, and ιμριλ ^ receptor ligand.

INSTITUTO MEXICANO 1J

I heard the industrial froredao&gt; nZ * J5 * cell surface. A targeted ligand can be linked to the dipeptide through a linker such as a PEG linker.

Non-limiting examples of suitable membrane-active polymers for use with the invention have been previously described in US Patent Publications 20080152661, 20090023890, 20080287630, and 20110207799. Suitable antipathic membrane-active polyamine can also be small peptides such as a melittin peptide.

The polymeric amines were reversibly modified using the enzyme separation linkers described herein. An amine is reversibly modified if removal of the modifying group results in regeneration of the amine. Reaction of the activated carbonate of the masking agent with a polymeric amine connects a targeted ligand or spherical stabilizer to the polymer through a removable peptidase through dipeptide-amidobenzyl carbamate coupling as shown:

or

<img file="MX347298B_D0018.tif" />

R¹ comprises a targeted ligand (either with or without protecting groups) or a PEG,

R² it is an active polyamine in the antipathic membrane,

<img file="MX347298B_D0019.tif" />

IMPI

MtX'CANO INSTITUTE OF INDUSTRIAL PROPERTY

ΑΆ is a dipeptide (either with _ or without__.^nriprotectors), and

Z is an amine reactive carbonate.

Protecting groups can be used during the synthesis of dipeptide masking agents. If present, the protecting groups can be removed before or after modification of the active polyamine in the antipathic membrane.

The reversible modification of a sufficient percentage of the polymeric amines with the dipeptide masking agents inhibits the membrane activity of the membrane-active polyamine. Modification of the polymeric amines with dipeptide masking agents which also preferably neutralize the amine charge. The dipeptide-amidobenzylcarbamate coupling is susceptible to protease (or peptidase) cleavage. In the presence of protease, the anilide bond is separated, resulting in an intermediate that immediately undergoes a 1,6-elimination reaction to release the free polymer:

<img file="MX347298B_D0020.tif" />

1.6-h elimination₂nr²

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OF INDUSTRIAL PROPERTY

In the above reaction scheme, ΆΑ is a dipeptide, R¹ comprises a targeted ligand or steric stabilizer, and R² It is an active polyamine in the antipathic membrane. Importantly, the free polymer is unmodified and therefore membrane activity is restored.

In the masked state, the polyamine active in the irreversibly masked membrane does not exhibit membrane disrupting activity. The reversible modification of more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90 % of the amines in the polyamine with dipeptide masking agents may be required to inhibit membrane activity and provide cell targeting function, ie, of a reversibly masked membrane active polymer (delivery polymer).

The present invention also provides a method of delivering a biologically active substance into the cell. More specifically the present invention is not directed to compounds, compositions, and methods useful for delivering iRNA polynucleotide mammalian cells in vivo.

In one embodiment, the iRNA polynucleotide is linked to the delivery polymer of the invention through a

<img file="MX347298B_D0022.tif" />

INSTITUTO MEXICANO '0 £ INDUSTRIAL PROPERTY

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physiologically labile covalent bond. Through the use of a physiologically labile bond, the polynucleotide can be separated from the polymer, releasing the polynucleotide to connect in functional interactions with cellular components.

The invention includes conjugated delivery systems of the general structure:

NLP ' ^XM²z, where N is an iRNA polynucleotide, L¹ is a physiologically labile bond, P is an amphipathic membrane active polyamine, M¹ is an activating ligand linked to P through a dipeptide-amidobenzylcarbamate coupling, and M² is a spherical stabilizer linked to P through a dipeptide-amidobenzyl-carbamate coupling. yez are each integer greater than or equal to zero as long as y + z has a value greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90% of the primary amines of the polyamine P, as determined by the amount of amines in P in the absence of any masking agent. In its unmodified state, P is a membrane-active polyamine. Supply polymer M¹yPM²z is not active on the membrane. The reversible modification of the primary amines P, through the coupling of M¹ and / or M², reversibly inhibits or inactivates P membrane activity. Some small antipathic membrane active polyamines, such as melittin peptide, are observed to contain as little as 3-5 primary amines. Modification of a percentage of amines means that it reflects the modification of a percentage of amines in a population of polymers. After separation from M¹ and M², the amines of the polyamine are therefore collected by reverting P to its membrane-active state, unmodified.

In another embodiment, the iRNA polynucleotide is co-administered in vivo with a delivery polymer of the invention. Thus, the invention includes compositions of the general structure:

Μ \ -Ρ-Μ²_ζ plus NT, where N is an iRNA polynucleotide, T is an activating group, P is an amphipathic membrane active polyamine, M¹ is an activating ligand linked to P through a dipeptide-amidobenzyl-carbamate coupling, and M²It is a spherical stabilizer linked to P through a dipeptide-amidobenzyl-carbamate coupling. yez are integers greater than or equal to zero as long as y + z have a value greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90% of the primary amines in polyamine P , as determined by the amount of amines in P in the absence of any masking agent. In its modified state, P is a membrane-active polyamine. The

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From the hrofieoa:&gt; INDUSTRIAL .— »· * supply polymer Μ \ -Ρ-Μ²ζ is not active on the membrane. The reversible modification of the primary amines ”p7 through the coupling of M¹ and / or M², reversibly inhibits or inactivates P membrane activity. Some small antipathic membrane-active polyamines, such as the militin peptide, are found to contain as little as 3-5 primary amines. Accordingly, a modification of a percentage of the amines means that it reflects the modification of a percentage of amines of a population of polymers. After separation from M¹ and M², the polyamine amines are therefore regenerated by reverting P to its membrane active state, unmodified. N is linked to T through a covalent bond to form a conjugate of the target group iRNA polynucleotide using standard methods in the art. A preferred covalent bond is a physiologically labile bond. NT. The supply polymer and NT are synthesized or made separately. Neither T nor N are covalently linked directly or indirectly to P, M¹ or M². Electrostatic or hydrophobic association of the polynucleotide or polynucleotide conjugate with the masked or unmasked polymer is not required for in vivo liver delivery of the polynucleotide. The masked polymer and the polynucleotide conjugate can be supplied in the same container or in separate containers. I know

<img file="MX347298B_D0024.tif" />

INSTITUTO MEXICANO DE LA RROHEDAD INDUSTRIAL can be combined before administration, co-administered. _____ or administered sequentially.

For hepatocyte delivery, if the iRNA polynucleotide binds to the delivery polymer through a covalent bond or is co-administered with the delivery polymer, and has a value greater than 50% and up to 100% of the amines primary amines in polymer P. z therefore has a value greater than or equal to zero percentage (0%) but less than 50% in the primary amines in polymer P. 1

For delivery to liver tumor cells, z can be greater than 100% of the primary amines in polymer P. In a preferred embodiment, to deliver tumor cells, z is greater than 50%, greater than 60% , greater than 70%, greater than 80% or greater than 90% of the primary amines in the polyamine P ey is zero.

Membrane-active polyamines are capable of disrupting plasma membranes or lysosomal / endocytic membranes. This membrane activity is an essential feature for cellular delivery of the polynucleotide. The membrane activity, however, leads to toxicity when the polymer is administered in vivo. Polyamines also readily interact with many anionic components in vivo, leading to unwanted biodistribution. Consequently, reversible masking of the membrane activity of the

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX347298B_D0025.tif" />

Polyamine is required for in vivo use.

This masking is accomplished through the reversible coupling of the described dipeptide masking agents to the membrane-active polyamine to form a reversibly masked membrane-active polymer, ie, a delivery polymer. In addition to inhibiting membrane activity, masking agents protect the polymer from non-specific interactions, reduce serum interactions, neutralize the amine to reduce positive charge, and form a near-neutrally charged polymer, increase circulation time, and / or or they provide cell-specific interactions, i.e., direction.

It is an essential characteristic of masking agents that, in aggregates, inhibit the activity on the polymer membrane. Many masking agents can protect the polymer from non-specific interactions (reduce serum interactions, increase time in circulation) Membrane-active polyamine is membrane-active in the unmodified (unmasked) and non-active (inactivated) state in the modified (masked) state. A sufficient number of masking agents bind to the polymer to obtain the desired level of inactivation. The desired level of polymer modification through coupling of agent (s) is readily determined using appropriate polymer activity assays. For²⁶ IMPI

INSTITUTO MEXICANO DI LA TRONEDAO INDUSTRIAL For example, if the polymer possesses membrane activity in a given assay, a sufficient level of masking agent binds to the polymer to obtain the desired level of inhibition of membrane activity in this assay. Masking requires modification of ^ 50%, ^ 60%, ^ 70%, ^ 80%, or ^ 90% of the primary amine groups in a polymer population, as determined by the amount of primary amines in the polymer in the absence of any masking agent. It is also a preferred characteristic of masking agents that their coupling to the polymer reduces the positive charge of the polymer, thus producing a more neutral delivery polymer. It is desired that the masked polymer retain aqueous solubility.

The membrane-active polyamine can be conjugated to masking agents in the presence of excess masking agents. Excess masking agent can be removed from the conjugated delivery polymer prior to administration of the delivery polymer.

As used herein, a spherical stabilizer is a non-ionic hydrophilic polymer (whether natural, synthetic, or non-natural) that prevents or inhibits intramolecular or intermolecular interactions of a polymer to which it is coupled relative to the polymer that does not contain the sterile stabilizer. A spherical stabilizer inhibits the

<img file="MX347298B_D0026.tif" />

<img file="MX347298B_D0027.tif" />

connection of the polymer to which it is coupled in electrostatic interactions. Electrostatic interaction is the non-covalent association of two or more substances due to the attractive forces between positive and negative charges. Steric stabilizers can inhibit interaction with blood components and therefore opsonization, phagocytosis, and absorption through the reticulum endothelial system. Steric stabilizers in this way can increase the circulating time of the molecules to which they are attached. Steric stabilizers can also inhibit the aggregation of a polymer. A preferred spherical stabilizer is a polyethylene glycol (PEG) or PEG derivative. As used herein, a preferred PEG can have from about 1-500 ethylene glycol monomers, 225 monomers. As used herein, a preferred PEG can also have an average molecular weight of about 85-20,000 Dathons (Da), about 85-1000 Da. As used herein, steric stabilizers prevent or avoid intramolecular or intermolecular interactions of a polymer to which they are coupled relative to the polymer that does not contain spherical stabilizer in aqueous solution.

Targeted ligands enhance the pharmacokinetic or biodistribution properties of a conjugate to which they are coupled to enhance cell or tissue specific distribution and cell specific uptake of the conjugate. As used herein, for clarity, the term target-directed ligand is used to denote a target-directed ligand that is coupled to a dipeptide masking agent, and a target group is an activating ligand that is bound to an iRNA polynucleotide in a target group-iRNA polynucleotide conjugate. Targeted ligands enhance the association of molecules with a target cell. In this way, targeted ligands can enhance the pharmacokinetic or biodistribution properties of a conjugate to which they are coupled to enhance cellular distribution and cellular uptake of the conjugate. Binding of a targeted ligand to a cell or cellular receptor can initiate endocytosis. Targeted ligands can be monovalent, divalent, trivalent, tetravalent, or have a higher valence. Targeted ligands can be selected from the group comprising: compounds with an affinity to the cell surface molecule, cell receptor ligands, antibody, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to the surface molecules of the cell. A preferred targeting ligand comprises a receptor ligand

INSTITUTO MEXICANO DE LA PUNEDAD INDUSTRIAL cellular. A variety of ligands have been used to target drugs and genes to cells and to specific cellular receptors. Cell receptor ligands can be selected from the group comprising: carbohydrates, glycans, saccharides (including, but not limited to: galactose, galactose derivatives, mannose, and mannose derivatives), vitamins, folate, biotin, aptamers, and peptides ( including, but not limited to: RGD-containing peptides, insulin, EGF, and transferrin).

For targeting a hepatic hepatocyte, a preferred targeting ligand is a saccharide having affinity for the asialoglycoprotein receptor (ASGPr). Galactose and galactose derivatives have been used to target molecules to hepatocytes in vivo through their binding to ASGPr expressed on the surface of hepatocytes. As used herein, an ASGPr targeting ligand comprises a galactose or a galactose derivative having an affinity for ASGPr equal to or greater than that of galactose. Binding of the galactose targeted ligand to ASGPr facilitates specific targeting of the hepatocyte delivery polymer to the cell and endocytosis of the delivery polymer into hepatocytes.

Ligands targeting an ASGPr target can be selected from the group consisting of: lactose, galactose, ³⁰ IMPI®

INSTITUTO MEXICANO OF. THE C'wwZSBL INDUSTRIAL PROPERTY

N-acetylgalactosamine (GalNAc), galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, Npropionylgalactosamine, Nn-butanoylgalactosamine, and N-isobutanoyl-galactosamine (Lobst, ST and Drickamer, KJBC 1996, 271, 6686). The ASGPr targeting portions can be monomeric (eg, having a single galactosamine) or multimeric (eg, having multiple galactosamines).

In one embodiment, the membrane-active polyamine is reversibly masked through the coupling of the targeted ligand masking agents of ASGPr at&gt; 50%,&gt; 60%,&gt; 70%,&gt; 80%, or&gt; 90%. of the primary amines in the polyamine. In another embodiment, the membrane-active polyamine is reversibly masked through the coupling of the targeted ligand masking agents ASGPr and the PEG masking agents to&gt; 50%,&gt; 60%,&gt; 70%,&gt; 80% or&gt; 90% of the primary amines in the polymer. When both targeting ligand masking agents and PEG masking agents, a ratio of PEG targeting ligand to ASGPr is about 0-4: 1; more preferably 0.52: 1.

Amphipathic, or amphiphilic, polymers are well known and recognized in the art and both have hydrophilic (polar, water-soluble) and hydrophobic (not

<img file="MX347298B_D0028.tif" />

polar, lipophilic, insoluble in water).

Hydrophilic groups indicate in terms of quality that the preferred chemical portion is water. Typically, such chemical groups are soluble in water, and are donors or acceptors of hydrogen bonds with water. A hydrophilic group can be charged or uncharged. Charged groups can be positively charged (anionic) or negatively charged (cationic) or both (zwitterionic). Examples of hydrophilic groups include the following chemical moieties: carbohydrates, polyoxyethylene, certain peptides, oligonucleotides, amines, amides, alkoxy amides, carboxylic acids, sulfurs, and hydroxyls.

Hydrophobic groups indicate in terms of quality that the chemical portion avoids water. Typically, such chemical groups are not soluble in water and tend not to form hydrogen bonds. Lipophilic groups dissolve in nonpolar fats, oils, lipids, and solvents and have little to no ability to form hydrogen bonds. Hydrocarbons containing two (2) or more carbon atoms, certain substituted hydrocarbons, cholesterol, and cholesterol derivatives are examples of hydrophobic groups and compounds.

Hydrophobic groups are preferably hydrocarbons, containing only carbon and hydrogen atoms. However, nonpolar substitutions or³² IMPIAS

Mexican INSTITUTE

FROM INDUSTRIAL PROPERTY Nonpolar heteroatoms that maintain hydrophobicity, and include, for example, fluorine, can be allowed. The term includes aliphatic groups, aromatic groups, acyl groups, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of which can be linear, branched, or cyclic. The term hydrophobic group also includes: sterols, spheroids, cholesterol and derivatives of spheroids and cholesterol.

As used herein, with respect to unfriendly polymers, a part is defined as a molecule derived when a covalent bond is broken and replaced by hydrogen. For example, in butylamine, a break between the carbon and nitrogen bonds, and a replacement with hydrogens, results in ammonia (hydrophilic) and butane (hydrophobic). If 1,4-diaminobutane is separated at the nitrogen-carbon bonds, and replaced with hydrogens, the resulting molecules are again ammonia (2x) and butane. However, 1,4-diaminobutane is not considered unsympathetic due to the formation of the hydrophobic part that requires the breaking of two bonds.

As used herein, a surface active polymer lowers the surface tension of water and / or the interfacial tension with other phases, and therefore is positively absorbed at the liquid / vapor interface. The property of the activity in fact of the molecules in

<img file="MX347298B_D0029.tif" />

The surface is usually due to the substance only unsympathetic or amphiphilic.

As used herein, membrane-active polymers are unsympathetic, surface-active polymers that are capable of inducing one or more of the following effects on a biological membrane: an alteration or interference of the membrane that allows permeable molecules membrane failure to enter a cell or pass through the membrane, membrane pore formation, membrane fission, or membrane disruption or dissolution. As used herein, a membrane, or a cell membrane, comprises a lipid bilayer. Membrane disruption or interference can be functionally defined by polymer activity in at least one of the following assays: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, lysis cellular, and endosomal release. Membrane-active polymers that can cause lysis of cell membranes are also called libic membrane polymers. Polymers that preferentially cause alteration of endosomes or lysosomes on the plasma membrane are considered ensosomolytic. The effect of active polymers on the cell membrane membrane can 'γτγτγ

WICKED WSH! rase? Temporary INBUSVmal. The active polymers affinity for the membrane and cause in the membrane possess a denaturation or deformation of the bilayer structures. The membrane active polymers can be synthetic or non-natural amphipathic polymers.

As used herein, membrane-active polymers are different from a class of polymers termed peptides or cell-penetrating polymers represented by compounds such as arginine-rich peptide derived from the HIV TAT protein, the antenapedia peptide, the VP22 peptide, transportan, arginine-rich artificial peptides, small guanidinium-rich ratifícal polymers, and the like. Although cell-penetrating compounds appear to transport some molecules across a membrane, from one side of the lipid bilayer to the other side of the lipid bilayer, apparently without requiring endocytosis, and without altering the integrity of the membrane. its mechanism is not understood.

Delivery of a polynucleotide to a cell is mediated by disruption of the active polymer in the membrane or by destabilization of the plasma membrane or an inner vesicle membrane (such as an endosome or lysosome), including the formation of a pore in the cell. membrane, by the alteration of the endosomal or lysosomal vesicles therefore allowing the release of the content of the

IMPI

MEXICAN INSTITUTE

DÉ LA RRONÉDA · INDUSTRIAL

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vesicle within the cytoplasm of the cell.

The antipathic membrane active polyamine copolymers of the invention are the product of the copolymerization of two or more monomeric species. In one embodiment, the antipathic membrane active heteropolymers of the invention have the general structure:

-(TO)_to- (B)_bwherein A contains a pendant primary or secondary amine functional group and B contains a pendant hydrophobic group, a and b are integers greater than 0. The polymers can be random, block or alternative. The incorporation of additional monomers is permissible.

Endosomolytic polymers are polymers that, in response to specific endosomal environmental factors, such as the presence of lytic enzymes, are capable of causing alteration or lysis of an endosome or providing release of the normally cell membrane compound, such as polynucleotide, from a vesicle wrapped in an inner cell membrane, such as an endosome or lysosome. Endosomolytic polymers undergo a change in their physicochemical properties in the endosome. This change can be a change in the solubility or ability of the polymer to interact with other compounds or membranes as a result of a change in charge, hydrophobicity or hydrophilicity. The active polyamine in the reversibly masked membrane of the invention is endosomolytic.

considers

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MEXICAN INSTITUTE, /

OF INDUSTRIAL PROPERTY · --as being a polymer

Melittin is a small antipathetic membrane active peptide that occurs naturally in bee venom. Melittin can be isolated from a biological source or it can be synthetic. Synthetic polymer is formulated or manufactured through a chemical process by man and is not created through a naturally occurring biological process. As used herein, melittin encompasses the naturally occurring bee venom peptides of the melittin family that can be found in, for example, the venom of the species: Apis mellifera, Apis cerana, Vespula maculifrons, Vespa magnifica, Vespa velutina nigrithorax, Polistas sp. HQL-2001, Apis florae, Apis dorsata. Apis cerana, Polistes hebraeus. As used herein, melittin also encompasses synthetic peptides that have an amino acid sequence identical to or similar to naturally occurring melittin peptides. Specifically, the amino acid sequence of melittin encompasses those shown in Table 1. Synthetic melittin peptides may contain naturally occurring L-form amino acids or enantiomeric (reverse) D-form amino acids. However, a melittin peptide should either contain essentially all amino acids of the L form or all amino acids of the D form but may have amino acids of the

INSTITUTO MEXICANO &lt;'^ “= 25' kíü PE LA FkOPIEDAD C« κπ.-81¾¾ ^ INDUSTRIAL stereocenter opposite attached to the carboxy or amino terminal. The amino acid sequence of melittin can also be reversed (reversed). Reverse melittin can have amino acids of the L form or amino acids of the D form (retro-inverted). Two melittin peptides can also be covalently linked to form a melittin dimer. Melittin can have modifying groups, other masking agents, that improve tissue targeting or facilitate in vivo circulation coupled to either amino-terminal or carboxy-terminal end.

A bond or linker is a connection between two atoms that link a chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds. For example, one linker can connect a masking agent or polynucleotide to a polymer. A labile coupling contains a labile bond. A coupling can optionally include a spacer that increases the distance between two bonded atoms. A separator can also add flexibility and / or length to the links. Spacers can include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl groups; each of which contains one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the

<img file="MX347298B_D0031.tif" />

The preceding list is not intended to limit the scope of the invention.

A labile bond is a covalent bond other than a covalent bond to a hydrogen atom that is capable of selectively separating or splitting under conditions that will not alter or separate other covalent bonds in the same molecule. More specifically, a labile bond is a covalent bond that is less stable (thermodynamically) or that breaks more easily (kinetically) under appropriate conditions than other non-labile covalent bonds in the same molecule. The separation of the labile bond within a molecule can result in the formation of two molecules. For those skilled in the art, bond separation or lability is generally explained in terms of bond separation half-life (the time required for half of the bonds to separate). In this way, labile bonds encompass bonds that can selectively separate more quickly than the other bonds in a molecule.

As used herein, "physiologically labile bond" is a labile bond that can be cleaved under conditions normally encountered or analogous to those found within a mammalian body. Physiologically labile linking groups are selected in such a way that they undergo chemical transformation (e.g.

<img file="MX347298B_D0032.tif" />

IMPI

INSTITUTO MEXICANO DE LA FROFIFDAU INDUSTRIAL example, separation) when they are present under certain physiological conditions.

As used herein, a cellular physiologically labile bond is a labile bond that separates under mammalian intracellular conditions. Mammalian intracellular conditions include chemical conditions such as pH, temperature, oxidizing or reducing conditions or agents, and salt concentration found in or analogous to those found in mammalian cells. Mammalian intracellular conditions include the presence of an enzyme activity normally present in the mammalian cell such as proteolytic or hydrolytic enzymes. A physiologically labile cell bond can also be severed in response to administration of a pharmaceutically acceptable exogenous agent.

Target group-interfering iRNA conjugates: A target group can bind to the 3 'or 5' end of the iRNA polynucleotide. For siRNA polynucleotides, the targeted portion can be linked to either the sense strand structure or the antisense strand structure, through the sense strand structure is preferred.

In one embodiment, the targeted group consists of a hydrophobic group. More specifically, the targeted group consists of a hydrophobic group that

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OF INDUSTRIAL PROPERTY · -has at least 20 carbon atoms. Hydrophobic groups used as polynucleotide targeting portions herein are referred to as hydrophobic targeting portions. Illustrative suitable hydrophobic groups can be selected from the group comprising: cholesterol, dicolesterol, tocopherol, ditocopherol, didecyl, didodecyl, dioctadecyl, didodecyl, dioctadecyl, isoprenoid, and choleamide. Hydrophobic groups have 6 or fewer carbon atoms and are not effective as polynucleotide targeting portions, even though hydrophobic groups have 8 to 18 carbon atoms as long as they increase the supply of the polynucleotide with increasing size. of the hydrophobic group (that is, an increasing number of carbon atoms). Coupling of a hydrophobic target group to an iRNA polynucleotide does not enhance efficient functional in vivo delivery of the iRNA polynucleotide in the absence of co-administration of the delivery polymer. Although siRNA-cholesterol conjugates have been reported by others to deliver siRNA (siRNA-cholesterol) to liver cells in vivo, in the absence of any additional delivery vehicle, high concentrations of siRNA are required and efficacy supply is poor. When combined with the delivery polymers described herein, the delivery of the polynucleotides is greatly improved. To the

<img file="MX347298B_D0033.tif" />

IMPI (HTHTUTO MEXICANO rw THE INDUSTRIAL PROPERTY provide siRNA-cholesterol together gqb ...... w supply of the invention, the efficacy of siRNA-cholesterol increases by approximately 100 times.

Hydrophobic groups useful as polynucleotide targeting portions can be selected from the group consisting of: an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, an aralkenyl group, and an aralkynyl group, each one of which can be linear, branched or cyclic, cholesterol derived from cholesterol, sterol, spheroid, and derived from spheroid. Hydrophobic targeting groups are preferably hydrocarbons, containing only carbon and hydrogen atoms. However, substitutions or heteroatoms that maintain hydrophobicity, eg, fluorine, may be allowed. The hydrophobic target group can be attached to the 3 'or 5' end of the iRNA polynucleotide using methods known in the art. For iRNA polynucleotides having two strand structures, such as siRNA, the hydrophobic group can be coupled to any strand structure.

In another embodiment the targeted group comprises a galactose aggregate (targeted portion of the galactose aggregate). As used herein, a galactose aggregate comprises a molecule having two to four terminal galactose derivatives.

IMPI INSTITUTO MEXICANO DE LA PROPERTY INDUSTRIAL ------- As used herein, the term galactose derivative includes both galactose and galactose derivatives that have an affinity for ASGPr equal to or greater than that of galactose. A terminal galactose derivative attaches to a molecule through its Cl carbon. A preferred galactose aggregate has three terminal galactosamines or galactosamine derivatives, each with affinity for the asialoglycoprotein receptor. A more preferred galactose aggregate has three N-terminal galactosamines. Other terms common in the art include tri-antennial galactose, tri-valent galactose, and trimers of galactose. Aggregates of tri-antenary galactose derivatives are known to bind ASGPr with higher affinity than structures of bi- ntenary or mono-antenary galactose derivatives (Baenziger and Fiete, 1980, Cell, 22, 611-620 ; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Multivalence is required to obtain nM affinity. Coupling of a single galactose derivative having affinity for the asialoglycoprotein receptor does not allow functional delivery of the iRNA polynucleotide to hepatocytes in vivo when co-administered with the delivery polymer.

A galactose aggregate contains two to four, preferably three galactose derivatives each linked to a central branch point. The galactose derivatives are coupled to the central branch point at

WICKED

INSTITUTO MEXICANO DE LA PROPERTY through the Cl carbons of saccharides. The lactose ^ 'd &amp; iíl'vaab'-of the lactose was preferably linked ^ -— «1 hand of branching through linkers or spacers. A preferred spacer is a flexible hydrophilic spacer (US Patent 5885968; Biessen et al., J. Med. Chem.

nineteen ninety five Vol. 39 p. 1538-1546). A preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG3 spacer. The branch point can be any small molecule that allows the coupling of the three galactose derivatives and further allows the coupling of a branch point to the iRNA polynucleotide. An illustrative branch point group is a di-lysine. A di-lysine molecule contains three amine groups through which the three galactose derivatives can be coupled and a reactive carboxyl group through which the di-lysine can be coupled to the polynucleotide of the iRNA. Branch point coupling to the iRNA polynucleotide can occur through a linker or spacer. A preferred spacer is a flexible hydrophilic spacer. A preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG3 spacer (three ethylene units). The merged aggregate of galactose can be coupled to the 3 '05' end of the iRNA polynucleotide using methods known in the art. For iRNA polynucleotides that have 2

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INSTITUTO MEXICANO S. US Patent 20110207799.

The term polynucleotide, or nucleic acid or polynucleic acid, is a term in the art that refers to a polymer containing at least two nucleotides. Nucleotides are monomeric units of polynucleotide polymers. Polynucleotides with less than 120 monomer units are generally called oligonucleotides. Natural nucleic acids have a deoxyribose- or ribose-phosphate backbone. A non-natural or synthetic polynucleotide is a polynucleotide that is polymerized in vitro or a cell-free system and contains the same or similar bases but contains a structure of a different type than the natural ribose or the deoxyribose phosphate structure. Polynucleotides can be synthesized using any technique known in the art. Polynucleotide structures known in the art include PNAs (Peptide Nucleic Acids), phosphorothioates, phosphorodiamidates, morpholinos, and other variants of the phosphate backbone of native nucleic acids. The bases include purines and pyrimidines, which further include the natural components adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs. Synthetic derivatives of purines and

IMPIO pyrimidines include, but are not limited to, modifications that place new reactive groups on the nucleotide such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. The term "base" encompasses any of the base analogs of DNA and RNA. A polynucleotide can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination. Polynucleotides can be polymerizable in vitro, they can be recombinant, contain chimeric sequences or derivatives of these groups. A polynucleotide can include a cap portion at the 5 'end, the 3' end, or both the 5 'and 3' ends. The cap portion may be, but is not limited to, an inverted deoxy abasic portion, an inverted deoxy-thymidine portion, a thymidine portion, or a 3 'glyceryl modification.

An RNA interference polynucleotide (iRNA) is a molecule capable of inducing RNA interference through interaction with mammalian cell RNA interference pathway machinery to degrade or inhibit translation of messenger RNA transcripts. (MRNA) of a transgene in a sequence-specific form. Two primary iRNA polynucleotides are small (or short) interfering RNA (siRNA) and micro-RNA (siRNA). IRNA polynucleotides can be selected from the group that "ΙΜΡΙ®5, ^{, HS} It consists of: siRNA, micro-RNA, biratenary RNA (AR.N¿UU-r-A-RtJ · small hairpin (shRNA) and expression cassettes encoding RNA capable of inducing RNA interference. SiRNA comprises a double-stranded structure typically containing 15-50 base pairs and preferably 21-25 base pairs and has a nucleotide sequence identical (preferably complementary) or nearly identical (partially complementary) to a coding sequence in an expressed target gene or RNA within of the cell. An siRNA can have a 3 'dinucleotide overhang. An siRNA can be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. An siRNA molecule of the invention comprises a sense region and an antisense region. In one embodiment, the siRNA of the conjugate is assembled from two oligonucleotide fragments wherein one fragment comprises the nucleotide sequence of the antisense strand structure of the siRNA molecule and a second fragment comprises the nucleotide sequence of the sense region of the molecule. SiRNA. In another embodiment, the sense strand structure is connected to the antisense strand structure through a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. Micro-RNAs (miRNAs) are small noncoding RNA gene products approximately 22 nucleotides in length that direct the

<img file="MX347298B_D0034.tif" />

destruction or translational repression of targets If the complementarity between the miRNA and the target mRNA is partial, the translation of the target mRNA is repressed. If the complementarity is extensive, the target mRNA is separated. For miRNA, the complex binds to target sites usually located in the 3 'UTR of mRNAs that typically share only partial homology with miRNA. A seeding region - an elongation of approximately seven (7) consecutive nucleotides at the 5 'end of the miRNA that forms the perfect base match for its target, plays a key role in the specificity of the miRNA. Binding of the RISC / miRNA complex to mRNA can lead to either repression of protein translation or separation and degradation of mRNA. Recent data indicates that mRNA separation occurs preferentially if there is perfect homology along the full length of the miRNA and its target rather than showing a perfect base combination only in the seeding region (Pillai et al., 2007).

The iRNA polynucleotide expression cassettes can be transcribed in the cell to produce small hairpin RNAs that can function as siRNAs, linear siRNAs of separate sense and antisense strand structure, or miRNA. RNA polymerase III transcribed DNAs contain promoters selected from the list that

INSTITUTO MEXICANO DE LA MONEDAD INDUSTRIAL comprises: U6 promoters, Hl promoters, and tRNA promoters. Polymerase II promoters include Ul, U2, U4, and U5 promoters, snRNA promoters, micro-RNA promoters, and mRNA promoters.

Lists of known miRNA sequences can be found in databases maintained by research organizations such as Wellcome Trust Sanger Institute, Penn Center for Bioinformatics, Memorial Sloan Kettering Cancer Center, and European Molecule Biology Laboratory, among others. Known effective siRNA sequences and cognate binding sites are well represented in the relevant literature. IRNA molecules are easily designed and produced through technologies known in the art. In addition, there are computational tools that increase the probability of finding effective and specific sequence motifs (Peí and others, 2006, Reynolds and others, 2004, Khvorova and others, 2003, Schwarz and others, 2003, Ui-Tei and others, 2004 , Heale and others, 2005, Chalk and others, 2004, Amarzguioui and others, 2004).

The polynucleotides of the invention can be chemically modified. Non-limiting examples of such chemical modifications include: phosphorothioate internucleotide linkages, 2'-0-methyl ribonucleotides, 2'-deoxy-2'fluoro ribonucleotides, 2'-deoxy ribonucleotides, universal base nucleotides, 5-C-methyl nucleotides, and the

INSTITUTO MEXICANO Dt LA NOFIEDAD INDUSTRIAL incorporation of inverted deoxybasic waste. These chemical modifications, when used in various polynucleotide constructs, are shown to preserve the activity of the polynucleotide in cells while at the same time increasing the serum stability of these compounds. Chemically modified siRNA may also minimize the possibility of activation of interferon activity in humans.

In one embodiment, a chemically modified iRNA polynucleotide of the invention comprises a duplex having two strand structures, one or both of which can be chemically modified, wherein each strand structure is from about 19 to about 29 nucleotides. In one embodiment, an iRNA polynucleotide of the invention comprises one or more modified nucleotides while retaining the ability to mediate iRNA within a cell or reconstitute in an in vitro system. An iRNA polynucleotide can be modified wherein the chemical modification comprises one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the nucleotides. An iRNA polynucleotide of the invention may comprise modified nucleotides as a percentage of the total number of nucleotides present in the iRNA polynucleotide. That is, an iRNA polynucleotide of the invention may generally comprise nucleotides⁵⁰ IMPI

MEXICAN INSTITUTE OF PROPERTY '¿-¿¡¿fós

INDUSTRIAL modified from about 5 to about 100% of _ .__l.__l_j__n_ · .ιΐίΐ · ι ιιιιιιιιιι ··· ι »&gt; —IWi the nucleotide positions (for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotide positions). The actual percentage of modified nucleotides present in a given iRNA polynucleotide depends on the total number of nucleotides present in the iRNA polynucleotide. If the iRNA polynucleotide is single-stranded structure, the percentage modification may be based on the total number of nucleotides present in the single-stranded iRNA polynucleotide. Likewise, if the iRNA polynucleotide is double stranded, the percentage modification can be based on the total number of nucleotides present in the sense strand structure, the antisense strand structure, or both sense and antisense structures. Furthermore, the actual percentage of modified nucleotides present in a given iRNA polynucleotide may also depend on the total number of purine and pyrimidine nucleotides present in the iRNA polynucleotide.

For example, where all pyrimidine nucleotides and / or all purine nucleotides present in the iRNA polynucleotide are modified.

An iRNA polynucleotide modulates the expression of RNA encoded by a gene. Because multiple genes may share some degree of sequence homology between

IMPI

MEXICAN INJTrtVTO

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yes, the iRNA polynucleotide can be designed to target a class of genes with sufficient sequence homology. In this way, an iRNA polynucleotide can contain a sequence that has complementarity to sequences that are shared between different gene targets or are unique to a specific gene target. Accordingly, the iRNA polynucleotide can be designed to target conserved regions of an RNA sequence that has multiple gene homology thus targeting multiple genes in a gene family (e.g., different gene isoforms, splice variants, mutant genes, etc.). In another embodiment, the iRNA polynucleotide can be designed to target a single sequence to a specific RNA sequence of a single gene.

The term "complementarity" refers to the ability of a polynucleotide to form a hydrogen bond (s) with another polynucleotide sequence through either traditional Watson-Crick or other non-traditional types. With reference to the polynucleotide molecules of the present invention, the energy without binding for a polynucleotide molecule to its target (effector binding site) or complementary sequence is sufficient to allow the relevant function of the polynucleotide to advance, for example, the enzymatic mRNA separation or translation inhibition. Determining the energies at the binding for nucleic acid molecules is well (Frier et al., 1986, Turner et al.,

<img file="MX347298B_D0036.tif" />

known in the art

1987). A percent complementarity indicates the percentage of bases, in a contiguous chain structure, in a first polynucleotide molecule that can form hydrogen bonds (eg, Watson-Crick base combination), with a second polynucleotide sequence (eg, 5, 6, 7, 8, 9, 10 of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). Perfect complementarity means that all bases in a contiguous strand structure of one polynucleotide sequence will hydrogen bond with the same number of contiguous bases in a second polynucleotide sequence.

By inhibition, downregulation, or destruction of gene expression, it means that the expression of the gene, as measured by the level of transcribed RNA of the gene or the level of the translated polypeptide, protein or protein subunit of the RNA is reduced below that observed in the absence of the conjugates of the blocking polynucleotide of the invention, down regulation or destruction of gene expression, with a polynucleotide supplied by the compositions of the invention, it is preferably below the level observed in the presence of a control inactive nucleic acid, a nucleic acid with a disordered sequence or with inactivating incompatibilities, or the absence of conjugation of the polynucleotide to the polymer ⁵³ IMPI ^

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Stabilization of siRNA against degradation via localized endosomal / lysosomal nucleases such as DNAse II has been found to strongly enhance target killing. Such stabilization can directly affect the amount of siRNA released into the cytoplasm where the iRNA machinery is located. Only the portion of siRNA available in the cytoplasm will activate the iRNA effect.

In addition to poor pharmacokinetic characteristics, siRNAs are susceptible to nucleases in the biological environment when administered as such in circulation without a protective delivery vehicle. Consequently, many siRNAs are rapidly degraded either extracellularly in tissue and the bloodstream or after intracellular absorption (endosome). Nuclease cleavage can be inhibited through nucleosides lacking a 2'-0H group such as 2'-deoxy, 2'-0-methyl (2'OMe) or 2'-deoxy-2'-f nucleotides luoro (2'-F) and through polynucleotides of non-5'-terminal nucleotide portions, such as, for example, cholesterol, aminoalkyl linker or a phosphorothioate at the first internucleotide coupling. Preferably, the iRNA polynucleotide lacks any 2'-OH nucleotide within the strand structure, starting with a 2'-OMe nucleotide at the 5 'end connected through

IMPIAS

MEXICAN INSTITUTE

DC THE PROPERTY of a phosphorothioate (PTO) bond to the second li ^^^ tidoT

The ANSI can be stabilized signif'iwrrt ^ OTiériE ^ 'when the following design is used, where an oligonucleotide is provided with an antisense strand structure with the modification pattern: 5' - (w) - (Zl) - (Z2) - (Z3) n_to-3 'and a sense strand structure with the modification pattern 5' - (Z3) n_s-3 ', where w is independently a 5'-phosphate or 5'-phosphothioate or H,

Zl is independently a 2'-modified nucleoside.

Z2 is independently a 2'-deoxy nucleoside or a 2'-fluoro-modified nucleoside,

Z3 is independently a 2'-modified nucleoside, n_to is 8-23 yn_s is 8-25.

In a preferred embodiment an oligonucleotide with an antisense strand structure is provided with the modification pattern: 5 '- (w) - (Zl) - (Z2) - (Z3) n_to-3 'and a sense strand structure with the modification pattern 5'- (Z3) n_s-3 ', where Zl is a 2'-Fluoromodified nucleoside or a 2-deoxy-nucleoside and all the remaining substituents as well as the variables n_to and n_s have the meaning given above.

In a preferred embodiment an oligonucleotide with an antisense strand structure is provided with the modification pattern: 5 '- (w) - (Zl) - (Z2) - (Z3) n_to-3 'and a

<img file="MX347298B_D0037.tif" />

sense strand structure with modification pattern 5 '- (Z3) n_s-3 ', where Z3 is a modified 2'-0-Methyl nucleoside, a 2'-Fluoro-modified nucleoside or a 2-deoxy nucleoside and all remaining substituents as well as the variables n_to and n_s have the meaning given above.

In a preferred embodiment an oligonucleotide with an antisense strand structure is provided with the modification pattern: 5 '- (w) - (Zl) - (Z2) - (Z3) n_to-3 'and a sense strand structure with the modification pattern 5' - (Z3) n_s-3 ', where Zl is a 2'-Fluoromodified nucleoside or a 2-deoxy-nucleoside and Z3 is a modified 2'O-Methyl nucleoside, a 2'-Fluoro-modified nucleoside or a 2-deoxy-nucleoside and all remaining substituents as well as variables n_to and n_s have the meaning given above.

Nucleosides in the nucleic acid sequence of the oligonucleotide with the new modification pattern can either be linked via 5'-3 'phosphorodiesters or 5'-3' phosphorothioates.

As used herein, the antisense structure is the chain structure of siRNA that is complementary to the target mRNA and that will bind to the mRNA once the siRNA is untied. The sense strand structure of siRNA comprises the new modification pattern that is complementary to the antisense strand structure.

In principle a nuclease cleavage site, between the iRNA polynucleotide and the targeted portion or delivery polymer to which it is covalently coupled can be introduced through the 3 'or 5' projections containing at least one nucleotide 2 '-0H in either sense or antisense structures. The final active siRNA species are generated through intracellular nuclease processing. Also, the use of fluid separation sites implemented by 2'-OH nucleotides within the combined base regions is possible. This can be done by using at least one 2'-OH nucleotide complementary to the opposite strand structure or by introducing either of at least one 2'-OH nucleotide or a hairpin / protrusion containing at least one 2 'nucleotide. -0H.

Coupling the polynucleotide to the delivery polymer

In one embodiment, the iRNA polynucleotide is linked to the delivery polymer through a physiologically labile linker or linker. The physiologically labile linker is selected such that it undergoes chemical transformation (e.g. separation) when it is present under certain physiological conditions (e.g. split sulfide bond in the

<img file="MX347298B_D0038.tif" />

cell cytoplasm depletion environment). Release of the polynucleotide from the polymer, through separation of the physiologically labile bond, facilitates the interaction of the polynucleotide with the associated components for activity.

The polynucleotide-polymer conjugate is formed by covalently linking the polynucleotide to the polymer. The polymer is polymerized or modified in such a way that it contains a reactive group A. The polynucleotide is also polymerized or modified in such a way that it contains a reactive group B. The reactive groups A and B are selected such that they can be linked through the covalent bonding using methods known in the art.

Conjugation of the polynucleotide to the polymer can be carried out in the presence of excess polymer. Because the polynucleotide and polymer can be oppositely charged during conjugation, the presence of excess polymer can reduce or eliminate aggregation of the conjugate. Alternatively an excess of the carrier polymer, such as a polycation can be used. Excess polymer can be removed from the conjugated polymer prior to administration of the conjugate to the animal or cell culture. Alternatively the excess polymer can be co-administered with the conjugate to the animal or cell culture.

In Vivo Administration

In pharmacology and toxicology, a —— il — III 'route. Hl I

INSTITUTO MEXICANO UE LA PROHEDAD administration is the trajectory through cu ^ - ^ 'er drug, fluid, poison or other substance that is put in uuiiL'dettr' '·· with the body. In general, methods of administering drugs and nucleic acids for the treatment of a mammal are well known in the art and can be applied in the administration of the compositions of the invention. The compounds of the present invention can be administered by any suitable route, most preferably parenterally, in a preparation appropriately adapted to this route. Thus, the compounds of the present invention can be administered via injection, for example intravenously, intramuscularly, intercutaneously, subcutaneously, or intraperitoneally. Accordingly, the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient.

Routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumoral, intraperitoneal, intrathecal, subdural, epidural, and intralymphatic injection using a syringe and needle and catheter. Intravascular as used herein means within a tubular structure called vessels that connects to a tissue or organ within the body. Within the cavity of the tubular structure, a body fluid flows

<img file="MX347298B_D0039.tif" />

ΙΜΡΠ

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY to or from the body part. Examples of bodily fluids include blood, cerebrospinal fluid (CSF), lymphatic fluid, or bile. Examples of vessels include arteries, arterioles, capillaries, small veins, sinusoids, veins, lymphatics, bile ducts, and ducts of the salivary or other exocrine glands. The intravascular route includes delivery through blood vessels such as an artery or a vein. The blood circulatory system provides the systemic dissemination of the pharmacist.

The disclosed compositions are injected into pharmaceutically acceptable carrier solutions. Pharmaceutically acceptable refers to those properties and / or substances that are acceptable to the mammal 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 produce an allergic or other unwanted or toxic reaction when administered to a mammal. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the US Pharmacopeia. and other generally recognized pharmacopoeia for use in animals and more particularly in humans.

This carrier may also contain adjuvants such

IMPI

INSTITUTO MEXICANO DE LA PROPERTY INDUSTRIAL as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both through sterilization procedures, supra, γ through the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, ascorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be obtained through the inclusion of oil that delays absorption such as aluminum monostearate and gelatin.

In one embodiment, the iRNA polynucleotide target group conjugate is co-administered with a delivery polymer of the invention. Co-administration means that the iRNA polynucleotide and the delivery polymer are administered to the mammal such that they are both present in the mammal at the same time. The target group-iRNA polynucleotide conjugate and delivery polymer can be administered simultaneously or can be delivered sequentially. For simultaneous administration, they can be mixed prior to administration. For sequential administration, any conjugate of the target portion-iRNA polynucleotide or delivery polymer can be administered first.

⁶¹ ΙΜΡΙ »$ • WnVTO MUICANO

OF U OWNED *** Λ .INDUSTRIAL

Therapeutic effect

The iRNA polynucleotides can be delivered for research purposes or to produce a change in the cell that is therapeutic. In vivo delivery of iRNA polynucleotides is useful for investigating reagents and for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic modification, and pharmacogenomic applications. RNA polynucleotide delivery has been described resulting in inhibition of endogenous gene expression in hepatocytes. Levels of a reporter (marker) gene expression measured after delivery of the polynucleotide indicates the reasonable expectation of similar levels of gene expression after delivery of other polynucleotides. Levels of treatment considered beneficial by one of ordinary skill in the art differ from disease to disease. For example, hemophilia A and B are caused by deficiencies in X-linked coagulation factors VIII and IX, respectively. Its clinical course is largely influenced by the percentage of normal serum levels of factor VIII or IX: less than 2%, severe; 2-5%, moderate; and 5-30% light. Thus, an increase of 1% to 2% of the normal level of the circulating factor in severe patients can be considered beneficial. Levels of 6% prevent spontaneous bleeding but not those that are secondary to surgery or damage.

<img file="MX347298B_D0040.tif" />

Similarly, the inhibition of a gene need not be 100% to provide a therapeutic benefit. One skilled in the art of gene therapy will reasonably anticipate beneficial levels of expression of a disease-specific gene based on sufficient levels of marker gene outcomes. In the example of hemophilia, if the marker genes are expressed to produce a protein at a level comparable in volume to 2% of the normal level of factor VIII, it can reasonably be expected that the gene encoding factor VIII will also be expressed at similar levels. In this way, reporter or marker genes serve as useful paradigms for the expression of intracellular proteins in general.

The liver is one of the most important target tissues for gene therapy given its central role in metabolism (eg, lipoprotein metabolism in various hypercholesterolems) and secretion of circulating proteins (eg, clotting factors in hemophilia). In addition, acquired disorders such as chronic hepatitis and cirrhosis are common and are also potentially treated through polynucleotide-based liver therapies. A number of diseases or conditions that affect or are affected by the liver are potentially treated through the destruction (inhibition) of the gene expression of the liver. Such liver diseases and conditions

IMPI

MEXICAN INSTITUTE

OF INDUSTRIAL PROPERTY

<img file="MX347298B_D0041.tif" />

they can be selected from the list comprising: liver cancers (including hepatocellular carcinoma, HCC), viral infections (including hepatitis), metabolic disorders (hyperlipidemia and diabetes), fibrosis, and acute liver damage.

The current dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied in order to obtain an amount of the active ingredient that is effective to obtain the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic. for the patient. The dose level selected will depend on a variety of pharmacokinetic factors such as the activity of the particular compositions of the present invention used, the route of administration, the time of administration, the degree of expression of the particular compound being used, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions used, age, sex, weight, condition, the general health and past medical history of the patient being treated, and similar factors well known in the medical arts.

The amount (dose) of delivery polymer and iRNA polynucleotide conjugate to be administered

<img file="MX347298B_D0042.tif" />

can be determined empirically. Effective destruction of gene expression has been demonstrated using 0.1-10 mg / kg body weight of the siRNA conjugate and 1.5-60 mg / kg body weight of the delivery polymer. A preferred amount in mice is 0.25-2.5 mg / kg of siRNA conjugate and 10-40 mg / kg of the delivery polymer. More preferably, about 1.5-20 mg / kg of the delivery polymer is administered. The amount of the iRNA polynucleotide conjugate is easily increased since it is typically non-toxic in larger doses.

As used herein, in vivo means that it takes place within an organism and more specifically the process is carried out within or on the living tissue of a whole living multicellular organism (animal), such as a mammal, as opposed to one partial or dead.

As used herein, pharmaceutical composition includes the conjugates of the invention, a pharmaceutical carrier or diluent, and any other medium or agent necessary for formulation.

As used herein, pharmaceutical carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, via injection or infusion.

EXAMPLES

Example 1. Synthesis of protease cleavable masking agents (peptidase).

All reactions, except amino acid coupling in aqueous NaHCOs and deprotection of the silyl group, were carried out under anhydrous conditions using fresh anhydrous solvents. Column purification was done on silica gel using specified eluents. The mass spectrum (MS) was taken using electrospray ionization.

In the preparation of active pnitrophenyl-p-acylamidobenzyl carbonate derivatives of NAG and PEG (NAG-L-AAPABC-PNP and PEG-AA-PABC-PNP) the respective PEG NHS ester or derivatives containing PEG were used to acylate the term amino of the dipeptide-p-acylaminobenzyl alcohol precursor.

In the following steps the benzylic hydroxy group was converted to p-nitrophenyl carbonate followed by removal of amino acid protecting groups and the NAG portion. In some applications, when paranitrophenol (PNP) -carbonates were used for the modification of certain polymers, the protecting groups before the polymer modification.

<img file="MX347298B_D0043.tif" />

<img file="MX347298B_D0044.tif" />

<img file="MX347298B_D0045.tif" />

<img file="MX347298B_D0046.tif" />

η-α'-α²-ραρα ra'-a²-paba

<img file="MX347298B_D0047.tif" />

<img file="MX347298B_D0048.tif" />

Deprotection r-a'-a²-pabc-pnp

R comprises an ASGPr ligand (protected or unprotected) or a PEG, and

TO¹ already² are amino acids (either protected or unprotected)

Synthesis starts from the preparation of HA derivatives¹TO²-PABA (Table 1). These adducts were obtained using the synthetic scheme described by Dubowchik et al. (2002) with some modifications. The amino acids protected with Fmoc, Fmoc-A¹-0H, were activated by conversion to N-hydroxyuccinimide esters, Fmoc-A¹-NHS, in reaction with dicyclohexylcarbodiimide (DCC) and N-hydroxyuccinimide (NHS). These reactive HNS esters were coupled with amino acids A²protected in the presence of NaHC0₃ aqueous added to maintain the amino group reagent. For the preparation of le and lf (Table 1), instead of NHS esters, we used

IMPI

INSTITUTO MEXICANO M LA PROPERTY; ·

INDUSTRIAL commercially available pentafluorophenyl esters (OPfp) for coupling.

Synthesis of Dipeptides Fmoc la-h.

a) AA NHS asters were prepared from respective amino acids with NHS and DCC and used without further purification.

Fmoc — A — OH ► Fmoc — A — NHS

Conditions: (i) N-hydroxysuccinimide (NHS), NN¹dicyclohexylcarbodiimide (DCC), 0-20 ° C.

For Fmoc-Ala-NHS, DCC (286 mg, 1.38 mmol) was added to an ice-cold solution of Fmoc-Ala-OH (412 mg, 1.32 mmol) and NHS (160 mg, 1.38 mmol) in DCM (13 ml), stirred for 30 min, and then at 20 ° C for 16 h. Solid dicyclohexylurea (DCU) was filtered and the solvent was removed in vacuo.

For Fmoc-Asn (DMCP) -NHS, DCC (148 mg, 0.72 mmol) was added to an ice-cold solution of Fmoc-Asn (DMCP) -OH (298 mg, 0.68 mmol) and NHS (83 mg, 0.72 mmol) in DCM (13 ml), stirred for 30 min, and then at 20 ° C for 16 h. The solid DCU was filtered off and the solvent was removed in vacuo.

For Fmoc-Gly-NHS, Fmoc-Gly-OH (891 mg, 3 mmol) and NHS (380 mg, 3.3 mmol) were stirred in THF (10 ml) at 0 ° C for 5 min and treated with a DCC solution ( 650 mg, 3.15 mmol) in THF (5 ml). The cooling bath was stirred in 30 min and the reaction mixture was stirred at 20 ° C for 10 h. The solid DCU was filtered, washed with THF, the solvent was removed in the

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX347298B_D0049.tif" />

rotary evaporator.

The product was weighed and dissolved in DME to form a 0.2 mM solution.

For Fmoc-Glu (O-2PhiPr) -NHS, DCC (217 mg,

1.05 mmol) to an ice-cold solution of Fmoc-Glu (O-2PhiPr) -OH (487 mg, 1 mmol) and NHS (127 mg, 1.1 mmol) in THF (5 ml), stirred for 15 min and then at 2 0 ° C for 10 h. The tests were done as described for Fmoc-Gly-NHS.

For Fmoc-Phe-NHS, DCC (1,181 g, 5.72 mmol) was added to an ice-cold solution of Fmoc-Phe-OH (2.11 g, 5.45 mmol) and NHS (664 mg, 5.77 mmol) in DCM (50 ml), stirred for 30 min, and then at 20 ° C for 10 h. The solid DCU was filtered off and the solvent was removed in vacuo.

For Fmoc-Val-NHS, DCC (227 mg, 1.1 mmol) was added to an ice-cold solution of Fmoc-Val-OH (339 mg, 1 mmol) and NHS (127 mg, 1.1 mmol) in DCM (13 ml), stirred for 30 min, and then at 20 ° C for 16 h. The solid DCU was filtered off and the solvent was removed in vacuo.

b) The amino acids Η-Asn (DMCP) -OH and H-Lys (MMT) -OH were prepared from available Fmoc-protected derivatives

<img file="MX347298B_D0050.tif" />

Terms:

(i)

Dimethylformamide triethylamine (DMF).

H-Asn (DMCP) -OH

Fmoc-Asn (DMCP) -OH (576 mg, 1.32 mmol) was stirred in DMF (9 mL) with Et₃N (3.7 ml, 26.4 mmol) for 15 h. All volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The residue was triturated with ether (30 ml) three times and dried in vacuo. Yield 271 mg (96%). MS: 643.6 [3M + 1]⁺; 451.3 [2M + Na]⁺; 429.5 [2M + 1]⁺; 236.7 [M + Na]⁺; 215.3 [M + l]⁺; 132.8 [M-DMCP + 1]⁺.

H-Lys (MMT) -OH. Fmoc-Lys (MMT) -OH (4.902 g, 7.65 mmol) was stirred in DMF (100 mL) with Et₃N (32 ml, 30 eq. 229.4 mmol) for 10 h. All volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The residue was triturated with ether twice and dried in vacuo. Yield 3. lg (97%). MS (modoneg.): 455, 453.3 [M + Cl] '; 417.8 [ml] '.

c) Synthesis of Fmoc-AiA₂-0H.

Fmoc — A¹-NHS

Fmoc — A¹—A — OH

Fmoc — A —OPfp ah

A = Ala, Phe

TO^x= Gly, Glu (2PhiPr), Asn (DMCP), Phe, Ala, Val.

TO²= Gly, Lys (MMT), Cit, Asn (DMCP), Lys (CH₃)₂.

Conditions: (i) HA₂-OH, NaHCO₃, mixture of dimethoxyethane (DME), tetrahydrofuran (THF) and H₂0. (ii) HA₂25

OH, NaHCO₃, DME / THF / H₂O. (iii) H-Cit-OH, NaHCO₃, THF in H₂OR.

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX347298B_D0051.tif" />

For Fmoc-GlyGly-OH

NaHCO₃ (100 mg, 1.2 mmol) la, Glycine (75 mg, 1 mmol) and dissolved in H2O (10 ml) and dimethoxyethane (DME) (5 ml). Fmoc-GlyNHS solution in DME (5 ml, 1 mmol) was added. THF (2.5 ml) was added, the mixture was sonicated to make it homogeneous and stirred for 20 h.

All volatiles were removed on a rotary evaporator, the residue was treated with EtOAc and 5% KHCO3 solution in

H2O. The product was extracted four times with EtOAc, washed with brine to pH = 3, dried (Na2SO4), concentrated and dried in vacuo.

Yield 321 mg (90%). MS: 775.0 [2M + 2Na]⁺; 377.4 [M + Na]⁺;

355.1 [M + l]⁺.

For Fmoc-Glu (O-2PhiPr) Gly-OH Ib, Glycine (75 mg, 1 mmol) and NaHCO₃ (84 mg, 1 mmol) were dissolved in a mixture of H2O (2 ml), THF (4 ml) and DME (5 ml). Fmoc-Glu (O-2PhiPr) -NHS solution in DME (5 ml, lmmol) was added and stirred for 10 h. All volatiles were removed on a rotary evaporator, 20 ml of 0.1M pH buffer MES (pH = 5) were added followed by EtOAc (25 ml). The reaction mixture was stirred on ice and made acidic to pH = 5 with 5% KHSO4 solution. The product was extracted four times with EtOAc, rinsed with brine at pH = 5, dried (Na2SO4), concentrated and dried in vacuo. Yield 528mg (96%). MS: 567 [M + Na]⁺; 562 [Μ + ΝΗ₄] ⁺ ; 545.0 [M + l]⁺; 427.1 [M-2PhiPr]⁺.

For Fmoc-Asn (DMCP) Gly-OH it was prepared from FmocAsn (DMCP) -NHS and H-Gly-OH as described above for

<img file="MX347298B_D0052.tif" />

IMPIG MEXICAN INSTRUMENT OF INDUSTRIAL CURRENCY

Ib. Yield 96%. MS: 987.4 [2M + 1]

516.3 [M + Na] ⁺; 494.4

[M + l]⁺; 412.2 [M-DMCP + 1]⁺.

For Fmoc-PheLys (MMT) -OH Id was prepared from Fmoc-Phe-NHS and H-Lys (MMT) -OH as described above for Ib.

Yield 94%. MS: 788.5 [M + l]⁺, 273.1 [M-MMT + 1]⁺.

For Fmoc-PheCit-OH you:

i) To Fmoc-Phe-NHS (4.96 g, 10.26 mmol) in DME (40 ml) was added to solution containing L-citrulline (1.80 g, 10.26 mmol) and NaHCO₃ (0.86 g, 10.26 mmol) in a mixture of H2O (40 ml) and THF (20 ml). The reaction was stirred for 15 hr. The residual DCC from the activation was filtered off and the organic solvent was removed on a rotary evaporator. H2O (100 ml) and iPrOH (10 ml) were added to the residue. The solution was made acidic to pH = 3 with 5% KHSO4, the product was extracted with a solution of EtOAc: iPrOH = 9: 1 (3x, 500 ml), washed with a mixture of brine: iPrOH = 9: 1 ( 2x, 50 mi), dried (Na₂SO4), filtered and concentrated, and dried with an oil pump. Trituration with ether gave the pure product. Yield 3.84 g (68%). MS: 545.6 [M + Na]⁺; 528.5 [M-H2O]⁺; 306.3 [M-Fmoc + H20]⁺.

ii) A solution of Fmoc-Phe-OPfp (553 mg, 1 mmol) in THF (5 ml) was added to a solution of H-Cit-OH (184 mg, 1.05 mmol) and NaHCCh (88.2 mg, 1.05 mmol) in H₂Or (2.6 mi). THF (2 ml) was added to make the solution homogeneous and stirred for 10 h. THF was removed on a rotary evaporator, the residue was diluted with H2O (10 ml) and iPrOH (1 ml) and made "ΙΜ ΡI

Mexican prruro Μ Μ CURRENCY Ο.

iHÓfrruAL acid at ρΗ = 1 with 3% of HC1. The product was extracted five times with a solution of EtOAc: iPrOH = 9: 1, rinsed with a mixture of brine: iPrOH = 9: 1, dried (Na2SO4) and concentrated in vacuo. Trituration with ether gave 313 mg of the pure product (57%).

Fmoc-AlaCit-OH If was prepared from Fmoc-Ala-NHS and H-Cit-OH as described above for le- (a). Yield 77%. MS: 959.8 [2M + Na]⁺; 938.1 [2M + 1]⁺; 491.4 [M + Na]⁺; 469.9 [M + l]⁺.

Crude Fmoc-ValCit-OH ig was prepared from Fmoc-Val-NHS and HCit-OH as described above for Ib. Final purification was done by trituration with ether. Total yield 76%. MS: 1060.3 [2M + 3Na]⁺; 1015.7 [2M + Na]⁺; 519.7 [M + Na]⁺; 497.9 [M + l]⁺.

Fmoc-Ala-Asn (DMCP) -OH lh was prepared from Fmoc-Ala-NHS and H-Asn (DMCP) -OH as described above for Ib. 95% yield. MS: 530.2 [M + Na]⁺; 508.2 [M + l]⁺; 426.0 [MDMCP + 1]⁺.

Coupling with p-aminobenzyl alcohol, preparation of Fmoc-AA-PABA and Fmoc-Ά-ΡΑΒΑ 2a-m.

The la-h products were coupled with paminobenzyl alcohol (PABA) in the presence of 2-ethoxy-letoxycarbonyl-1,2-dihydroquinoline (EEDQ) to form 2a-h. Four 3 j-1 representatives with only one amino acid that were coupled to the PABA portion were also prepared

<img file="MX347298B_D0053.tif" />

Fmoc — A¹—²-ΟΗ ah

<img file="MX347298B_D0054.tif" />

IMPI

<img file="MX347298B_D0055.tif" />

ah

TO¹= Gly, Glu (2Ph¡Pr), Asn (DMCP), Phe, Ala, Val. TO^z= Gly, Lys (MMT), Cit, Asn (DMCP), Lys (CH₃)₂

Fmoc — A¹—OH

<img file="MX347298B_D0056.tif" />

il

<img file="MX347298B_D0057.tif" />

TO¹= Lys (CH₃)₂, Leu, Asn (DMCP), Cit

Conditions: (i) PABA, EEDQ, THF

For Fmoc-GlyGly-PABA 2a, a solution of the (318 mg, 0.9 mmol) and PABA (220 mg, 1.8 mmol) in DCM (17 ml) and MeOH (6 ml) was stirred with EEDQ (444 mg, 1.8 mmol) ) for 10 h. All volatiles were removed on a rotary evaporator, the residue was triturated with Et20 and the product was filtered and dried in vacuo. Yield 348 mg (84%).

For Fmoc-Glu (O-2PhiPr) Gly-PABA 2b, a solution of Ib (524 mg, 0.96 mmol) and PABA (142 mg, 1.55 mmol) in DCM (10 ml) was stirred with EEDQ (357 mg, 1.44 mmol ) for 10 h. The tests were done as described above for 2a. Yield 462 mg (74%).

Fmoc-Asn (DMCP) Gly-PABA 2c, prepared as described above for 2a. Yield 64%. MS: 621.5 [M + 22]⁺; 599.3 [M + l]⁺.

Fmoc-PheLys (MMT) -PABA 2d, prepared as described

<img file="MX347298B_D0058.tif" />

above for 2b. Yield 70%.

For Fmoc-PheCit-PABA 2e, a solution of le (5.98 g, 10.97 mmol) and PABA (2.70 g, 21.95 mmol) in DCM (150 ml) and MeOH (50 ml) was treated with EEDQ (5.43 g, 21.95 mmol) ) and stirred for 15 h. The tests were done as described above for 2a. Yield 6.14 g (86%). MS: 650.7 [M + l]⁺; 527.3 [M-PABA + 1]⁺.

For Fmoc-AlaCit-PABA 2f, a solution of lf (2.89 g, 6.17 mmol) and PABA (1.52 g, 12.34 mmol) in DCM (45 ml) and MeOH (15 ml) was treated with EEDQ (3.05 g, 12.34 mmol) ) and stirred for 15 h. The tests were done as described above for 2a. Yield 4.56 g (74%). MS (ES, neg mode): 307.4 [M-263.6-1] 349.9 [M-Fmoc-1] 610, 608.4 [M + HC1-1] -.

Fmoc-ValCit-ΡΆΒΑ 2g was prepared as described above for 2b. (98%).

Fmoc-AlaAsn (DMCP) -PABA 2h was prepared as described above for 2a. Yield 59%. MS: 613.2 [M + l]⁺;

531.4 [M-DMCP + 1]⁺; 408.2 [M-205 + l]⁺.

For Fmoc-Lys (CH₃) a-PABA 2i, the salt of Fmoc-Lys (CH₃) aOH HC1 (433 mg, lmmol) and PABA (246 mg, 2 mmol) were dissolved in DCM (10 ml) and MeOH (1.5 ml), cooled to 5 ° C and EEDQ (495 mg, 2 mmol) was added. The cooling bath was removed and the mixture was stirred for 10 h at room temperature. All volatiles were removed on a rotary evaporator, the residue was triturated with EtaO, and the crude product was filtered.

It was redissolved in a mixture of DCM (2 ml) and MeOH (1 ml) and precipitated again by dropwise addition in Et₂Or (40 mi). The product was filtered and dried under vacuum. Yield 448 mg (83%).

For Fmoc-Leu-PABA 2j, a solution of Fmoc-Leu-OH (353 mg, 1 mmol), EEDQ (495 mg, 2 mmol) and PABA (222 mg, 1.8 mmol) in DCM (10 ml) was stirred for 10 h. All volatiles were removed on a rotary evaporator, the residue was dissolved in Et₂O (40 ml), chilled on ice dried for 2h and the solid was separated by centrifugation. The crude material obtained was purified on a column, gradient eluent of MeOH (1-2%) in CHCl3. Yield 444mg (97%). MS: 459.4 [M + l]⁺.

Fmoc-Asn (DMCP) -PABA 2k was prepared as described for 2j. In tests instead of column purification after removal of DCM the residue was triturated with Et₂Or, cooled to 0 ° C and the crude product was filtered. This treatment was repeated one more time followed by vacuum drying. Yield 77%. MS: 542.5 [M + l]⁺.

For Fmoc-Cit-PABA 21, a solution of Fmoc-Cit-OH (345.7 mg, 0.87 mmol) and PABA (214 mg, 1.74 mmol) in DCM (10 ml) and MeOH (4 ml) was treated with EEDQ (43 0 mg, 1.74 mmol) and stirred for 15 h. The solid product was triturated three times with ether, and the product was filtered and dried. Yield 288mg (67%). MS: 502.3 [M + l]⁺; 485.5 [M-H2O + 1]⁺; 263 [M-Fmoc-H20 + 1]⁺ ; 179.0 [M-306 + 1]⁺; 120.2 [M-365.3 + 1]⁺ .

^lb IMPI®3

MEXICAN INSTITUTE „« 3

OF THE INDUSTRIAL PROPERTY -The 2m product was prepared using a different scheme: coupling of the derivative H-Lys (CH3) 2-PABA 3 with Fmoc-Phe-NHS.

(i) ^(¡) &gt;

i ----- ► H — Lys (CH₃)₂—NH — ά ΌΗ

3i

Fmoc — Phe — Lys (CH₃)₂-NH —---^0H2m

Conditions: (i) triethylamine (Et₃N) in DMF, 10 h. (ii) Fmoc-Phe-NHS, diisopropylethylamine (DIEA), DMF.

For Fmoc - PheLys (CH₃) -PABA 2m, Fmoc-Lys (CH₃) 2-PABA (2i) (448 mg, 0.83 mmol) was deprotected with Fmoc by shaking with Et₃N (3.5 mi) in DMF (llmL) for 10 h. All volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum to obtain crude product 3i. This product was dissolved in DMF (7 ml), Fmoc-Phe-NHS (482 mg, 0.996 mmol) was added followed by DIEA (0.42 ml, 2.2 mmol) and the mixture was stirred for 10 h. The DIEA solvent was removed on a rotary evaporator at 40 ° C / oil pump vacuum to obtain 2m crude which was used without further purification. MS: 549.4 [M + l]⁺.

Preparation of Η-ΆΆ-ΡΑΒΑ 3a-h, m and HA-PABA 3j-l.

<img file="MX347298B_D0059.tif" />

<img file="MX347298B_D0060.tif" />

IMPI

MEXICAN INSTITUTE of INDUSTRIAL FROF1EDAD

<img file="MX347298B_D0061.tif" />

(i)

<img file="MX347298B_D0062.tif" />

<img file="MX347298B_D0063.tif" />

3¡-l

Conditions: (i) Et₃N in DMF, 10.

The Fmoc derivatives 2a-h, j-1 were treated with Et₃N in

DMF as described above for 3i followed by concentration and drying in vacuo. The crude products were dissolved in DMF to form 0.1 M solution and used without further purification.

Table 1. H-AA-PABA Intermediaries (1-3)

<td></td><td>Ai</td><td>TO²</td>

<td>1, 2, 3a</td><td>Gly</td><td>Gly</td>

<td>1, 2, 3b</td><td>Glu (2PhiPr)</td><td>Gly</td>

<td>1, 2, 3c</td><td>Asn (DMCP)</td><td>Gly</td>

<td>1, 2, 3d</td><td>Phe</td><td>Lys (MMT)</td>

<td>1, 2, 3e</td><td>Phe</td><td>Cit</td>

<td>1/2, 3f</td><td>To</td><td>Cit</td>

<td>1, 2, 3g</td><td>Val</td><td>Cit</td>

<td>1, 2, 3h</td><td>To</td><td>Asn (DMCP)</td>

<td>1/2, 3i</td><td>Lys (CH₃)₂</td><td></td>

<td>1, 2, 3j</td><td>Leu</td><td> —</td>

<td>1, 2, 3k</td><td>Asn (DMCP)</td><td></td>

<td> 1, 2, 31</td><td>Cit</td><td> -</td>

<td>2.3m</td><td>Phe</td><td>Lys (CH₃)₂</td>

<img file="MX347298B_D0064.tif" />

IMPI Mexican institute

INDUSTRY¹-

<img file="MX347298B_D0065.tif" />

2PhiPr DMCP MMP

Preparation of protease cleavable NAG masking reagents.

Preparation of NAG (R¹, R², R³) -L-AA-PABC-PNP (Tables 2, 3) or

H II

R¹ °&gt; oo ^l2' ^NHAc_Λ oMfv or

Preparation of NAG (R³, R², R³) -L-AA-PABC-PNP where R¹, R² and R³ are protecting groups and L is a bond between the portion of galactosamine (NAG) and the dipeptide (AA) begins in the preparation of acids NAG-L-CO₂H 6, 10a, b, 13 and 17 which, after conversion to the NHS ester, were used to acylate H-AA-PABA 2. In carbonates 21a-f designated for base sensitive polymers the protecting groups had to be removed prior to polymer modification. For this purpose in the preparation of 10a, b, 13 and 17 the Ac protecting groups in the GAL portion were replaced with triethylsilyl (TES) and tert-butyldimethylsilyl (TBDMS) leaving groups. Those groups can

<img file="MX347298B_D0066.tif" />

stir using a 70% solution of trifluoroacetic acid (TFA) in H2O at 0 ° C without compromising the base sensitive PNP carbonate portion.

a) In the preparation of NAG-L¹-CO₂H 6 where NAGtetraacetate 4 [3-5] protected with ZR¹= R²= R³= OAc was deprotected with Z (H2, Pd / C (10%), MeOH, CHCl3 (20%) to obtain NAG-amine 5 which was then acyl with succinic anhydride (succinic anhydride, EtsN, DCM, Ih).

<img file="MX347298B_D0067.tif" />

<img file="MX347298B_D0068.tif" />

NHAc

Conditions: (i) H₂, Pd / C (10%), MeOH, CHCI3, (20%), (ii), Succinic anhydride, Et₃N, CDM, Ih.

NAG-amine 5: For the preparation of 5 a solution of NAG 4 (6.74 g, 11.85 mmol) in MeOH (144 ml) and CHC1₃ (36 ml) was hydrogenated in the presence of 10% Pd / C (674 mg) at 1 atm. for 10 h. The catalyst was filtered through celite, the product was concentrated and dried in vacuo. Yield 5.04 g (98%).

NAG-LA-OH 6: For the preparation of 6 a solution of succinic anhydride (966 mg, 9.65 mmol) in DCM (30 ml) was _ _ MI ^ II - * added to NAG-amine 5 (4g, 9.15 mmol) in DCM (50 mi) followed by Et₃N (1964 ml, 14 mmol). After 1 h the reaction mixture was concentrated and dried in vacuo. The product was purified on a column, gradient eluent of MeOH (5-7%) in CHC1₃. Yield 3.1 g (63%). MS: 535.3 [M + l]⁺ ; 330.3 [deglycosylation product]⁺.

b) NAG derivatives with easily removable silyl ether protecting groups were prepared by Odeocetylation of 4 in a mixture of triethylamine in aqueous methanol followed by treatment with trialkylsilyl chlorides.

i) NAG-Li-OH 10a, b. 10a: R¹= OTES and OTBDMS, R²= 0H, R³= OTES; 10b: R¹= R³= OTBDMS, R²= OH.

Preparation of NAG 8a, b.

8a

NHAc

8b

R¹·

H ^N\.

8a: R₁₌OTBDMS _v OTES, R₂= OH, R₃= OTES

8b: R- | = R₃= OTBDMS, R₂= OH

NHAc

Conditions: (i) Et₃N, MeOH, H₂Or (5: 7: 6) 10. (ii)

<img file="MX347298B_D0069.tif" />

TBDMSC1 in DMF.

(1 eq.), Imidazole, lh followed by TESC1 (3 eq.), 10 h (iii) TBDMSC1 (3 eq.), Imidazole, 10 h in DMF.

Derivative NAG 7.

Preparation of 10a, b.

<img file="MX347298B_D0070.tif" />

9.10 a: R ^ OTBDMS and OTES, R₂= OH, R₃= OTES

9.10 b: R₁₌R3 = OTBDMS, R₂= OH

Conditions: (i) H₂, Pd / C (10%), THF. (ii) succinic anhydride, Et₃N, DCM, lh.

For the preparation of 7 NAG 4 (2g, 3.52 mmol), Odesacetylated by stirring in a solution of MeOH (10 ml), H₂0 (32 mi), and Et₃N (25 mi) for 10 h. All volatiles were removed on a rotary evaporator at 40 ° C and the residue was dried by two evaporations of toluene from the reaction mixture. Product 7 used directly in the next step. MS: 544.3 [M + Et₃N + l]⁺; 443.7 [M + l]⁺; 204 [deglycosylation product]⁺.

For the preparation of 8a of product 7 (1.76 mmol) in DMF (15 ml) it was treated with imidazole (718 mg, 10.54 mmol) and TBDMSC1 (265 mg, 1.76 mmol), stirred for 2 h and the reaction mixture was cooled to 0 ° C. TESC1 (531 mg, 3.52 mmol) was added, stirred for 10 h, concentrated and dried in vacuo. The residue *

<img file="MX347298B_D0071.tif" />

IMPI

INSTITUTO MEXICANO Dt LA PROPERTY INDUSTRIAL was collected in a mixture of EtOAc (110 ml) and H2O (30 ml).

The organic layers were separated, cooled to 5 ° C, washed with citric acid (5%), H₂0, NaHC0₃ and dried (Na2SO₄). The crude product was passed through a column, eluent 2% MeOH in CHCl3 to give a mixture of di-protected NAG derivatives with Si TBDMS and TES 8a. Yield 575 mg (49%). MS: 672.0 [M + l]⁺; 432.5 [deglycosylation product]⁺.

For the preparation of 8b, a solution of 7 (1.76 mmol) in DMF (15 ml) was stirred with imidazole (718 mg, 10.56 mmol) and TBDMSC1 (1061 g, 7 mmol) for 10 h. The reaction mixture was processed as described above for the preparation of 8a. Yield after column purification 767 mg (65%). M: 672.0 [M + l]⁺; 432.7 [deglycosylation product]⁺.

Product 10a was prepared as a mixture of NAG di-protected derivatives with Si TBDMS and TES following the procedure described for 10b below.

For the preparation of 10b, compound 8b (920 mg, 1.37 mmol) was hydrogenated in THF (20 ml) in the presence of 10% Pc / C (150 mg) at 1 atm for 10 h. The catalyst was filtered through celite, product 9b was concentrated and dried in vacuo.

The NAG-amine 9b without further purification was dissolved in DCM (12 ml), succinic anhydride solution was added

<img file="MX347298B_D0072.tif" />

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX347298B_D0073.tif" />

(140 mg, 1.40 mmol) in DCM (7 ml) followed by ppr Et₃N (0.236 mi,

1,676 mmol) and stirred for 2 h. The solvent was removed on a rotary evaporator and the product was purified on a column, eluent 1% AcOH, 10% MeOH in CHC1₃.

Yield 614 mg (72%).

ii) NAG-L²-OH 13. R¹= R³= OTBDMS, R²= OH. NAG derivatives with longer PEG spacer.

For analogs with longer PEG spacers, precursor 5 was first deprotected with acetyl to yield 11 (Et₃N, MeOH, H₂0 (5: 7: 6) 10). 11 was then acylated with half-benzyl half bis-dPEGs NHS ester (Product of Quanta cat. # 10237) to produce benzyl ester 12 (NHS-PEG5CO₂Bn, Et₃N, DCM). 12 was subsequently bis-silylated with TBDMSC1 (TBDMSC1 (3 eq.), Imidazole, 10 h in DMF) and debenzylated by hydrogenation (H2, Pd / C (10%), THF) to obtain acid 13.

Preparation of the NAG derivative 12.

<img file="MX347298B_D0074.tif" />

Conditions: (i) Et₃N, MeOH, H₂Or (5: 7: 6) 10. (ii) NHSPEG₅-CO₂Bn, Et₃N, DCM.

Preparation of NAG-L²-0H 13.

<img file="MX347298B_D0075.tif" />

IMPI

I WRMRRO MEXICAN OF INDUSTRIAL PROPERTY

<img file="MX347298B_D0076.tif" />

<img file="MX347298B_D0077.tif" />

<img file="MX347298B_D0078.tif" />

Conditions: TBDMSC1 (3 eq.), Imidazole, 10 in DMF. (vi) H₂, Pd / C (10%), THF.

Benzyl ester NAG-PEGe-SA 12. For the preparation of amine NAG 11, amine NAG 5 (0.381 mmol) was O-deacetylated as described for precursor 7 (procedure for 8a, b). Product 11 was dried by two evaporations of toluene on a rotary evaporator and dissolved in DMF (25 ml). NHS half benzyl ester Bis-dPEGs (200 mg, 0.381 mmol) was added to the reaction mixture followed by DIEA (0.079 ml, 0.457 mmol), stirred for 8 h, and concentrated on a rotary evaporator at 40 ° C / pump vacuum. oil. Crude product 12 was used in the next step without further purification. MS: 719.4 [M + l]⁺; 516.4 [deglycosylation product]⁺.

For the preparation of NAG-L²-0Bnm, dry product 12 was dissolved in DMF (5 ml), treated with TBDMSC1 (230 mg, 1524 mmol) followed by imidazole (156 mg, 2.29 mmol). The reaction mixture was stirred for 10 h, all volatiles were removed

<img file="MX347298B_D0079.tif" />

IMPI

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL in a rotary evaporator at 40 ° C / oil pump vacuum and the residue was collected in EtOAc (85 ml) and washed with HC1 (1%),

H2O. The aqueous phases were combined and re-extracted with EtOAc. The combined organic solutions were dried (Na2SO₄), concentrated and purified on a column, gradient eluent of MeOH (3-6%) in CHCl3. Yield of benzyl ester 291 mg (80%). MS: 965.3 [M + NH₄]⁺; 948.0

[M + l]⁺; 516.4 [deglycosylation product]⁺.

For the preparation of NAG-L²-OH 13 the aster NAG-L²-OBn was dehydrogenated as described for 9b (procedure for 10b). Yield 98%. MS: 858.0 [M + l]⁺; 426.1 [deglycosylation product]⁴·. The product was used without further purification.

iii) NAG-L³-OH 17. R¹= R³= OTBDMS, R²= 0H. NAG derivatives with longer PEG spacer.

was prepared by glycosylation of pentaacetate 14 [35] with mono-tBu PEG aster₄ (TMSOTf, DCE / HO-PEG₄-CO₂tBu, SnCl₄, DCM) to produce 15. 15 was hydrolyzed (HCO2H, 10h) to obtain acid 16, which after O-deacetylated (EtsN, MeOH, H₂0 (5: 7: 6) 10h) and treated with TBDMSC1 (TBDMSC1 (3 eq.), Imidazole, 10h in DMF) to obtain bis-silylated NAG acid 17.

Preparation of NAG (OAc) 3-L ester³-O-tBu 15.

<img file="MX347298B_D0080.tif" />

Oxazoline

<img file="MX347298B_D0081.tif" />

IMPI

INSTITUTO MEXICANO PE LA INDUSTRIAL PROPERTY

Conditions: (i) trimethylsilyl tri f-1 uu i uπιgΓaΊϊS1TTTδnaüo ~ ^^,(TMSOTf), dichloroethane (DCE). (ii) t-butyl 12-hydroxy-4,7,10-trioxadodecanoate (H0-PEG₄-C0₂tBu), SnCl₄, dichloromethane (DCM).

<img file="MX347298B_D0082.tif" />

<img file="MX347298B_D0083.tif" />

NHAc

Conditions: (i) HCO₂H, lOh. (ii) Et₃N, MeOH, H₂Or (5: 7: 6) 10. (iii) TBDMSC1 (3 eq.), imidazole, 10h in DMF.

For NAG ester (OAc) ₃-L³-O-tBu 15 pentacetyl, the galactosamine derivative 14 (10 g, 25.64 mmol) was dried by two evaporations of toluene. The resulting white glass was treated with TMSTf (5.18 ml, 28.6 mmol) in DCE (223 ml), and stirred at 60 ° C for 16 h. The reaction mixture was cooled to 0 ° C, quenched with TEA (2.6 ml), diluted with CHC1₃(3 00 mi) and washed twice with NaHCO₃ solution and with brine. The separated organic solution was treated with MgSO₄, concentrated, and dried under vacuum. The crude oxazoline derivative was used without further purification. Yield 8.14 g (96%).

MS: 368.1 [M + K]⁺; 352.2 [M + Na]⁺; 330.2 [M + l]⁺ .

IMPI MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

A stirred mixture of the oxazoline derivative (5.28 g, 16 mmol), t-butyl 12-hydroxy-4,7,10-trioxadodecanoate (5.12g, 18.4 mmol) and CaSO4 (20g) in DCM (270 ml) was SnC14 (0.84 ml, 0.84 mmol) was added dropwise. The solution was stirred for 16 h, filtered, diluted with CHCl3 (250 ml), washed twice with NaHCOs solution and brine. The product was dried with MgSCU, and concentrated. The crude was purified on a column, gradient eluent of MeOH (0-7%) in ethyl acetate. Yield 4.83 g (50%). MS: 630.8 [M + Na]⁺; 625.5 [M + NH₄]⁺; 608.4 [M + l]⁺;

552.6 [Mt-Bu + l]⁺; 330.2 [deglycosylation product]⁺.

For NAG (OAc) 3-L³-OH 16, tert-butyl ester 15 (1.99 g, 3.27 mmol) was stirred in clean formic acid (54 ml) for 16 h and all volatiles were removed in vacuo followed by three evaporations of toluene. The product was dried with an oil pump under vacuum for 2 h and used without further purification. Yield 1.77 g (98%). MS: 330.2 [deglycosylation product]⁺; 590.4 [M + K]⁺; 574.6 [M + Na]⁺; 569.6

[M + NH₄]⁺; 552.6 [M + l]⁺ .

For NAG (R¹, R², R³) -L³-OH 17 (R¹= R³= OTBDMS, R²= OH), product 16 was O-deacetylated, treated with TBDMSC1 as in the preparation of 8b and purified on a column, eluent 3% MeOH, 0.5% AcOH in CHCl3 - Yield 18%. MS: 1228.7

[M + l]⁺, 796.7 [deglycosylation product]⁺.

The five acids obtained 6, 10a, b, 13, 17 are

<img file="MX347298B_D0084.tif" />

converted to the NHS 18a-e ester in reaction with NHS and

DCC (NHS, DCC, DCM, 10h).

6,10,13,17

<img file="MX347298B_D0085.tif" />

<img file="MX347298B_D0086.tif" />

ae

<img file="MX347298B_D0087.tif" />

18th R¹= R²= R³= OAc, L¹

18b R¹= OTBDMS and OTES, R²= OH, R³= TES, L¹18c R '= R³= OTBDMS, R²= OH, L '

18d R¹= R³= OTBDMS, R²= OH, L²

18e R¹= R³= OTBDMS, R²= OH, L³

Conditions: (i) NHS, DCC, DXCM, 10.

For the preparation of NAG-L-NHS 18a-e the procedure described for 18c below was used. For product 18c, an ice-cold solution of 10b (614 mg, 0.964 mmol) and NHS (122 mg, 1061 mmol) in DCM (15 ml) was treated with DCC (219 mg, 1061 mmol), stirred for 30 min on ice and 8 h at 20 ° C. The reaction mixture was cooled to 0 ° C, DCU was filtered, the residue was concentrated and dried in vacuo. The crude product was dissolved in DMF to form 0.05 M solution and used without further purification.

Products 18b-e were prepared as described for 18a.

c) Formation of 20a-l, 3a-h was acylated with NHS ester of

<img file="MX347298B_D0088.tif" />

IMPI

ΙΗΤΠΤυΤΌ MEXICAN OF INDUSTRIAL PROPERTY hydroxyl protected NAG derivatives 18a-e (DIEA, _ DMF, 5 10h) to give 19a-1. Products 19a-1 were then treated with 5 equivalents of bis (p-nitrophenyl) carbonate ((PNPjaCO) ((PNP) 2CO, dioxane or DCM, 40-50 ° C, 15-24h) to produce the PNP carbonate derivatives protected with O-Acetyl 20a-l.

Products 20a-e were used directly for peptide modification. Acetyl groups and 2PhiPr, DMCP, MMT protecting groups of amino acids were removed after modification during consecutive treatment of DPC with TFA and EtaN.

3a-g + I8a-e

<img file="MX347298B_D0089.tif" />

NAG (R, R₂R₃)-THE A₂--NHNAG-LA, A₂-PABA

19a-l

<img file="MX347298B_D0090.tif" />

<img file="MX347298B_D0091.tif" />

20a-e

Conditions: (i) DIEA, dioxane or DCM, 25-60 ° C, 16-48h

NAG (R¹R²R³) -L-AA-PABA 19a

M

DMF, 5-10h. (ii) (PNP)₂CO,

1.

For product 19a (R¹= R²= R³= OAc, L = L¹, AA = GlyGly), a solution of NAG-NHS 18a ester in DMF (0.05M) (0.282 mmol) was treated with 0.1 M solution of 3a (0.282 mmol) in DMF and DIEA (59 μΐ, 0.338 mmol). In 3 h all volatiles were removed on a rotary evaporator at 40 ° C / pump vacuum

<img file="MX347298B_D0092.tif" />

IMPI INSTITUTO MEXICANO DE LA PROPERTY INDUSTRIAL oil, crushed with Et₂O and purified on a column, eluent: EtOAc gradient: CHC13: MeOH = 8: 7: 5-8: 7: 6. Yield 114mg (53%). MS: 754.4 [M + l]⁺.

The product 19b (Ri = R2 = R³= OAc, L = L¹, AA = Glu (2PhiPr) Gly) was prepared as described for 19a and purified on a column, eluent EtOAc: CHC13: MeOH = 8: 7: 3. Yield 64%. MS: 944.5 [M + l]⁺.

For product 19c (R¹= R²= R³= OAc, Ε = Ε²

AA = Asn (DMCP) Gly), to a solution of 3c (0.43 mmol) and DIEA (83 μΐ, 0.4 76 mmol) in DMF (2.15 ml) was added a solution of 18a (0.43 mmol) in DMF (2.15 ml) . The mixture was stirred for 16 h, filtered and all volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The crude product was triturated with Et₂O and purified on a column, eluent CHCI3: acetone: MeOH (5: 5: 1). Yield 242mg (62%). MS: 915.3 [M + Na]⁺; 910.6 [M + NH₄]⁺; 893.6 [M + l]⁺ .

Product 19d (R¹= R²= R³= OAc, L = L¹ AA = PheLys (MMT)) was prepared as described for 19a and purified on a column, gradient eluent of MeOH (5-6%) in CHCl3. Yield 56%. MS: 1187.9 [M + l]⁺.

For product 19e (R¹= R²= R³= OAc, L = L¹, AA = PheCit), to a solution of 3e (0.57 mmol) and DIEA (119 μΐ, 0.684 mmol) in DMF (3 ml) a solution of 18a (0.57 mmol) in DMF (3 ml) was added. The mixture was stirred for 16 h, filtered and all volatiles were removed on a rotary evaporator at⁹¹'NSTTTVW MEXICANO ---- de la noeiu&gt; Ar&gt; F ^ INDUSTRIAL

40 ° C / oil pump vacuum. The crude product was precipitated from Et₂O (45 ml) of CHC13: MeOH (5 ml) and used without further purification. Yield 392 mg (73%). MS: 966.8 [M + Na]⁺; 944.7 [M + l]⁺; 926.8 [MH₂OR]⁺; 821.5 [M-PABA + 1]⁺; 615.6 [M-NAcGal + 1]⁺; 492.3 [M-PABA-NAcGal + 1]⁺ .

The product 19f (R¹= R³= 0TBDMS, R²= 0H, L = L³, AA = AlaCit) was prepared as described for 19e and used without further purification. Yield 50%. MS: 993.2 [M + Na]⁺; 971.0 [M + l]⁺; 539.6 [deglycosylation product]⁺.

The product 19g (R¹= R³= OTBDMS, R²= OH L = L¹, AA = ValCit) was prepared as described for 19f and used in the next step without further purification. Efficiency 67%: 998.9

[M + l] ⁺ .

The product 19h (R¹= R³= OTBDMS, R²= OH L = L¹, AA = Glu (2PhiPr) Gly) was prepared as described for 19a and purified on a column, eluent solution of 3% NH4OH and 7.5% MeOH in DCM. Yield 15%. MS: 1047.2 [M + l]⁺, 615.7, 432.6 [deglycosylation product] ⁺.

The 19i (R¹= R³= OTBDMS, R²= 0H, L = L¹, AA = PheCit) was prepared as described for the preparation of 19e and used without further purification. Yield 50%. MS: 1068.7 [M + Na]⁺; 1047.3 [M + l]⁺; 615.4 [deglycosylation product]⁺; 432.5 [deglycosylation product]⁺.

The product 19j (R ^ OTBDMS and OTES, R₂= OH, R³= OTES, L = L¹, AA = PheCit) was prepared from 3e and 18b as a mixture of

INSTITUTO MEXICANO Di LA PROPERTY protected NEG derivatives C-3 and C-6 O-TBDMS and β ^ ΤΕθ¹- coftteM is described for the preparation of 19e y — ufe i ligaren. — en? - • gl · - ”next step without further purification. Yield 76%.

MS: 1047.4 [M + l]⁺, 615.8 [deglycosylation product]⁺.

The 19k R product¹= R³= OTBDMS, R²= 0H, L = L², AA = PheCit was prepared as described for 19e and used in the next step without further purification. Efficiency 67%: 1268.2

[M + l]⁺; 835.9 [deglycosylation product]⁺.

Product 191 R¹= R³= OTBDMS, R²= OH, L = L³, AA = PheCit was prepared as described for 19e and purified on a column, eluent 5% MeOH solution in CHCl3 Yield 60%. MS: 1064.0 [M + l]⁺; 632.7 [deglycosylation product]⁺.

NAG-AA-PABC-PNP 20a-1

For product 20a (R¹= R²= R³= OAc, L = L¹, AA = GlyGly), a suspension of 19a (100 mg, 0.132 mmol), (PNP) 2CO (202 mg, 0.663 mmol) and DIEA (0.07 ml, 0.396 mmol) in dioxane (5 ml) were stirred for 8 h in darkness at 40 ° C. Another portion of (PNP)₂CO (121 mg, 0.397 mmol) and DIEA (0.04 ml, 0.226 mmol) were added and stirring continued for another 8 h at 40 ° C. All volatiles were removed on a rotary evaporator and the product was purified on a column, eluent: CHCl3: EtOAc: MeOH = 7: 8: 3. Yield 84mg (69%).

For product 20b (R³= R²= R³= OAc, L = L¹,

AA = Glu (2PhiPr) Gly), a solution of 19b (160 mg, 0.169 mmol), (PNP)₂CO (258 mg, 0.847 mmol) and DIEA (0.09 ml, 0.507 mmol) in

DCM (10 ml) was stirred at ___. , _,._Λ "THE FBOMEDAD darkness for 10 h, oewCTont

IMPI

MEXICAN INSTITUTE

<img file="MX347298B_D0093.tif" />

a rotary evaporator and the product was —- purified ^iiria column, eluent: 5-6% MeOH solution in CHCl3. Yield 174mg (92%).

For product 20c (R¹= R²= R³= OAc, L = L¹

AA = Asn (DMCP) Gly), a solution of 19c (127 mg, 0.142 mmol), (PNP)₂CO (216 mg, 0.710 mmol) and DIEA (74 μΐ, 0.426 mmol) in DCM (5 ml) were stirred in the dark for 16 h, concentrated on a rotary evaporator and the product was purified on a column, eluent: CHCI3: EtOAc: MeOH (7: 2.2: 0.8). Yield 110.6 mg (74%). MS: 1080.9 [M + Na]⁺; 1058.7 [M + l]⁺.

The product 20d (R¹= R2 = R3 = OAc, L = L¹ AA = PheLys (MMT)) was prepared as described by 20b and purified on a column, eluent: CHCl3: EtOAc: MeOH = 9: 7: 1 Yield 76 mg (47%).

For product 20e (R¹= R²= R³= OAc, L = L¹, AA = PheCit), a solution of 19e (164 mg, 0.173 mmol), (PNP)₂CO (528 mg,

1.73 mmol) and DIEA (182 µΐ, 1.04 mmol) in dioxane (17 ml) was stirred in the dark at 60 ° C for 16 h and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by two consecutive evaporations of DMF on a rotary evaporator at 40 ° C / oil pump vacuum and the product was purified on a column, eluent CHCI3: EtOAc: MeOH (8: 1.5: 0.5) followed by CHCl₃: MeOH (7: 1). Yield 85mg (44%). MS: 1132.0 [M + Na]⁺; 1110.1 [M + l]⁺ ; 780.8 [deglycosylation product]⁺.

<img file="MX347298B_D0094.tif" />

IMPI

MEXICAN INSTITUTE

OF INDUSTRIAL PROPERTY

The product 20f (R¹= R³= 0TBDMS, R²= 0H, L = L¹, AA = AlaCit) was prepared as described by 20e and purified on a column, eluent: CHCl3: EtOAc: MeOH = 9: 10: 1. Performance

153 mg (36%).

The product 20g (R^X= R³= OTBDMS, R²= 0H, L = L¹, AA = ValCit) was prepared as described by 20e and purified on a column, eluent CHCl3: EtOAc: MeOH = 16: 3: 1. Yield 44%.

MS: 1164.5 [M + l]⁺.

The product 20h (R¹= r3 = 0TBDMS, R²= OH, L = L¹,

AA = Glu (2PhiPr) Gly) was prepared as described by 20b and purified on a column, eluent CHCl3: EtOAc: MeOH = 8: 7: 1. Yield 77%.

For the 20i (R¹= R³= OTBDMS, R²= OH, L = L¹, AA = PheCit), a solution of 191 (316 mg, 0.297 mmol), (PNP)₂CO (913mg, 2.97 mmol) and DIEA (310 µΐ, 1.78 mmol) in dioxane (7 ml) were stirred in the dark at 65 ° C for 40h and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by two consecutive evaporations of DMF on a rotary evaporator at 40 ° C / oil pump vacuum and the product was purified on a column, eluent CHCI3: EtOAc: MeOH (8: 1.5: 0.5) followed by CHCl₃: MeOH (92:08). Yield 297mg (81%). MS: 1246.7 [M + NH₄]⁺; 1228.7 [M + l]⁺; 797.6 [deglycosylation product]⁺; 432.7

[deglycosylation product]⁴·.

The product 20j (R¹= OTBDMS and OTES, R₂= OH, R³= OTES, L = L¹,

<img file="MX347298B_D0095.tif" />

AA = PheCit), the product 20 j as a mixture of protected derivatives C-3 and C-6 O-TBDMS and O-TES 'was prepared as described by 20e and purified on a column, eluent CHC1₃: EtOAc: MeOH = 16: 3: 1 followed by 10% MeOH in CHCl3.

Yield 50%. MS: 1212.0 [M + l]⁺, 480.0 [deglycosylation product] ⁺.

The product 20k (R¹= R³= OTBDMS, R²= OH, L = L², AA = PheCit) was prepared as described by 20e and purified on a column, gradient eluent of MeOH (8-10%) in CHCl3. Yield 85%. The product was used directly in the next step.

For product 201 (R¹= R³= OTBDMS, R²= OH, L = L³, AA = PheCit), solution of 191 (316 mg, 297 mmol), (PNP)₂CO (912 mg, 3 mmol) and DIEA (0.31 ml, 1.78 mmol) in dioxane (8 ml) were stirred under Ar in the dark at 60 ° C for 48h. All volatiles were removed on a rotary evaporator and the product was purified on a column, eluent:

CHC1₃ : EtOAc: MeOH = 16: 3: 1. Yield 297mg (81%).

NAG-L-AA-PABC-PNP 21a-f (R¹= R²= R³= OH), deprotection of

20f-l

Formation of 21a-f. For modification of base sensitive polyacrylates, the protecting groups on NAG and AA dipeptide were removed prior to coupling to the polymer by treating 20f-l with a mixture of TFA: H₂O = 3: 1 (TFA / H₂O = 3: 1, 5 ° C, 2-3h) to give the reagents of ⁹⁶ IMPI

MEXICAN INSTITUTE • PE LA N1OPIEOA »

INDUSTRIAL masking of dipeptide NAG 21a-f.

or

M ------ ► NAG (OH)₃-THE A₂ NH — Q ^ Aq- ^ g — N0₂

21a-f

Conditions: TFA / H₂O = 3: 1, 5 ° C, 2-3h

For product 21a (AA = AlaCit, L = L¹) compound 20f (150 mg, 0.132 mmol) was stirred in ice-cold solution of TFA: H2O = 3: 1 (2 ml) for 4 h and added dropwise in Et₂Or in agitation (20 mi). The precipitate was separated and dried by evaporation of toluene on a rotary evaporator / 30 ° C and then under vacuum. Yield 112 mg (94%). MS: 907.2 [M + l]⁺; 704.4 [deglycosylation product] *.

For product 21b (AA = ValCit, L = L¹), compound 20g (305 mg, 0.26 mmol) was stirred in ice-cold TFA: H solution₂O = 3: 1 (5 mi) per lh and added drip to Et₂Or in agitation (45 mi). The solid product was separated and dried by evaporation of toluene on a rotary evaporator / 30 ° C and then under vacuum. Yield 193 mg (79%). MS: 935.8 [M + 1] *, 732.7 [deglycosylation product] *.

The product 21c (AA = GluGly, L = L¹) was prepared as described for 21b. Yield 55mg (98%). MS: 865.5

[M + l]⁺, 662.3 [deglycosylation product] *.

The product 21d (AA = PheCit, L ^ L¹) was prepared as described for 21b. MS: 983.7 [M + l]⁺, 780.9 [deglycosylation product] ⁺.

<img file="MX347298B_D0096.tif" />

«RsF

<img file="MX347298B_D0097.tif" />

For product 2le (AA = PheCit, L = L²), the compueS-tO—. ·, .....,

20k was stirred in ice-cold TFA: H2O = 3: 1 (5 ml) for 1.5h under the conditions described for 21b. Yield 25% counting from 19k. MS: 1203.9 [M + l]⁺, 1001.0 [deglycosylation product] ⁺.

The product 21f (AA = PheCit L = L³) was prepared from 201 using 3 h of deprotection under the conditions described for 21b. Yield 75%. MS: 1203.9 [M + l]⁺, 1001.0 [deglycosylation product]⁴.

Table 2. Intermediaries NAG-L-AA-PABA (19) and NAG-LAA PABC (20)

<td>compound</td><td>Ai</td><td>to₂</td><td>L</td><td>Ri</td><td>r₂</td><td>r₃</td>

<td>19th</td><td>Gly</td><td>Gly</td><td>Li</td><td>OAc</td><td>OAc</td><td>OAc</td>

<td>19b</td><td>Glu (2PhiPr)</td><td>Gly</td><td>Li</td><td>OAc</td><td>OAc</td><td>OAc</td>

<td>19c</td><td>Asn (DMCP)</td><td>Gly</td><td>Li</td><td>OAc</td><td>OAc</td><td>OAc</td>

<td>19d</td><td>Phe</td><td>Lys (MMT)</td><td>Li</td><td>OAc</td><td>OAc</td><td>OAc</td>

<td>19e</td><td>Phe</td><td>Cit</td><td>Li</td><td>OAc</td><td>OAc</td><td>OAc</td>

<td>19, 20f</td><td>To</td><td>Cit</td><td>Li</td><td>OTBDMS</td><td>Oh</td><td>OTBDMS</td>

<td>19, 20g</td><td>Val</td><td>Cit</td><td>Li</td><td>OTBDMS</td><td>Oh</td><td>OTBDMS</td>

<td>19, 20h</td><td>Glu (2PhiPr)</td><td>Gly</td><td>Li</td><td>OTBDMS</td><td>Oh</td><td>OTBDMS</td>

<td>19, 20i</td><td>Phe</td><td>Cit</td><td>Li</td><td>OTBDMS</td><td>Oh</td><td>OTBDMS</td>

<td>19, 20j</td><td>Phe</td><td>Cit</td><td>Li</td><td>OTBDMS, TES</td><td>Oh</td><td>OTES</td>

<td>19, 20k</td><td>Phe</td><td>Cit</td><td>l₂</td><td>OTBDMS</td><td>Oh</td><td>OTBDMS</td>

<td> 19, 201</td><td>Phe</td><td>Cit</td><td>Li</td><td>OTBDMS</td><td>Oh</td><td>OTBDMS</td>

Table 3. NAG-L-AiA₂-Final PABC used for the

<img file="MX347298B_D0098.tif" />

IMPI SP nSTITUTO MEXICANO

Dt INDUSTRIAL PROPERTY preparation CPD (20, 21)

<td>compound</td><td>Ai</td><td>to₂</td><td>L</td>

<td>20 a</td><td>Gly</td><td>Gly</td><td>Li</td>

<td>20b</td><td>Glu</td><td>Gly</td><td>Li</td>

<td>20c</td><td>Asn</td><td>Gly</td><td>Li</td>

<td>20d</td><td>Phe</td><td>Lys</td><td>Li</td>

<td>20e</td><td>Phe</td><td>Cit</td><td>Li</td>

<td>21a</td><td>To</td><td>Cit</td><td>Li</td>

<td>21b</td><td>Val</td><td>Cit</td><td>Li</td>

<td>21c</td><td>Glu</td><td>Gly</td><td>Li</td>

<td>2 Id</td><td>Phe</td><td>Cit</td><td>Li</td>

<td>21e</td><td>Phe</td><td>Cit</td><td>L₂</td>

<td>21f</td><td>Phe</td><td>Cit</td><td>l₃</td>

Preparation of Protease Divisible PEG Masking Reagents

The amino group of any of H-AA-PABA 3b, e, g, h, j, km was acylated with a PEG acid ester NHS (DIEA, DMF, 5-10h) to produce 22a-k. The hydroxyl group in product 22a-k was then converted to pnitrophenyl carbonate ((PNP)₂CO, dioxane or THF, 40-60 ° C, 10h) to produce 23a-k. For 23a, d, g, the protecting groups of Asn and Glu were removed by treatment with aqueous TFA (TFA / H₂O = 3: 1.5 ° C, 2-3h) to obtain the desired products 24a-c. (Can consistency be retained by converting from 23a, d, g to 24a, d, g?). Also, is there a consistency between amino acids for each letter between the NAG and PEG versions?

Preparation of

PEGn-AA-PABA 22a-k.

<img file="MX347298B_D0099.tif" />

Its b, e, g, h, jm

<img file="MX347298B_D0100.tif" />

OR

<img file="MX347298B_D0101.tif" />

22a-k

PEG-AA (Prot) -PABA

Conditions: (i) DIEA, DMF, 5-10h.

Product 22a (n = ll, AA = GluGly). A 0.1M solution of 3b in DMF (3.5 ml, 0.35 mmol) was stirred for 10 h with PEGn-NHS ester (240 mg, 0.35 mmol) and DIEA (0.061 ml, 0.35 mmol). All volatiles were removed on a rotary evaporator at 40 ° C / oil pump and the product was purified on a column, eluent: CHCl3: MeOH: AcOH = 38: 2: 1. Yield 274 mg (78%) MS: 1015.6 [M + NH₄]⁺, 998.7 [M + l]⁺.

Product 22b (n = ll, AA = PheCit). To a solution of 3e (0.88 mmol) and DIEA (167 µΐ, 0.96 mmol) in DMF (3 ml) was added a solution of PEGn-NHS ester (0.80 mmol) in DMF (3 ml). The mixture was stirred for 16 h, filtered and all volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The crude was precipitated in EtzO (45 ml) of CHClsiMeOH (5 ml) and purified on a column, eluent at a gradient of MeOH (10-16%) in CHC1₃.

¹⁰⁰industrial * · --— Yield 42 0 mg (53%). MS: 1015.9 [Μ + Η₂Ο]⁺; 998.8 [M + l]⁺; _ • ΐβν. ·· I · Ι Ι · · &lt;! ·¹ ' .....

981.1 [Μ-Η₂Ο]⁺.

The product 22c (η = 11, AA = ValCit). Product 22f was prepared from crude 3g (obtained from 300mg, 0.5mmol from 2g), PEGn-NHS ester (298mg, 0.435mmol) and DIEA (0.09ml, 0.522mmol) as described for 22a. After concentration on a rotary evaporator at 40 ° C / oil pump, the product was suspended in a MeOH: DCM = 1: 1 (6 ml) mixture, sonicated, filtered and Et precipitated.₂Or (50 mi). The solid was separated and the procedure was repeated again. Residual solvents were removed in vacuo. Yield 283mg (60%). MS: 951.5 [M + l]⁺.

The product 22d (n = ll, AA = AlaAsn (DMCP)). A solution of PEGn-NHS ester (0.56 mmol) in DMF (3 ml) was added to a solution of 3h (0.56 mmol) and DIEA (116 μ és, 0.67 mmol) in DMF (3 ml). The mixture was stirred for 16 h, filtered and all volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The residue was dissolved in a mixture of CHCl3: MeOH = 1: 1 (5 ml) and precipitated in Et₂0 frozen (0 ° C) (45 ml). The solid was purified on a column, gradient eluent of MeOH (3-14%) in DCM. Yield 261 mg (49%). MS: 983.7 [M + Na]⁺; 979.1 [M + NH4]⁺, - 961.8 [M + l] ⁺ ; 943.9 [M-H20 + l]⁺.

The product 22e (n = ll, AA = PheLys (Me₂)). Product 22e was prepared as described for 22a. The purification was done

101

IMPI

MEXICAN INSTITUTE

OF INDUSTRIAL PROPERTY

<img file="MX347298B_D0102.tif" />

using Nucleodur C-18 HPLC column, 250 x 4.6, eluent

ACN-H2O (0.1% TFA), ramp 15-30%. MS: 998.1 [M + l]⁺. The isolated product was desalted on Dowex 1x8 resin, eluent H2O. Yield 40%.

The product 22f (n = ll, AA = Leu). Product 22f was prepared as described for 22a and purified on a column, eluent: CHCl3: EtOAc: MeOH: AcOH = 9: 7: 2: 0.04. Yield 48%. MS: 824.9 [M + NHd *.

The product 22g (n = ll, AA = Asn (DMCP). The crude 3k (obtained from 419 mg, 0.77 mmol from 2k), PeguNHS ester (200 mg, 0.292 mmol) and DIEA (0.06 ml, 0.35 mmol) were stirred in DCM (5 ml) for 10 h. The solvent was removed on a rotary evaporator and the product was purified on a column, eluent CHCI3: EtOAc: MeOH AcOH = 4.5: 3.5: 1: 0.02. Yield 254 mg (37%) MS: 891.1 [M + l]⁺.

The product 22h (n = ll AA = Cit). To a solution of 31 (0.50 mmol) and DIEA (104 μΐ, 0.60 mmol) in DMF (2.5 ml) was added a solution of PEGn-NHS ester (0.50 mmol) in DMF (2.5 ml). The mixture was stirred for 16 h, filtered and all volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The residue was dissolved in a mixture of CHCl3: MeOH = 1: 1 (5 ml) and precipitated in Et₂Or (45 mi). The precipitation was repeated two more times and the product was used without further purification. Yield 340mg (80%). MS: 869.4 [M + NH₄]⁺; 851.9 [M + l]⁺.

<img file="MX347298B_D0103.tif" />

The product 22i (n = 23, AA = PheCit). To a solution of 3e (0.72 mmol) and DIEA (130 µl, 0.74 mmol) in DMF (3 ml) was added a solution of PEG23-NHS (0.60 mmol) in DMF (3 ml). The mixture was stirred for 16 h, filtered and all volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The residue was dissolved in a mixture of CHCl3: MeOH = 1: 1 (5 ml) and precipitated in Et₂Or (45 mi). The solid product was purified on a column, gradient eluent of MeOH (7-12%) in CHC1₃. Yield 487mg (53%). MS: 1555.2 [M + Na]⁺; 1544.7 [M + NH₄]⁺; 1527.7 [M + l]⁺ .

The product 22 j (PEG with average MW of 1000. AA = PheCit). A mixture of mPEG-1000 alcohol (Fluka) (0.173g, 0.173 mmol), N, N-disuccinimidyl carbonate (62 mg, 0.242 mmol), and TEA (0.101 ml, 0.726 mmol) was stirred in MeCN (1 ml) for 16 h. All volatiles were removed on a rotary evaporator and the crude residue was dissolved in CHCl3 (10 mL). The organic layer was washed with H2O (1 ml, pH = 5), then brine, dried over Na2SO₄ and concentrated to give PEG-1000-NHS carbonate. This product was stirred for 16 h with 3e (0.121 mmol) and DIEA (30 µl, 0.173 mmol) in DMF (1 ml), filtered and all volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum . The residue was dissolved in a mixture of CHCl3: MeOH = 1: 1 (5 ml) and precipitated in Et₂Or (45 ml). The precipitation was repeated two more times and the product was used without further purification.

103

Yield 134mg (79%).

IMPI 0¾¾

MEXICAN INSTITUTE

0E THE PRORIEPAl 'industrial -The product 22k (n = 23, AA = ValCit). To SUIucIÓri 3e3g '(1.0 mmol) and DIEA (183 μΐ, 1.04 mmol) in DMF (4 ml) a solution of PEG23-NHS ester (0.87 mmol) in DMF (4 ml) was added. The mixture was stirred for 16 h, filtered and all volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The residue was dissolved in a mixture of CHCl3: MeOH = 1: 1 (5 ml) and precipitated in Et₂Or (45 mi). The precipitation was repeated two more times and the product was used without further purification. Yield 1.0 g (77%). MS: 1496.1 [M + NH₄]⁺; 1479.3 [M + l]⁺ .

PEG-AA-PABC-PNP 23a-k

<img file="MX347298B_D0104.tif" />

Or ak

PEG-AA (Prot) -PABC-PNP

Condition: (i) (PNP) 2C0, dioxane or THF, 40-60 ° C, 10h.

For product 23a (n = ll, AA = Glu (2PhiPr) Gly), product 22a (274 mg, 0.274 mmol) in DCM (15 ml) was shaken in the dark with (PNP)₂CO (418 mg, 1,372 mmol) and DIEA (0.143 ml, 0.823 mmol) for 15 h. The solvent was removed on a rotary evaporator and the product was purified on a column, eluent 4% MeOH, 0.2% AcOH in CHCl3. Yield 260mg (81%). MS: 1180.7 [M + NH₄]⁺.

For product 23b (n = ll, AA = PheCit), a solution of

104

<img file="MX347298B_D0105.tif" />

22b (419 mg, 0.42 mmol), (PNP)₂CO (766 mg, 2.52 mmol) and DIEA (263 µΐ, 1.52 mmol) in dioxane (4 ml) were stirred in the dark at 50 ° C for 15h and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by two consecutive evaporations of DMF on a rotary evaporator at 40 ° C / oil pump vacuum and the product was purified on a column, eluent CHC1₃: EtOAc: MeOH (4.5: 5: 0.5) followed by CHCl₃: MeOH (9: 1). Yield 390mg (80%). MS: 1181.2 [M + NH₄] ⁺, 1164.2 [M + l] ⁺ .

For product 23c (n = ll, AA = ValCit), a solution of 22c (273 mg, 0.287 mmol), (PNP)₂C0 (874 mg, 2.88 mmol) and DIEA (0.3 ml, 1.72 mmol) in 1,4-dioxane (22 ml) were stirred in the dark for 24 h at 50 ° C. The solvent was removed on a rotary evaporator at 40 ° C / oil pump and the product was purified on a column, eluent: CHCI3: EtOAc: MeOH = 16: 3: 1 followed by 12-15% MeOH in CHCI3 Yield 163 mg (51%). MS: 1116.0 [M + l]⁺.

Product 23d (n = ll, AA = AlaAsn (DMCP)) was prepared as described in preparation of 23b. The product was purified on a column, eluent CHCl3: EtOAc: MeOH (9: 2: 1). Yield 77%. MS: 1144.0 [M + NH₄]⁺; 1127.3 [M + l]⁺.

The product 23e (n = ll, AA = PheLys (Me) ₂) was prepared as described for 23a and purified on a column, eluent: 10% MeOH, 0.2% AcOH in CHC1₃. Yield 63%. MS: 1163.1 [M + l]⁺.

Product 23f (n = ll, AA = Leu) was prepared as

105

<img file="MX347298B_D0106.tif" />

<img file="MX347298B_D0107.tif" />

described for 23c using only 5 entities of (FOCUS and 3 equivalents of DIEA aplicands ^ caier-'pülf'24h. The product was purified on a column, gradient eluent of MeOH (7-12%) in CHC1₃. Yield 75%. MS: 972 [M + l]⁺ .

The 23g product (n = ll, AA = Asn (DMCP)) was prepared as described for 23f and the crude product was used in the next step without further purification. MS: 1073.4 [M + 18]⁺ .

For product 23h (n = ll, AA = Cit), 22h solution (340 mg, 0.40 mmol), (PNP)₂CO (608 mg, 2.00 mmol) and DIEA (208 µΐ, 1.20 mmol) in DCM (4 ml) were stirred in the dark at 30 ° C for 15h and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by two consecutive evaporations of DMF on a rotary evaporator at 40 ° C / oil pump vacuum and the product was purified on a column, eluent CHCI3: EtOAc: MeOH (7: 2.5: 0.5) followed by a gradient of MeOH (8-14%) in CHCI3. Yield 390mg (80%). MS: 1034.3 [M + NH₄]⁺; 1016.9 [M + l]⁺.

The product 23i (n = 23, AA = PheCit) was prepared as described in the preparation of 23b and purified on a column, eluent CHCI3: EtOAc: MeOH (4.5: 5: 0.5) followed by a gradient of MeOH (6- 12%) in CHC1₃. Yield 86%. MS: 1711.4 [M + NH₄]⁺; 1694.4 [M + l]⁺ .

Product 23j (PEG 1000K AA = PheCit) was prepared as described in Preparation 23b and purified on a

106

<img file="MX347298B_D0108.tif" />

IMPI

INSTITUTO MEXICANO ot LA FRONF.UAO INDUSTRIAL column, eluent CHCI3: EtOAc: MeOH (4.5: 5: 0.5) followed by a gradient of MeOH (6-12%) in CHCI3. Yield 72%.

The 23k product (n = 23, AA = ValCit) was prepared as described in preparation of 23b, and the product purified with HPLC. Column: Luna (Phenomenex) 5u, C-8, 100 A. Mobile Phase: ACN-H2O (F3CO2H 0.01%), ACN gradient 30-37%, 31 minutes. Yield: 530 mg (48%). MS: 1666.4 [M + Na]⁺; 1644.2 [M + l]⁺ .

PEG-AA-PABC-PNP 24a-c, AA deprotection.

or

<img file="MX347298B_D0109.tif" />

Or ac

PEG-AA-PABC-PNP

Conditions: (i) TFA / H₂O = 3: 1.5 ° C, 2-3h.

Product 24a (n = ll, AA = GluGly). Product 23a (250 mg, 0.215 mmol) was stirred in a 3% TFA solution of CHCl3 (16 mL) for 35 min, concentrated on a rotary evaporator, and dried in vacuo. Yield 224 mg (100%) (MS: 1062.6 [M + NH₄]⁺; 1045.9 [M + l]⁺.

Product 24b (n = ll, AA = AlaAsn-PABC-PNP). Compound 23d was stirred for 1.5 h in a TFA: DCM (3: 1) mixture and all volatiles were removed on a rotary evaporator at 20 ° C. The product was purified on a column, gradient eluent of MeOH (6-12%) in CHCl3. Yield 30%. MS: 1066.7 [M + Na]⁺, 1062.0 [M + NH₄]⁺; 1045.2 [M + l]⁺.

Product 24c (n = ll, AA = Asn). A reaction flask with

<img file="MX347298B_D0110.tif" />

23g (160mg

0.143 mmol)

107 cooled to

IMPI

INSTITUTO MEXICANO M LA PKOfBOAD INDUSTRIAL

0 ° C and a cold mixture of TFA: H was added₂Or (9: 1) (12.5 mi). The mixture was stirred for 1.5 h and diluted with H₂Or cold (50 mi). Stirring continued for 20 min at 20 ° C. The precipitate was filtered and rinsed with H₂0. All volatiles were removed on a rotary evaporator at 40 ° C and the product was purified on a column, eluent CHC1₃ : EtOAc: MeOH: AcOH = 4.5: 3.5: 1.2: 0.02. Yield 43mg (30%). MS: 974.0 [M + l]⁺.

Table 4. Final PEG-L-A1A2-PABC used for the preparation of DPC.

<td colspan="2">compound</td><td colspan="2">AA</td><td rowspan="2">size</td>

<td>PEGn-AA-PABA</td><td>PEGn-AA- (PNP)</td><td>TO¹</td><td>TO²</td>

<td> 22</td><td>23a</td><td>Glu (2PhiPr)</td><td>Gly</td><td>n = 11</td>

<td> 22</td><td>23b</td><td>Phe</td><td>Cit</td><td>n = 11</td>

<td> 22</td><td>23c</td><td>Val</td><td>Cit</td><td>n = 11</td>

<td> 22</td><td>23d</td><td>To</td><td>Asn (DMCP)</td><td>n = 11</td>

<td> 22</td><td>23e</td><td>Phe</td><td>Lys (CH₃)₂</td><td>n = 11</td>

<td> 22</td><td>23f</td><td>Leu</td><td> -</td><td>n = 11</td>

<td> 22</td><td>23g</td><td>Asn (DMCP)</td><td> -</td><td>n = 11</td>

<td> 22</td><td>23h</td><td>Cit</td><td> -</td><td>n = 11</td>

<td> 22</td><td>23i</td><td>Phe</td><td>Cit</td><td>n = 23</td>

<td> 22</td><td>23j</td><td>Phe</td><td>Cit</td><td>1 kDa</td>

<td> 22</td><td>23k</td><td>Val</td><td>Cit</td><td>n = 23</td>

<td></td><td>24th</td><td>Glu</td><td>Gly</td><td>n = 11</td>

<td></td><td>24b</td><td>To</td><td>Asn</td><td>n = 11</td>

<td></td><td>24c</td><td>Asn</td><td> -</td><td>n = 11</td>

108

IMPIAS

INSTITUTO MUICANCtH LA rRONLUAC · „,, _ _,,, 1NDUSTMM / ** t4-s—

Example 2. Binding of protease cleavable masking agents to polymers with amine-forming agents of p-acylamidobenzyl carbamate.

A. Modification of Melittin with protease cleavable masking agents. Ix mg of melittin peptide and 10 mg of HEPES base at 1-10 mg / ml peptide was masked by the addition of 2-6x mg of amine-reactive p-nitrophenyl carbamate derivatives or N-hydroxysuccinimide carbonate of divisible substrate of protease containing NAG. The solution was then incubated at least ih at room temperature (RT) before injection into animals.

B. Modification of polyamine with protease cleavable masking agents. The activated carbonates (reactive to amine) of derivatives of p-acylaminobenzyl alcohol were reacted with amino groups of active polyamine in amphipathic membranes at Η₂0 at pH&gt; 8 to produce a pacylamidobenzyl carbamate. .

or

R¹—AA-NH —— Z ₊ H₂N — R² --- ► R¹—AA-NH —— CH ^ O ^^ NH — R²

R¹ comprises an ASGPr ligand (whether protected or unprotected) or a PEG,

R² is an antipathetic membrane active polyamine,

AA is a dipeptide (either protected or unprotected), and

Z is an amine reactive carbonate.

For x mg of polymer, 12x mg of base were added

109

<img file="MX347298B_D0111.tif" />

free of HEPES in isotonic glucose. To the buffer polymer solution, 2x to 16x mg of 200 mg / ml dipeptide masking agent in DMF was added. In some applications, the polymer was modified with 2x mg dipeptide masking agent followed by siRNA coupling. The polymer-siRNA conjugate was then further modified with 6x to 8x mg of dipeptide masking agent.

Example 3. ARNsis. The siRNAs have the following sequences: siRNA Factor VII sense: (Chol) -5 'GfcAfaAfgGfcGfuGfcCfaAfcUfcAf (invdT) 3' (Seq ID 1) antisense: 5 'pdTsGfaGfuUfgGfcAfcGfcCfudUfGfCf' sense 3 GfCUfCfUfCfCf 3 'GfcGfcCfuDUfGfCfCf 3' PdTsGfaGfuUfgGfcAfcGfcCfuDUfGAfCfCf ' Seq ID 3) antisense: 5 'GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT 3' (Seq ID 4) or sense: (NH₂Cg) GfuUfgGfuGfaAfuGfgAfgCfuCfaGf (invdT) 3 '(Seq ID 5) antisense: pCfsUfgAfgCfuCfcAfuüfcAfcCfaAfcdTsdT 3' (Seq ID 6) or sense: 5 '(NH₂Cg) uGuGfcAfaAfgGfcGfuGfcCfaAfcUfcAf (invdT) 3 '(Seq ID 23) antisense: 5' pdTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT 3 '(Seq ID 24) ARNfufCfuF (prGuFuG 3) -siFAG Factor VII (prGfuGfuG 3) -siFGFUF VII (prGfuG FuGFuG) -si5AfG factor VII (prGfuGfuG) antisense: 5 'pCfsUfgAfgCfuCfcAfuUfcAfcCfaAfcdTsdT 3' (Seq ID 8) ApoB siRNA:

110

<img file="MX347298B_D0112.tif" />

sense: (cholC6SSC6) -5 'GGAAUCuuAuAuuuGAUCcAsA 3' (Seq ID 9) antisense: 5 'uuGGAUcAAAuAuAAGAuUCcscsU 3' (Seq ID 10)

Ahal siRNA:

sense: (NH₂C₆) GfgAfuGfaAfgUfgGfaGfaUfuAfgUf (invdT) 3 '(Seq ID 11) antisense: pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 3' (Seq ID 12) Luc siRNA sense: (chol) 5'-uGfuTFUcfu (Chol) 5'-uGfuTUcfug

13) antisense: 5'-UfcGfaAfgUfaCfuCfaGfcGfuAfaGfdTsdT-3 '(Seq ID 14) or

sense: (NH₂C₆) cuuAcGcuGAGuAcuucGAdTsdT 3 '(Seq ID 15) antisense: UCGAAGuACUcAGCGuAAGdTsdT 3' (Seq ID 16)

Eg5-KSP sense: (NH₂Cs) UfcGfaGfaAfuCfuAfaAfcUfaAfcUf (invdT) 3 '(Seq ID 17) antisense: pAfGfuUfaGfuUfuAfgAfuUfcUfcGfadTsdT 3' (Seq ID 18) or sense: AGUuAGUUuAGAUUCdT 19 'Seq ID: 19UCTUCGAdT (Antisense ID 19)₂Cs) ucGAGAAucuAAAcuAAcudTsdT 3 '(Seq ID 20)

EGFP sense: 5 '(NH₂Cg) AuAucAuGGccGAcAAGcAdTsdT 3 '(Seq ID 21) antisense: 5' UGCUUGUCGGCcAUGAuAUdTsdT 3 '(Seq ID 22) lowercase letters = 2'-O-CH substitution₃ s = phosphorothioate bond f after nucleotide = 2'-F substitution

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OF THE INDUSTRIAL PROPERTY

<img file="MX347298B_D0113.tif" />

d before nucleotide = 2'-deoxy

RNA synthesis was performed in solid phase using conventional phosphoramidite chemistry on ÁKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany) and controlled pore glass (CPG) as solid support.

Example 4. Administration of polynucleotide iRNAs in vivo, and delivery to hepatocytes. DPCs were prepared as described above. Six to eight week old mice (strain C57BL / 6 or ICR, -18-20 g each) were obtained from Harían Sprague Dawley (Indianapolis IN). Mice were housed for at least 2 days prior to injection. Feeding was carried out ad libitum with the Harían Teklad Rodent Diet (Harían, Madison WI). The DPCs were synthesized as described herein. Conjugate solutions (0.4 ml) were infused into the tail vein. The compositions were soluble and did not aggregate under physiological conditions. Injection into other vessels, eg, retro-orbital injection, was predicted to be equally effective. Wistar Han rats, 175-200g were obtained from Charles River (Wilmington, MA). The rats were housed for at least 1 week prior to injection. The injection volume for rats was typically 1 ml. Unless otherwise stated, serum samples were taken and / or liver samples were harvested 48 hours after injection.

112

<img file="MX347298B_D0114.tif" />

Determination of ApoB levels in serum. Mice were fasted for 4 h (16 h for rats) before serum collection by sub-mandibular bleeding. For the blood of rats, the collection was from the jugular vein. ApoB protein levels in serum were determined by standard sandwich ELISA methods. Briefly, a polyclonal goat anti-mouse ApoB antibody and a rabbit anti-mouse ApoB antibody (Biodesign International) were used as capture and detection antibodies respectively. HRP-conjugated goat anti-rabbit IgG antibody (Sigma) was then applied to bind the ApoB / antibody complex. The absorbance of the colorimetric development of tetramethylbenzidine (TMB, Sigma) was then measured by a Tecan Safire2 plate reader (Austria, Europe) at 450 nm.

Measurements of plasma Factor VII (F7) activity. Mouse plasma samples were prepared by collecting blood (9 volumes) by submandibular bleeding into microfuge tubes containing 0.109 mol / L sodium citrate anticoagulant (1 volume) following standard procedures. F7 activity in plasma was measured with a chromogenic method using a BIOPHEN VII kit (Hyphen BioMed / Aniara, Mason, OH) following the manufacturer's recommendations. The absorbance of the colorimetric development was measured using a microplate reader.

<img file="MX347298B_D0115.tif" />

Tecan Safire2 at 405 nm.

Example 5. Delivery of siRNA to liver cSTuTas in vivo using a membrane active polyacrylate polyamine reversibly modified with dipeptide cleavable masking agents. Ant41658-111 polyacrylate in 100 mM pH 7.5 HEPES buffer was modified 0.5% by weight with the activated succinimidyloxycarbonyl-alpha-methyl-alpha (2-pyridyldithio) toluene (SMPT) disulfide reagent (Pierce) to give thiol reactive groups for subsequent siRNA coupling. The thiol-reactive polymer was then diluted to 5 mg / ml in 60 mg / ml HEPES base. To this solution, 10 mg / ml of various described enzyme divisible masking reagents were added. This amount represented a molar ratio of 1 polymer amine to 2 masking reagents. For the polymer modification reaction, a preferred molar ratio of polymer amines to masking reagents is 1: 1 to 1: 5. A more preferred ratio is 1: 2 to 1: 4. A more preferred ratio is 1: 2. After 1 hour, acetate-protected thiol endogenous rodent Factor VII siRNA (0.1 to 0.2 weight equivalents relative to polymer) was added to the polymer solution. After overnight incubation, the conjugates were further modified by the addition of a Nacetylgalactosamine derivative of maleic anhydride (NAG-CDM; Table 5).

¹¹⁴ IMPI

INSTITUTO MEXICANO 0t IA INDUSTRIAL CURRENCY

NAG-CDM was added at 25 mg / ml and incubated for 30 minutes at 4

Four. .i », |» M »» · '* ····· · * ··· ** · hours.

For the modification of the NAG-CDM polymer, NAG-CDM was lyophilized from a 0.1% aqueous glacial acetic acid solution. To the dry NAG-CDM a polymer solution was added. After complete dissolution of the anhydride, the solution was incubated for at least 30 min at RT before administration to the animal. The reaction of d NAG-CDM with the polymer produced:

<img file="MX347298B_D0116.tif" />

<img file="MX347298B_D0117.tif" />

Or where R is the polymer and R1 comprises an ASGPr ligand (eg N-acetylgalactosamine).

As shown in Table 5, Factor VII expression was reduced to 49-85% in animals treated with dipeptide masking agent DPC.

Table 5. Destruction of Factor Vil in vivo in mice treated with PEG-AA-p-nitrophenyl-carbamate + NAG-CDM-DPCs.

<td>Dipeptide masking agent</td><td>Polymer Dose (mg / kg) ^to</td><td>SiRNA dose (mg / kg) ^to</td><td>% fVII activity ^b</td>

<td>PEG12-AlaAsn</td><td> 15</td><td> 2</td><td> 32</td>

<td>PEG12-PheCit</td><td> 15</td><td> 2</td><td> 15</td>

<td>PEG12-AsnGly</td><td> 15</td><td> 2</td><td> 51</td>

<td colspan="4">¹¹⁵ IMPI®? INSTITUTO MEXICANO Wl-ltj iwniKTklAL --------</td><td> 1</td>

<td rowspan="2">Dipeptide entnascator agent</td><td rowspan="2">Polymer Dose (mg / kg) ^to</td><td>SiRNA dose</td><td>% of activity.</td><td rowspan="5"> —— -</td>

<td>(mg / kg) ^to</td><td>by fVII ^b</td>

<td>PEG24-PheCit</td><td> 1.5</td><td> 0.25</td><td> 23</td>

<td>PEG12-Asn</td><td> 1.5</td><td> 0.25</td><td> 34</td>

<td>PEG24-ValCit</td><td> 1.5</td><td> 0.25</td><td> 23</td>

<td colspan="4">a mg of polymer or siRNA per kg of animal weight</td><td></td>

b relative to inexperienced control

Example 6. Delivery of siRNA in vivo using NAG / PEG-AA-p-nitrophenyl-carbamate poly (acrylate) DPCs.

A) PEG plus NAG modification. Ant-41658111 polyacrylate in 100 mM pH 7.5 buffer HEPES was modified to 0.5% by weight with Pierce's succinimidyloxycarbonyl-alpha-methyl-alpha (2-pyridyldithio) toluene (SMPT) activated disulfide reagent. The thiol-reactive polymer was diluted to 5 mg / ml in 60 mg / ml of HEPES base. To this solution, 10 mg / ml of various PEG-AA-p-nitrophenyl carbonate masking reagents were added. After 1 hour, acetate-protected thiol Factor VII siRNA was added to the polymer solution at a siRNA ratio range of 5-10 to 1. After overnight incubation, NAG masking reagents were added. -AA-p-nitrophenyl-carbonate at 40 mg / ml. After incubation of at least 30 minutes, but not more than 4 hours, the DPC was injected into the tail vein of ICR mice.

116

<img file="MX347298B_D0118.tif" />

of 20 g. At 48 hours after injection, a serum sample was harvested and fVII levels were measured.

B) Modification of NAG only. ANT-41658 111 polyacrylate in 100 mM pH 7.5 buffer HEPES was modified 0.5% by weight with Pierce's Succinimidyloxycarbonyl 1-alpha-methyl-alpha (2-pyridyldithio) toluene (SMPT) activated disulfide reagent. The thiol-reactive polymer was diluted to 5 mg / ml in 60 mg / ml of HEPES base. The acetate-protected thiol factor VII siRNA was added to the polymer solution at a polymer to siRNA ratio range of 5-10 to 1. After overnight incubation, NAG-AA-pnitrophenyl-carbonate masking reagents were added at 50 mg / ml. After incubation for at least 30 minutes, but not more than 4 hours, the polymer-siRNA conjugate was injected into the tail vein of 2 0 gm ICR mice. At 48 hours after injection, a serum sample was harvested and fVII levels were measured. Table 6. Destruction of Factor VII in vivo in mice treated with PEG / NAG-AA-p-nitrophenyl-carbamate DPC

<td>Masking Agent</td><td>amount⁰</td><td>Polymer Dose (mg / kg) ^to</td><td>Dose of SiRNA (mg / kg) ^to</td><td>% fVII activity ^b</td>

<td>12 units of PEG12-PheCit</td><td>2x</td><td> 15</td><td> 2</td><td> 27</td>

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<td>followed by NAG-PEG4-PheCit</td><td>8x</td><td></td><td></td><td></td><td></td>

<td>24 units of PEG24-PheCit followed by NAG-PEG4-PheCit</td><td>2x 8x</td><td> 15</td><td> 2</td><td> 27</td><td></td>

<td>PEG24-PheCit followed by NAG-PEG4-PheCit</td><td>2x 8x</td><td> 1.5</td><td> 2.5</td><td> 23</td><td></td>

<td>NAG-PEG4-PheCit</td><td>10</td><td> 15</td><td> 2</td><td> 44</td><td></td>

<td>NAG-PEG2-GluGly</td><td>10</td><td> 1.5</td><td> 2.5</td><td> 27</td><td></td>

<td>NAG-PEG2-PheCit</td><td>10</td><td> 1.5</td><td> 2.5</td><td> 72</td><td></td>

^to mg of polymer or siRNA per animal weight ^b relative to inexperienced control ^c equivalents by weight

Example 7. Delivery of siRNA in vivo using DPC of NAG / PEG-AA-p-nitrophenyl-carbamate poly (vinyl ether). Modified amphipathic membrane active poly (vinyl ether) polyamine DW1360 with 10 weight equivalents of dipeptide cleavable masking agents as described above for Polyacrylate Ant-41658-111 except that protected groups retain masking agents during polymer modification . After polymer modification, amino acid protecting groups were removed by acetate protecting groups TFA and NAG by incubation in the presence of a solution of 30% by volume of triethylamine, 50% of methanol and

20% water. The acetate deprotection solution was removed by rotary evaporation. The masked polymer was co-injected into mice with cholesterol-siRNA conjugate.

<img file="MX347298B_D0119.tif" />

Table 7. Destruction of ApoB in vivo in mice co-injected with conjugate of NAG-AA-p-nitrophenylcarbamate poly (vinyl ether) and cholesterolApoB siRNA.

<td>Masking agent</td><td>Dose of Polymer (mg / kg) ^to</td><td>Dose of SiRNA (mg / kg) ^to</td><td>% of ApoB^b</td>

<td>NAG-AsnGly</td><td> 25</td><td> 5</td><td> 67</td>

<td>NAG-PheLys</td><td> 25</td><td> 5</td><td> 70</td>

<td>NAG-GluGly</td><td> 25</td><td> 5</td><td> 48</td>

^to mg polymer or siRNA per kg of animal weight ^b relative to inexperienced control

Example 8. In vivo destruction of endogenous ApoB levels after delivery of ApoB siRNA with melittin delivery peptide in mice, enzymatically cleavable masking agents. Melittin was reversibly modified with the indicated amount of enzymatically cleavable masking agents as described above. Then 200-300 pg of masked melittin was co-injected with 50-100 pg of the ApoB siRNA-cholesterol conjugate. The effect on ApoB levels was determined as described above. Protease cleavable dipeptide amidobenzyl carbamate modified melittin was an effective siRNA delivery peptide. The use

Melithma peptide D IMPI is preferred in combination with enzymatically "cleavable" masking agents. Although more peptide was required for the same level of target gene destruction, because the masking of the peptide was more stable, the therapeutic index was either not altered or improved (compared to masking the same peptide with CDM -NAG).

Table 8. Inhibition of Factor VII activity in normal liver cells in mice treated with the Factor Vll-cholesterol siRNA conjugate and GIL-Melittin (form D) (Seq ID 25) reversibly inhibited with the enzymatically cleavable masking agent.

<td rowspan="2">Peptide</td><td colspan="2">NAG link</td><td rowspan="2">P9 peptide</td><td rowspan="2">pg SiRNA</td><td rowspan="2">percentage destruction</td>

<td>amount³</td><td>kind</td>

<td></td><td>5x</td><td>CDM-NAG</td><td> 200</td><td> 100</td><td> 97</td>

<td></td><td>5x</td><td>NAG-AlaCit</td><td> 200</td><td> 50</td><td> 96</td>

<td></td><td>5x</td><td>NAG-GluGly</td><td> 200</td><td> 50</td><td> 96</td>

<td>GIL d-Mel</td><td>5x</td><td>NAG-PEG₄-PheCit</td><td> 200</td><td> 50</td><td> 94</td>

<td>(Seq ID 25)</td><td>5x</td><td>NAG-PEG₇-PheCít</td><td> 200</td><td> 50</td><td> 86</td>

<td></td><td>5x</td><td>CDM-NAG</td><td> 300</td><td> 50</td><td> 98</td>

<td></td><td>2x</td><td>NAG-GluGly</td><td> 300</td><td> 50</td><td> 95</td>

<td></td><td>4x</td><td>NAG-GluGly</td><td> 300</td><td> 50</td><td> 95</td>

<td></td><td>6x</td><td>NAG-GluGly</td><td> 300</td><td> 50</td><td> 82</td>

Amount of masking agent per amine

<img file="MX347298B_D0120.tif" />

Melittin used in the masking reaction.

Example 9. Tumor targeting with protease DPSOTTvTsi'Sles.

A) Measurement of target gene destruction. For all the studies presented below a specific siRNA for the transcript of the Ahal gene, the target gene. An siRNA for green fluorescent protein (EGFP) was used off-target control. The siRNA Ahal was complementary to a sequence motif in Ahal that is 100% homologous to both the human and mouse gene. Accordingly, the delivery of siRNA Ahal either within the host cells or within the tumor cells in the human xenograft results in the division and degradation of the mRNA. Using different sequence motifs in mouse and human Ahal genes, PCR primers were designed that allow quantitative measurement of both human Ahal and mouse Ahal mRNA levels in tissue samples that contained a mixed population of cell types. At 24, 48, or 72 hours after siRNA delivery, tumors were harvested with some healthy mouse liver tissue and processed in Tri Reagent (Invitrogen) for total RNA isolation. Both human and mouse Ahal mRNA levels were then measured by qPCR assays, using human Cyc-A and mouse β-actin as internal reference genes. The levels of ARAm Ahal in animals of

121

<img file="MX347298B_D0121.tif" />

Sham injected animals, or mice receiving off-target control GFP siRNA are considered to be 100%. The results were expressed as a percentage of the Ahal mRNA level relative to the control and are shown in the following Tables.

B) Mouse model with orthotopic hepatocellular carcinoma (HCC) tumor. HegG2, Hep3B, or HuH7 hepatocellular cell carcinoma was co-transfected with 2 expression vectors, pMIR85 a human placental secreted alkaline phosphatase (SEAP) vector and pMIR3 a neomycin / kanamycin resistant gene vector, to develop lines of Cells expressing SEAP cells were cultured in DMEM supplemented with 10% FBS and 300 ug / ml G418), collected, counted and mixed with matrigel (BD Biosciences) (50% by volume). The nude nude nude mice or Scid were anesthetized with ~ 3% isoflurane and placed in a reclining position on the sternum. A small 1–2 cm midline abdominal incision was made just below the xiphoid. Using a damp cotton swab, the left lobe of the liver was gently extruded. The left lobe of the liver was delicately retracted and a syringe needle was inserted into the middle of the left lobe. The syringe needle was inserted bevel down approximately 0.5 cm just below the liver capsule. 10 were injected

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FROM INDUSTRIAL PROPERTY ----- μΐ of the cell / matrigel mixture, containing 100,000 _ L 1 L »L ^{J 11} · *** · cells, in the liver using a syringe pump. The needle was left in the liver for a few moments (15-20 seconds) to ensure the injection was complete. SEAP-HepG2 cells were injected into athymic nude mice. SEAP-Hep3B and SEAP-HuH7 cells were injected into Scid beige mice. The syringe was then removed from the liver needle and a cotton swab was placed over the injection site to prevent cell leakage or bleeding. The Matrigel / cells mixture formed a mass that was visible and did not disappear after removal of the needle. The liver lobe was then delicately placed on the abdomen and the abdominal wall closed. Serum was collected once a week after tumor implantation and SEAP was tested to monitor tumor growth. For most of the studies, tumor-bearing mice were used 4-5 weeks after implantation, when tumor measurements were predicted to be around 4-8 mm based on SEAP values.

C) Colorectal Metastatic Tumor Model. HT29 cells were grown in McCoy's 5a medium supplemented with 10% FBS, harvested, counted and mixed with matrigel (BD Biosciences) (50% by volume). The athymic nude mice were anesthetized with ~ 3% isoflurane and placed in a reclining position on the sternum. It made a small

<img file="MX347298B_D0122.tif" />

123 IMPI tlKTtWFO MEXICAN B &lt;U MOLDAD iHDUSTUAL midline abdominal incision 1-2 cm, just below the xiphoid. Using a damp cotton swab, the left lobe of the liver was gently extruded. The left lobe of the liver was delicately retracted and a syringe needle was inserted into the middle of the left lobe. The syringe needle was inserted bevel down approximately 0.5 cm just below the liver capsule. 5 µΐ of the cell / matrigel mixture, containing 40,000 cells, was injected into the liver using a syringe pump. The needle was left in the liver for a few moments (15-20 seconds) to ensure the injection was complete. The syringe was then removed from the liver needle and a cotton swab was placed over the injection site to prevent cell leakage or bleeding. The Matrigel / cell mixture formed a mass that was visible and did not disappear after removal of the needle. The liver lobe was then delicately placed back on the abdomen and the abdominal wall closed. Tumor mice were used 4-5 weeks after implantation.

Example 10. In vivo destruction of target gene expression in the HepG2-SEAP orthotopic hepatocellular carcinoma (HCC) model after administration of DPC PEG24Val-Cit. Ant-129-1 polymer DPCs (2011062805) were modified (masked) with either 18x excess weight¹²⁴ IMPIAS,

INSTITUTO MEXICANO • '-í *' '* t DE LA PRCi'ISI'A' · 'V ·' j · &gt;&gt; * INDUSTRIAL ------------ of PEG2 masking agent₄-Phe-Cit (or the masking agent

PEG2₄-Val-Cit) or with 7x PEG550-CDM as described above. Ahal-siRNA (RD-09070) or GFP-siRNA (RD-05814; out of control) was coupled to the polymer as described above (4: 1 weight ratio). The DPCs were not purified by gel filtration prior to delivery, and no targeted ligand was added. A 320 pg (polymer weight) DPC conjugate in 200 µΐ isotonic glucose per animal was administered by tail vein injections (n ​​= 3 per group). After 24 hours, the animals received a second injection of 320 pg (polymer weight) of DPC conjugate in 200 µl of isotonic glucose. Forty-eight hours after the second injection, serum samples were collected to assess toxicity by measuring levels of liver enzyme (ALT and AST) and blood urea nitrogen (BUN), followed by tissue harvesting, and qPCR analysis.

Using DPV PEG₂₄-Val-Cit-Ant-129-l-siRNA to deliver Ahal siRNA, resulted in 46% destruction of Ahal gene in human tumor cells (Table 9). In contrast to the levels of destruction of human Ahal, mouse Ahal was destroyed by 70% in response to the administration of DPC PEG2.₄-Val-Cit-Ant-129-l-siRNA (Table 1). Compared to similar DPCs made with disubstituted maleic anhydride (PEG550-CDM) masking agents, the destruction of Ahal of

125

<img file="MX347298B_D0123.tif" />

endogenous hepatocyte decreased. As indicated by the levels

ALT, AST and BUN, the DPC PEG₂4-Val-Cit were well tolerated and exhibited no toxicity (Table).

Table 9. Destruction of Ahal in HepG2 liver tumor model by modified maleic anhydride vs. cleavable peptide modified Ahal siRNA DPC.

<td rowspan="2"></td><td></td><td>PEG550-CDM DPC</td><td>PEG₂₄-Val-Cit DPC</td>

<td>Control siRNA</td><td>SiRNA Ahal</td><td>SiRNA Ahal</td>

<td>Ahal levels human (tumor)</td><td> 100 ± 8.0</td><td> 54.4 ± 7.3</td><td> 54.9 + 7.6</td>

<td>Mouse Ahal levels (hepatocytes)</td><td> 100 ± 9.2</td><td> 7.5 ± 0.9</td><td> 30.4 ± 3.1</td>

Table 10. Toxicity markers in blood chemistry after administration of modified maleic anhydride or DPC siRNA Ahal modified with cleavable peptide.

<td></td><td></td><td>PEG550-CDM DPC</td><td>PEG₂₄-Val-Cit DPC</td>

<td></td><td>SiRNA control</td><td>SiRNA Ahal</td><td>SiRNA Ahal</td>

<td>ALT</td><td> 44.3 ± 5.1</td><td> 58.0 ± 37.5</td><td> 35.0 ± 11.3</td>

<td>AST</td><td> 81.7 ± 4.0</td><td> 102.3 ± 57.5</td><td> 67.7 ± 14.4</td>

<td>BUN</td><td> 25.3 ± 4.2</td><td> 23.0 ± 3.5</td><td> 21.3 ± 1.2</td>

Example 11. Destruction of gene expression

INDUSTRIAL - DPC target targeting bispecific antibody (bsAb) (2011090701). The Ant-129-1 ~ polymer was modified with 5x Dig-PheCit masking agent (Dig-FCit) as described above. The siRNA was then coupled to the conjugate. Finally, the Dig-FCit-Ant-129-l-siRNA conjugate was further modified with 8x (weight) of PEGiz-FCit. Ahal-siRNA (RD-09070) or GFP siRNA (RD-05814) was coupled at a 4: 1 ratio of polymer: siRNA by weight. PEGi2-FCit DPCs were purified on Sephadex G50 spinning columns to remove uncoupled reagents.

Bi-specific antibodies directed at a cell target (bsAb) were made specific for the heparan sulfate proteoglycan Glypican-3 (GPC3), a cell surface heparan sulfate proteoglycan that is known to be highly expressly in HepG2-SEAP cells, and digoxigenin (Dig). As a control, bispecific antibodies specific for CD33 protein (bone marrow derived hematopoietic stem cell marker) and Dig were made. CD33 is not expressed by HepG2-SEAP cells. BsAbs were complexed with modified DPCs at a weight ratio of 1.25: 1 to provide an estimated molar ratio of 1: 1. Complexes were formed in PBS at least 30 minutes prior to delivery.

DPCs were administered to mice with HepG2-SEAP tumors, either with or without the targeted agent.

<img file="MX347298B_D0124.tif" />

127

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INSTITUTO MEXICANO DI LA NOPIEDAD INDUSTRIAL bsAb. Each animal (n = 3 per group) received a single dose of

250 pg (weight of polymer) of DPC. The DPCs were injected into the tail vein of the mice in 200 µl of sterile PBS. Serum and tissue samples were harvested 48 hours later and analyzed as described above. As shown in Table 11, a single dose of bsAb targeted DPC (250 pg polymer, 62.5 pg siRNA) resulted in 21-32% target gene kill.

Table 11. Destruction of Ahal in HepG2 liver tumor model by targeted DPCs siRNA Ahal modified with peptide cleavable masking agent using bi-specific antibodies.

<td rowspan="3"></td><td colspan="4">Dig-FCit + PEGi masking agent₂-FCit</td>

<td colspan="2"></td><td>GPC-Dig bsAb</td><td>CD33-Dig bsAb</td>

<td>SiRNA control</td><td colspan="3">SiRNA Ahal</td>

<td>Levels of Human ahal (tumor)</td><td> 100 ± 10.1</td><td> 78.6 + 11.5</td><td> 72.7 ± 2.4</td><td> 68.1 ± 6.4</td>

<td>Levels of Mouse ahal (hepatocytes)</td><td> 100 ± 9.5</td><td> 65.1 ± 8.1</td><td> 73.7 + 10.5</td><td> 91.3 + 8.3</td>

Example 12. Destruction of target gene expression with DPC targeting bispecific antibody (bsAb). DPCs were prepared as above except that a) PEG₂4-FCit was used instead of PEGi₂-FCit and

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<img file="MX347298B_D0125.tif" />

128 „...........1

b) Dig-PEGi2-NHS was used to couple Dig to^INfirst. The PEG24-FCit DPCs were less aggregated than the PEGiz-FCit DPCs — DPCs and were smaller and more homogeneous. In addition to being a non-labile coupling, Dig-PEGi2-NHS also contained a longer PEG. DPCs were complexed with bsAb and injected into animals as described above. The serum and tissue harvest was performed either 24 or 48 hours post-injection. As shown in Table 12, a single dose of DPC (250 pg polymer weight) resulted in human Ahal destruction of 46-56% at 24 hours post injection.

Table 12. Destruction of Ahal in HepG2 liver tumor model by DPC siRNA Ahal modified using length increased PEG peptide cleavable masking agents.

<td rowspan="3"></td><td colspan="3">Dig-PEGi2-NHS + PEG2 masking agent₄-FCit</td>

<td colspan="2">GPC-Dig bsAb</td><td>CD33-Dig bsAb</td>

<td>control siRNA</td><td colspan="2">Ahal siRNA</td>

<td>Human Ahal (tumor) levels</td><td> 100 ± 3.1</td><td> 54.1 + 15.1</td><td> 43.5 ± 6.6</td>

Example 13. Targeted DPC for Human Colorectal Adenocarcinoma Metastatic Liver Tumor Tissue by bsAb Targeted DPC. Polymer

Ant-129-1 was modified with 5x molar excesses of Dig-PEGi₂-NHS

129

ΙΜΡΪ® ^ -β® and 8χ excess by weight of PEG24-FCit. SiRNA Ahal or siRNA GFP were coupled to the modified polymer at a 4: 1 weight ratio of polymer: siRNA. DPCs were purified on a Sephadex G50 spinning column to remove uncoupled reagents. Dig-DPCs were complexed with an equimolar amount of IGFIR-Dig bsAb or CD33-Dig bsAb or no bsAb in sterile PBS at least 30 minutes before injections to the animals containing tumor cell HT29 (human colorectal adenocarcinoma; Number ATCC HTB38) and injected with the DPCs. HT29 cells overexpress the insulin-like growth factor 1 receptor (IGF1R) protein, and can bind and internalize into the bi-specific antibody IGFIRDig. Animals (n = 3) received DPC (320 pg of polymer). The injections were repeated after 24 hours. Serum and tissue samples were collected 48 hours after the second dose. The destruction of human Ahal in tumor cells was 26-38% (Table 13). Compared to CDM-DPC, FCit-DPC showed less off-target liver Ahal destruction (78-83% compared to 24-36%). FCit-DPCs also demonstrated decreased liver accumulation compared to CDM-DPCs.

Table 13. Destruction of Ahal in HT29 colorectal adenocarcinoma metastatic liver tumor by DPC

Dipeptide divisible siRNA Ahal.

130

ΙΜΡΪ ^ ^η iNnuSTRIAt -----

<td></td><td></td><td>polymer</td><td>Ant-129-1</td><td>_r___ r »- ​​'</td>

<td></td><td colspan="3">Dig-PEGis-ÑHS + Masking Agent</td><td>PEG₂₄-FCit</td>

<td></td><td colspan="2">bsAb IG FIR-Dig</td><td>bsAb CD33- Dig</td><td></td>

<td></td><td>Control siRNA</td><td>SiRNA Ahal</td><td></td><td></td>

<td>Human Ahal (tumor) levels</td><td> 100 ± 4.9</td><td> 72.3 ± 6.1</td><td> 73.7 ± 3.2</td><td> 62.0 ± 9.0</td>

<td>Mouse Ahal levels (liver)</td><td> 100.0 ± 11</td><td> 64.0 ± 5.0</td><td> 75.7 ± 11</td><td> 66.6 ± 5.8</td>

Example 14. In vivo destruction of endogenous Ahal in liver tumor. 400 pg of Lau41648-106 were modified with 8x (weight) of PEGi₂-ValCit or 16x PEG₂₄-PheCit. 100 pg of Ahal siRNA or 100 pg of Eg5 control siRNA were coupled to the modified polymer as described above. Animals containing Hep3B-SEAP tumor cells were injected with the DPCs. Serum and tissue samples were collected 48 hours after injection. The destruction of human Ahal in tumor cells was 26-38%.

Table 14. Destruction of Ahal in Hep3B-SEAP liver tumor by peptide divisible DPC siRNA Ahal.

<td>Polymer (400 pg)</td><td>modification</td><td>SiRNA (100 pg)</td><td>Ahal KD</td>

<td>Lau41648-106</td><td>8x PEG12-ValCit</td><td>Ahal</td><td> 38 ± 0.02 %</td>

IMPI

<img file="MX347298B_D0126.tif" />

131

<td>Polymer (400 pg)</td><td>modification</td><td>SiRNA (100 pg) ^</td><td>..... Ahal, KT) --—</td>

<td rowspan="2"></td><td rowspan="2">16x PEG24-Phecit</td><td>Ahal</td><td> 41 ± 0.07 %</td>

<td>EG5</td><td> 13 + 0.11 %</td>

Example 15. In vivo circulation and cellular targeting of the masked polymer. Lau24AB polyacrylate (100 pg) was treated with reagent¹²⁵I-Bolton-Hunter (BH) (50 pCi) in 50 mM HEPES pH 8.0 buffer for 1 hr at RT. The labeled polymer was purified on 2 ml of the 10 Sephadex QEA spin column in water. The labeled polymer solution was stored at 4 ° C. The unlabeled polymer was supplemented with polymer labeled with¹²⁵I-labeled to inject approx. 1 mg of polymer having 0.2 pCi per 200 g of rat. The mixture of the labeled and unlabeled polymers (calculated for -3.5 15 animals) was modified as described above with PEG24-FCit or PEG-CDM (2 mg / ml polyacrylate, 14 mg / ml PEGCDM reagent, 14 mg / ml ml of NAG-PEG-CDM reagent, 16 mg / ml of PEG24-FCit reagent). Incubation time 1 hr. The reaction mixture was then diluted with isotonic glucose to produce the injection dose per animal in a volume of 1 ml. 3 animals / group were injected. The animals were bled (0.1 - 0.2 ml) at the given times. The amount of polymer present in the samples was determined by counting in a gamma counter. As shown in FIG. 4, the modified polymer

132

<img file="MX347298B_D0127.tif" />

The protease cleavable masking agent cleared less rapidly from the serum than the polymer masked with the pH labile maleic anhydride masking agent. Increased circulation time is beneficial in targeting non-liver tissue.

Example 16. Amphipathic membrane active polymer synthesis.

A) Poly (vinyl acrylate)

i) RAFT copolymerization of N-Boc-ethylethoxy acrylate and

<img file="MX347298B_D0128.tif" />

where:

A is an ethyl-ethoxy amino acrylate

IMPI

MEXICAN INSTITUTE

Say LA rXOMEDAO

INDUSTRIAL ^ * «._-- protected with boc

B is a propyl methacrylate

C is an acidic RAFT CPCPA agent (4-Cyano-4 (phenylcarbontioylthio) pentanoic)

And n and m are integers.

Removal of the boc protecting group after synthesis produces the amine monomers.

For other membrane active polymers, A can also be protected ethyl-, propyl-, or butyl-amino acrylates. B can be higher hydrophobic acrylate (10-24 carbon atoms, C18 shown), lower hydrophobic acrylate (1-6 carbon atoms, C4 shown), or a combination of lower and higher hydrophobic acrylates.

Copolymers consisting of Amine acrylate / C3were methacrylate were synthesized as follows. The monomers and the RAFT agent were weighed and brought to butyl acetate in the indicated proportions. AIBN (azobis-isobutyronitrile) was added and nitrogen was bubbled through the reaction at RT for ih. The reaction mixture was then placed in an oil bath at 80 ° C for 15h. The polymer was then precipitated with hexane, and further functionally precipitated using a DCM / Hexane solvent system (see below). The polymer was then dried under reduced pressure. The polymer was deprotected with 7 ml 2M of HCl in Acetic Acid for 30 min at RT. After 30 min, 15 ml were added.

<img file="MX347298B_D0129.tif" />

134

IMPI Mexican nwrmrro DI LA INDUSTRIAL NOREDAD of water to the reaction mixture, and the mixture was transferred in 3.5 kDa of MWCO dialysis tubing. The polymer was dialyzed overnight against NaCl and then another day against dH₂O. The water was then removed via lyophilization, and the polymer was dissolved in dH₂OR.

Monomer Synthesis for (Ant 41658-111). 2,2'Azobis (2-methylpropionitrile) (AIBN, radical initiator), 4-Cyano-4- (phenylcarbontioylthio) pentanoic acid (CPCPA, RAFT agent) and butyl acetate were purchased from Sigma Aldrich. The propyl methacrylate monomer (Alfa Aesar) was filtered to remove the inhibitors.

In a 2L round bottom flask equipped with a stir bar, 2- (2-aminoethoxy) ethanol (21.1 g, 202.9 mmol, Sigma Aldrich) was dissolved in 350 ml of dichloromethane. In a separate IL flask, anhydrous BOC (36.6 g, 169.1 mmol) was dissolved in 660 ml dichloromethane. The 2L round bottom flask was fitted with an addition funnel and anhydrous BOC solution was added to the flask over 6 h. The reaction was allowed to stir overnight. In a 2L separatory funnel, the product was washed with 300 ml each of 10% citric acid, 10% eK₂CO3, NaHCOa sat. , and NaCl sat. The product, 2- (2-aminoethoxy) ethanol protected with BOC, was dried over Na₂SW₄, filtered by gravity, and the DCM was evaporated using rotary evaporation and high vacuum.

In a 500 ml round bottom flask fitted with a

IMPIé&gt;

MEXICAN INSTITUTE

OF. LA FROrlEDAI · ÍA »» - JK INDUSTRIAL stirring bar and rinsing with argon, BOC-protected 2- (2-aminoethoxy) ethanol (27.83g ^ ..... 135.8 mmol) was added, followed by 240 ml anhydrous dichloromethane. Diisopropylethylamine (35.5 ml, 203.7 mmol) was added, and the system was placed in a dry ice / acetone bath. The acryloyl chloride (12.1 ml, 149.4 mmol) was diluted using 10 ml of dichloromethane, and added dropwise to the argon flush system. The system was kept under argon and allowed to come to room temperature and stirred overnight. The product was washed with 100 ml each of dHsO, 10% citric acid, 10% K2CO3, NaHCO₃ sat., and saturated NaCl. The product, BOC-amino ethyl ethoxy acrylate (BAEEA), was dried over NazSOi, gravity filtered, and the DCM was evaporated using rotary evaporation. The product was purified through column chromatography on 29 cm of silica using a 7.5 cm diameter column. The solvent system used was 30% ethyl acetate in hexane. Rf: 0.30. The fractions were collected and the solvent was removed using rotary evaporation and high vacuum. BAEEA, was obtained with 74% yield. BAEEA was stored in the freezer.

<img file="MX347298B_D0130.tif" />

BAEEA

Polymer Ant-41658-111: Solutions of AIBN (1.00 mg / ml) and the acid RAFT agent (4-Cyano-

<img file="MX347298B_D0131.tif" />

136

IMPI iwsrnvro mexicanc of INDUSTRIAL property

4 (phenylcarbontioylthio) pentanoic (CPCPA), 10.0 mg / ml) in butyl acetate were prepared. The monomer molar feed ratio was 75 BAEEA: 25 propyl methacrylate (CAS: 2210-28-8) with 0.108 of RAFT CPCPA agent and 0.016 of AIBN catalyst (0.00562 of total moles).

BAEEA (1.09 g, 4.21 mmol) (A), propyl methacrylate (.180 g, 1.41 mmol) (B), CPCPA solution (.170 ml, 0.00609 mmol) (C), AIBN solution (0.150 ml, 0.000915 mmol), and butyl acetate (5.68 ml) were added to a 20 ml glass flask with a stir bar. The bottle was sealed with a rubber cap and the solution was flushed with nitrogen using a long syringe needle with a second short syringe needle as the outlet for one hour. The syringe needles were removed and the system was heated at 80 ° C for 15 h using an oil bath. The solution was allowed to cool to room temperature and was transferred to a 50 ml centrifuge before adding the hexane (35 ml) to the solution. The solution was centrifuged for 2 min at 4,400 rpm. The supernatant layer was carefully decanted and the bottom layer (solid or gel-like) was rinsed with hexane. The bottom layer was then redissolved in DCM (7 ml), precipitated in hexane (35 ml) and centrifuged once more. The supernatant was decanted and the bottom layer was rinsed with hexane before drying the polymer under reduced pressure for several hours. Molecular weight obtained through MALS: 73,000 (PDI 1.7);

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MEXICAN INSTITUTE OF INDUSTRIAL CURRENCY

Polymer Composition obtained using H¹NMR: 69:31 Amine: Alkyl.

Fractional Precipitation. The dry, precipitated product was dissolved in DCM (100 mg / ml). Hexane was added until just before the cloudy point (~ 20 ml) was reached. The resulting milky solution was centrifuged. The bottom layer (thick liquid representing ~ 60% of the polymer) was removed and completely precipitated in hexane. The remaining upper solution was also completely precipitated by the further addition of hexane. Both fractions were centrifuged, after which the polymer was isolated and dried under vacuum. Fraction 1: Mw 87,000 (PDI 1.5); Fraction 2: Mw 52,000 (PDI 1.5-1.6).

MALS analysis. About 10 mg of the polymer was dissolved in 0.5 ml 89.8% dichloromethane, 10% tetrahydrofuran, 0.2% triethylamine. Molecular weight and polydispersity (PDI) were measured using a Wyatt Helos II multi-angle light scattering detector coupled to Shimadzu Prominence HPLC using a Jordi 5μ 7.8x300 Mixed Bed column. Crude Polymer: PM: 73,000 (PDI 1.7), Fraction 1: PM 87,000 (PDI: 1.5), Fraction 2: PM 52,000 (PDI 1.5-1.6)

The purified BOC protected polymer was reacted with 2M HC1 in Acetic Acid (7 ml) for 0.5 h to remove the BOC protecting groups and produce the amines. I know

138

WICKED

INSTITUTO MEXICANO OI LA PROPERTY added 15 ml of dHaO to the reaction, the ^IN^ olucioh ~ a cellulose tube was transferred with 3 5 0ΰ MW of "re cut'e ^ dialyzed with high salt for 24 h, then against dHzO for 18 h. The contents were lyophilized, then dissolved in DI H2O at a concentration of 20 mg / ml. The polymer solution was stored at 2-8 ° C.

ii) The Lau24B Polymer was prepared as above except that the monomer feed ratio was 72.5 BAEEA: 27.5 propyl methacrylate.

iii) Ant-129-1 was made essentially as described above except that the following monomers were used: or

NHBoc or

<img file="MX347298B_D0132.tif" />

Table 15. Reagents for the synthesis of the Ant polymer

129-1.

<td>'j w I 1 &lt;a * 1 * ·&gt; us · - i wgc 1 _t u ** - Z Sil IH * I m</td><td></td>

<td>PM mass% reaction volume, moles —------ ' (g / mol) moles (g) (mi) moles</td><td rowspan="6"> - ·</td>

<td>Monomers</td>

<td>N-Boc-amino- 229.27 70 3.94X10-³ 0.9031 0.005627 propyl acrylate butyl methacrylate 142.2 25 1.41xl0-³ 0.2000 0.224 0.005627 C18 methacrylate 338.54 5 2.81xl0-⁴ 0.0952 0.005627 ethylene glycol 170.16 5 2.81X10-⁴ 0.0479 0.44 0.005627 diacrylate</td>

<td>other reagent</td>

<td>CPCPA (reactive 279.38 0.213 1.2xl0 ~⁵ 0.0033 0.335 0.005627 RAFT) AIBN (initiator) 164.21 0.032 1.8xl0-⁶ 0.0003 0.295 0.005627 butyl acetate 5,272</td>

<td>target molecular weight 100000 Total Units 469.56 by CTA% CTA 0.213</td>

<td>For N-Boc-Amino-Propyl-Acrylate (ΒΑΡΑ). In a flask</td><td></td>

500 ml round bottom equipped with a stir bar and rinsed with argon, 3- (BOC-amino) 1-propanol (TCI) (135.8 mmol) was added, followed by 240 ml of anhydrous dichloromethane. Diisopropylethylamine (203.7 mmol) was added, and the system was placed in a dry ice / acetone bath. Acryloyl chloride (149.4 mmol) was diluted using 10 ml of dichloromethane, and added dropwise to the argon flushed system. The system

<img file="MX347298B_D0133.tif" />

it was kept under argon and allowed to come to room temperature and stirred overnight. The product was washed with 100 ml each of dH₂O, 10% citric acid, 10% K₂CO3, sat. NaHCO3, and saturated NaCl. The product, BOC-amino propyl acrylate (ΒΑΡΑ), was dried over Na₂SO4, gravity filtered, and DCM evaporated using rotary evaporation. The product was purified through column chromatography on 29 cm of silica using a 7.5 cm diameter column. The solvent system used was 30% ethyl acetate in hexane. Rf: 0.30. The fractions were collected and the solvent was removed using rotary evaporation and high vacuum. ΒΑΡΑ was obtained with 74% yield. ΒΑΡΑ was stored in the freezer.

iv) Random copolymerization of N-Boc-ethylethoxy acrylate and propyl methacrylate. Copolymers consisting of Acrylate Amine / C_n methacrylate were synthesized as follows. The weighed monomers were brought into dioxane at the indicated proportions. AIBN (azobis-isobutyronitrile) was added and nitrogen was bubbled through the reaction at RT for Ih. The reaction mixture was then placed in a 60 ° C oil bath for 3h. The polymer was then dried under reduced pressure. The polymer was purified by GPC. The polymer fractions were then deprotected with 7 ml 2M HCl in Acetic acid for 30 min at RT. After 30 min, 15 ml of water was added to the reaction mixture, and the

ΙΗίΙΙΜΙ ι »MU ι · mi ι m μ&gt; · ι

INSTITUTO MEXICANO DE LA nOFIWAD mixture was transferred in 3.5 kDa diaYi'sis tubing MWC &lt;57 The polymer was dialyzed overnight against'NaCl and then another day against dH₂O. The water was then removed via lyophilization, and the polymer was dissolved in dH₂OR.

Lau41648-106 polymer. The monomer molar feed ratio was 80 BAEEA: 20 propyl methacrylate (CAS: 2210-28-8) and 3% AIBN catalyst based on total moles of monomer. BAEEA (6.53 g, 25.2 mmol) (A), propyl methacrylate (0.808 g, 6.3 mmol) (B), AIBN (0.155 g, 0.945 mmol), and dioxane (34.5 ml) were added to a 50 ml glass tube. with stirring bar. Compounds A and B were prepared as described above in Example 16Ai. The reaction was determined in triplicate. Each solution was flushed with nitrogen using a long pipette for 1 hour. The pipette was shaken and each tube was carefully capped. Each solution was then heated at 60 ° C for 3 h using an oil bath. Each solution was allowed to cool to room temperature and combined on a round bottom. The crude polymer was dried under reduced pressure. Molecular weight obtained through MALS: 55,000 (PDI 2.1); polymer composition obtained using H ^ MR: 74:26 Amine: Alkyl.

Lau41648-106

GPC fractionation. The dry crude polymer was made up to 50 mg / ml in 75% dichloromethane, 25% tetrahydrofuran, and 0.2% triethylamine. The polymer was then fractionated on a Jordi de Gel DVB 10 column.⁴ Á 500mm / 22mm using a flow rate of ml / min and injections of 10 ml. A 15-17 minute earlier fraction was collected, and a 17-19 minute later fraction was collected. Fraction 15-17: Mw 138,000 (PDI 1.1); Fraction 1719: Mw 64,000 (PDI 1.2).

MALS analysis. Approximately 10 mg of the polymer was dissolved in 0.5 ml 89.8% dichloromethane, 10% tetrahydrofuran, 0.2% triethylamine. Molecular weight and polydispersity (PDI) were measured using a Wyatt Helos II multi-angle light scattering detector coupled to Prominencia Shimadzu HPLC using a DVB LS column from

143

WICKED.

Mexican INSTITUTE

OF THE PtOFlEOAC ·,, _, INDUSTRIAL

Jordi Mixed Bed 5μ 7.8x300. Crude Polymer: PM: 55,000 (PDI 2.1), Fraction 15-17: PM 138,000 (PDI: 1.IH Fraction 17 ^ 19: PM 64,000 (PDI 1.2)

The purified BOC protected polymer was reacted in 2M HC1 in acetic acid (7 ml) for 0.5 h to remove the BOC protecting groups and produce the amines. Added 15 ml of dH₂Or to the reaction, the solution was transferred to a cellulose pipe with 3500 MW cut-off, dialyzed with high salt for 24 h, then against dH₂Or for 18 h. The contents were lyophilized, then dissolved in H₂Or DI at a concentration of 20 mg / ml. The polymer solution was stored at 2-8 ° C.

v) Synthesis of amphipathic, water-soluble, membrane-active poly (vinyl ether) polyamine terpolymers. X mole% amine protected vinyl ether (eg, 2-vinyloxy ethyl phthalimide) was added to an oven dried round bottom flask under a nitrogen blanket in anhydrous dichloromethane. To this solution Y mole% lower hydrophobic group (eg propyl, butyl) of vinyl ether and optionally Z mole% higher hydrophobic group (eg dodecyl, octadecyl) vinyl ether were added (FIG. 1). The solution was placed in a -50 to -78 ° C bath, and the 2-vinyloxy ethyl phthalimide was allowed to precipitate. To this solution 10 mol% of BF were added₃ (OCH2CH3) ₂ and the

<img file="MX347298B_D0134.tif" />

Reaction was allowed to proceed for 2-3 h at -50 to -78 ° C. The polymerization was terminated by the addition of ammonium hydroxide in methanol solution. The polymer was brought to dryness under reduced pressure and then developed in 1,4-dioxane / methanol (2/1). 20 molar equivalents of hydrazine per phthalimide were added to remove the protecting group from the amine. The solution was refluxed for 3 h and then brought to dryness under reduced pressure. The resulting solid was dissolved in 0.5 mol / L HCl and refluxed for 15-min to form the hydrochloride salt of the polymer, diluted with distilled water, and refluxed for an additional hour. The solution was then neutralized with NaOH, cooled to room temperature (RT), transferred to a molecular cellulose tubing, dialyzed against distilled water, and lyophilized. The polymer can further be purified using size exclusion chromatography or other. The molecular weight of the polymers is estimated using columns according to standard procedures, including analytical size exclusion chromatography and multi-angle light scattering size exclusion chromatography (SEC-MALS).

DW1360 polymer. An amine / butyl / octadecyl poly (vinyl ether) terpolymer, was synthesized from the monomers of 2-vinyloxy ethyl phthalimide (5 g, 23.02 mmol), butyl vinyl ether (0.665 g, 6.58 mmol), and octadecyl ether.

145

OF THE PROPERTY í&gt; ra!.? *. Z INDUSTRIAL vinyl (0.488 g, 1.64 mmol).

phthalimide 200 ml from a flask

Oven dried round bottom 2-vinyloxy ethyl was added with a magnetic stir bar under a blanket of

Argon in 36 ml of anhydrous dichloromethane. Butyl vinyl ether and n-octadecyl vinyl ether were added to this solution. The monomers were completely dissolved at room temperature (RT) to obtain a clear, homogeneous solution. The reaction vessel with the clear solution was then placed in a -50 ° C bath generated by the addition of dry ice to 1: 1 solution of denatured ACS grade alcohol and ethylene glycol and a visible precipitation of the monomer was allowed to form. phthalimide. After cooling for about 1.5 min, BF was added₃ · (OCH2CH3) 2 (0.058 g, 0.411 mmol) to start the polymerization reaction. The phthalimide monomer dissolved after the start of polymerization. The reaction was allowed to proceed for 3 h at -50 ° C. The polymerization was stopped by the addition of 5 ml of 1% ammonium hydroxide in methanol. The solvents were then removed by rotary evaporation.

The polymer was then dissolved in 30 ml of 1,4-dioxane / methanol (2/1). Hydrazine (0.147 g, 46 mmol) was added to this solution and the mixture was heated under reflux for 3 h. Then the solvents were removed by rotary evaporation and the resulting solid was then developed in 20 ml of 0.5 mol / L of HC1 and refluxed for 15 minutes, diluted with 20 ™ IMPI ¡ΝΤΠΤυΤΟ MEXICANO

FROM INDUSTRIAL PROPERTY my distilled water, and refluxed for an additional hour. This solution was then neutralized with NaOH, cooled to RT, transferred to 3,500 molecular weight cellulose tubing, dialyzed for 24 h (2x20L) against distilled water, and lyophilized.

B) Melittin. All melittin peptides were made using standard peptide synthesis techniques in the art. Suitable melittin peptides can be all amino acids of the L form, all amino acids of the D (reverse) form. Regardless of the L or D form, the melittin peptide sequences can be reversed (retro).

It is noted that in relation to this date, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention.