JP5941926B2 - In vivo polynucleotide delivery conjugates having enzyme-sensitive linkages - Google Patents

Description

  Delivery of polynucleotides and other substantially cell membrane impermeable compounds to living cells is highly limited by the complex membrane system of cells. Drugs used in antisense, RNAi, and gene therapy are relatively large hydrophilic polymers and are often highly negatively charged. Both of these physical characteristics severely limit their direct diffusion across the cell membrane. Thus, a major barrier to polynucleotide delivery is the delivery of the polynucleotide across the cell membrane to the cytoplasm or nucleus of the cell.

  One means that has been used to deliver small nucleic acids in vivo has been to bind nucleic acids to small targeting molecules or either lipids or sterols. Although some delivery and activity has been observed with these conjugates, the nucleic acid doses required by these methods are enormous.

  A number of transfection reagents have also been developed that achieve reasonably efficient delivery of polynucleotides to cells in vitro. However, in vivo delivery of polynucleotides using these same transfection reagents is complicated and ineffective due to in vivo toxicity, deleterious serum interactions, and poor targeting. Transfection reagents, cationic polymers and lipids that work well in vitro typically form large cationic electrostatic particles and destabilize the cell membrane. The positive charge of the in vitro transfection reagent promotes binding to the nucleic acid through charge-charge (electrostatic) interactions, thus forming a nucleic acid / transfection reagent complex. Positive charges are also beneficial for non-specific binding of media to cells and membrane fusion, destabilization, or destruction. Membrane destabilization facilitates delivery of substantially cell membrane impermeable polynucleotides across the cell membrane. These properties promote nucleic acid migration in vitro, but they cause toxicity and ineffective targeting in vivo. The cationic charge results in interaction with serum components, which causes destabilization of polynucleotide-transfection reagent interactions, poor bioavailability, and poor targeting. The membrane activity of transfection reagents, which can be effective in vitro, often leads to toxicity in vivo.

  For in vivo delivery, the vehicle (nucleic acid and related delivery material) should be small, less than 100 nm in diameter, preferably less than 50 nm. Even smaller complexes, less than 20 nm or less than 10 nm would be more useful. Delivery vehicles larger than 100 nm have little access to cells other than vascular cells in vivo. Complexes formed by electrostatic interactions tend to aggregate or degrade when exposed to physiological salt concentrations or serum components. Furthermore, the cationic charge on the in vivo delivery vehicle leads to harmful serum interactions and thus poor bioavailability. Interestingly, high negative charges can also inhibit targeted in vivo delivery by interfering with the interaction essential for targeting, ie binding of the targeting ligand to the cellular receptor. Therefore, near neutral media are desirable for in vivo distribution and targeting. If not carefully regulated, membrane disruption or destabilizing activity is toxic when used in vivo. Balancing vehicle toxicity and nucleic acid delivery is more easily achieved in vitro than in vivo.

  Rozema et al. In US Patent Application Publication No. 20080152661 provided a means of reversibly modulating membrane disruption activity of membrane active polyamines using disubstituted maleic anhydride modifications. The maleamate linkage formed by the reaction of maleic anhydride with an amine is pH labile in a pH range suitable for in vivo delivery. This process allowed membrane active polymers to be used for in vivo delivery or nucleic acids. We now provide modified membrane active polymers with dipeptide-amidobenzyl-carbamate linkages. The dipeptide-amidobenzyl-carbamate linkage is reversible and physiologically reactive. Unlike the pH labile maleamate linkage by modification with disubstituted maleic anhydride, the polymer modifier linkage described herein results in a more stable, enzymatically cleavable linkage in circulation in vivo.

US Patent Application Publication No. 20080152661

In a preferred embodiment, the inventors have reversibly modified an amphiphilic membrane active polyamine and a masking agent for inhibiting its membrane activity: a dipeptide masking agent or a protease cleavable masking agent herein A masking agent comprising a steric stabilizer or targeting ligand linked to a dipeptide-aminobenzyl-carbonate is described. The dipeptide masking agent has the general formula:
In the RA ¹ A ² -amidobenzyl-carbonate formula, R is a steric stabilizer or targeting ligand, A ¹ is an amino acid, and A ² is an amino acid. The reaction of the masking agent carbonate with the polymeric amine results in a carbamate linkage. The masking agent is stable until the dipeptide is cleaved in vivo by an endogenous protease and thus cleaves the steric stabilizer or targeting ligand from the polyamine. After enzymatic cleavage after dipeptide (between A ² and an amide benzyl) amide Benzyl - carbamate undergoes spontaneous metastasis, resulting polymeric amines is to play. Preferably R is neutral. More preferably, R is uncharged. A preferred steric stabilizer is polyethylene glycol (PEG). The targeting ligand may be selected from a list comprising haptens such as digoxigenin, vitamins such as biotin, antibodies, monoclonal antibodies, and cell surface receptor ligands. The targeting ligand may be linked to the dipeptide via a linker such as a PEG linker. A preferred cell surface receptor ligand is an asialoglycoprotein receptor (ASGPr) ligand. A preferred ASGPR ligand is N-acetylgalactosamine (NAG). Preferred dipeptides consist of a hydrophobic amino acid linked to a hydrophilic uncharged amino acid via an amide bond. A preferred amidobenzyl group is a p-amidobenzyl group. A preferred carbonate is an activated amine-reactive carbonate.

  In a preferred embodiment, the present invention is a composition for delivering an RNA interference (RNAi) polynucleotide to a cell in vivo, wherein the polyamine is masked by reversible modification with a dipeptide masking agent as described herein. A composition comprising a masked amphiphilic membrane active polyamine (delivery polymer) and an RNAi polynucleotide. The delivery polymer can be covalently linked to the RNAi polynucleotide. A preferred linkage for covalent attachment of the delivery polymer to the RNAi polynucleotide is a physiologically labile linkage. In one embodiment, the linkage is orthogonal to the dipeptide masking agent linkage. Alternatively, the delivery polymer is not covalently bound to the RNAi polynucleotide and the RNAi polynucleotide is covalently bound to the targeting molecule.

  In preferred embodiments, the inventors: a) via a dipeptide-amidobenzyl-carbamate linkage to multiple targeting ligands or steric stabilizers; and b) one or more reversible to one or more polynucleotides. A composition comprising an amphiphilic membrane active polyamine is described that is covalently bonded through a covalent linkage. In one embodiment, the dipeptide-amidobenzyl-carbamate linkage is orthogonal to the polynucleotide reversible covalent bond. The polynucleotide-polymer conjugate is administered to the mammal in a pharmaceutically acceptable carrier or diluent.

  In a preferred embodiment, the inventors: a) an amphiphilic membrane active polyamine covalently linked via a dipeptide-amidobenzyl-carbamate linkage to multiple targeting ligands or steric stabilizers; and b) 20 Described is a composition comprising an RNAi polynucleotide covalently linked to a targeting group selected from the list consisting of a hydrophobic group having the above carbon atoms and a galactose cluster. In this embodiment, the RNAi polynucleotide is not covalently linked to the modified amphiphilic membrane active polyamine. The modified polyamine and RNAi polynucleotide targeting group conjugate can be synthesized separately and supplied in separate containers, or supplied in one container. The modified polyamine and RNAi polynucleotide-targeting group conjugate are administered to a mammal together or separately in a pharmaceutically acceptable carrier or diluent.

式中、R4は標的指向リガンドまたは立体安定化剤を含み、R3はアミン反応性カルボナート部分を含み、かつR1およびR2はアミノ酸側鎖である。好ましい活性化カルボナートはパラ-ニトロフェノールである。しかし、当技術分野において公知の他のアミン反応性カルボナートもパラ-ニトロフェノールの代わりに容易に用いられる。活性化カルボナートとアミンとの反応により、標的指向リガンドまたは立体安定化剤が膜活性ポリアミンに、ペプチダーゼ切断可能なジペプチド-アミドベンジルカルバマート連結を介して連結される。アミノ酸とアミドベンジル基との間でのジペプチドの酵素切断により、R4がポリマーから除去されて脱離反応が誘発され、これはポリマーアミンの再生を引き起こす。 Preferred dipeptide masking agents include protease (peptidase) cleavable dipeptide-p-amidobenzylamine reactive carbonate derivatives. The protease-cleavable masking agent of the present invention uses a dipeptide linked to an amide benzyl activated carbonate moiety. A targeting ligand or steric stabilizer is attached to the amino terminus of the dipeptide. The amidobenzyl activated carbonate moiety is attached to the carboxy terminus of the dipeptide. Protease cleavable linkers suitable for use according to the present invention have the following structure:

Where R4 contains a targeting ligand or steric stabilizer, R3 contains an amine reactive carbonate moiety, and R1 and R2 are amino acid side chains. A preferred activated carbonate is para-nitrophenol. However, other amine-reactive carbonates known in the art are readily used in place of para-nitrophenol. Reaction of the activated carbonate with an amine couples the targeting ligand or steric stabilizer to the membrane active polyamine via a peptidase cleavable dipeptide-amidobenzyl carbamate linkage. Enzymatic cleavage of the dipeptide between the amino acid and the amidobenzyl group removes R4 from the polymer and induces an elimination reaction, which causes the regeneration of the polymeric amine.

The dipeptide masking agents of the present invention are useful for reversible modification / inhibition of amphiphilic membrane active polyamines. Covalent bonds are generated by reaction of activated carbonates of dipeptide masking agents with polymeric amines, particularly primary amine groups. Accordingly, provided herein are conjugates comprising a dipeptide-amidobenzyl-carbonate masking agent as described herein and the following amphiphilic membrane active polyamine:

式中、
Xは-NH-、-O-、または-CH ₂ -であり、
Yは-NH-または-O-であり、
R1は-CH ₂ -フェニル、-CH-(CH ₃ ) ₂ 、-CH ₂ -CH-(CH ₃ ) ₂ 、-CH(CH ₃ )-CH ₂ -CH ₃ 、-CH ₃ 、

であり、
R2は水素、-(CH ₂ ) ₃ -NH-C(O)-NH ₂ 、-(CH ₂ ) ₄ -N-(CH ₃ ) ₂ 、または-CH ₂ -C(O)-NH ₂ であり、
R4は非荷電であって、ポリエチレングリコールまたは標的指向リガンドを含み、
R5は2、4、または6位にあって、-CH ₂ -O-C(O)-O-Zであり、ここでZは
-ハロゲン化物、

であり、かつ、
R6は、R5が占有している位置を除き、2、3、4、5、または6位のそれぞれで独立に水素、アルキル、-(CH ₂ ) _n -CH ₃ （ここでn＝0〜4）、-(CH ₂ )-(CH ₃ ) ₂ 、またはハロゲン化物である。
[本発明1003]
R4が立体安定化剤を含む、本発明1002の化合物。
[本発明1004]
立体安定化剤がポリエチレングリコール（PEG）である、本発明1003の化合物。
[本発明1005]
標的指向リガンドがアシアロ糖タンパク質受容体（ASGPr）リガンドを含む、本発明1002の化合物。
[本発明1006]
ASGPrリガンドが、ラクトース、ガラクトース、N-アセチルガラクトサミン、ガラクトサミン、N-ホルミルガラクトサミン、N-プロピオニルガラクトサミン、N-n-ブタノイルガラクトサミン、およびN-イソ-ブタノイル-ガラクトサミンからなるリストより選択される、本発明1005の化合物。
[本発明1007]
XおよびYが-NH-であり、かつZが

である、本発明1001の化合物。
[本発明1008]
R1が-CH ₂ -フェニルである、本発明1007の化合物。
[本発明1009]
R2が-(CH ₂ ) ₃ -NH-C(O)-NH ₂ である、本発明1007の化合物。
[本発明1010]
下記を含む、インビボでRNA干渉（RNAi）ポリヌクレオチドを細胞に送達するための送達ポリマー：

式中、
Pは両親媒性膜活性ポリアミンであり、
M ¹ はジペプチド-アミドベンジル-カルバマート連結を介してPに連結された標的指向リガンドであり、
M ² はジペプチド-アミドベンジル-カルバマート連結を介してPに連結された立体安定化剤であり、
yおよびzはそれぞれゼロ以上の整数であり、かつ
y＋zは、マスキング剤の非存在下でのポリアミンP上のアミンの量により判定して、P上の一級アミンの50％よりも大きい値を有する。
[本発明1011]
立体安定化剤がPEGである、本発明1010の送達ポリマー。
[本発明1012]
標的指向リガンドがASGPrリガンドを含む、本発明1010の送達ポリマー。
[本発明1013]
ASGPrリガンドが、ラクトース、ガラクトース、N-アセチルガラクトサミン、ガラクトサミン、N-ホルミルガラクトサミン、N-プロピオニルガラクトサミン、N-n-ブタノイルガラクトサミン、およびN-イソ-ブタノイル-ガラクトサミンからなるリストより選択される、本発明1012の送達ポリマー。
[本発明1014]
両親媒性膜活性ポリアミンが、ランダム、ブロック、または交互合成ポリマーからなるリストより選択される、本発明1010の送達ポリマー。
[本発明1015]
両親媒性膜活性ポリアミンがメリチンペプチドである、本発明1010の送達ポリマー。
[本発明1016]
ジペプチド-アミドベンジル-カルバマート連結が下記によって表される構造を有する、本発明1010の送達ポリマー：

式中、
Xは-NH-、-O-、または-CH ₂ -であり
Yは-NH-または-O-であり
R1は-CH ₂ -フェニル、-CH-(CH ₃ ) ₂ 、-CH ₂ -CH-(CH ₃ ) ₂ 、-CH(CH ₃ )-CH ₂ -CH ₃ 、-CH ₃ 、

であり；
R2は水素、-(CH ₂ ) ₃ -NH-C(O)-NH ₂ 、-(CH ₂ ) ₄ -N-(CH ₃ ) ₂ 、または-CH ₂ -C(O)-NH ₂ であり、
R4はM ¹ の標的指向リガンドまたはM ² の立体安定化剤を含み、かつ
ポリアミンは両親媒性膜活性ポリアミンである。
[本発明1017]
両親媒性膜活性ポリアミンがRNAiポリヌクレオチドにさらに共有結合している、本発明1016の送達ポリマー。
[本発明1018]
インビボでRNAiポリヌクレオチドを哺乳動物の細胞に送達する方法であって、RNAiポリヌクレオチドを哺乳動物に本発明1010の送達ポリマーと同時投与する段階を含む、方法。
[本発明1019]
RNAiポリヌクレオチドが送達ポリマーに共有結合している、本発明1018の方法。
[本発明1020]
細胞が肝実質細胞または肝臓癌細胞である、本発明1018の方法。
本発明の化合物は一般に、有機または医薬品化学分野の当業者には公知の方法を用いて得ることができる。本発明のさらなる目的、特徴、および利点は、添付の図面と一緒に読めば、以下の詳細な説明から明らかになるであろう。
[Invention 1001]
A compound for reversibly modifying an amphiphilic membrane active polyamine comprising a steric stabilizer or targeting ligand covalently linked to a dipeptide-amidobenzyl-carbonate.
[Invention 1002]
Compounds of the invention 1001 having the structure represented by:

Where
X is —NH—, —O—, or —CH ₂ —;
Y is -NH- or -O-
R1 is -CH ₂ - _{phenyl, -CH- (CH 3) 2,} -CH 2 -CH- (CH 3) 2, -CH (CH 3) -CH 2 -CH 3, -CH 3,

And
R2 is _{hydrogen, - (CH 2) 3 -NH} -C (O) -NH 2, - (CH 2) 4 -N- (CH 3) 2, or be a -CH ₂ -C (O) -NH ₂ ,
R4 is uncharged and comprises polyethylene glycol or a targeting ligand,
R5 is in the 2, 4, or 6 position and is —CH ₂ —OC (O) —OZ, where Z is
-Halides,

And
R6 is independently hydrogen, alkyl, — (CH ₂ ) _n —CH ₃ (where n = 0-4 ) at each of the 2, 3, 4, 5, or 6 positions, except where R5 occupies ),-(CH ₂ )-(CH ₃ ) ₂ , or a halide.
[Invention 1003]
The compound of the present invention 1002 wherein R4 comprises a steric stabilizer.
[Invention 1004]
The compound of the present invention 1003, wherein the steric stabilizer is polyethylene glycol (PEG).
[Invention 1005]
The compound of the invention 1002, wherein the targeting ligand comprises an asialoglycoprotein receptor (ASGPr) ligand.
[Invention 1006]
The present invention 1005, wherein the ASGPr ligand is selected from the list consisting of lactose, galactose, N-acetylgalactosamine, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, Nn-butanoylgalactosamine, and N-iso-butanoyl-galactosamine Compound.
[Invention 1007]
X and Y are -NH- and Z is

The compound of the invention 1001.
[Invention 1008]
R1 is -CH ₂ - phenyl, the compounds of the present invention 1007.
[Invention 1009]
R2 is - (CH _{2) 3 -NH-C (O) -NH} 2, compound of the present invention 1007.
[Invention 1010]
Delivery polymers for delivering RNA interference (RNAi) polynucleotides to cells in vivo, including:

Where
P is an amphiphilic membrane active polyamine,
M ¹ is a targeting ligand linked to P via a dipeptide-amidobenzyl-carbamate linkage,
M ² is a steric stabilizer linked to P via a dipeptide-amidobenzyl-carbamate linkage,
y and z are each integers greater than or equal to zero, and
y + z has a value greater than 50% of the primary amine on P as judged by the amount of amine on polyamine P in the absence of a masking agent.
[Invention 1011]
The delivery polymer of the invention 1010, wherein the steric stabilizer is PEG.
[Invention 1012]
The delivery polymer of invention 1010, wherein the targeting ligand comprises an ASGPr ligand.
[Invention 1013]
The present invention 1012 wherein the ASGPr ligand is selected from the list consisting of lactose, galactose, N-acetylgalactosamine, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, Nn-butanoylgalactosamine, and N-iso-butanoyl-galactosamine Delivery polymer.
[Invention 1014]
The delivery polymer of the invention 1010, wherein the amphiphilic membrane active polyamine is selected from the list consisting of random, block, or alternating synthetic polymers.
[Invention 1015]
The delivery polymer of the invention 1010 wherein the amphiphilic membrane active polyamine is a melittin peptide.
[Invention 1016]
The delivery polymer of the invention 1010 wherein the dipeptide-amidobenzyl-carbamate linkage has the structure represented by:

Where
X is -NH -, - O-, or -CH ₂ -
Y is -NH- or -O-
R1 is -CH ₂ - _{phenyl, -CH- (CH 3) 2,} -CH 2 -CH- (CH 3) 2, -CH (CH 3) -CH 2 -CH 3, -CH 3,

Is;
R2 is _{hydrogen, - (CH 2) 3 -NH} -C (O) -NH 2, - (CH 2) 4 -N- (CH 3) 2, or be a -CH ₂ -C (O) -NH ₂ ,
R4 comprises a steric stabilizing agent for targeting ligand or M ² of M ^1, and
The polyamine is an amphiphilic membrane active polyamine.
[Invention 1017]
The delivery polymer of the invention 1016, wherein the amphiphilic membrane active polyamine is further covalently linked to the RNAi polynucleotide.
[Invention 1018]
A method of delivering an RNAi polynucleotide to a mammalian cell in vivo, comprising co-administering the RNAi polynucleotide to a mammal with a delivery polymer of the invention 1010.
[Invention 1019]
The method of the present invention 1018, wherein the RNAi polynucleotide is covalently linked to the delivery polymer.
[Invention 1020]
The method of the present invention 1018 wherein the cells are hepatocytes or liver cancer cells.
The compounds of the present invention can generally be obtained using methods known to those skilled in the organic or medicinal chemistry arts. Further objects, features and advantages of the present invention will become apparent from the following detailed description when read in conjunction with the accompanying drawings.

FIG. 1 shows the structure of a dipeptide masking agent, where R1 and R2 are R groups of amino acids, R4 is a steric stabilizer targeting ligand, and X is —NH—, —O -, Or -CH2-, Y is -NH- or -O- and R5 is in the 2, 4, or 6 position and is -CH2-OC (O) -OZ, wherein Z 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. FIG. 2 shows the structure of a dipeptide masking agent linked to a polyamine, where R1 and R2 are the R groups of amino acids, R4 is the targeting ligand of the steric stabilizer, and X is —NH—, —O—, or —CH ₂ —, and Y is —NH— or —O—. FIG. 3 shows the structures of various dipeptide masking agents. FIG. 4 is a graph showing the circulation time of a polymer modified with a dipeptide masking agent or two different maleic anhydride based masking agents.

Detailed Description of the Invention Described are masking agents useful for reversible modification and inhibition of amphiphilic membrane active polyamines and delivery polymers produced by modification of polyamines with dipeptide masking agents. The peptidase cleavable linkage is stable to hydrolysis in the absence of enzyme, is electrically neutral, and provides long-term DPC stability during storage and in vivo circulation. Improved (and longer) half-life in circulation promotes an expanded window of opportunity for ligand-mediated accumulation in tissues such as tumor tissue. Polymer delivery is particularly useful for in vivo delivery of RNAi polynucleotides. In vivo delivery of RNAi polynucleotides is useful for therapeutic inhibition (knockdown) of gene expression.

The dipeptide masking agent has the general formula:
RA ¹ A ² -amidobenzyl-carbonate, where R is a steric stabilizer or targeting ligand, A ¹ is an amino acid, A ² is an amino acid, and carbonate is an activated amine-reactive carbonate . R is preferably uncharged. The reaction of the masking agent carbonate with the polymeric amine results in a carbamate linkage. The masking agent is stable until the dipeptide is cleaved in vivo by an endogenous protease and thus cleaves the steric stabilizer or targeting ligand from the polyamine. After enzymatic cleavage after dipeptide (between A ² and an amide benzyl) amide Benzyl - carbamate undergoes spontaneous metastasis, resulting polymeric amines is to play. A preferred steric stabilizer is polyethylene glycol (PEG). A preferred targeting ligand for hepatic delivery is the ASGPr ligand. A preferred ASGPr ligand is N-acetylgalactosamine (NAG). A preferred amidobenzyl group is a p-amidobenzyl group.

A dipeptide of a dipeptide masking agent represented by A ¹ A ² (or AA) is a dimer of amino acids linked via amide bonds. Amino acids, including α and β amino acids, are well known in biology and chemistry and are molecules that contain amine groups, carboxylic acid groups, and side chains that vary between different amino acids. Preferred amino acids are α-amino acids having the general formula H ₂ NCHRCOOH, where R is an organic substituent. Preferred α-amino acids are uncharged natural amino acids. In preferred dipeptides, A1 is a hydrophobic amino acid and A2 is an uncharged hydrophilic amino acid. Preferred hydrophobic amino acids are phenylalanine, valine, isoleucine, leucine, alanine, or tryptophan. Preferred uncharged hydrophilic amino acids are asparagine, glutamine, or citrulline. More preferably the hydrophobic amino acid is phenylalanine or valine. A more preferred uncharged hydrophilic amino acid is citrulline. Although dipeptides are preferred, additional amino acids can be inserted between A ¹ and R. It is also possible to use one amino acid in place of the dipeptide by leaving the amino acid A ^1. Any natural amino acids used in the present invention are referred to herein by their common abbreviations. Although charged amino acids can be used, the masking agent is preferably uncharged.

  In a preferred embodiment, amphiphilic membrane active polyamines are reversibly modified by reaction with a dipeptide-amidobenzyl-carbonate masking agent of the present invention to yield membrane inactive delivery polymers. Dipeptide masking agents can shield the polymer from non-specific interactions, prolong circulation time, enhance specific interactions, inhibit toxicity, or alter the charge of the polymer.

式中：
Xは-NH-、-O-、または-CH₂-であり
Yは-NH-または-O-であり
R1は好ましくは
-(CH₂)_k-フェニル（kは1、2、3、4、5、6であり；k＝1ではフェニルアラニン）、
-CH-(CH₃)₂（バリン）、
-CH₂-CH-(CH₃)₂（ロイシン）、
-CH(CH₃)-CH₂-CH₃（イソロイシン）、
-CH₃（アラニン）、
-(CH₂)₂-COOH（グルタミン酸）、
または

であり；
R2は好ましくは
水素（グリシン）
-(CH₂)₃-NH-C(O)-NH₂（シトルリン）、
-(CH₂)₄-N-(CH₃)₂（リジン(CH₃)₂）、
-(CH₂)_k-C(O)-NH₂；（kは1、2、3、4、5、6である）、
-CH₂-C(O)-NH₂（アスパラギン）、
-(CH₂)₂-C(O)-NH₂（グルタミン）、
-CH₂-C(O)-NR¹R²（アスパラギン酸アミド）、
-(CH₂)₂-C(O)-NR¹R²（グルタミン酸アミド）、
-CH₂-C(O)-OR¹（アスパラギン酸エステル）、または
-(CH₂)₂-C(O)-OR¹（グルタミン酸エステル）であり、
R¹およびR²はアルキル基であり
R4はポリエチレングリコールまたは標的指向リガンドを含み；かつ
ポリアミンは両親媒性膜活性ポリアミンである。 The reversibly masked polymer of the present invention comprises the following structure:

In the formula:
X is -NH-, -O-, or -CH _2-
Y is -NH- or -O-
R1 is preferably
-(CH ₂ ) _k -phenyl (k is 1, 2, 3, 4, 5, 6; phenylalanine at k = 1),
-CH- (CH ₃ ) ₂ (valine),
-CH ₂ -CH- (CH ₃ ) ₂ (leucine),
-CH (CH ₃ ) -CH ₂ -CH ₃ (isoleucine),
-CH ₃ (alanine),
-(CH ₂ ) ₂ -COOH (glutamic acid),
Or

Is;
R2 is preferably hydrogen (glycine)
-(CH ₂ ) ₃ -NH-C (O) -NH ₂ (citrulline),
-(CH ₂ ) ₄ -N- (CH ₃ ) ₂ (lysine (CH ₃ ) ₂ ),
_{- (CH 2) k -C (} O) -NH 2; (k is 1,2,3,4,5,6),
-CH ₂ -C (O) -NH ₂ (asparagine),
-(CH ₂ ) ₂ -C (O) -NH ₂ (glutamine),
-CH ₂ -C (O) -NR ¹ R ² (aspartic acid amide),
-(CH ₂ ) ₂ -C (O) -NR ¹ R ² (glutamic acid amide),
-CH ₂ -C (O) -OR ¹ (aspartate), or
- (CH ₂₎ a ₂ -C (O) -OR ¹ (glutamate),
R ¹ and R ² are alkyl groups
R4 contains polyethylene glycol or a targeting ligand; and the polyamine is an amphiphilic membrane active polyamine.

  Although the above structure shows one dipeptide masking agent linked to the polymer, in the practice of the invention, 50% to 90% or more of the polymeric amine is modified by the dipeptide masking agent.

式中、R1、R2、R4およびポリアミンは前述のとおりである。 In a preferred embodiment, the reversibly masked polymer of the present invention comprises the following structure:

In the formula, R1, R2, R4 and polyamine are as described above.

式中：
X、Y、R1、R2、およびR4は前述のとおりであり
R5は2、4、または6位にあって、-CH₂-O-C(O)-O-Zであり、ここでZは
-ハロゲン化物、

R6は、R5が占有している位置を除き、2、3、4、5、または6位のそれぞれで独立に水素、アルキル、-(CH₂)_n-CH₃（ここでn＝0〜4）、-(CH₂)-(CH₃)₂、またはハロゲン化物である。 The reversibly masked polymer of the present invention is formed by reaction of the dipeptide masking agent of the present invention with an amine on the polymer. The dipeptide masking agent of the present invention has the following structure:

In the formula:
X, Y, R1, R2, and R4 are as described above.
R5 is a 2, 4 or 6-position, a -CH ₂ -OC (O) -OZ, where Z is
-Halides,

R6 is independently hydrogen, alkyl, — (CH ₂ ) _n —CH ₃ (where n = 0-4) at each of the 2, 3, 4, 5, or 6 positions, except where R5 occupies ),-(CH ₂ )-(CH ₃ ) ₂ , or a halide.

。 In a preferred embodiment, X is —NH—, Y is —NH—, R 4 is uncharged, R 5 is in position 4 and R 6 is hydrogen, as shown by the following:

.

In another embodiment, R4 is:
R- (O-CH ₂ -CH ₂ ) _s -O-Y1-
R is hydrogen, methyl or ethyl; and s is an integer from 1 to 150, and Y1 is selected from the list comprising:
-O-Y2-NH-C (O)-(CH ₂ ) ₂ -C (O)-, where Y2 is
-(CH ₂ ) _{3-
-C (O) -N- (CH 2} -CH 2 -O) p -CH 2 -CH 2 - (p is an integer of 1 to 20), and
-O-.

  The targeting ligand may be selected from a list comprising haptens, vitamins, antibodies, monoclonal antibodies, and cell surface receptor ligands. The targeting ligand may be linked to the dipeptide via a linker such as a PEG linker.

  Non-limiting examples of membrane active polymers suitable for use according to the present invention have been previously described in US Patent Application Publication Nos. 20080152661, 20090023890, 20080287630, and 20110207799. Suitable amphiphilic membrane active polyamines may be small peptides such as melittin peptides.

R¹は標的指向リガンド（保護基と共に、または保護基なしのいずれか）またはPEGを含み、
R²は両親媒性膜活性ポリアミンであり、
AAはジペプチド（保護基と共に、または保護基なしのいずれか）であり、かつ
Zはアミン反応性カルボナートである。 The polymeric amine was reversibly modified with an enzyme cleavable linker as described herein. When the amine is regenerated by cleaving the modifying group, the amine is reversibly modified. As shown below, reaction of the masking agent's activated carbonate with a polymeric amine couples the targeting ligand or steric stabilizer to the polymer via a peptidase-cleavable dipeptide-amidobenzylcarbamate linkage:

R ¹ comprises a targeting ligand (either with or without a protecting group) or PEG,
R ² is an amphiphilic membrane active polyamine,
AA is a dipeptide (either with or without protecting groups), and
Z is an amine reactive carbonate.

  Protecting groups may be used during the synthesis of the dipeptide masking agent. If present, the protecting group may be removed before or after modification of the amphiphilic membrane active polyamine.

上記反応スキームにおいて、AAはジペプチドであり、R¹は標的指向リガンドまたは立体安定化剤を含み、かつR²は両親媒性膜活性ポリアミンである。重要なことに、遊離ポリマーは脱修飾され、したがって膜活性を復旧する。 A reversible modification of a sufficient percentage of the polymeric amine with a dipeptide masking agent inhibits the membrane activity of the membrane active polyamine. Also, modification of the polymeric amine with a dipeptide masking agent preferably neutralizes the charge of the amine. The dipeptide-amidobenzyl-carbamate linkage is sensitive to protease (or peptidase) cleavage. In the presence of a protease, the anilide bond is cleaved to yield an intermediate that immediately undergoes a 1,6 elimination reaction to release the free polymer:

In the above reaction scheme, AA is a dipeptide, R ¹ contains a targeting ligand or steric stabilizer, and R ² is an amphiphilic membrane active polyamine. Importantly, the free polymer is demodified, thus restoring membrane activity.

  In the masked state, reversibly modified membrane active polyamines do not exhibit membrane disruption activity. More than 50% and more than 55% of amines on polyamines to inhibit membrane activity and provide cell targeting function, ie generate reversibly masked membrane active polymer (delivery polymer) , More than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90%, reversible modification with a dipeptide masking agent is required sell.

  The present invention also provides a method of delivering a bioactive substance into a cell. More specifically, the present invention is directed to compounds, compositions, and methods useful for delivering RNAi polynucleotide mammalian cells in vivo.

  In one embodiment, the RNAi polynucleotide is linked to the delivery polymer of the invention via a physiologically labile covalent bond. By using physiologically labile linkages, the polynucleotide can be cleaved from the polymer to release the polynucleotide that participates in the functional interaction with cellular components.

式中、NはRNAiポリヌクレオチドであり、L¹は生理的に不安定な連結であり、Pは両親媒性膜活性ポリアミンであり、M¹はジペプチド-アミドベンジル-カルバマート連結を介してPに連結された標的指向リガンドであり、かつM²はジペプチド-アミドベンジル-カルバマート連結を介してPに連結された立体安定化剤である。yおよびzはそれぞれ、いかなるマスキング剤も非存在下でのポリアミンP上のアミンの量により判定して、y＋zがP上の一級アミンの50％よりも大きい、60％よりも大きい、70％よりも大きい、80％よりも大きい、または90％よりも大きい値を有するとの条件で、ゼロ以上の整数である。その非修飾状態で、Pは膜活性ポリアミンである。送達ポリマーM¹ _y-P-M² _zは膜活性ではない。P一級アミンのM¹および/またはM²の結合による可逆的修飾は、Pの膜活性を可逆的に阻害または不活化する。メリチンペプチドなどの、いくつかの小さい両親媒性膜活性ポリアミンは、3〜5つという少数の一級アミンを含むことに留意される。アミンのあるパーセンテージの修飾は、ポリマーの集団におけるアミンのあるパーセンテージの修飾を反映することになる。M¹およびM²の切断後、ポリアミンのアミンが再生され、それによりPはその非修飾の膜活性状態に戻る。 The present invention includes conjugate delivery systems of the following general structure:

Where N is an RNAi polynucleotide, L ¹ is a physiologically unstable linkage, P is an amphiphilic membrane active polyamine, and M ¹ is linked to P via a dipeptide-amidobenzyl-carbamate linkage. a linked targeting ligand, and M ² is a dipeptide - is through carbamate linked linked to P steric stabilizer - amide benzyl. y and z are each greater than 50%, greater than 60%, greater than 70% of the primary amine on P, as determined by the amount of amine on polyamine P in the absence of any masking agent Is an integer greater than or equal to zero on condition that it has a value greater than, greater than 80%, or greater than 90%. In its unmodified state, P is a membrane active polyamine. The delivery polymer M ¹ _y -PM ² _z is not membrane active. Reversible modification of P primary amines by M ¹ and / or M ² binding reversibly inhibits or inactivates P membrane activity. It is noted that some small amphiphilic membrane active polyamines, such as melittin peptides, contain as few as 3-5 primary amines. A percentage modification of the amine will reflect a percentage modification of the amine in the polymer population. After cleavage of M ¹ and M ² , the amine of the polyamine is regenerated, thereby returning P to its unmodified membrane active state.

In another embodiment, the RNAi polynucleotide is co-administered with the delivery polymer of the invention in vivo. Accordingly, the present invention includes compositions of the following general structure:
M ¹ _y -P-M ² _z plus NT,
Where N is an RNAi polynucleotide, T is a target-directing group, P is an amphiphilic membrane active polyamine, and M ¹ is target-directed linked to P via a dipeptide-amidobenzyl-carbamate linkage. It is a ligand and M ² is a steric stabilizer linked to P via a dipeptide-amidobenzyl-carbamate linkage. y and z are each greater than 50%, greater than 60%, greater than 70% of the primary amine on P, as determined by the amount of amine on polyamine P in the absence of any masking agent Is an integer greater than or equal to zero on condition that it has a value greater than, greater than 80%, or greater than 90%. In its unmodified state, P is a membrane active polyamine. The delivery polymer M ¹ _y -PM ² _z is not membrane active. Reversible modification of P primary amines by M ¹ and / or M ² binding reversibly inhibits or inactivates P membrane activity. It is noted that some small amphiphilic membrane active polyamines, such as melittin peptides, contain as few as 3-5 primary amines. Thus, a percentage modification of the amine will reflect a percentage modification of the amine in the polymer population. After cleavage of M ¹ and M ² , the amine of the polyamine is regenerated, thereby returning P to its unmodified membrane active state. N is covalently linked to T using standard methods in the art to generate RNAi polynucleotide-targeting group conjugates. Preferred covalent bonds are physiologically labile bonds. NT. The delivery polymer and NT are synthesized or manufactured separately. Neither T nor N is directly or indirectly covalently bound to P, M ¹ or M ² . Electrostatic or hydrophobic binding of a polynucleotide or polynucleotide conjugate to a masked or unmasked polymer is not necessary for in vivo hepatic delivery of the polynucleotide. The masked polymer and polynucleotide conjugate can be shared in the same container or separate containers. These may be mixed prior to administration, may be administered simultaneously, or may be administered sequentially.

  For hepatocyte delivery, y is greater than 50% of the primary amine on polymer P, whether the RNAi polynucleotide is covalently linked to the delivery polymer or co-administered with the delivery polymer. Is large and has a value of up to 100%. Thus, z is greater than zero percent (0%) but has a value less than 50% of the primary amine on polymer P.

  For delivery to liver tumor cells, z may be larger and have a value of up to 100% of the primary amine on polymer P. In preferred embodiments, z is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of primary amines on polyamine P for delivery to tumor cells. Also has a large value and y is zero.

  Membrane active polyamines can disrupt the plasma membrane or lysosome / endocytosis membrane. This membrane activity is an essential feature for cellular delivery of polynucleotides. However, membrane activity causes toxicity when the polymer is administered in vivo. Polyamines also readily interact with many anionic components in vivo, causing undesirable biodistribution. Therefore, reversible masking of polyamine membrane activity is essential for in vivo use.

  Masking is accomplished by reversibly coupling the described dipeptide masking agent to a membrane active polyamine to produce a reversibly masked membrane active polymer, ie a delivery polymer. In addition to inhibiting membrane activity, masking agents shield the polymer from non-specific interactions, reduce serum interactions, neutralize polyamines, reduce positive charges, and produce nearly neutral charge polymers. Prolongs circulation time and / or provides cell-specific interaction, ie targeting.

  Overall, it is an essential feature of masking agents that the masking agent inhibits the membrane activity of the polymer. Masking agents can shield the polymer from non-specific interactions (reduce serum interactions and increase circulation time). A membrane active polyamine is membrane active in an unmodified (not masked) state and is not membrane active (inactivated) in a modified (masked) state. A sufficient number of masking agents are linked to the polymer to achieve the desired level of inactivation. The desired level of modification of the polymer by conjugation of the masking agent is readily determined using an appropriate polymer activity assay. For example, if the polymer has membrane activity in a given assay, a sufficient level of masking agent is linked to the polymer to achieve the desired level of inhibition of membrane activity in that assay. Masking is ≧ 50%, ≧ 60%, ≧ 70%, ≧ 80% or ≧ 90 of the primary amine groups on the polymer population, as judged by the amount of primary amine on the polymer in the absence of any masking agent. % Modification is required. It is also a preferred feature of the masking agent that the binding of the masking agent to the polymer reduces the polymer's positive charge and thus produces a more neutral delivery polymer. It is desirable that the masked polymer retains water solubility.

  The membrane active polyamine can be coupled to the masking agent in the presence of excess masking agent. The delivery polymer may be administered after the excess masking agent is removed from the bound delivery polymer.

  As used herein, a “steric stabilizer” is a nonionic that prevents or inhibits intramolecular or intermolecular interactions of the polymer to which it is attached compared to a polymer that does not contain a steric stabilizer. A hydrophilic polymer (either natural, synthetic or non-natural). Steric stabilizers prevent the polymer to which they are attached from participating in electrostatic interactions. An electrostatic interaction is a non-covalent bond of multiple substances due to the attractive force between positive and negative charges. Steric stabilizers can inhibit interactions with blood components and thus can inhibit opsonization, phagocytosis, and uptake by the reticuloendothelial system. Thus, steric stabilizers can extend the circulation time of molecules to which they are attached. Steric stabilizers can also inhibit polymer aggregation. Preferred steric stabilizers are polyethylene glycol (PEG) or PEG derivatives. Preferred PEGs used herein may have about 1 to 500 ethylene glycol monomers, or 2 to 25 ethylene glycol monomers. Preferred PEGs used herein may also have a molecular weight average of about 85-20,000 Daltons (Da), about 85-1000 Da. The steric stabilizer used herein prevents or inhibits intramolecular or intermolecular interactions of the polymer to which it is attached in aqueous solution compared to a polymer that does not contain a steric stabilizer.

  “Targeting ligands” enhance the pharmacokinetic or biodistribution properties of the conjugates to which they are bound, improving the cell or tissue-specific distribution and cell-specific uptake of the conjugates. For clarity, the term “targeting ligand” as used herein is used to indicate a targeting ligand that is bound to a dipeptide masking agent, and “targeting group” is an RNAi polynucleotide-target. A targeting ligand linked to an RNAi polynucleotide in a directing group conjugate. Targeting ligands enhance the binding of molecules to target cells. Thus, targeting ligands can enhance the pharmacokinetic or biodistribution properties of the conjugates to which they are bound, improving the cellular distribution and cellular uptake of the conjugates. Binding of the targeting ligand to the cell or cell receptor can initiate endocytosis. The targeting ligand can have a monovalent, divalent, trivalent, tetravalent, or higher valency. The targeting ligand may be selected from the group comprising: compounds having affinity for cell surface molecules, cell receptor ligands, antibodies, monoclonal antibodies, antibody fragments, and antibody-like substances having affinity for cell surface molecules. Preferred targeting ligands include cell receptor ligands. Various ligands have been used to target drugs and genes to cells and specific cellular receptors. Cell receptor ligands include: carbohydrates, glycans, carbohydrates (including but not limited to galactose, galactose derivatives, mannose, and mannose derivatives), vitamins, folate, biotin, aptamers, and peptides (RGD Containing peptide, insulin, EGF, and transferrin) may be selected from the group comprising, but not limited to.

  For hepatocyte targeting, preferred targeting ligands are carbohydrates that have an 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, “ASGPr targeting ligand” includes galactose and galactose derivatives that have an affinity for ASGPr that is equal to or greater than galactose. Binding of the galactose targeting ligand to ASGPr promotes cell-specific targeting of the delivery polymer to hepatocytes and endocytosis of the delivery polymer to hepatocytes.

  ASGPr targeting ligand consists of lactose, galactose, N-acetylgalactosamine (NAG), galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, Nn-butanoylgalactosamine, and N-isobutanoylgalactosamine You may choose from a group (Iobst, ST and Drickamer, KJBC 1996, 271, 6686). The ASGPr targeting moiety can be a monomer (eg, having one galactosamine) or a multimer (eg, having multiple galactosamines).

  In one embodiment, the membrane active polyamine is reversibly reversible by binding to a ≧ 50%, ≧ 60%, ≧ 70%, ≧ 80%, or ≧ 90% of the primary amine on the polyamine of the ASGPr targeting ligand masking agent. Mask it. In another embodiment, the membrane active polyamine is reduced to ≧ 50%, ≧ 60%, ≧ 70%, ≧ 80%, or ≧ 90% of the primary amine on the polymer of the ASGPr targeting ligand masking agent and the PEG masking agent. Mask reversibly by binding. In the case of both the ASGPr targeting ligand masking agent and the PEG masking agent, the ratio of PEG to ASGPr targeting ligand is about 0-4: 1, more preferably about 0.5-2: 1.

  Amphipathic or amphiphilic polymers are well known and recognized in the art and include hydrophilic (polar, water-soluble) and hydrophobic (nonpolar, lipophilic, water-insoluble) groups. Or have both parts.

  “Hydrophilic group” is a qualitative term that indicates that the chemical moiety prefers water. Typically, such chemical groups are water soluble and are hydrogen bond donors or acceptors with water. The hydrophilic group can be charged or uncharged. The charged group can be positively charged (anion) or negatively charged (cation) or both (zwitterion). Examples of hydrophilic groups include the following chemical moieties: hydrocarbons, polyoxyethylenes, certain peptides, oligonucleotides, amines, amides, alkoxyamides, carboxylic acids, sulfur, and hydroxyls.

  “Hydrophobic group” is a qualitative term that indicates that the chemical moiety avoids water. Typically, such chemical groups are not water soluble and tend not to form hydrogen bonds. Lipophilic groups dissolve in fats, oils, lipids, and nonpolar solvents and have little or no ability to form hydrogen bonds. Hydrocarbons containing multiple carbon atoms, certain substituted hydrocarbons, cholesterol, and cholesterol derivatives are examples of hydrophobic groups and compounds.

  The hydrophobic group is preferably a hydrocarbon containing only carbon and hydrogen atoms. However, nonpolar substituted or nonpolar heteroatoms that remain hydrophobic and include, for example, fluorine are acceptable. The term includes aliphatic groups, aromatic groups, acyl groups, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, which are each linear, branched, or cyclic. There may be. The term hydrophobic group also includes sterols, steroids, cholesterol, and steroids and cholesterol derivatives.

  For amphiphilic polymers, a moiety used herein is defined as a molecule that is derived when one covalent bond is broken and replaced with hydrogen. For example, in butylamine, breakage of the bond between carbon and nitrogen, and substitution with hydrogen yields ammonia (hydrophilic) and butane (hydrophobic). When 1,4-diaminobutane is cleaved at the nitrogen-carbon bond and replaced with hydrogen, the resulting molecules are again ammonia (2 ×) and butane. However, 1,4-diaminobutane is not considered amphiphilic because the formation of a hydrophobic moiety requires the cleavage of two bonds.

  The surface active polymer used herein reduces the surface tension of water and / or the interfacial tension with other phases and is therefore actively adsorbed at the liquid / vapor interface. The nature of the surface activity is usually due to the fact that the molecule of the substance is amphipathic or amphiphilic.

  A “membrane active” polymer as used herein is a surface active, amphiphilic polymer that can induce one or more of the following effects on biological membranes: Changes or breaks in the membrane, pore formation in the membrane, splitting of the membrane, or breakage or dissolution of the membrane that allows it to enter or pass through. As used herein, a membrane, or cell membrane, includes a lipid bilayer. Membrane change or disruption can be functionally defined by polymer activity in at least one of the following assays: erythrocyte lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis, and endosome release. Membrane active polymers that can cause cell membrane lysis are also referred to as membrane soluble polymers. Polymers that cause disruption of endosomes or lysosomes preferentially over the plasma membrane are considered endosomal lytic. The effect of the membrane active polymer on the cell membrane may be temporary. Membrane activity has an affinity for the membrane and causes denaturation or deformation of the bilayer structure. The membrane active polymer can be a synthetic or non-natural amphiphilic polymer.

  The membrane active polymer used in the present specification is a compound such as a peptide rich in arginine derived from HIV TAT protein, antennapedia peptide, VP22 peptide, transportan, artificial peptide rich in arginine, artificial polymer rich in small guanidinium, etc. Different from the class of polymers called cell penetrating peptides or polymers represented by Cell penetrating compounds allow some molecules to move from one side of the lipid bilayer to the other without apparently requiring endocytosis and without interfering with membrane integrity. It seems that they are transported across the membrane to the back, but their mechanism is unknown.

  Delivery of polynucleotides to cells involves the formation of pores in the membrane or disrupts endosomal or lysosomal vesicles, thereby releasing the contents of the vesicles into the cytoplasm, or plasma membranes or internal small cells. It is mediated by membrane active polymers that disrupt or destabilize the cell membrane (such as endosomes or lysosomes).

The amphiphilic membrane active polyamine copolymer of the present invention is the product of the copolymerization of two or more monomer species. In one embodiment, the amphiphilic membrane active heteropolymer of the present invention has the following general structure:
-(A) _a- (B) _b-
Where A contains primary or secondary amine functional side groups and B contains hydrophobic side groups, and a and b are integers> 0. The polymer may be random, block, or alternating. Incorporation of additional monomers is allowed.

  An “endosome-soluble polymer” is an endosome or compound of a compound that is normally cell membrane impermeable, such as a polynucleotide, such as in response to an endosome-specific environmental factor, such as the presence of a lytic enzyme, causing disruption or lysis of the endosome A polymer that can provide release from membrane-encapsulated vesicles inside cells, such as lysosomes. Endosome-soluble polymers cause a shift in their physicochemical properties in the endosome. This shift can be a change in the solubility of the polymer or the ability to interact with other compounds or membranes as a result of the shift in charge, hydrophobicity, or hydrophilicity. The reversibly masked membrane active polyamines of the present invention are believed to be endosomal soluble polymers.

  “Melittin” is a small amphiphilic membrane active peptide found in natural bee venom. Melittin can be isolated from biomaterials or can be a synthetic product. Synthetic polymers are formulated or manufactured by “human” chemical processes and are not produced by natural biological processes. As used herein, melittin includes, for example, the natural bee venom peptides of the melittin family that can be found in the following species of venom: Apis mellifera, Apis cerana, a species of cross-beetle (Vespula maculifrons), a kind of wasp (Vespa magnifica), a kind of wasp (Vespa velutina nigrithorax), a kind of wasp (Polistes sp. HQL-2001), a bee (Apis florae), a bee (Apis dorsta), Apis cerana cerana), Polistes hebraeus. As used herein, melittin includes synthetic peptides having the same or similar amino acid sequence as a natural melittin peptide. Specifically, melittin amino acid sequences include those shown in Table 1. Synthetic melittin peptides can include natural L-form amino acids or enantiomeric D-form amino acids (inverso). However, melittin peptides should basically contain either all L-type or all D-type amino acids, but may have opposite stereogenic amino acids attached to either the amino or carboxy terminus. The melittin amino acid sequence can also be reversed (reverso). Level somelitin can have L-type amino acids or D-type amino acids (retroinverso). Two melittin peptides can also be covalently linked to produce a melittin dimer. Melittin may have modifying groups other than masking agents attached to either the amino terminus or carboxy terminus that enhance tissue targeting or promote in vivo circulation.

  A linkage or “linker” is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. . For example, the linkage can connect the masking agent or polynucleotide to the polymer. Unstable linkages include unstable bonds. The linkage may optionally include a spacer that extends the distance between the two joined atoms. The spacer may further add flexibility and / or length to the connection. Spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl groups; each of these includes one or more heteroatoms, It can include rings, amino acids, nucleotides, and sugars. Spacer groups are well known in the art and the foregoing 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 can be selectively broken down or cleaved under conditions that do not break or break other covalent bonds in the same molecule. More specifically, labile bonds are less stable (thermodynamic) or more rapidly broken (power) than other non-labile covalent bonds in the same molecule under appropriate conditions. (Scientifically) a covalent bond. Breaking unstable bonds within a molecule can cause the formation of two molecules. For those skilled in the art, bond breakage or instability is generally discussed by bond break half-life (t _1/2 ) (the time it takes for half of the bond to break). Thus, labile bonds include bonds that can be selectively cleaved more rapidly than other bonds in the molecule.

  As used herein, a “physiologically labile bond” is a labile bond that is cleavable under conditions normally encountered in a mammalian body or under conditions similar to those encountered. A physiologically labile linking group is selected to undergo a chemical transformation (eg, cleavage) when under certain physiological conditions.

  As used herein, a cytophysiologically unstable bond is an unstable bond that is cleavable under mammalian intracellular conditions. Mammalian intracellular conditions include chemical conditions such as pH, temperature, oxidation or reduction conditions or substances, and salt concentrations similar to those found or encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally present in mammalian cells, such as proteolytic or hydrolytic enzymes. Cell physiologically unstable bonds may be cleaved in response to administration of a pharmaceutically acceptable exogenous substance.

  RNAi interference targeting group conjugate: The targeting group may be linked to the 3 ′ or 5 ′ end of the RNAi polynucleotide. For siRNA polynucleotides, the targeting moiety may be linked to either the sense strand or the antisense strand, but the sense strand is preferred.

  In one embodiment, the targeting group consists of a hydrophobic group. More specifically, the targeting group consists of a hydrophobic group having at least 20 carbon atoms. A hydrophobic group used as a polynucleotide targeting moiety is referred to herein as a hydrophobic targeting moiety. Suitable exemplary hydrophobic groups may be selected from the group comprising cholesterol, dicholesterol, tocopherol, ditocopherol, didecyl, didodecyl, dioctadecyl, didodecyl, dioctadecyl, isoprenoids, and cholamide. Hydrophobic groups having 6 or fewer carbon atoms are not effective as polynucleotide targeting moieties, but hydrophobic groups having 8 to 18 carbon atoms increase the size of the hydrophobic group (ie, the number of carbon atoms Incremental polynucleotide delivery with increasing) is provided. Attachment of the hydrophobic targeting group to the RNAi polynucleotide does not provide efficient functional in vivo delivery of the RNAi polynucleotide without co-administration of the delivery polymer. Although siRNA-cholesterol conjugates have been reported by other researchers to deliver siRNA (siRNA-cholesterol) to hepatocytes in vivo, high levels of siRNA are required and delivered without any additional delivery vehicle The effect is bad. When combined with the delivery polymers described herein, the delivery of polynucleotides is greatly improved. By providing siRNA-cholesterol with the delivery polymer of the present invention, the effectiveness of siRNA-cholesterol is increased by about 100 times.

  Hydrophobic groups useful as polynucleotide targeting moieties may be selected from the group consisting of alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of which is linear, It may be branched or cyclic, cholesterol, cholesterol derivatives, sterols, steroids, and steroid derivatives. The hydrophobic targeting group is preferably a hydrocarbon containing only carbon and hydrogen atoms. However, substituted or heteroatoms that maintain hydrophobicity, such as fluorine, are acceptable. The hydrophobic targeting group may be attached to the 3 ′ or 5 ′ end of the RNAi polynucleotide using methods known in the art. For RNAi polynucleotides having two strands, such as siRNA, the hydrophobic group may be attached to either strand.

  In another embodiment, the targeting group comprises a galactose cluster (galactose cluster targeting moiety). As used herein, a galactose cluster includes molecules having 2 to 4 terminal galactose derivatives. The term galactose derivative as used herein includes both galactose and derivatives of galactose that have an affinity for ASGPr that is equal to or greater than galactose. A terminal galactose derivative is attached to the molecule through its C-1 carbon. Preferred galactose clusters have three terminal galactosamines or galactosamine derivatives, each with affinity for the asialoglycoprotein receptor. A more preferred galactose cluster has three terminal N-acetylgalactosamines. Other terms common in the art include tri-branched galactose, trivalent galactose and galactose trimer. Tribranched galactose derivative clusters bind to ASGPr with greater affinity than bibranched or single chain galactose derivative structures (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol Chem., 257, 939-945). Multivalent is required to achieve nM affinity. The binding of one galactose derivative with affinity for the asialoglycoprotein receptor does not allow functional delivery of RNAi polynucleotides to hepatocytes in vivo when co-administered with a delivery polymer.

The galactose cluster comprises 2 to 4, preferably 3 galactose derivatives each linked to a central branch point. A galactose derivative is attached to the central branch point through the C-1 carbon of the sugar. The galactose derivative is preferably linked to the branch point via a linker or spacer. A preferred spacer is a soft hydrophilic spacer (US Pat. No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 1538-1546). A preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG ₃ spacer. A branch point is any small molecule that allows the binding of three galactose derivatives and further allows the branch point to bind to an RNAi polynucleotide. An exemplary branch point group is dilysine. The dilysine molecule contains three amine groups through which three galactose derivatives are attached, and carboxyl reactive groups through which dilysine can be attached to RNAi polynucleotides. The attachment of branch points to RNAi polynucleotides can occur through linkers or spacers. A preferred spacer is a soft hydrophilic spacer. A preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG ₃ spacer (3 ethylene units). The galactose cluster may be attached to the 3 ′ or 5 ′ end of the RNAi polynucleotide using methods known in the art. For RNAi polynucleotides having two strands, such as siRNA, the galactose cluster may bind to either strand. Suitable galactose clusters are described in US Patent Publication No. 20110207799.

  The term “polynucleotide” or the term nucleic acid or polynucleic acid is a term of the art that means a polymer comprising at least two nucleotides. A nucleotide is a monomeric unit of a polynucleotide polymer. Polynucleotides having less than 120 monomer units are often referred to as 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 in a cell-free system and contains the same or similar bases as the natural ribose or deoxyribose-phosphate backbone, but may contain a different type of backbone. Polynucleotides can be synthesized using any technique known in the art. Polynucleotide backbones known in the art include PNA (peptide nucleic acids), phosphorothioates, phosphorodiamidates, morpholinos, and other variants of the phosphate backbone of natural nucleic acids. Bases include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs. Synthetic derivatives of purines and pyrimidines include modifications that place new reactive groups on nucleotides, including but not limited to amines, alcohols, thiols, carboxylates, and alkyl halides. However, it is not limited to them. The term base includes any known base analog of DNA and RNA. A polynucleotide may comprise ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination. A polynucleotide may be polymerized in vitro, may be recombinant, and includes a chimeric sequence or a derivative of these groups. The polynucleotide may include an end cap moiety at the 5 ′ end, 3 ′ end, or both 5 ′ and 3 ′ ends. The cap moiety can be, but is not limited to, a reverse deoxyabasic moiety, a reverse deoxythymidine moiety, a thymidine moiety, or a 3 ′ glyceryl modification.

  “RNA interference (RNAi) polynucleotides” are through the interaction of mammalian cell RNA interference pathway mechanisms to degrade or inhibit transgene messenger RNA (mRNA) transcripts in a sequence-specific manner. A molecule that can induce RNA interference. The two main RNAi polynucleotides are small (or short) interfering RNA (siRNA) and microRNA (miRNA). The RNAi polynucleotide can be selected from the group comprising siRNA, microRNA, double stranded RNA (dsRNA), short hairpin RNA (shRNA), and an expression cassette encoding RNA that can induce RNA interference. The siRNA typically contains 15-50 base pairs, preferably 21-25 base pairs, and is the same (fully complementary) or nearly the same (partial) as the coding sequence in the target gene or RNA expressed in the cell (Complementary to) a double-stranded structure having a nucleotide sequence. The siRNA may have a dinucleotide 3 ′ overhang. The siRNA may consist of two annealed polynucleotides or one polynucleotide that forms a hairpin structure. The siRNA molecules of the present invention include a sense region and an antisense region. In one embodiment, the conjugate siRNA is constructed from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siRNA molecule and the second fragment is the nucleotide sequence of the sense region of the siRNA molecule. including. In another embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. MicroRNAs (miRNAs) are small non-coding RNA gene products about 22 nucleotides in length that induce destruction of their mRNA targets or translational repression. When the complementarity between the miRNA and the target mRNA is partial, translation of the target mRNA is suppressed. If the complementarity is high, the target mRNA is cleaved. For miRNAs, the complex binds to a target site that is typically located in the 3 ′ UTR of an mRNA that typically shares only partial homology with the miRNA. The “seed region” —a sequence of approximately seven consecutive nucleotides at the 5 ′ end of the miRNA that forms a complete base pair with its target—plays an important role in miRNA specificity. Binding of the RISC / miRNA complex to mRNA can result in either repression of protein translation or cleavage and degradation of the mRNA. Recent data show that mRNA cleavage occurs preferentially when there is complete homology over the entire length of the miRNA and its target, instead of showing complete base pairing only in the seed region (Pillai et al. al. 2007).

  RNAi polynucleotide expression cassettes can be transcribed intracellularly to produce small hairpin RNAs that can function as siRNAs, separate sense and antisense strand linear siRNAs, or miRNAs. The RNA polymerase III transcribed DNA comprises a promoter selected from the list comprising U6 promoter, H1 promoter, and tRNA promoter. RNA polymerase II promoters include U1, U2, U4, and U5 promoters, snRNA promoters, microRNA promoters, and mRNA promoters.

  A list of known miRNA sequences can be found in databases maintained by research organizations such as the 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 also detailed in the relevant literature. RNAi molecules are readily designed and produced by techniques known in the art. In addition, there are computer tools that increase the chances of finding effective and specific sequence motifs (Pei et al. 2006, Reynolds et al. 2004, Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004 Heale et al. 2005, Chalk et al. 2004, Amarzguioui et al. 2004).

  The polynucleotides of the present invention can be chemically modified. Non-limiting examples of such chemical modifications include phosphorothioate internucleotide linkages, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 2′-deoxy ribonucleotides, “universal Includes “base” nucleotides, 5-C-methyl nucleotides, and reverse deoxyabasic residue incorporation. These chemical modifications have been shown to preserve polynucleotide activity in cells when used in various polynucleotide constructs, while at the same time increasing the serum stability of these compounds. Chemically modified siRNAs can also minimize the possibility of activating interferon activity in humans.

  In one embodiment, a chemically modified RNAi polynucleotide of the invention comprises a duplex having two strands, one or both of which may be chemically modified, wherein each strand is about 19 to about 29 nucleotides. In one embodiment, an RNAi polynucleotide of the invention comprises one or more modified nucleotides, but retains the ability to mediate RNAi within a cell or reconstituted in vitro system. RNAi polynucleotides can be modified, where chemical modifications are made to one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides Including. The RNAi polynucleotides of the invention can include modified nucleotides as a percentage of the total number of nucleotides present in the RNAi polynucleotide. Thus, RNAi polynucleotides of the invention generally have from about 5 to about 100% of the nucleotide positions (eg, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the nucleotide positions). %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) modified nucleotides. The actual percentage of modified nucleotides present in a given RNAi polynucleotide depends on the total number of nucleotides present in the RNAi polynucleotide. If the RNAi polynucleotide is single stranded, the percent modification may be based on the total number of nucleotides present in the single stranded RNAi polynucleotide. Similarly, if the RNAi polynucleotide is double stranded, the percent modification may be based on the total number of nucleotides present in the sense strand, antisense strand, or both sense and antisense strands. In addition, the actual percentage of modified nucleotides present in a given RNAi polynucleotide can also depend on the total number of purine and pyrimidine nucleotides present in the RNAi polynucleotide. For example, here all pyrimidine nucleotides and / or all purine nucleotides present in the RNAi polynucleotide are modified.

  RNAi polypeptides regulate the expression of RNA encoded by a gene. Since multiple genes can share some sequence homology with each other, RNAi polynucleotides can be designed to target classes of genes with sufficient sequence homology. Thus, an RNAi polynucleotide can comprise a sequence having complementarity to a sequence that is shared between different gene targets or that is unique to a particular gene target. Thus, RNAi polynucleotides target conserved regions of RNA sequences that have homology between several genes, thereby enabling several genes within the gene family (eg, different gene isoforms, splice variants, mutants Gene, etc.) can be designed to target. In another embodiment, RNAi polynucleotides can be designed to target sequences that are unique to a particular RNA sequence of one gene.

  The term “complementarity” refers to the ability of a polynucleotide to form a hydrogen bond with another polynucleotide sequence, either by traditional Watson-Crick or other non-traditional types. With respect to the polynucleotide molecules of the present invention, the free energy of binding of the polynucleotide molecule to its target (effector binding site) or complementary sequence may advance the associated function of the polynucleotide, eg, enzymatic mRNA cleavage or translation inhibition. Enough. Determination of the binding free energy of nucleic acid molecules is well known in the art (Frier et al. 1986, Turner et al. 1987). The percent complementarity is the percentage of bases (eg, out of 10) of the first polynucleotide molecule that can form hydrogen bonds (eg, Watson-Crick base pairing) with the second polynucleotide sequence. 5, 6, 7, 8, 9, 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementarity). Full complementarity means that all bases in the adjacent strands of the polynucleotide sequence hydrogen bond with the same number of adjacent bases in the second polynucleotide sequence.

  Inhibiting, down-regulating, or knocking down gene expression is the expression of a gene as assessed by the level of RNA transcribed from the gene, or the level of a polypeptide, protein or protein subunit translated from RNA. Is reduced from that observed in the absence of the blocking polynucleotide conjugates of the present invention. Inhibition, downregulation, or knockdown of gene expression by a polynucleotide delivered by a composition of the invention is preferably a control inactive nucleic acid, a nucleic acid containing a disrupted sequence or inactivation mismatch, or a masked polymer Lower than the level observed in the absence of polynucleotide binding to

  It was found that siRNA stabilization against degradation by endosomal / lysosomal localized nucleases such as DNase II strongly improves target knockdown. Such stabilization can directly affect the amount of siRNA released into the protoplasm where there is a cellular RNAi mechanism. Only the siRNA part available in the protoplasm will induce the RNAi effect.

  In addition to poor pharmacokinetic properties, siRNAs are sensitive to nucleases in the living environment when administered as such in the circulation without a protective delivery vehicle. Thus, many siRNAs are rapidly degraded either extracellularly in tissues and bloodstream, or after intracellular uptake (endosomes). Nuclease cleavage is by nucleotides lacking a 2′-OH group, such as 2′-deoxy, 2′-O-methyl (2′-OMe) or 2′-deoxy-2′-fluoro (2′-F) nucleotides, And can be inhibited by a polynucleotide 5'-terminal non-nucleotide moiety such as, for example, cholesterol, aminoalkyl linker or phosphothioate at the first internucleotide linkage. Preferably, the RNAi polynucleotide does not have any 2'-OH nucleotides in the strand, starts with a 5'-terminal 2'-OMe nucleotide and is linked to the second nucleotide by a phosphorothioate (PTO) linkage .

siRNA can be significantly stabilized when using the following design, wherein the oligonucleotide is modified pattern: 5 _{'- (Z3) n a -3} (w) - (Z1) - - (Z2)' Provided with an antisense strand having and a sense strand having a modification pattern 5 ′-(Z3) n _s -3 ′, wherein
w is independently 5′-phosphate or 5′-thiophosphate or H;
Z1 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 _a is 8 to 23 and n _s is 8 to 25.

In one preferred embodiment, the oligonucleotide comprises an antisense strand having _a modification pattern: 5 ′-(w)-(Z1)-(Z2)-(Z3) na-3 ′ and a modification pattern 5 ′-(Z3) n _s -3 'is provided with a sense strand having, wherein Z1 is 2'-fluoro - modified nucleoside or 2-deoxy - a nucleoside, and all remaining substituents and variables n _a and n _s above meanings Have

In one preferred embodiment, the oligonucleotide comprises an antisense strand having _a modification pattern: 5 ′-(w)-(Z1)-(Z2)-(Z3) na-3 ′ and a modification pattern 5 ′-(Z3) n provided with a sense strand having _s- 3 ', wherein Z3 is a 2'-O-methyl modified nucleoside, 2'-fluoro-modified nucleoside or 2-deoxy-nucleoside, and all remaining substituents, as well as variables n _a and n _s have the aforementioned meanings.

In one preferred embodiment, the oligonucleotide comprises an antisense strand having _a modification pattern: 5 ′-(w)-(Z1)-(Z2)-(Z3) na-3 ′ and a modification pattern 5 ′-(Z3) n provided with a sense strand having _s- 3 ′, wherein Z1 is a 2′-fluoro-modified nucleoside or 2-deoxy-nucleoside, and Z3 is a 2′-O-methyl modified nucleoside, 2′-fluoro-modified nucleoside or 2-deoxy - a nucleoside, and the remaining substituents all, and variables n _a and n _s have the abovementioned meaning.

  Nucleosides in the nucleic acid sequence of oligonucleotides having a novel modification pattern can be linked by either 5′-3 ′ phosphodiester or 5′-3 ′ phosphorothioate.

  As used herein, an “antisense” strand is a siRNA strand that is complementary to a target mRNA and that will bind to the mRNA once it has been unwound. The sense strand of the siRNA containing the novel modification pattern is complementary to the antisense strand.

  As a general rule, the nuclease cleavage site between an RNAi polynucleotide and the targeting moiety or delivery polymer to which it is covalently linked comprises at least one 2'-OH nucleotide in either the sense strand or the antisense strand It can be introduced by 3'- or 5'-overhangs. The final active siRNA species is generated by intracellular nuclease treatment. It is also possible to use a defined cleavage site given by a 2′-OH nucleotide in the base pair region. This is either using at least one 2'-OH nucleotide complementary to the opposite strand, or either a hairpin / bulge containing at least one mismatched 2'-OH nucleotide or at least one 2'-OH nucleotide. Can be done by introduction.

Linking a polynucleotide to a delivery polymer In one embodiment, an RNAi polynucleotide is linked to a delivery polymer via a physiologically labile bond or linker. Physiologically labile linkers are selected to undergo chemical transformations (eg, cleavage) when under certain physiological conditions (eg, disulfide bonds that are cleaved in the reducing environment of the cytoplasm). Release of the polynucleotide from the polymer by breakage of a physiologically unstable linkage facilitates the interaction of the polynucleotide with the appropriate cellular components for activity.

  A polynucleotide-polymer conjugate is formed by covalent attachment of a polynucleotide to a polymer. The polymer is polymerized or modified so that it contains a reactive group A. A polynucleotide is also polymerized or modified such that it contains a reactive group B. The reactive groups A and B are selected using methods known in the art so that they can be linked via a reversible covalent bond.

  Binding of the polynucleotide to the polymer can be performed in the presence of excess polymer. Since polynucleotides and polymers can be of opposite charge during conjugation, the presence of excess polymer can reduce or eliminate conjugate aggregation. Alternatively, an excess carrier polymer such as a polycation can be used. After the excess polymer is removed from the bound polymer, the conjugate can be administered to the animal or cell culture. Alternatively, excess polymer can be co-administered with the conjugate to the animal or cell culture.

In Vivo Administration In pharmacology and toxicology, the route of administration is the way the drug, liquid, poison, or other substance comes into contact with the body. In general, methods of administering drugs and nucleic acids to treat mammals are well known in the art and can be applied to administration of the compositions of the present invention. The compounds of the present invention can be administered via any suitable route, most preferably parenterally, in a formulation tailored to that route. Thus, the compounds of the invention can be administered by injection, eg, intravenous, intramuscular, intradermal, subcutaneous, or intraperitoneal. Accordingly, the present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

  Parenteral routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumoral, intraperitoneal, subarachnoid, using syringes and needles or catheters, Subdural, epidural and intralymphatic injections are included. In the present specification, the term “intravascular” means the inside of a tubular structure called a blood vessel that is connected to a tissue or organ in the body. Within the lumen of the tubular structure, body fluid flows to or from the body part. Examples of body fluids include blood, cerebrospinal fluid (CSF), lymph fluid, or bile. Examples of blood vessels include arteries, arterioles, capillaries, venules, sinusoid vessels, veins, lymphatic vessels, bile ducts, and ducts of salivary or other exocrine glands. Intravascular routes include delivery through blood vessels such as arteries or veins. The blood circulatory system provides systemic diffusion of the drug.

  The described composition is injected in a pharmaceutically acceptable carrier solution. Pharmaceutically acceptable means properties and / or substances that are acceptable to mammals from a pharmacological / toxicological point of view. The term pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and typically do not cause allergic or other adverse or toxic reactions when administered to a mammal. To do. Preferably, the term pharmaceutically acceptable as used herein is approved by federal or state government regulatory agencies for use in animals, particularly humans, or is recognized by the United States Pharmacopeia or other generally accepted. Means that it is listed in the designated pharmacopoeia.

  These carriers may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by both the sterilization procedures described above and the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may be desirable to include isotonic agents such as sugars, sodium chloride in the composition. In addition, prolonged absorption of the injectable form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

  In one embodiment, the RNAi polynucleotide-targeting group conjugate is co-administered with the delivery polymer of the invention. Co-administration means that the RNAi polynucleotide conjugate and delivery polymer are administered to the mammal such that both are present in the mammal simultaneously. The RNAi polynucleotide-targeting group conjugate and delivery polymer may be administered simultaneously, or they may be administered sequentially. These may be mixed prior to administration for simultaneous administration. For sequential administration, either the RNAi polynucleotide-targeting moiety conjugate or the delivery polymer may be administered first.

Therapeutic effect
RNAi polynucleotides may be delivered for research purposes or to produce changes that are therapeutic in cells. In vivo delivery of RNAi polynucleotides is useful for research reagents and for various therapeutic, diagnostic, target validation, genome discovery, genetic engineering, and pharmacogenomic applications. The inventors have disclosed RNAi polynucleotide delivery that results in inhibition of endogenous gene expression in hepatocytes. The level of reporter (marker) gene expression measured after delivery of the polynucleotide indicates a reasonable expectation that gene expression after other polynucleotide delivery will be at a similar level. The level of treatment deemed beneficial by those skilled in the art varies from disease to disease. For example, hemophilia A and B are caused by deletion of X-linked factor VIII and factor IX, respectively. These clinical courses are greatly influenced by the percentage of normal serum levels of factor VIII or factor IX: <2%, severe; 2-5%, moderate; and 5-30% mild. Thus, an increase in the normal level of circulating factors from 1% to 2% in severe patients can be considered beneficial. Levels higher than 6% prevent spontaneous bleeding but not secondary to surgery or injury. Similarly, gene inhibition need not be 100% to provide a therapeutic benefit. Those skilled in gene therapy will reasonably predict beneficial levels of disease-specific gene expression based on sufficient levels of marker gene results. In the case of hemophilia, if the marker gene is expressed to produce a protein at a level comparable to 2% by volume of the normal level of factor VIII, the gene encoding factor VIII will be expressed at a similar level. I can reasonably anticipate deafness. Thus, reporter or marker genes generally serve as useful representatives of intracellular protein expression.

  The liver is the most important target tissue for gene therapy given its central role in its metabolism (eg, lipoprotein metabolism in various hypercholesterolemias) and secretion of circulating proteins (eg, coagulation factors in hemophilia) One of them. In addition, acquired disorders such as chronic hepatitis and cirrhosis are common and may be treated by polynucleotide-based liver therapy. Some diseases or conditions that affect or are affected by the liver may be treatable through knockdown (inhibition) of gene expression in the liver. Such liver diseases and conditions include liver cancer (including hepatocellular carcinoma, HCC), viral infection (including hepatitis), metabolic disorders (including hyperlipidemia and diabetes), fibrosis, and acute liver A list containing injuries can be selected.

  The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is effective to achieve the desired therapeutic response for the particular patient, composition, and mode of administration without becoming toxic to the patient. The amount of active ingredient can be varied to obtain the desired amount. The dose level chosen will depend on the activity of the particular composition of the invention used, the route of administration, the time of administration, the rate of elimination of the particular compound being used, the duration of treatment, and other factors used in combination with the particular composition used. Depends on various pharmacokinetic factors, including the drugs, compounds and / or materials of the patient, age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field It will be.

  The amount (dose) of the delivery polymer, RNAi polynucleotide-targeting group conjugate or delivery polymer-RNAi polynucleotide conjugate to be administered can be determined empirically. The inventors have shown effective knockdown of gene expression using 0.1-10 mg siRNA per kg animal weight and 1.5-60 mg delivery polymer per kg animal weight. Preferred amounts in mice are 0.25-2.5 mg / kg siRNA-conjugate and 10-40 mg / kg delivery polymer. More preferably, about 1.5-20 mg / kg of delivery polymer is administered. Since RNAi polynucleotide-conjugates are typically not toxic at high doses, the amount is easily increased.

  As used herein, in vivo means something that occurs inside an organism, more specifically, living whole cells, such as whole living tissue, mammals, etc., against partially or dead things A process that takes place in or on an organism (animal).

  As used herein, “pharmaceutical composition” includes a conjugate of the invention, a pharmaceutical carrier or diluent, and any other media or substances required for formulation.

  “Pharmaceutical carrier” as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).

Example 1. Synthesis of a protease (peptidase) cleavable masking agent
All reactions were carried out under anhydrous conditions using fresh anhydrous solvent, except for the coupling of amino acids in NaHCO ₃ aqueous solution and silyl group deprotection. Column purification was performed on silica gel using the specified eluent. Mass spectra (MS) were obtained using electrospray ionization.

RはASGPrリガンド（保護または未保護）またはPEGを含み、かつ
A¹およびA²はアミノ酸（保護または未保護のいずれか）である。 In the preparation of active p-nitrophenyl-p-acylamide benzyl carbonate derivatives of NAG and PEG (NAG-L-AA-PABC-PNP and PEG-AA-PABC-PNP), the inventors included PEG or NAG, respectively. The amino terminus of the dipeptide-p-acylaminobenzyl alcohol precursor was acylated using the NHS ester of the derivative. In the following steps, the benzylic hydroxyl group was converted to p-nitrophenyl carbonate, followed by removal of the protecting group from the amino acid and NAG moieties. In some applications, protecting groups prior to polymer modification when paranitrophenol (PNP) -carbonate is used to modify certain polymers.

R contains an ASGPr ligand (protected or unprotected) or PEG, and
A ¹ and A ² are amino acids (either protected or unprotected).

The synthesis begins with the preparation of the HA ¹ A ² -PABA (Table 1) derivative. These adducts were obtained using the synthetic scheme described by Dubowchik et al. (2002) with some modifications. Fmoc-protected amino acid, Fmoc-A ¹ -OH, is converted to N-hydroxycuccinimide ester, Fmoc-A ¹ -NHS in a reaction with dicyclohexylcarbodiimide (DCC) and N-hydroxycuccinimide (NHS). It was activated by conversion. These reactive NHS esters were coupled with protected amino acid A ^{2 in the} presence of an aqueous NaHCO ₃ solution added to keep the amino group reactive. In the preparation of 1e and 1f (Table 1), commercially available pentafluorophenyl ester (OPfp) was used for coupling instead of NHS ester.

条件：（i）N-ヒドロキシスクシンイミド（NHS）、N-N'-ジシクロヘキシルカルボジイミド（DCC）、0〜20℃。 Synthesis of Fmoc dipeptide 1a-h
a) NHS ester of AA was prepared from each amino acid using NHS and DCC and used without further purification.

Conditions: (i) N-hydroxysuccinimide (NHS), N-N′-dicyclohexylcarbodiimide (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). Stir for 1 min and then at 20 ° C. for 16 h. Solid dicyclohexylurea (DCU) was removed by filtration and the solvent was removed under reduced pressure.

  For Fmoc-Asn (DMCP) -NHS, DCC (148 mg, 0.72 mmol) of Fmoc-Asn (DMCP) -OH (298 mg, 0.68 mmol) and NHS (83 mg, 0.72 mmol) in DCM (13 mL). Added to ice-cold solution, stirred for 30 minutes, then stirred at 20 ° C. for 16 hours. Solid DCU was filtered off and the solvent was removed under reduced pressure.

  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 a DCC solution in THF (5 mL) ( 650 mg, 3.15 mmol). The cooling bath was removed in 30 minutes and the reaction mixture was stirred at 20 ° C. for 10 hours. Solid DCU was removed by filtration, washed with THF, and the solvent was removed on a rotary evaporator. The product was weighed and dissolved in DME to make a 0.2 mM solution.

  For Fmoc-Glu (O-2PhiPr) -NHS, DCC (217 mg, 1.05 mmol) was added to Fmoc-Glu (O-2PhiPr) -OH (487 mg, 1 mmol) and NHS (127 mg, 1.1 mmol) in THF (5 mL). ) And was stirred for 15 minutes, and then stirred at 20 ° C. for 10 hours. Post-treatment was performed as described for Fmoc-Gly-NHS.

  For Fmoc-Phe-NHS, add DCC (1.181 g, 5.72 mmol) 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). For 30 minutes and then at 20 ° C. for 10 hours. Solid DCU was filtered off and the solvent was removed under reduced pressure.

  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) for 30 minutes. Stir then stirred at 20 ° C. for 16 h. Solid DCU was filtered off and the solvent was removed under reduced pressure.

条件：（i）ジメチルホルムアミド（DMF）中のトリエチルアミン（Et₃N）。 b) Amino acids, H-Asn (DMCP) -OH and H-Lys (MMT) -OH were prepared from available Fmoc protected derivatives.

Conditions: (i) Triethylamine (Et ₃ N) in dimethylformamide (DMF).

H-Asn (DMCP) -OH
Fmoc-Asn (DMCP) -OH (576 mg, 1.32 mmol) was stirred with Et ₃ N (3.7 mL, 26.4 mmol) in DMF (9 mL) for 15 hours. All volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The residue was triturated 3 times with ether (30 mL) and dried under reduced pressure. 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 with Et ₃ N (32 mL, 30 eq, 229.4 mmol) in DMF (100 mL) for 10 hours. All volatiles were removed on a rotary evaporator at 40 ° C / oil pump vacuum. The residue was triturated twice with ether and dried under reduced pressure. Yield 3.1 g (97%). MS (negative mode): 455, 453.3 [M + Cl] ⁻ ; 417.8 [M−1] ⁻ .

A¹＝Gly、Glu(2PhiPr)、Asn(DMCP)、Phe、Ala、Val。
A²＝Gly、Lys(MMT)、Cit、Asn(DMCP)、Lys(CH₃)₂。
条件：（i）H-A₂-OH、NaHCO₃、ジメトキシエタン（DME）、テトラヒドロフラン（THF）およびH₂Oの混合物。（ii）H-A₂-OH、NaHCO₃、DME/THF/H₂O。（iii）H-Cit-OH、NaHCO₃、H₂O中のTHF。 c) Synthesis of Fmoc-A ₁ A ₂ -OH

A ¹ = Gly, Glu (2PhiPr), Asn (DMCP), Phe, Ala, Val.
A ² = Gly, Lys (MMT), Cit, Asn (DMCP), Lys (CH ₃ ) ₂ .
Conditions: (i) A mixture of HA ₂ —OH, NaHCO ₃ , dimethoxyethane (DME), tetrahydrofuran (THF) and H ₂ O. _{(Ii) HA 2 -OH, NaHCO} 3, DME / THF / H 2 O. (Iii) THF in H-Cit-OH, NaHCO ₃ , H ₂ O.

For Fmoc-GlyGly-OH 1a, glycine (75 mg, 1 mmol) and NaHCO ₃ (100 mg, 1.2 mmol) were dissolved in H ₂ O (10 mL) and dimethoxyethane (DME) (5 mL). Fmoc-Gly-NHS solution (5 ml, 1 mmol) in DME was added. THF (2.5 mL) was added and the mixture was sonicated to homogeneity and stirred for 20 hours. All volatiles were removed on a rotary evaporator and the residue was treated with EtOAc and 5% KHCO ₃ solution in H ₂ O. The product was extracted 4 times with EtOAc, washed with brine at pH = 3, dried (Na ₂ SO ₄ ), concentrated and dried under reduced pressure. Yield 321 mg (90%). MS: 775.0 [2M + 2Na] ^< +>; 377.4 [M + Na] ^< +>; 355.1 [M + 1] ^< +>.

For Fmoc-Glu (O-2PhiPr) Gly-OH 1b, glycine (75 mg, 1 mmol) and NaHCO ₃ (84 mg, 1 mmol) into a mixture of H ₂ O (2 mL), THF (4 mL) and DME (5 mL). Dissolved. Fmoc-Glu (O-2PhiPr) -NHS solution (5 mL, 1 mmol) in DME was added and stirred for 10 hours. All volatiles were removed on a rotary evaporator and 20 mL of 0.1 M MES buffer (pH = 5) was added followed by EtOAc (25 mL). The reaction mixture was stirred on ice and acidified with a 5% solution of KHSO ₄ to pH = 5. The product was extracted four times with EtOAc, washed with brine at pH = 5, dried (Na ₂ SO ₄ ), concentrated and dried under reduced pressure. Yield 528 mg (96%). MS: 567 [M + Na] ⁺ ; 562 [M + NH ₄ ] ⁺ ; 545.0 [M + 1] ⁺ ; 427.1 [M-2PhiPr] ⁺ .

Fmoc-Asn (DMCP) Gly-OH 1c was prepared from Fmoc-Asn (DMCP) -NHS and H-Gly-OH as described above for 1b. Yield 96%. MS: 987.4 [2M + 1] ⁺ ; 516.3 [M + Na] ⁺ ; 494.4 [M + 1] ⁺ ; 412.2 [M-DMCP + 1] ⁺ .

Fmoc-PheLys (MMT) -OH 1d was prepared from Fmoc-Phe-NHS and H-Lys (MMT) -OH as described above for 1b. Yield 94%. MS: 788.5 [M + 1] ⁺ , 273.1 [M-MMT + 1] ⁺ .

For Fmoc-PheCit-OH 1e:
i) Fmoc-Phe-NHS (4.96 g, 10.26 mmol) in DME (40 mL) was mixed with L-citrulline (1.80 g, 10.26 mmol) and NaHCO _{3 in} a mixture of H ₂ O (40 mL) and THF (20 mL). 0.86 g, 10.26 mmol). The reaction mixture was stirred for 15 hours. Residual DCC from activation was filtered and the organic solvent was removed on a rotary evaporator. To the residue was added H ₂ O (100 mL) and iPrOH (10 mL). The suspension was acidified with 5% KHSO ₄ to pH = 3, the product was extracted with EtOAc: iPrOH = 9: 1 solution (3 ×, 500 mL), and a mixture of brine: iPrOH = 9: 1 (2 × , 50 mL), dried (Na ₂ SO ₄ ), filtered and concentrated, and dried with an oil pump. Trituration with ether gave pure product 1e. Yield 3.84 g (68%). MS: 545.6 [M + Na] ⁺ ; 528.5 [MH ₂ O] ⁺ ; 306.3 [M-Fmoc + H ₂ O] ⁺ .

ii) A solution of Fmoc-Phe-OPfp (553 mg, 1 mmol) in THF (5 mL) was added H-Cit-OH (184 mg, 1.05 mmol) and NaHCO ₃ (88.2 mg, 1.05 mmol) in H ₂ O (2.6 mL). ). THF (2 mL) was added to make the solution homogeneous and stirred for 10 hours. The THF was removed on a rotary evaporator and the residue was diluted with H ₂ O (10 mL) and iPrOH (1 mL) and acidified with 3% HCl to pH = 1. The product was extracted 5 times with EtOAc: iPrOH = 9: 1 solution, washed with a mixture of brine: iPrOH = 9: 1, dried (Na ₂ SO ₄ ) and concentrated under reduced pressure. Trituration with ether gave 313 mg of pure product 1e (57%).

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

Crude Fmoc-ValCit-OH 1 g was prepared from Fmoc-Val-NHS and H-Cit-OH as described above for 1b. Final purification was performed by trituration with ether. Total yield 76%. MS: 1060.3 [2M + 3Na] ⁺ ; 1015.7 [2M + Na] ⁺ ; 519.7 [M + Na] ⁺ ; 497.9 [M + 1] ⁺ .

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

条件：（i）PABA、EEDQ、THF Coupling with p-aminobenzyl alcohol, preparation of Fmoc-AA-PABA and Fmoc-A-PABA 2a-m Products 1a-h were converted to p-aminobenzyl alcohol (PABA) and 2-ethoxy-1-ethoxycarbonyl- Coupling in the presence of 1,2-dihydroquinoline (EEDQ) produced 2a-h. Four representative compounds 3j-l in which only one amino acid is bound to PABA were also prepared.

Conditions: (i) PABA, EEDQ, THF

For Fmoc-GlyGly-PABA 2a, a solution of 1a (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 hours. did. All volatiles were removed on a rotary evaporator, the residue was triturated with Et ₂ O, the product was filtered and dried under reduced pressure. Yield 348 mg (84%).

  For Fmoc-Glu (O-2PhiPr) Gly-PABA 2b, a solution of 1b (524 mg, 0.96 mmol) and PABA (142 mg, 1.55 mmol) in DCM (10 mL) with EEDQ (357 mg, 1.44 mmol) for 10 hours. Stir. Post-treatment was performed as described above for 2a. Yield 462 mg (74%).

Fmoc-Asn (DMCP) Gly-PABA 2c was prepared as described above for 2a. Yield 64%. MS: 621.5 [M + 22] ⁺ ; 599.3 [M + 1] ⁺ .

  Fmoc-PheLys (MMT) -PABA 2d was prepared as described above for 2b. Yield 70%.

For Fmoc-PheCit-PABA 2e, a solution of 1e (5.98 g, 10.97 mmol) and PABA (2.70 g, 21.95 mmol) in DCM (150 mL) and MeOH (50 mL) with EEDQ (5.43 g, 21.95 mmol). Treated and stirred for 15 hours. Post-treatment was performed as described above for 2a. Yield 6.14 g (86%). MS: 650.7 [M + 1] ⁺ ; 527.3 [M-PABA + 1] ⁺ .

For Fmoc-AlaCit-PABA 2f, a solution of 1f (2.89 g, 6.17 mmol) and PABA (1.52 g, 12.34 mmol) in DCM (45 mL) and MeOH (15 mL) with EEDQ (3.05 g, 12.34 mmol). Treated and stirred for 15 hours. Post-treatment was performed as described above for 2a. Yield 4.56g (74%). MS (ES, negative mode): 307.4 [M-263.6-1] ⁻ ; 349.9 [M-Fmoc-1] ⁻ ; 610, 608.4 [M + HCl-1] ⁻ .

  Fmoc-ValCit-PABA 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 + 1] ⁺ ; 531.4 [M-DMCP + 1] ⁺ ; 408.2 [M-205 + 1] ⁺ .

For Fmoc-Lys (CH ₃ ) ₂ -PABA 2i, Fmoc-Lys (CH ₃ ) ₂ -OH HCl salt (433 mg, 1 mmol) and PABA (246 mg, 2 mmol) in DCM (10 mL) and MeOH (1.5 mL) And cooled to 5 ° C. and EEDQ (495 mg, 2 mmol) was added. The cooling bath was removed and the mixture was stirred at room temperature for 10 hours. All volatiles were removed on a rotary evaporator, the residue was triturated with Et ₂ O and the crude product was filtered. This was redissolved in a mixture of DCM (2 mL) and MeOH (1 mL) and precipitated again by dropwise addition into Et ₂ O (40 mL). The product was filtered and dried under reduced pressure. 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 hours. All volatiles were removed on a rotary evaporator, the residue was dissolved in Et ₂ O (40 mL), cooled on dry ice for 2 hours, and the solid was separated by centrifugation. The resulting crude material was purified by column with an eluent gradient of MeOH (1-2%) in CHCl ₃ . Yield 444 mg (97%). MS: 459.4 [M + 1] ⁺ .

Fmoc-Asn (DMCP) -PABA 2k was prepared as described for 2j. After removal of DCM, in a workup instead of column purification, the residue was triturated with Et ₂ O, cooled to 0 ° C. and the crude product was filtered. This process was repeated once more followed by drying under reduced pressure. Yield 77%. MS: 542.5 [M + 1] ⁺ .

For 2 liters of Fmoc-Cit-PABA, 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 EEDQ (430 mg, 1.74 mmol). ) And stirred for 15 hours. The solid product was triturated three times with ether and the product was filtered and dried. Yield 288 mg (67%). MS: 502.3 [M + 1] ⁺ ; 485.5 [MH ₂ O + 1] ⁺ ; 263 [M-Fmoc-H ₂ O + 1] ⁺ ; 179.0 [M-306 + 1] ⁺ ; 120.2 [M-365.3 + 1] ⁺ .

条件：（i）DMF中、トリエチルアミン（Et₃N）、10時間。（ii）Fmoc-Phe-NHS、ジソプロピルエチルアミン（DIEA）、DMF。 The product 2m was prepared using a different scheme: coupling of H-Lys (CH ₃ ) ₂ -PABA derivative 3 with Fmoc-Phe-NHS.

Conditions: (i) DMF, triethylamine (Et ₃ N), 10 hours. (Ii) Fmoc-Phe-NHS, disopropylethylamine (DIEA), DMF.

For Fmoc-PheLys (CH ₃ ) -PABA 2m, Fmoc-Lys (CH ₃ ) ₂ -PABA (2i) (448 mg, 0.83 mmol) with Et ₃ N (3.5 mL) in DMF (11 mL) for 10 hours Fmoc deprotection was achieved by stirring. All volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum to give 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 hours. Solvent and DIEA were removed on a rotary evaporator under reduced pressure at 40 ° C./oil pump to give crude 2 m which was used without further purification. MS: 549.4 [M + l] ^< +>.

条件：（i）DMF中、Et₃N、10時間。 Preparation of H-AA-PABA 3a-h, m and HA-PABA 3j-l

Conditions: (i) in DMF, Et ₃ N, 10 hours.

Fmoc derivatives 2a-h, j-l were treated with Et ₃ N in DMF as described above for 3i, followed by concentration and drying under reduced pressure. The crude product was dissolved in DMF to make a 0.1M solution and used without further purification.

(Table 1) Intermediates of H-AA-PABA (1-3)

R¹、R²およびR³が保護基であり、Lがガラクトサミン部分（NAG）とジペプチド（AA）との間の連結である、NAG(R¹,R²,R³)-L-AA-PABC-PNPの調製を、NAG-L-CO₂H酸6、10a、b、13および17の調製から開始し、これらをNHSエステルに変換した後、H-AA-PABA 2をアシル化するために用いた。塩基感受性ポリマーのために設計したカルボナート21a〜fにおいて、保護基をポリマー修飾の間に除去しなければならなかった。このために、10a、b、13および17の調製において、GAL部分のAc-保護基を不安定なトリエチルシリル（TES）およびtert-ブチルジメチルシリル（TBDMS）基で置き換えた。これらの基は、塩基感受性PNP-カルボナート部分を損なうことなく、H₂O中70％トリフルオロ酢酸（TFA）溶液を0℃で用いて除去することができる。 Preparation of protease-cleavable NAG masking reagent
Preparation of ^{NAG (R 1, R 2,} R 3) -L-AA-PABC-PNP ( Tables 2 and 3)

N ¹ (R ¹ , R ² , R ³ ) -L-AA-, wherein R ¹ , R ² and R ³ are protecting groups, and L is a linkage between a galactosamine moiety (NAG) and a dipeptide (AA) Preparation of PABC-PNP starts with the preparation of NAG-L-CO ₂ H acids 6, 10a, b, 13 and 17 to convert them to NHS esters and then acylate H-AA-PABA 2 Used for. In carbonates 21a-f designed for base sensitive polymers, protecting groups had to be removed during polymer modification. For this purpose, in the preparation of 10a, b, 13 and 17, the Ac-protecting group of the GAL moiety was replaced with labile triethylsilyl (TES) and tert-butyldimethylsilyl (TBDMS) groups. These groups can be removed using a 70% trifluoroacetic acid (TFA) solution in H ₂ O at 0 ° C. without compromising the base sensitive PNP-carbonate moiety.

条件：（i）H₂、Pd/C（10％）、MeOH、CHCl₃、(20％）、（ii）無水コハク酸、Et₃N、CDM、1時間。 a) In the preparation of NAG-L ¹ -CO ₂ H 6 where R ¹ = R ² = R ³ = OAc, Z-protected NAG-4 acetate 4 [3-5] is Z-deprotected (H ₂ , Pd / C (10%), MeOH, CHCl ₃ (20%) gave NAG-amine 5, which was then acylated with succinic anhydride (succinic anhydride, Et ₃ N, DCM, 1 hour).

_{Conditions: (i) H 2, Pd} / C (10%), MeOH, CHCl 3, (20%), (ii) succinic anhydride, Et ₃ N, CDM, 1 hour.

For the preparation of NAG-amine 5: 5, a solution of NAG 4 (6.74 g, 11.85 mmol) in MeOH (144 mL) and CHCl ₃ (36 mL) in the presence of 10% Pd / C (674 mg) at 1 atm. Hydrogenated for 10 hours. The catalyst was filtered off through celite and the product was concentrated and dried under reduced pressure. Yield 5.04g (98%).

For the preparation of NAG-L ¹ -OH 6: 6, succinic anhydride (966 mg, 9.65 mmol) in DCM (30 mL) was added to NAG-amine 5 (4 g, 9.15 mmol) in DCM (50 mL), Subsequently Et ₃ N (1.964 mL, 14 mmol) was added. After 1 hour, the reaction mixture was concentrated and dried under reduced pressure. The product was purified by column with an eluent gradient of MeOH (5-7%) in CHCl ₃ . Yield 3.1 g (63%). MS: 535.3 [M + 1] ⁺ ; 330.3 [deglycosylated product] ⁺ .

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

条件：（i）Et₃N、MeOH、H₂O（5：7：6）10時間。（ii）DMF中TBDMSCl（1当量）、イミダゾール、1時間の後、TESCl（3当量）、10時間。（iii）DMF中TBDMSCl（3当量）、イミダゾール、10時間。 i) NAG-L ¹ -OH 10a, b. 10a: R ¹ = OTES and OTBDMS, R ² = OH, R ³ = OTES; 10b: R ¹ = R ³ = OTBDMS, R ² = OH
Preparation of NAG 8a, b

_{Conditions: (i) Et 3 N,} MeOH, H 2 O (5: 7: 6) 10 hours. (Ii) TBDMSCl (1 eq) in DMF, imidazole, 1 h, then TESCl (3 eq), 10 h. (Iii) TBDMSCl (3 equivalents) in DMF, imidazole, 10 hours.

条件：（i）H₂、Pd/C（10％）、THF。（ii）無水コハク酸、Et₃N、DCM、1時間。
7の調製のために、NAG 4（2g、3.52mmol）をMeOH（10mL）、H₂O（32mL）、およびEt₃N（25mL）の溶液中で10時間撹拌することによりO-脱アセチル化した。すべての揮発性物質をロータリーエバポレーターにより40℃で除去し、残渣を反応混合物からトルエンの蒸発を2回行って乾燥した。生成物7を次の段階で直接用いた。MS: 544.3 [M+Et₃N+1]⁺; 443.7 [M+1]⁺; 204 [脱グリコシル化生成物]⁺。 NAG derivative 7
Preparation of 10a and b

_{Conditions: (i) H 2, Pd} / C (10%), THF. (Ii) Succinic anhydride, Et ₃ N, DCM, 1 hour.
For the preparation of 7, NAG 4 (2 g, 3.52 mmol) was O-deacetylated by stirring in a solution of MeOH (10 mL), H ₂ O (32 mL), and Et ₃ N (25 mL) for 10 hours. did. 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 was used directly in the next step. MS: 544.3 [M + Et ₃ N + 1] ⁺ ; 443.7 [M + 1] ⁺ ; 204 [deglycosylated product] ⁺ .

For the preparation of 8a, product 7 (1.76 mmol) in DMF (15 mL) was treated with imidazole (718 mg, 10.54 mmol) and TBDMSCl (265 mg, 1.76 mmol), stirred for 2 hours and the reaction mixture was cooled to 0 ° C. Until cooled. TESCl (531 mg, 3.52 mmol) was added, stirred for 10 hours, concentrated and dried under reduced pressure. The residue was dissolved in a mixture of EtOAc (110 mL) and H ₂ O (30 mL). The organic layer was separated, cooled to 5 ° C., washed with citric acid (5%), H ₂ O, NaHCO ₃ and dried (Na ₂ SO ₄ ). The crude product was passed through the column with 2% MeOH in CHCl ₃ to obtain a mixture of TBDMS and TES di-Si-protected NAG derivative 8a. Yield 575 mg (49%). MS: 672.0 [M + 1] ⁺ ; 432.5 [deglycosylated 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 TBDMSCl (1.061 g, 7 mmol) for 10 hours. The reaction mixture was processed as described above for the preparation of 8a. Yield 767 mg (65%) after column purification. M: 672.0 [M + 1] ⁺ ; 432.7 [deglycosylated product] ⁺ .

  Product 10a was prepared as a mixture of TBDMS and TES di-Si-protected NAG derivative according to 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% (150 mg) Pc / C at 1 atmosphere for 10 hours. The catalyst was filtered off through celite and the product 9b was concentrated and dried under reduced pressure.

Dissolve NAG-amine 9b in DCM (12 mL) without further purification and add a solution of succinic anhydride (140 mg, 1.40 mmol) in DCM (7 mL) followed by Et ₃ N (0.236 mL, 1.676 mmol). And stirred for 2 hours. The solvent was removed on a rotary evaporator and the product was purified by column with an eluent of 1% AcOH, 10% MeOH in CHCl ₃ . Yield 614 mg (72%).

^{ii) NAG-L 2 -OH 13} . R ¹ = R ³ = OTBDMS, R ² = OH. NAG derivative with longer PEG spacer. For analogs with longer PEG spacers, precursor 5 was first acetyl deprotected to give 11 (Et ₃ N, MeOH, H ₂ O (5: 7: 6) 10 hours). 11 was then acylated with bis-dPEG ₅ half benzyl half NHS ester (Quanta product catalog number 10237) to give benzyl ester 12 (NHS-PEG ₅ —CO ₂ Bn, Et ₃ N, DCM). 12 is then bis-silylated with TBDMSCl (TBDMSCl in DMF (3 eq), imidazole, 10 h) and debenzylated by hydrogenation (H ₂ , Pd / C (10%), THF) to give acid 13. It was.

条件：（i）Et₃N、MeOH、H₂O（5：7：6）10時間。（ii）NHS-PEG₅-CO₂Bn、Et₃N、DCM。 Preparation of NAG-derivative 12

_{Conditions: (i) Et 3 N,} MeOH, H 2 O (5: 7: 6) 10 hours. (Ii) NHS-PEG ₅ —CO ₂ Bn, Et ₃ N, DCM.

条件：DMF中TBDMSCl（3当量）、イミダゾール、10時間。（vi）H₂、Pd/C（10％）、THF。 Preparation of NAG-L ² -OH 13

Conditions: TBDMSCl (3 eq) in DMF, imidazole, 10 hours. _{(Vi) H 2, Pd /} C (10%), THF.

NAG-PFG ₈ -SA benzyl ester 12. For the preparation of NAG-amine 11, NAG-amine 5 (0.381 mmol) was O-deacetylated as described for precursor 7 (procedure for 8a, b). Product 11 was dried by evaporating toluene twice on a rotary evaporator and dissolved in DMF (25 mL). To the reaction mixture was added bis-dPEG ₅ half benzyl half NHS ester (200 mg, 0.381 mmol), followed by DIEA (0.079 mL, 0.457 mmol), stirred for 8 hours and concentrated on a rotary evaporator at 40 ° C./oil pump vacuum. did. The crude product 12 was used in the next step without further purification. MS: 719.4 [M + 1] ⁺ ; 516.4 [deglycosylated product] ⁺ .

For the preparation of NAG-L ² -OBn, the dried product 12 was dissolved in DMF (5 mL) and treated with TBDMSCl (230 mg, 1.524 mmol) followed by imidazole (156 mg, 2.29 mmol). The reaction mixture was stirred for 10 h, all volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum, the residue was dissolved in EtOAc (85 mL) and washed with HCl (1%), H ₂ O. . The aqueous phases were combined and back extracted with EtOAc. The combined organic solutions were dried (Na ₂ SO ₄ ), concentrated and purified by column with an eluent gradient of MeOH (3-6%) in CHCl ₃ . Yield 291 mg (80%) of benzyl ester. MS: 965.3 [M + NH ₄ ] ⁺ ; 948.0 [M + 1] ⁺ ; 516.4 [deglycosylated product] ⁺ .

For the preparation of NAG-L ² —OH 13 the ester NAG-L ² —OBn was hydrogenated as described for 9b (procedure for 10b). Yield 98%, MS: 858.0 [M + 1] ⁺ ; 426.1 [deglycosylated product] ⁺ . The product was used without further purification.

iii) NAG-L ³ —OH 17 R ¹ = R ³ = OTBDMS, R ² = OH. NAG derivative with longer PEG spacer.
17 was prepared by glycosylation of pentaacetate ester 14 [3-5] with PEG ₄ mono-tBu ester to give 15 (TMSOTf, DCE / HO-PEG ₄ -CO ₂ tBu, SnCl ₄ , DCM). Hydrolysis of 15 (HCO ₂ H, 10 h) gave acid 16, which was then O-deacetylated (Et ₃ N, MeOH, H ₂ O (5: 7: 6) 10 h) and TBDMSCl Treatment (TBDMSCl in DMF (3 eq), imidazole, 10 h) gave bis-silylated NAG-acid 17.

条件：（i）トリフルオロメタンスルホン酸トリメチルシリル（TMSOTf）、ジクロロエタン（DCE）（ii）12-ヒドロキシ-4,7,10-トリオキサドデカン酸t-ブチル（HO-PEG₄-CO₂tBu）、SnCl₄、ジクロロメタン（DCM）。 Preparation of ester NAG (OAc) ₃ -L ³ -O-tBu 15

Conditions: (i) Trimethylsilyl trifluoromethanesulfonate (TMSOTf), dichloroethane (DCE) (ii) t-butyl 12-hydroxy-4,7,10-trioxadodecanoate (HO-PEG ₄ -CO ₂ tBu), SnCl ₄ , dichloromethane (DCM).

条件：（i）HCO₂H、10時間。（ii）Et₃N、MeOH、H₂O（5：7：6）10時間。（iii）DMF中TBDMSCl（3当量）、イミダゾール、10時間。

Conditions: (i) HCO ₂ H, 10 hours. _{(Ii) Et 3 N, MeOH} , H 2 O (5: 7: 6) 10 hours. (Iii) TBDMSCl (3 equivalents) in DMF, imidazole, 10 hours.

For the ester NAG (OAc) ₃ -L ³ -O-tBu 15, the pentaacetyl derivative 14 of galactosamine (10 g, 25.64 mmol) was dried by evaporation twice from 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 hours. The reaction mixture was cooled to 0 ° C., quenched with TEA (2.6 mL), diluted with CHCl ₃ (300 mL) and washed twice with NaHCO ₃ solution and brine. The separated organic solution was treated with MgSO ₄ , concentrated and dried under reduced pressure. The crude oxazoline derivative was used without further purification. Yield 8.14g (96%). MS: 368.1 [M + K] ⁺ ; 352.2 [M + Na] ⁺ ; 330.2 [M + 1] ⁺ .

Stir a mixture of oxazoline derivative (5.28 g, 16 mmol), t-butyl 12-hydroxy-4,7,10-trioxadodecanoate (5.12 g, 18.4 mmol) and CaSO ₄ (20 g) in DCM (270 mL). To this was added SnCl ₄ (0.84 mL, 0.84 mmol) dropwise. The solution was stirred for 16 hours, filtered, diluted with CHCl ₃ (250 mL), and washed twice with NaHCO ₃ solution and brine. The product was dried over MgSO ₄ and concentrated. The crude product was purified by column with an eluent gradient of MeOH (0-7%) in ethyl acetate. Yield 4.83 g (50%). MS: 630.8 [M + Na] ⁺ ; 625.5 [M + NH ₄ ] ⁺ ; 608.4 [M + 1] ⁺ ; 552.6 [Mt-Bu + 1] ⁺ ; 330.2 [deglycosylated product] ⁺ .

For NAG (OAc) ₃ -L ³ -OH 16 tert-butyl ester 15 (1.99 g, 3.27 mmol) was stirred in neat formic acid (54 mL) for 16 h and all volatiles were removed under reduced pressure. Removal was followed by evaporation of toluene three times. The product was dried with a vacuum oil pump for 2 hours and used without further purification. Yield 1.77 g (98%). MS: 330.2 [deglycosylated product] ⁺ ; 590.4 [M + K] ⁺ ; 574.6 [M + Na] ⁺ ; 569.6 [M + NH ₄ ] ⁺ ; 552.6 [M + 1] ⁺ .

For NAG (R ¹ , R ² , R ³ ) -L ³ -OH 17 (R ¹ = R ³ = OTBDMS, R ² = OH), product 16 was O-deacetylated and as in the preparation of 8b. Treated with TBDMSCl and purified by column with eluent of 3% MeOH, 0.5% AcOH in CHCl ₃ . Yield 18%. MS: 1228.7 [M + 1] ⁺ , 796.7 [deglycosylated product] ⁺ .

条件：（i）NHS、DCC、DXCM、10時間。 All five resulting acids 6, 10a, b, 13, 17 were converted to NHS esters 18a-e in a reaction with NHS and DCC (NHS, DCC, DCM, 10 hours).

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

  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, 1.061 mmol) in DCM (15 mL) was treated with DCC (219 mg, 1.061 mmol) and on ice for 30 min and 20 Stir at 0 ° C. for 8 hours. The reaction mixture was cooled to 0 ° C., DCU was filtered off, the residue was concentrated and dried under reduced pressure. The crude product was dissolved in DMF to make a 0.05M solution and used without further purification.

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

条件：（i）DIEA、DMF、5〜10時間。（ii）（PNP)₂CO、ジオキサンまたはDCM、25〜60℃、16〜48時間。 c) Generation of 20a-l, 3a-h was acylated with NHS ester of hydroxyl protected NAG derivatives 18a-e (DIEA, DMF, 5-10 hours) to give 19a-l. The product 19a-l is then treated with 5 equivalents of bis (p-nitrophenyl) carbonate ((PNP) ₂ CO) ((PNP) ₂ CO, dioxane or DCM, 40-50 ° C., 15-24 hours) O-acetyl protected PNP carbonate derivatives 20a-l were obtained. Products 20a-e were used directly for peptide modification. The protecting groups 2PhiPr, DMCP, MMT from the acetyl group and amino acids were removed after modification during sequential treatment of DPC with TFA and Et ₃ N.

Conditions: (i) DIEA, DMF, 5-10 hours. (Ii) (PNP) ₂ CO, dioxane or DCM, 25-60 ° C., 16-48 hours.

NAG (R ¹ R ² R ³ ) -L-AA-PABA 19a ~ l
For product 19a (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA = GlyGly), NAG-NHS ester 18a solution (0.05M) (0.282 mmol) in DMF was added to 3a in DMF. Treated with 0.1 M solution (0.282 mmol) and DIEA (59 μL, 0.338 mmol). In 3 hours, all volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum, triturated with Et ₂ O, and column eluent: EtOAc: CHCl ₃ : MeOH = 8: 7: 5 Purified with an 8: 7: 6 gradient. Yield 114 mg (53%). MS: 754.4 [M + 1] ⁺ .

The product 19b (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA = Glu (2PhiPr) Gly) was prepared as described for 19a and the column was eluted with EtOAc: CHCl ₃ : MeOH = Purified at 8: 7: 3. Yield 64%. MS: 944.5 [M + 1] ⁺ .

For product 19c (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA = Asn (DMCP) Gly) 3c (0.43 mmol) and DIEA (83 μL, 0.476) in DMF (2.15 mL) To a solution of 18) (0.43 mmol) in DMF (2.15 mL) was added. The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum. The crude product was triturated with Et ₂ O and purified by column with eluent CHCl ₃ : acetone: MeOH (5: 5: 1). Yield 242 mg (62%). MS: 915.3 [M + Na] ⁺ ; 910.6 [M + NH ₄ ] ⁺ ; 893.6 [M + 1] ⁺ .

The product 19d (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA = PheLys (MMT)) was prepared as described for 19a, and the column was run with MeOH (5-6% in CHCl ₃ ) Eluent gradient. Yield 56%. MS: 1187.9 [M + 1] ⁺ .

For product 19e (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA = PheCit) into a solution of 3e (0.57 mmol) and DIEA (119 μL, 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 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum. The crude product was precipitated from CHCl ₃ : MeOH (5 mL) into Et ₂ O (45 mL) and used without further purification. Yield 392 mg (73%). MS: 966.8 [M + Na] ⁺ ; 944.7 [M + 1] ⁺ ; 926.8 [MH ₂ O] ⁺ ; 821.5 [M-PABA + 1] ⁺ ; 615.6 [M-NAcGal + 1] ⁺ ; 492.3 [M- PABA-NAcGal + 1] ⁺ .

The product 19f (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ¹ , AA = AlaCit) was prepared as described for 19e and used without further purification. Yield 50%. MS: 993.2 [M + Na] ⁺ ; 971.0 [M + 1] ⁺ ; 539.6 [deglycosylated product] ⁺ .

The product 19g (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ¹ , AA = ValCit) was prepared as described for 19f and used in the next step without further purification. Yield 67%: 998.9 [M + 1] ⁺ .

The product 19h (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ¹ , AA = Glu (2PhiPr) Gly) was prepared as described for 19a and the column was NH ₄ OH 3 in DCM. And eluent of 7.5% and MeOH 7.5% solution. Yield 15%. MS: 1047.2 [M + 1] ⁺ , 615.7, 432.6 [deglycosylated product] ⁺ .

The product 19i (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ¹ , 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 + 1] ⁺ ; 615.4 [deglycosylated product] ⁺ ; 432.5 [deglycosylated product] ⁺ .

The product 19j (R ¹ = OTBDMS and OTES, R ² = OH, R ³ = OTES, L = L ¹ , AA = PheCit), C-3 and C-6 O-TBDMS and O-TES protected NAG derivatives Prepared as described for the preparation of 19e from 3e and 18b as a mixture and used in the next step without further purification. Yield 76%. MS: 1047.4 [M + 1] ⁺ , 615.8 [deglycosylated product] ⁺ .

The product 19k R ¹ = R ³ = OTBDMS, R ² = OH, L = L ² , AA = PheCit was prepared as described for 19e and used in the next step without further purification. Yield 67%: 1268.2 [M + 1] ⁺ ; 835.9 [deglycosylated product] ⁺ .

Product 19l R ¹ = R ³ = OTBDMS, R ² = OH, L = L ³ , AA = PheCit was prepared as described for 19e and purified by column with eluent of 5% MeOH solution in CHCl ₃ did. Yield 60%. MS: 1064.0 [M + 1] ⁺ ; 632.7 [deglycosylated product] ⁺ .

NAG-AA-PABC-PNP 20a ~ l
For product 20a (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA = GlyGly), 19a (100 mg, 0.132 mmol), (PNP) ₂ CO (202 mg, in dioxane (5 mL) 0.663 mmol) and DIEA (0.07 mL, 0.396 mmol) were stirred in the dark at 40 ° C. for 8 hours. Further (PNP) ₂ CO (121 mg, 0.397 mmol) and DIEA (0.04 mL, 0.226 mmol) were added and stirring was continued at 40 ° C. for a further 8 hours. All volatiles were removed on a rotary evaporator and the product was purified by column with eluent: CHCl ₃ : EtOAc: MeOH = 7: 8: 3. Yield 84 mg (69%).

For product 20b (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA = Glu (2PhiPr) Gly) 19b in DCM (10 mL) (160 mg, 0.169 mmol), (PNP) ₂ A solution of CO (258 mg, 0.847 mmol) and DIEA (0.09 mL, 0.507 mmol) is stirred in the dark for 10 hours, concentrated on a rotary evaporator and the product is passed through the column with eluent: 5-6% MeOH in CHCl ₃ Purified with solution. Yield 174 mg (92%).

For product 20c (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA = Asn (DMCP) Gly), 19c (127 mg, 0.142 mmol), (PNP) _{2 in} DCM (5 mL) A solution of CO (216 mg, 0.710 mmol) and DIEA (74 μL, 0.426 mmol) was stirred in the dark for 16 h, concentrated on a rotary evaporator and the product was passed through the column with eluent: CHCl ₃ : EtOAc: MeOH (7: 2.2: 0.8). Yield 110.6 mg (74%). MS: 1080.9 [M + Na] ^< +>; 1058.7 [M + 1] ^< +>.

The product 20d (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA-PheLys (MMT)) was prepared as described for 20b, and was eluent: CHCl ₃ : EtOAc: MeOH = by column. Purified 9: 7: 1. Yield 76 mg (47%).

For product 20e (R ¹ = R ² = R ³ = OAc, L = L ¹ , AA = PheCit) 19e (164 mg, 0.173 mmol) in dioxane (17 mL), (PNP) ₂ CO (528 mg, 1.73 mmol) and DIEA (182 μL, 1.04 mmol) were 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 successive evaporations of DMF on a rotary evaporator at 40 ° C./oil pump vacuum and the product was passed through the column with eluent CHCl ₃ : EtOAc: MeOH (8: 1.5: 0.5 ) Followed by purification with CHCl ₃ : MeOH (7: 1). Yield 85 mg (44%). MS: 1132.0 [M + Na] ⁺ ; 1110.1 [M + 1] ⁺ ; 780.8 [deglycosylated product] ⁺ .

The product 20f (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ¹ , AA = AlaCit) was prepared as described for 20e and was eluent: CHCl ₃ : EtOAc: MeOH = 9 by column. : 10: 1 purification. Yield 153 mg (36%).

20 g of product (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ¹ , AA = ValCit) was prepared as described for 20e and the column was prepared by eluent CHCl ₃ : EtOAc: MeOH = 16: Purified 3: 1. Yield 44%. MS: 1164.5 [M + 1] ⁺ .

The product 20h (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ¹ , AA = Glu (2PhiPr) Gly) was prepared as described for 20b, and the column was eluted with CHCl ₃ : EtOAc: Purified with MeOH = 8: 7: 1. Yield 77%.

For product 20i (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ¹ , AA = PheCit), 19i (316 mg, 0.297 mmol), (PNP) ₂ CO in dioxane (7 mL) ( A solution of 913 mg, 2.97 mmol) and DIEA (310 μL, 1.78 mmol) was stirred in the dark at 65 ° C. for 40 hours and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by two successive evaporations of DMF on a rotary evaporator at 40 ° C./oil pump vacuum and the product was passed through the column with eluent CHCl ₃ : EtOAc: MeOH (8: 1.5: 0.5 ), Followed by purification with CHCl ₃ : MeOH (92:08). Yield 297 mg (81%). MS: 1246.7 [M + NH ₄ ] ⁺ ; 1228.7 [M + 1] ⁺ ; 797.6 [deglycosylated product] ⁺ ; 432.7 [deglycosylated product] ⁺ .

Product 20j (R ¹ = OTBDMS and OTES, R ₂ = OH, R ³ = OTES, L = L ¹ , AA = PheCit), mixture of C-3 and C-6 O-TBDMS and O-TES protected NAG derivatives The product 20j as was prepared as described for 20e and purified by column with eluent CHCl ₃ : EtOAc: MeOH = 16: 3: 1 followed by 10% MeOH in CHCl ₃ . Yield 50%. MS: 1212.0 [M + 1] ⁺ , 480.0 [deglycosylated product] ⁺ .

The product 20k (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ² , AA = PheCit) was prepared as described for 20e, and the column was MeOH (8-10%) in CHCl ₃ Of eluent gradient. Yield 85%. The product was used directly in the next step.

For the product 20l (R ¹ = R ³ = OTBDMS, R ² = OH, L = L ³ , AA = PheCit) 19 l (316 mg, 297 mmol), (PNP) ₂ CO (912 mg) in dioxane (8 mL) , 3 mmol) and DIEA (0.31 mL, 1.78 mmol) were stirred for 48 hours in the dark at 60 ° C. under Ar atmosphere. All volatiles were removed on a rotary evaporator and the product was purified by column with eluent: CHCl ₃ : EtOAc: MeOH = 16: 3: 1. Yield 297 mg (81%).

条件：TFA/H₂O＝3：1、5℃、2〜3時間 NAG-L-AA-PABC- PNP 21a~f (R 1 = R 2 = R 3 = OH), deprotection of 20f~l
Generation of 21a-f. For modification of the base sensitive polyacrylate, after removing the protecting groups on NAG and dipeptide AA, the 20f ~ l was coupled to the polymer by treating with a mixture of TFA: H ₂ O = 3: 1 (TFA / H ₂ O = 3: 1, 5 ° C., 2-3 hours), NAG-dipeptide masking reagents 21a-f were provided.

Condition: TFA / H ₂ O = 3: 1, 5 ° C., 2-3 hours

For product 21a (AA = AlaCit, L = L ¹ ), compound 20f (150 mg, 0.132 mmol) was stirred in ice-cold TFA: H ₂ O = 3: 1 solution (2 mL) for 4 hours, with stirring Of Et ₂ O (20 mL) was added dropwise. The precipitate was separated and the toluene was evaporated to dryness on a rotary evaporator / 30 ° C. and then dried under reduced pressure. Yield 112 mg (94%). MS: 907.2 [M + 1] ⁺ ; 704.4 [deglycosylated product] ⁺ .

For product 21b (AA = ValCit, L = L ¹ ), compound 20 g (305 mg, 0.26 mmol) was stirred in ice-cold TFA: H ₂ O = 3: 1 solution (5 mL) for 1 hour, with stirring In Et ₂ O (45 mL). The solid product was separated and the toluene was evaporated to dryness on a rotary evaporator / 30 ° C. and then dried under reduced pressure. Yield 193 mg (79%). MS: 935.8 [M + 1] ⁺ , 732.7 [deglycosylated product] ⁺ .

Product 21c (AA = GluGly, L = L ¹ ) was prepared as described for 21b. Yield 55 mg (98%). MS: 865.5 [M + 1] ⁺ , 662.3 [deglycosylated product] ⁺ .

Product 21d (AA = PheCit, L = L ¹ ) was prepared as described for 21b. MS: 983.7 [M + 1] ⁺ , 780.9 [deglycosylated product] ⁺ .

For product 21e (AA = PheCit, L = L ² ), compound 20k was stirred in ice-cold TFA: H ₂ O = 3: 1 solution (5 mL) for 1.5 hours under the conditions described for 21b. 25% yield from 19k. MS: 1203.9 [M + 1] ⁺ , 1001.0 [deglycosylated product] ⁺ .

Product 21f (AA = PheCit, L = L ³ ) was prepared from 20l under the conditions described for 21b using 3 hours of deprotection. Yield 75%. MS: 1203.9 [M + 1] ⁺ , 1001.0 [deglycosylated product] ⁺ .

Table 2 Intermediates NAG-L-AA-PABA (19) and NAG-L-AA_PABC (20)

(Table 3) Final NAG-LA ₁ A ₂ -PABC (20, 21) used for DPC preparation

Preparation of protease-cleavable PEG masking reagent Any H-AA-PABA 3b, e, g, h, j, k-m amino group is acylated with NHS ester of PEG-acid (DIEA, DMF, 5-10 Time), 22a-k. The hydroxyl groups of products 22a-k were then converted to p-nitrophenyl carbonate ((PNP) ₂ CO, dioxane or THF, 40-60 ° C., 10 hours) to give 23a-k. For 23a, d, g, the protecting group is removed from Asn and Glu by aqueous TFA treatment (TFA / H ₂ O = 3: 1, 5 ° C., 2-3 h) to give the desired products 24a-c Obtained. (Can we maintain consistency by converting 23a, d, g to 24a, d, g?) Also, there is consistency between amino acids for each letter between NAG and PEG versions. ?

条件：（i）DIEA、DMF、5〜10時間。 Preparation of PEG _n -AA-PABA 22a-k

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

Product 22a (n = 11, AA = GluGly). A 0.1M solution of 3b in DMF (3.5 mL, 0.35 mmol) was stirred with PEG ₁₁ -NHS ester (240 mg, 0.35 mmol) and DIEA (0.061 mL, 0.35 mmol) for 10 hours. All volatiles were removed on a rotary evaporator at 40 ° C./oil pump and the product was purified by column with eluent: CHCl ₃ : MeOH: AcOH = 38: 2: 1. Yield 274 mg (78%) MS: 1015.6 [M + NH ₄ ] ⁺ , 998.7 [M + 1] ⁺ .

Product 22b (n = 11, AA = PheCit). To a solution of 3e (0.88 mmol) and DIEA (167 μL, 0.96 mmol) in DMF (3 mL) was added a solution of PEG ₁₁ -NHS ester (0.80 mmol) in DMF (3 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum. The crude product was precipitated from CHCl ₃ : MeOH (5 mL) into Et ₂ O (45 mL) and purified by column with an eluent gradient of MeOH (10-16%) in CHCl ₃ . Yield 420 mg (53%). MS: 1015.9 [M + H ₂ O] ⁺ ; 998.8 [M + 1] ⁺ ; 981.1 [MH ₂ O] ⁺ .

Product 22c (n = 11, AA = ValCit). Product 22f was prepared from crude 3 g (300 mg, obtained from 2 g of 0.5 mmol), PEG ₁₁ -NHS ester (298 mg, 0.435 mmol) and DIEA (0.09 mL, 0.522 mmol) 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 mixture (6 mL), sonicated, filtered and precipitated into Et ₂ O (50 mL) . The solid was separated and the procedure was repeated again. Residual solvent was removed under reduced pressure. Yield 283 mg (60%). MS: 951.5 [M + 1] ^< +>.

Product 22d (n = 11, AA = AlaAsn (DMCP)). To a solution of 3h (0.56 mmol) and DIEA (116 μL, 0.67 mmol) in DMF (3 mL) was added a solution of PEG ₁₁ -NHS ester (0.56 mmol) in DMF (3 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum. The residue was dissolved in a CHCl ₃ : MeOH = 1: 1 mixture (5 mL) and precipitated into cooled (0 ° C.) Et ₂ O (45 mL). The solid was purified by column with an eluent gradient of MeOH (3-14%) in DCM. Yield 261 mg (49%). MS: 983.7 [M + Na] ⁺ ; 979.1 [M + NH ₄ ] ⁺ ; 961.8 [M + 1] ⁺ ; 943.9 [MH ₂ O + 1] ⁺ .

Product 22e (n = 11, AA = PheLys (Me ₂ )). Product 22e was prepared as described for 22a. Purification was performed using an HPLC column Nucleodur C-18, 250 × 4.6, eluent ACN-H ₂ O (0.1% TFA), gradient 15-30%. MS: 998.1 [M + 1] ⁺ . The isolated product was desalted with Dowex 1 × 8 resin with eluent H ₂ O. Yield 40%.

Product 22f (n = 11, AA = Leu). Product 22f was prepared as described for 22a and purified by column with eluent: CHCl ₃ : EtOAc: MeOH: AcOH = 9: 7: 2: 0.04. Yield 48%. _{MS: 824.9 [M + NH 4} ] +.

22 g of product (n = 11, AA = Asn (DMCP). Crude 3k (obtained from 419 mg, 0.77 mmol 2k), Peg ₁₁ NHS ester (200 mg, 0.292 mmol) and DIEA (0.06 mL, 0.35 mmol) in DCM. The solvent was removed on a rotary evaporator and the product was purified by column with eluent CHCl ₃ : EtOAc: MeOH: AcOH = 4.5: 3.5: 1: 0.02 Yield 254 mg (37 MS): 891.1 [M + 1] ⁺ .

Product 22h (n = 11 AA = Cit). To a solution of 3 l (0.50 mmol) and DIEA (104 μL, 0.60 mmol) in DMF (2.5 mL) was added a solution of PEG ₁₁ -NHS ester (0.50 mmol) in DMF (2.5 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum. The residue was dissolved in a CHCl ₃ : MeOH = 1: 1 mixture (5 mL) and precipitated into Et ₂ O (45 mL). The precipitation was repeated two more times and the product was used without further purification. Yield 340 mg (80%). _{MS: 869.4 [M + NH 4} ] +; 851.9 [M + 1] +.

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 PEG ₂₃ -NHS ester (0.60 mmol) in DMF (3 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum. The residue was dissolved in a CHCl ₃ : MeOH = 1: 1 mixture (5 mL) and precipitated into Et ₂ O (45 mL). The solid was purified by column with an eluent gradient of MeOH (7-12%) in CHCl ₃ . Yield 487 mg (53%). MS: 1555.2 [M + Na] ⁺ ; 1544.7 [M + NH ₄ ] ⁺ ; 1527.7 [M + 1] ⁺ .

Product 22j (average MW 1000 PEG, AA = PheCit). A mixture of mPEG-1000-alcohol (Fluka) (0.173 g, 0.173 mmol), N, N-disuccinimidyl carbonate (62 mg, 0.242 mmol), and TEA (0.101 mL, 0.726 mmol) in MeCN (1 mL) For 16 hours. All volatiles were removed on a rotary evaporator and the crude residue was dissolved in CHCl ₃ (10 mL). The organic layer was washed with H ₂ O (1 mL, pH = 5) then brine, dried over Na ₂ SO ₄ and concentrated to give PEG-1000-NHS carbonate. The product was stirred for 16 hours with 3e (0.121 mmol) and DIEA (30 μL, 0.173 mmol) in DMF (1 mL), filtered and all volatiles removed by rotary evaporator at 40 ° C./oil pump vacuum. did. The residue was dissolved in a CHCl ₃ : MeOH = 1: 1 mixture (5 mL) and precipitated into Et ₂ O (45 mL). The precipitation was repeated two more times and the product was used without further purification. Yield 134 mg (79%).

Product 22k (n = 23, AA = ValCit). To a solution of 3 g (1.0 mmol) and DIEA (183 μL, 1.04 mmol) in DMF (4 mL) was added a solution of PEG ₂₃ -NHS ester (0.87 mmol) in DMF (4 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ° C./oil pump vacuum. The residue was dissolved in a CHCl ₃ : MeOH = 1: 1 mixture (5 mL) and precipitated into Et ₂ O (45 mL). The precipitation was repeated two more times and the product was used without further purification. Yield 1.0 g (77%). _{MS: 1496.1 [M + NH 4} ] +; 1479.3 [M + 1] +.

条件：（i）（PNP)₂CO、ジオキサンまたはTHF、40〜60℃、10時間。 PEG-AA-PABC-PNP 23a-k

Conditions: (i) (PNP) ₂ CO, dioxane or THF, 40-60 ° C., 10 hours.

For product 23a (n = 11, AA = Glu (2PhiPr) Gly), product 22a (274 mg, 0.274 mmol) in DCM (15 mL) was (PNP) ₂ CO (418 mg, 1.372 mmol) in the dark. And stirred with DIEA (0.143 mL, 0.823 mmol) for 15 h. The solvent was removed on a rotary evaporator and the product was purified by column with an eluent of 4% MeOH, 0.2% AcOH in CHCl ₃ . Yield 260 mg (81%). _{MS: 1180.7 [M + NH 4} ] +.

For product 23b (n = 11, AA = PheCit) of 22b (419 mg, 0.42 mmol), (PNP) ₂ CO (766 mg, 2.52 mmol) and DIEA (263 μL, 1.52 mmol) in dioxane (4 mL). The solution was stirred in the dark at 50 ° C. for 15 hours and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by evaporation of DMF twice on a rotary evaporator under reduced pressure at 40 ° C./oil pump, and the product was passed through the column with eluent CHCl ₃ : EtOAc: MeOH (4.5: 5: 0.5), Subsequently, purification was performed with CHCl ₃ : MeOH (9: 1). Yield 390 mg (80%). _{MS: 1181.2 [M + NH 4} ] +, 1164.2 [M + 1] +.

For product 23c (n = 11, AA = ValCit), 22c (273 mg, 0.287 mmol), (PNP) ₂ CO (874 mg, 2.88 mmol) and DIEA (0.3 mL) in 1,4-dioxane (22 mL) , 1.72 mmol) was stirred in the dark at 50 ° C. for 24 hours. The solvent was removed on a rotary evaporator at 40 ° C./oil pump and the product was purified by column with eluent CHCl ₃ : EtOAc: MeOH = 16: 3: 1 followed by 12-15% MeOH in CHCl ₃ . Yield 163 mg (51%). MS: 1116.0 [M + 1] ⁺ .

Product 23d (n = 11, AA = AlaAsn (DMCP)) was prepared as described in the preparation of 23b. The product was purified by column with the eluent CHCl ₃ : EtOAc: MeOH (9: 2: 1). Yield 77%. _{MS: 1144.0 [M + NH 4} ] +; 1127.3 [M + 1] +.

Product 23e (n = 11, AA = PheLys (Me) ₂ ) was prepared as described for 23a and purified by column with eluent: 10% MeOH in CHCl ₃ , 0.2% AcOH. Yield 63%. MS: 1163.1 [M + 1] ⁺ .

Product 23f (n = 11, AA = Leu) was prepared as described for 23c using only 5 equivalents of (PNP) ₂ CO and 3 equivalents of DIEA and applying heat for 24 hours. The product was purified by column with an eluent gradient of MeOH (7-12%) in CHCl ₃ . Yield 75%. MS: 972 [M + 1] ⁺ .

The product 23g (n = 11, 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 = 11, AA = Cit) 22h (340 mg, 0.40 mmol), (PNP) ₂ CO (608 mg, 2.00 mmol) and DIEA (208 μL, 1.20 mmol) in DCM (4 mL) The solution was stirred in the dark at 30 ° C. for 15 hours and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by evaporating DMF twice on a rotary evaporator under 40 ° C./oil pump vacuum and the product was passed through the column with eluent CHCl ₃ : EtOAc: MeOH (7: 2.5: 0.5), Subsequent purification with a gradient of MeOH (8-14%) in CHCl ₃ . Yield 390 mg (80%). _{MS: 1034.3 [M + NH 4} ] +; 1016.9 [M + 1] +.

The product 23i (n = 23, AA = PheCit) was prepared as described in the preparation of 23b and was columned with eluent CHCl ₃ : EtOAc: MeOH (4.5: 5: 0.5) followed by CHCl ₃ Purified with a gradient of MeOH (6-12%). Yield 86%. _{MS: 1711.4 [M + NH 4} ] +; 1694.4 [M + 1] +.

The product 23j (PEG 1000K AA = PheCit) was prepared as described in the preparation of 23b, and the column was prepared by eluent CHCl ₃ : EtOAc: MeOH (4.5: 5: 0.5) followed by MeOH in CHCl ₃ Purified with a gradient (6-12%). Yield 72%.

Product 23k (n = 23, AA = ValCit) was prepared as described in the preparation of 23b and the product was purified by HPLC. Column: Luna (Phenomenex) 5u, C-8, 100 A. Mobile _{phase: ACN-H 2 O (F} 3 CO 2 H 0.01%), ACN gradient 30-37%, 31 min. Yield: 530 mg (48%). MS: 1666.4 [M + Na] ^< +>; 1644.2 [M + 1] ^< +>.

条件：（i）TFA/H₂O＝3：1、5℃、2〜3時間。 PEG-AA-PABC-PNP 24a-c, AA deprotection

Conditions: (i) TFA / H ₂ O = 3: 1, 5 ° C., 2-3 hours.

Product 24a (n = 11, AA = GluGly). The product 23a (250 mg, 0.215 mmol) was stirred in 3% TFA solution of CHCl ₃ (16 mL) for 35 minutes, concentrated on a rotary evaporator and dried under reduced pressure. Yield 224 mg (100%) (MS: 1062.6 [M + NH ₄ ] ⁺ ; 1045.9 [M + 1] ⁺ .

Product 24b (n = 11, AA = AlaAsn-PABC-PNP). Compound 23d was stirred in a mixture of TFA: DCM (3: 1) for 1.5 hours and all volatiles were removed on a rotary evaporator at 20 ° C. The product was purified by column with an eluent gradient of MeOH (6-12%) in CHCl ₃ . Yield 30%. MS: 1066.7 [M + Na] +, 1062.0 [M + NH 4] +; 1045.2 [M + 1] +.

Product 24c (n = 11, AA = Asn). The reaction flask containing 23 g (160 mg, 0.143 mmol) was cooled to 0 ° C. and a cold mixture of TFA: H ₂ O (9: 1) (12.5 mL) was added. The mixture was stirred for 1.5 hours and diluted with cold H ₂ O (50 mL). Stirring was continued at 20 ° C. for 20 minutes. The precipitate was filtered and washed with H ₂ O. All volatiles were removed on a rotary evaporator at 40 ° C. and the product was purified by column with eluent CHCl ₃ : EtOAc: MeOH: AcOH = 4.5: 3.5: 1.2: 0.02. Yield 43 mg (30%). MS: 974.0 [M + 1] ⁺ .

Table 4 Final PEG-LA ₁ A ₂ -PABC used for DPC preparation

Example 2. Linkage of a protease-cleavable masking agent to an amine-containing polymer-generation of a p-acylamidobenzylcarbamate linkage
A. Modification of melittin with a protease-cleavable masking agent. Amine-reactive p-nitrophenyl carbonate or N-hydroxysuccinimide carbohydrate of 2-6 × mg NAG-containing protease cleavable substrate with 1-10 mg / mL peptide of 1 × mg melittin peptide and 10 × mg HEPES base Masked by addition of a nate derivative. The solution was then incubated at room temperature (RT) for at least 1 hour prior to injection into animals.

R¹はASGPrリガンド（保護または未保護のいずれか）またはPEGを含み、
R²は両親媒性膜活性ポリアミンであり、
AAはジペプチド（保護または未保護のいずれか）であり、かつ
Zはアミン反応性カルボナートである。 B. Modification of polyamine with a protease-cleavable masking agent. Activation of the p-acylamidobenzyl alcohol derivative (amine-reactive) The carbonate is reacted with the amino group of an amphiphilic membrane active polyamine in H ₂ O at pH> 8 to give p-acylamidobenzylcarbamate.

R ¹ contains an ASGPr ligand (either protected or unprotected) or PEG,
R ² is an amphiphilic membrane active polyamine,
AA is a dipeptide (either protected or unprotected), and
Z is an amine reactive carbonate.

  To xmg polymer was added 12 x mg HEPES free base in isotonic glucose. To the buffered polymer solution was added 2 × to 16 × mg 200 mg / ml dipeptide masking agent in DMF. In some applications, the polymer was modified with 2 × mg dipeptide masking agent followed by siRNA binding. The polymer-siRNA conjugate was then further modified with 6 × to 8 × mg dipeptide masking agent.

第VII因子siRNA（霊長類）

ApoB siRNA：

Aha1 siRNA：

Luc siRNA

Eg5-KSP

EGFP

小文字＝2'-O-CH₃置換
s＝ホスホロチオエート連結
ヌクレオチドの後のf＝2'-F置換
ヌクレオチドの前のd＝2'-デオキシ Example 3.siRNA
The siRNA had the following sequence:
Factor VII siRNA

Factor VII siRNA (primate)

ApoB siRNA:

Aha1 siRNA:

Luc siRNA

Eg5-KSP

EGFP

Lower case = 2'-O-CH ₃ substitution
s = d = 2′-deoxy before f = 2′-F substituted nucleotides after phosphorothioate linked nucleotides

  RNA synthesis was performed by conventional phosphoramidite chemistry on a solid phase using AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany) and porous glass (CPG) as a solid support.

Example 4. Administration of RNAi polynucleotides in vivo and delivery to hepatocytes
DPC was prepared as described above. Six to eight week old mice (C57BL / 6 or ICR strain, approximately 18-20 g each) were obtained from Harlan Sprague Dawley (Indianapolis IN). Mice were housed in cages for at least 2 days prior to injection. Feeding was performed as appropriate using Harlan Teklad Rodent Diet (Harlan, Madison WI). DPC was synthesized as described herein. The conjugate solution (0.4 mL) was infused by instillation into the tail vein. The composition was soluble and non-aggregating at physiological conditions. The solution was injected by instillation into the tail vein. Injection into other blood vessels, such as retro-orbital injection, is expected to be equally effective. Wistar Han rats, 175-200 g, were obtained from Charles River (Wilmington, Mass.). Rats were housed in cages for at least 1 week prior to injection. Rat injection volume was typically 1 ml. Unless otherwise stated, serum samples were collected 48 hours after injection and / or liver samples were collected.

Quantification of serum ApoB levels Serum was collected by submaxillary blood collection after mice were fasted for 4 hours (16 hours for rats). For rats, blood was collected from the jugular vein. Serum ApoB protein levels were quantified by standard sandwich ELISA methods. Briefly, polyclonal goat anti-mouse ApoB antibody and rabbit anti-mouse ApoB antibody (Biodesign International) were used as capture and detection antibodies, respectively. Subsequently, HRP-conjugated goat anti-rabbit IgG antibody (Sigma) was applied to bind the ApoB / antibody complex. The absorbance of tetramethylbenzidine (TMB, Sigma) color development was then measured at 450 nm with a Tecan Safire2 (Austria, Europe) microplate reader.

Measurement of plasma factor VII (F7) activity Plasma samples from animals are 0.109 mol of blood (9 volumes) (submandibular for mice or from the jugular vein for rats) according to standard procedures. / L Sodium citrate was prepared by collecting in a micro centrifuge tube containing sodium citrate anticoagulant (1 volume). Plasma F7 activity was measured by the chromogenic method using the BIOPHEN VII kit (Hyphen BioMed / Aniara, Mason, OH) according to the manufacturer's recommendations. Color absorbance was measured at 405 nm with a Tecan Safire2 microplate reader.

Example 5. Delivery of siRNA to hepatocytes in vivo using amphiphilic membrane active polyacrylate polyamine reversibly modified with a dipeptide cleavable masking agent
Succinimidyloxycarbonyl-alpha-methyl-alpha (2-pyridyl-dithio) toluene (SMPT) (Pierce) activated polyacrylate Ant-41658-111 in 100 mM pH 7.5 HEPES buffer Was modified with 0.5% by weight to provide a thiol reactive group for subsequent siRNA attachment. The thiol reactive polymer was then diluted to 5 mg / mL in 60 mg / mL HEPES base. To this solution was added 10 mg / mL of various described enzyme cleavable masking reagents. This amount was the molar ratio of masking agent 2 to polymer amine 1. For polymer modification reactions, the preferred molar ratio of polymeric amine to masking reagent is from 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 conjugate was further modified by adding an N-acetylgalactosamine derivative of maleic anhydride (NAG-CDM; Table 5). NAG-CDM was added to 25 mg / mL and incubated for 30 minutes to 4 hours.

式中、Rはポリマーであり、R1はASGPrリガンド（例えば、N-アセチルガラクトサミン）を含む。表5に示すとおり、第VII因子発現はジペプチド剤でマスクしたDPCで処置した動物では49〜85％低減した。 NAG-CDM was lyophilized from a 0.1% aqueous solution of glacial acetic acid for NAG-CDM polymer modification. The polymer solution was added to the dried NAG-CDM. After complete dissolution of the anhydride, the solution was incubated at room temperature for at least 30 minutes before being administered to the animals. d Reaction of NAG-CDM with the polymer gave:

Where R is a polymer and R1 contains an ASGPr ligand (eg, N-acetylgalactosamine). As shown in Table 5, Factor VII expression was reduced by 49-85% in animals treated with DPC masked with dipeptide agents.

^a動物体重1kgあたりのポリマーまたはsiRNAのmg
^b未処置対照に対して TABLE 5 Factor VII knockdown in vivo in mice treated with PEG-AA-p-nitrophenyl-carbamate + NAG-CDM-DPC

mg of ^a animal body weight per 1kg polymer or siRNA
^b vs untreated control

Example 6. In vivo delivery of siRNA using NAG / PEG-AA-p-nitrophenyl-carbamate poly (acrylate) DPC
A) PEG plus NAG modification
Polyacrylate Ant-41658-111 in 100 mM pH 7.5 HEPES buffer with succinimidyloxycarbonyl-alpha-methyl-alpha (2-pyridyldithio) toluene (SMPT), an activated disulfide reagent from Pierce 0.5 wt% modified. The thiol reactive polymer was diluted to 5 mg / mL in 60 mg / mL HEPES base. To this solution was added 10 mg / mL of various PEG-AA-p-nitrophenyl-carbonate masking reagents. After 1 hour, acetate protected thiol factor VII siRNA was added to the polymer solution in a ratio of polymer to siRNA = 5-10: 1. After overnight incubation, NAG-AA-p-nitrophenyl-carbonate masking reagent was added to 40 mg / mL. After incubation for at least 30 minutes but no more than 4 hours, DPC was injected into the tail vein of 20 g ICR mice. Serum samples were taken 48 hours after injection to determine the level of fVII.

B) Modification with NAG alone
Polyacrylate ANT-41658 111 in succinimidyloxycarbonyl-alpha-methyl-alpha (2-pyridyldithio) toluene (SMPT), an activated disulfide reagent from Pierce, 0.5 in 100 mM pH 7.5 HEPES buffer Modified by weight%. The thiol reactive polymer was diluted to 5 mg / mL in 60 mg / mL HEPES base. Acetate-protected thiol siRNA factor VII was added to the polymer solution in a ratio of polymer to siRNA = 5-10: 1. After overnight incubation, NAG-AA-p-nitrophenyl-carbonate masking reagent was added to 50 mg / mL. After incubation for at least 30 minutes but no more than 4 hours, polymer-bound siRNA was injected into the tail vein of 20 g ICR mice. Serum samples were taken 48 hours after injection to determine the level of fVII.

^a動物体重1kgあたりのポリマーまたはsiRNAのmg
^b未処置対照に対して
^c重量当量 TABLE 6 Factor VII knockdown in vivo in mice treated with PEG / NAG-AA-p-nitrophenyl-carbamate DPC

mg of ^a animal body weight per 1kg polymer or siRNA
^b vs untreated control
^c weight equivalent

Example 7. In vivo delivery of siRNA using NAG / PEG-AA-p-nitrophenyl-carbamate poly (vinyl ether) DPC Amphiphilic membrane active poly (vinyl ether) polyamine DW1360 can cleave 10 × weight equivalent dipeptide The masking agent was modified as described for polyacrylate Ant-41658-111 except that the masking agent retained a protecting group during polymer modification. After polymer modification, the amino acid protecting group was removed by TFA and the NAG acetate protecting group was removed by incubation in the presence of a solution of 30% by volume triethylamine, 50% methanol and 20% water. The acetate deprotection solution was removed by rotary evaporation. The masked polymer was co-injected into mice with cholesterol-ApoB siRNA conjugate.

^a動物体重1kgあたりのポリマーまたはsiRNAのmg
^b未処置対照に対して Table 7. ApoB knockdown in vivo in mice co-injected with NAG-AA-p-nitrophenyl-carbamate poly (vinyl ether) and cholesterol-ApoB siRNA conjugates

mg of ^a animal body weight per 1kg polymer or siRNA
^b vs untreated control

Example 8. Knockdown of endogenous ApoB levels after delivery of ApoB siRNA by melittin delivery peptide in vivo in mice, enzymatically cleavable masking agent An amount of enzymatically cleavable masking of melittin as indicated above Reversibly modified with agents. 200-300 μg of masked melittin was then co-injected with 50-100 μg of ApoB siRNA-cholesterol conjugate. The effect on ApoB levels was determined as described above. Peptidase cleavable dipeptide-amidobenzyl carbamate modified melittin was an effective siRNA delivery peptide. The use of D-type melittin peptides in combination with an enzymatically cleavable masking agent is preferred. Because peptide masking was more stable, more peptides were required for the same level of target gene knockdown, but the therapeutic index did not change or improve (for masking the same peptide with CDM-NAG) Compared to).

^aマスキング反応において用いたメリチンアミンあたりのマスキング剤の量。 Table 8: Normal hepatocytes in mice treated with Factor VII-siRNA cholesterol conjugate and GIL-melittin (type D) (Seq ID 25) reversibly inhibited with the indicated enzymatically cleavable masking agent. Of factor VII activity in food

The amount of masking agent per Merichin'amin used in the ^a masking reaction.

Example 9. Tumor targeting by protease-cleavable DPC
A) Target gene knockdown measurement siRNA specific to Aha1 gene transcript and target gene for all the tests shown below. SiRNA against high sensitivity green fluorescent protein (EGFP) was used as an off-target control. Aha1 siRNA was complementary to the sequence motif of Aha1 that is 100% homologous in both human and mouse genes. Thus, delivery of Aha1 siRNA to either host cells or tumor cells in human xenografts causes mRNA cleavage and degradation. Using different sequence motifs in the mouse and human Aha1 genes, PCR primers were designed that allow quantitative measurement of both human Aha1 and mouse Aha1 mRNA levels in tissue samples containing a mixed population of cell types. At 24, 48 or 72 hours after siRNA delivery, tumors with some healthy mouse liver tissue attached were harvested and processed in Tri-Reagent (Invitrogen) for total RNA isolation. Both human and mouse Aha1 mRNA levels were then measured by qPCR assay using human Cyc-A and mouse β-actin as internal reference genes. Aha1 mRNA levels in animals from mock injected animals or mice injected with off-target control GFP siRNA were considered 100%. Results are expressed as a percentage of Aha1 mRNA levels relative to controls and are shown in the table below.

B) Orthotopic hepatocellular carcinoma (HCC) tumor model mouse
HegG2, Hep3B, or HuH7 cell hepatocellular carcinoma co-transfected with two expression vectors, pMIR85 human placental secreted alkaline phosphatase (SEAP) vector and pMIR3 neomycin / kanamycin resistance gene vector to produce a stable SEAP cell line occured. Cells were cultured in DMEM supplemented with 10% FBS and 300 ug / ml G418, harvested, counted and mixed with Matrigel (BD Biosciences) (50% by volume). Athymic nude mice or Scid beige mice were anesthetized with approximately 3% isoflurane and placed in a supine position. A small 1-2 cm midline abdominal incision was made just below the xiphoid process. Using a damp cotton swab, the left liver lobe was slowly removed from the body. The liver left lobe was slowly retracted and a syringe needle was inserted in the middle of the left lobe. The oblique end of the syringe needle was inserted approximately 0.5 cm just below the liver capsule. 10 μl of a cell / matrigel mixture containing 100,000 cells was injected into the liver using a syringe pump. The needle was inserted into the liver for a while (15-20 seconds) to ensure complete injection. 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 needle from the liver and a cotton swab was placed over the injection site to prevent cell leakage or bleeding. The matrigel / cell mixture formed a visible mass and did not disappear after removal of the needle. Next, the liver lobe was slowly returned to the abdomen, and the abdominal wall was sutured. Serum was collected once a week after tumor implantation and monitored for tumor growth by SEAP assay. In most studies, tumor mice were used 4-5 weeks after transplantation, where tumor measurements were estimated to be about 4-8 mm based on SEAP values.

C) Colorectal metastasis tumor model
HT29 cells were cultured in McCoy's 5a medium supplemented with 10% FBS, harvested, counted and mixed with Matrigel (BD Biosciences) (50% by volume). Athymic nude mice were anesthetized with approximately 3% isoflurane and placed in the sternum position. A small 1-2 cm midline abdominal incision was made just below the xiphoid process. Using a damp cotton swab, the left liver lobe was slowly removed from the body. The liver left lobe was slowly retracted and a syringe needle was inserted in the middle of the left lobe. The oblique end of the syringe needle was inserted approximately 0.5 cm just below the liver capsule. 5 μl of a cell / matrigel mixture containing about 40,000 cells was injected into the liver using a syringe pump. The needle was inserted into the liver for a while (15-20 seconds) to ensure complete injection. The syringe was then removed from the liver and a cotton swab was placed over the injection site to prevent cell leakage or bleeding. The matrigel / cell mixture formed a visible mass and did not disappear after removal of the needle. Next, the liver lobe was slowly returned to the abdomen, and the abdominal wall was sutured. Tumor mice were used 4-5 weeks after transplantation.

Example 10. As knockdown aforementioned target gene expression in vivo after PEG ₂₄ -Val-Cit DPC administration in HepG2-SEAP orthotopic hepatocellular carcinoma (HCC) model, (2011062805) Ant-129-1 polymer DPC was modified (masked) with either an 18 × excess of PEG ₂₄ -Phe-Cit masking agent (or PEG ₂₄ -Val-Cit masking agent) or 7 × PEG ₅₅₀ -CDM. As described above, Aha1-siRNA (RD-09070) or GFP-siRNA (RD-05814; off-target control) was bound to the polymer (4: 1 weight ratio). DPC was not purified by gel filtration prior to delivery and no targeting ligand was added. For each animal, 320 μg of DPC conjugate (polymer weight) in 200 μl of isotonic glucose was administered by tail vein infusion (n = 3 per group). After 24 hours, the animals were given a second injection of 320 μg DPC conjugate (polymer weight) in 200 μl isotonic glucose. Forty-eight hours after the second infusion, serum samples are collected and evaluated for toxicity by measuring liver enzymes (ALT and AST) and blood urea nitrogen (BUN) levels, followed by tissue sampling and qPCR Analysis was carried out.

Using PEG ₂₄ -Val-Cit-Ant-129-1-siRNA DPC to deliver Aha1 siRNA caused a 46% knockdown of the Aha1 gene in human tumor cells (Table 9). In contrast to the human Aha1 knockdown level, mouse Aha1 showed 70% knockdown in response to PEG ₂₄ -Val-Cit-Ant-129-1-siRNA DPC administration (Table 1). Endogenous hepatocyte Aha1 knockdown was reduced compared to a similar DPC made with a disubstituted maleic anhydride masking agent (PEG ₅₅₀ -CDM). As indicated by ALT, AST and BUN levels, PEG ₂₄ -Val-Cit DPC was well tolerated and showed no toxicity (Table).

Table 9: Aha1 knockdown in HepG2 liver tumor model by maleic anhydride modified or peptide cleavable modified Aha1 siRNA DPC

Table 10: Blood chemistry toxicity markers after maleic anhydride modification or peptide cleavable modification Aha1 siRNA DPC administration

Example 11. Bispecific antibody (bsAb) -targeted DPC (2011090701) knockdown of target gene expression As previously described, Ant-129-1 polymer was masked with 5 × Dig-PheCit (Dig-FCit) masking agent. Qualified. The siRNA was then bound to the conjugate. Finally, the Dig-FCit-Ant-129-1-siRNA conjugate was further modified with 8 × (weight) PEG ₁₂ -FCit. Aha1-siRNA (RD-09070) or GFP siRNA (RD-05814) was conjugated at a 4: 1 polymer: siRNA weight ratio. PEG ₁₂ -FCit DPC was purified on a Sephadex G50 spin column to remove unbound reagents.

  Cell-targeting bispecific antibody specific for heparan sulfate proteoglycan glypican-3 (GPC3), a cell surface heparan sulfate proteoglycan known to be highly expressed in HepG2-SEAP cells, and digoxigenin (Dig) (BsAb) was created. As controls, bispecific antibodies specific for the protein CD33 (a marker for bone marrow-derived hematopoietic stem cells) and Dig were generated. CD33 is not expressed by HepG2-SEAP cells. BsAb was complexed with the modified DPC at a weight ratio of 1.25: 1 to provide an estimated 1: 1 molar ratio. The complex was formed in PBS for at least 30 minutes prior to delivery.

  DPC was administered to HepG2-SEAP tumor-bearing mice either with or without bsAb targeting agent. Each animal (n = 3 per group) received a single dose of 250 μg DPC (polymer weight). DPC was injected into the tail vein of mice in 200 μl sterile PBS. Serum and tissue samples were collected 48 hours later and analyzed as described above. As shown in Table 11, a single dose of bsAb targeted DPC (250 [mu] g polymer, 62.5 [mu] g siRNA) caused 21-32% target gene knockdown.

(Table 11) Aha1 knockdown in HepG2 liver tumor model by Aha1 siRNA DPC modified with peptide-cleavable masking agent using bispecific antibody

Example 12. Bispecific Antibody (bsAb) -Target Gene Expression Knockdown by Targeted DPC
DPC was prepared as described above except that a) PEG ₂₄ -FCit was used instead of PEG ₁₂ -FCit and b) Dig was coupled to the polymer using Dig-PEG ₁₂ -NHS. PEG ₂₄ -FCit DPC was less aggregated, smaller in size and more homogeneous than PEG ₁₂ -FCit DPC. In addition to the ligation not being unstable, Dig-PEG ₁₂ -NHS also contained longer PEG. DPC is a complex with bsAb and animals were injected as described above. Serum and tissue collection were performed either 24 or 48 hours after injection. As shown in Table 12, a single dose of DPC (250 μg polymer weight) caused 46-56% human Aha1 knockdown 24 hours after injection.

Table 12: Aha1 knockdown in HepG2 liver tumor model by Aha1 siRNA DPC modified with peptide cleavable masking agent with long PEG length

Example 13. Targeting of DPC to human colorectal adenocarcinoma metastasizing liver tumor tissue by bsAb targeting DPC
Ant-129-1 polymer was modified with 5 × molar excess of Dig-PEG ₁₂ —NHS and 8 × weight excess of PEG ₂₄ —FCit. Aha1 siRNA or GFP siRNA was conjugated to the modified polymer in a 4: 1 polymer: siRNA weight ratio. DPC was purified on a Sephadex G50 spin column to remove unbound reagents. Prior to injection, Dig-DPC was complexed in sterile PBS with equimolar amounts of IGF1R-Dig bsAb or CD33-Dig bsAb or without bsAb for at least 30 minutes. Animals containing HT29 tumor cells (human colorectal adenocarcinoma; ATCC number HTB-38) were injected with DPC. HT29 cells overexpress the insulin-like growth factor-1 receptor protein (IGF1R), bind to the IGF1R-Dig bispecific antibody, and can take it inside. Animals (n = 3) were administered DPC (320 μg polymer weight). The injection was repeated 24 hours later. Serum and tissue samples were collected 48 hours after the second dose. The knockdown of human Aha1 in tumor cells was 26-38% (Table 13). Compared to CDM-DPC, FCit-DPC showed lower off-target liver Aha1 knockdown (24-36% versus 78-83%). FCit-DPC also showed reduced liver accumulation compared to CDM-DPC.

Table 13: Aha1 knockdown in HT29 colorectal adenocarcinoma metastatic liver tumor by dipeptide-cleavable Aha1 siRNA DPC

Example 14. In vivo knockdown of endogenous Aha1 in liver tumors
400 μg of Lau41648-106 was modified with 8 × (weight) PEG ₁₂ -ValCit or 16 × PEG ₂₄ -PheCit. 100 μg Aha1 siRNA or 100 μg Eg5 control siRNA was conjugated to the modified polymer as described above. Animals containing Hep3B-SEAP tumor cells were injected with DPC. Serum and tissue samples were collected 48 hours after injection. Human Aha1 knockdown in tumor cells was 26-38%.

Table 14: Aha1 knockdown in Hep3B-SEAP liver tumors by dipeptide-cleavable Aha1 siRNA DPC

Example 15. In vivo circulation and tissue targeting of masked polymers
Lau24AB polyacrylate (100 μg) was treated with ¹²⁵ I-Bolton-Hunter (BH) reagent (50 μCi) in 50 mM HEPES pH 8.0 buffer for 1 hour at room temperature. The labeled polymer was purified on a 2 ml underwater Sephadex QEA spin column. The labeled polymer solution was stored at 4 ° C. The unlabeled polymer was supplemented with ¹²⁵ I-labeled polymer to inject about 1 mg of 0.2 μCi per 200 g rat. A mixture of labeled and unlabeled polymer (calculated for about 3.5 animals) was modified with PEG ₂₄ -FCit or PEG-CDM as described above (2 mg / ml polyacrylate, 14 mg / ml PEG-CDM reagent, 14 mg / ml NAG-PEG-CDM reagent, 16 mg / ml PEG ₂₄ -FCit reagent). Incubation time 1 hour. The reaction mixture was then diluted with isotonic glucose to give an infusion dose of 1 ml volume per animal. Three animals / group were injected. Blood was drawn from the animals at a given time (0.1-0.2 ml). The amount of polymer in the sample was determined by counting with a gamma counter. As shown in FIG. 4, the polymer modified with a protease cleavable masking agent was slower to drain than the polymer masked with a pH labile maleic anhydride masking agent. Prolonged circulation time is beneficial for targeting to non-liver tissue.

式中：Aはboc保護エチル-エトキシアミノアクリラートであり
Bはメタクリル酸プロピルであり
CはRAFT剤CPCPA（4-シアノ-4-(フェニルカルボノチオイルチオ)ペンタン酸）であり
かつnおよびmは整数である。
合成後のboc保護基の除去によりアミンモノマーを生じる。 Example 16. Synthesis of amphiphilic membrane active polymer
A) Poly (vinyl acrylate)
i) RAFT copolymerization of N-Boc-ethylethoxyacrylate and propyl methacrylate:

Where: A is boc protected ethyl-ethoxyaminoacrylate
B is propyl methacrylate
C is the RAFT agent CPCPA (4-cyano-4- (phenylcarbonothioylthio) pentanoic acid) and n and m are integers.
Removal of the boc protecting group after synthesis yields an amine monomer.

  For other membrane active polymers, A can be beprotected ethyl, propyl, or butylaminoacrylate. B is higher acrylate with higher hydrophobicity (10-24 carbon atoms, C18), less hydrophobic (1-6 carbon atoms, C4) acrylate, or higher acrylate with lower hydrophobicity It can be a combination with acrylate.

A copolymer consisting of amine acrylate / C3 methacrylate was synthesized as follows. Monomers and RAFT agent were weighed and added in the proportions shown in butyl acetate. AIBN (azobis-isobutyronitrile) was added and nitrogen was bubbled through the reaction mixture for 1 hour at room temperature. The reaction mixture was then placed in an 80 ° C. oil bath for 15 hours. The polymer was then precipitated with hexane and further fractionated using a DCM / hexane solvent system (see below). The polymer was then dried under reduced pressure. The polymer was deprotected with 7 ml of 2M HCl in acetic acid for 30 minutes at room temperature. After 30 minutes, 15 ml of water was added to the reaction mixture and the mixture was transferred to a 3.5 kDa MWCO dialysis tube. The polymer was dialyzed overnight against NaCl and then dialyzed against dH ₂ O for another day. The water was then removed by lyophilization and the polymer was dissolved in dH ₂ O.

Monomer synthesis for (Ant 41658-111)
2,2'-azobis (2-methylpropionitrile) (AIBN, radical initiator), 4-cyano-4- (phenylcarbonothioylthio) pentanoic acid (CPCPA, RAFT agent) and butyl acetate from Sigma Aldrich Purchased. Propyl methacrylate monomer (Alfa Aesar) was filtered to remove the inhibitor.

In a 2 L round bottom flask 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 1 L flask, BOC anhydride (36.6 g, 169.1 mmol) was dissolved in 660 mL dichloromethane. A 2 L round bottom flask was fitted with an addition funnel and the BOC anhydride solution was added to the flask over 6 hours. The reaction mixture was stirred overnight. In a 2 L separatory funnel, the product was washed with 300 ml each of 10% citric acid, 10% K ₂ CO ₃ , saturated NaHCO ₃ , and saturated NaCl. The product BOC protected 2- (2-aminoethoxy) ethanol was dried over Na ₂ SO ₄ , gravity filtered and DCM was evaporated using rotary evaporation and high vacuum.

In a 500 ml round bottom flask with a stir bar and flushed with argon, BOC protected 2- (2-aminoethoxy) ethanol (27.836 g, 135.8 mmol) was added followed by 240 mL of anhydrous dichloromethane. Diisopropylethylamine (35.5 ml, 203.7 mmol) was added and the system was placed in a dry ice / acetone bath. Acryloyl chloride (12.1 ml, 149.4 mmol) was diluted with 10 ml of dichloromethane and added dropwise to the system flushed with argon. The system was maintained under an argon atmosphere, allowed to warm to room temperature and stirred overnight. The product was washed with 100 mL each of dH ₂ O, 10% citric acid, 10% K ₂ CO ₃ , saturated NaHCO ₃ , and saturated NaCl. The product BOC-aminoethylethoxy acrylate (BAEEA) was dried over Na ₂ SO ₄ , gravity filtered and DCM was evaporated by rotary evaporation. The product was purified by column chromatography on 29 cm silica using a 7.5 cm diameter column. The solvent system used was 30% ethyl acetate in hexane. Rf: 0.30. Fractions were collected and the solvent was evaporated using rotary evaporation and high vacuum. BAEEA was obtained with a yield of 74%. BAEEA was stored in the freezer.

  Polymer Ant-41658-111: A solution of AIBN (1.00 mg / mL) and RAFT agent (4-cyano-4 (phenylcarbonothioylthio) pentanoic acid (CPCPA), 10.0 mg / mL) in butyl acetate was prepared . The molar feed ratio of the monomers was 75 BAEEA: 25 propyl methacrylate (CAS: 2210-28-8) and 0.108 CPCPA RAFT agent and 0.016 AIBN catalyst (total 0.00562 mol).

BAEEA (1.09 g, 4.21 mmol) (A), propyl methacrylate (0.180 g, 1.41 mmol) (B), CPCPA solution (0.170 ml, 0.00609 mmol) (C), AIBN solution (0.150 ml, 0.000915 mmol), and Butyl acetate (5.68 ml) was added to a 20 ml glass vial with a stir bar. The vial was sealed with a rubber stopper and nitrogen was bubbled through the solution for 1 hour using a long syringe needle and a second short syringe needle as the outlet. The syringe needle was removed and the system was heated at 80 ° C. for 15 hours using an oil bath. After returning the solution to room temperature and transferring to a 50 ml centrifuge tube, hexane (35 ml) was added to the solution. The solution was centrifuged at 4,400 rpm for 2 minutes. The supernatant layer was carefully decanted and the lower layer (solid or gel-like) was washed with hexane. The lower layer was then redissolved in DCM (7 mL), precipitated in hexane (35 mL), and centrifuged again. The supernatant was decanted and the lower layer was washed with hexane, and then the polymer was dried under reduced pressure for several hours. Molecular weight obtained by MALS: 73,000 (PDI 1.7); polymer composition obtained by H ¹ NMR: amine: alkyl = 69: 31.

Fractionated precipitation The dried and precipitated product was dissolved in DCM (100 mg / mL). Hexane was added until just after the cloud point was reached (about 20 ml). The resulting milky solution was centrifuged. The lower layer (a thick liquid that is about 60% polymer) was extracted and completely precipitated in hexane. The remaining upper solution was also completely precipitated by adding more hexane. Both fractions were centrifuged, after which the polymer was isolated and dried under reduced pressure. Fraction 1: Mw 87,000 (PDI 1.5); Fraction 2: Mw 52,000 (PDI 1.5-1.6).

MALS analysis Approximately 10 mg of polymer was dissolved in 0.5 mL of 89.8% dichloromethane, 10% tetrahydrofuran, 0.2% triethylamine. Molecular weight and polydispersity (PDI) were measured using a Jordi 5μ 7.8 × 300 Mixed Bed LS DVB column with a Wyatt Helos II multi-angle light dispersion detector connected to a Shimadzu Prominence HPLC. Crude polymer: MW: 73,000 (PDI 1.7), Fraction 1: MW 87,000 (PDI: 1.5), Fraction 2: MW 52,000 (PDI 1.5-1.6)

The purified BOC protected polymer was reacted with 2M HCl in acetic acid (7 ml) for 0.5 h to remove the BOC protecting group to yield the amine. 15 mL of dH ₂ O was added to the reaction mixture and the solution was transferred to a 3500 MW cut-off cellulose tube and dialyzed against high salt for 24 hours and then against dH ₂ O for 18 hours. The contents were lyophilized and then dissolved in DI H ₂ O at a concentration of 20 mg / ml. The polymer solution was stored at 2-8 ° C.

ii) Polymer Lau24B was prepared as described 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:

(Table 15) Ant-129-1 polymer synthesis reaction product

For N-Boc-amino-propyl-acrylate (BAPA), 3- (BOC-amino) 1-propanol (TCI) (135.8 mmol) in a 500 ml round bottom flask with stir bar and flushed with argon 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 with 10 ml of dichloromethane and added dropwise to the system flushed with argon. The system was maintained under an argon atmosphere, allowed to warm to room temperature and stirred overnight. The product was washed with 100 mL each of dH ₂ O, 10% citric acid, 10% K ₂ CO ₃ , saturated NaHCO ₃ , and saturated NaCl. The product BOC-aminopropyl acrylate (BAPA) was dried over Na ₂ SO ₄ , gravity filtered and DCM was evaporated by rotary evaporation. The product was purified by column chromatography on 29 cm silica using a 7.5 cm diameter column. The solvent system used was 30% ethyl acetate in hexane. Rf: 0.30. Fractions were collected and the solvent was evaporated using rotary evaporation and high vacuum. BAPA was obtained in 74% yield. BAPA was stored in the freezer.

iv) Random copolymerization of N-Boc-ethylethoxy acrylate and propyl methacrylate. The copolymer of amine acrylates / C _n methacrylate was synthesized as follows. Monomers were weighed and added in the proportions indicated in dioxane. AIBN (azobis-isobutyronitrile) was added and nitrogen was bubbled through the reaction mixture for 1 hour at room temperature. The reaction mixture was then placed in an oil bath at 60 ° C. for 3 hours. The polymer was then dried under reduced pressure. The polymer was purified by GPC. The polymer was then deprotected with 7 ml of 2M HCl in acetic acid for 30 minutes at room temperature. After 30 minutes, 15 ml of water was added to the reaction mixture and the mixture was transferred to a 3.5 kDa MWCO dialysis tube. The polymer was dialyzed overnight against NaCl and then dialyzed against dH ₂ O for another day. The water was then removed by lyophilization and the polymer was dissolved in dH ₂ O.

Polymer Lau41648-106
The monomer feed ratio was 80 BAEEA: 20 propyl methacrylate (CAS: 2210-28-8) and 3% AIBN catalyst based on the 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 50 ml of a stir bar. Added to glass tube. Compounds A and B were prepared as described above in Example 16Ai. The reaction was planned in triplicate. Nitrogen was bubbled through each solution for 1 hour using a long pipette. The pipette was removed and each tube was capped carefully. Each solution was then heated at 60 ° C. for 3 hours using an oil bath. Each solution was allowed to warm to room temperature and mixed in a round bottom. The crude polymer was dried under reduced pressure. Molecular weight obtained by MALS: 55,000 (PDI 2.1); polymer composition obtained by H ¹ NMR: amine: alkyl = 74: 26.

GPC Fractionation The dried crude polymer was dissolved at 50 mg / ml in 75% dichloromethane, 25% tetrahydrofuran, and 0.2% triethylamine. The polymer was then fractionated on a Jordi Gel DVB 10 ⁴ Å-500 mm / 22 mm column using a flow rate of 5 ml / min and an injection of 10 ml. The first fraction was collected at 15-17 minutes and the latter fraction was collected at 17-19 minutes. Fraction 15-17: Mw 138,000 (PDI 1.1); Fraction 17-19: Mw 64,000 (PDI 1.2).

MALS analysis Approximately 10 mg of polymer was dissolved in 0.5 mL of 89.8% dichloromethane, 10% tetrahydrofuran, 0.2% triethylamine. Molecular weight and polydispersity (PDI) were measured using a Jordi 5μ 7.8 × 300 Mixed Bed LS DVB column with a Wyatt Helos II multi-angle light dispersion detector connected to a Shimadzu Prominence HPLC. Crude polymer: MW: 55,000 (PDI 2.1), fraction 15-17: MW 138,000 (PDI: 1.1), fraction 17-19: MW 64,000 (PDI 1.2)

The purified BOC protected polymer was reacted with 2M HCl in acetic acid (7 ml) for 0.5 h to remove the BOC protecting group to yield the amine. 15 mL of dH ₂ O was added to the reaction mixture and the solution was transferred to a 3500 MW cut-off cellulose tube and dialyzed against high salt for 24 hours and then against dH ₂ O for 18 hours. The contents were lyophilized and then dissolved in DI H ₂ O at a concentration of 20 mg / ml. The polymer solution was stored at 2-8 ° C.

v) Synthesis of water-soluble, amphiphilic, membrane-active poly (vinyl ether) polyamine terpolymers
X mol% amine protected vinyl ether (eg, 2-vinyloxyethyl phthalimide) is added to anhydrous dichloromethane in a dry bottom round bottom flask under nitrogen atmosphere. To this solution is added Y mol% lower hydrophobic group (eg propyl, butyl) vinyl ether and optionally Z mol% higher hydrophobic group (eg dodecyl, octadecyl) vinyl ether (FIG. 1). The solution is placed in a -50 to -78 ° C bath to precipitate 2-vinyloxyethylphthalimide. To this solution is added 10 mol% BF _3. (OCH ₂ CH ₃ ) ₂ and the reaction is allowed to proceed at −50 to −78 ° C. for 2-3 hours. Polymerization is stopped by adding an ammonium hydroxide solution in methanol. The polymer is dried under reduced pressure and then added to 1,4-dioxane / methanol (2/1). The protecting group is removed from the amine by adding 20 mol equivalents of hydrazine to phthalimide. The solution is refluxed for 3 hours and then dried under reduced pressure. The resulting solid is dissolved in 0.5 mol / L HCl and refluxed for 15 minutes to produce the hydrochloride salt of the polymer, diluted with distilled water and refluxed for an additional hour. The solution is then neutralized with NaOH, cooled to room temperature (RT), transferred to a molecular cellulose tube, dialyzed against distilled water and lyophilized. The polymer can be further purified using size exclusion or other chromatography. The molecular weight of the polymer is estimated using a column according to standard procedures, including analytical size exclusion chromatography and size exclusion chromatography with multiple light dispersion (SEC-MALS).

Polymer DW1360
Amine / butyl / octadecyl poly (vinyl ether) terpolymers were synthesized from 2-vinyloxyethylphthalimide (5 g, 23.02 mmol), butyl vinyl ether (0.665 g, 6.58 mmol), and octadecyl vinyl ether (0.488 g, 1.64 mmol) monomers . 2-Vinyloxyethylphthalimide was added to 36 mL of anhydrous dichloromethane in a round bottom flask dried in a 200 mL drier containing a magnetic stir bar under a nitrogen atmosphere. To this solution was added butyl vinyl ether and n-octadecyl vinyl ether. The monomer was completely dissolved at room temperature (RT) to give a clear homogeneous solution. The reaction vessel containing the clear solution is then placed in a -50 ° C. bath generated by adding dry ice to a 1: 1 solution of ACS grade denatured alcohol and ethylene glycol to produce a visible precipitate of phthalimide monomer. It was. After cooling for about 1.5 minutes, BF _3. (OCH ₂ CH ₃ ) ₂ (0.058 g, 0.411 mmol) was added to initiate the polymerization reaction. The phthalimide monomer was dissolved after the start of polymerization. The reaction was allowed to proceed for 3 hours at -50 ° C. The polymerization was stopped by adding 5 mL of 1% ammonium hydroxide in methanol. The solvent was then removed by rotary evaporation.

  The polymer was then dissolved in 30 mL of 1,4-dioxane / methanol (2/1). To this solution was added hydrazine (0.147 g, 46 mmol) and the mixture was heated to reflux for 3 hours. The solvent was then removed by rotary evaporation and the resulting solid was then added to 20 mL 0.5 mol / L HCl and refluxed for 15 minutes, diluted with 20 mL distilled water and refluxed for an additional hour. The solution was then neutralized with NaOH, cooled to room temperature, transferred to a 3,500 molecular weight cellulose tube, dialyzed against distilled water for 24 hours (2 × 20 L), and lyophilized.

B) Melittin All melittin peptides were made using standard peptide synthesis techniques in the art. Suitable melittin peptides can be all L-amino acids, all D-amino acids (inverso). Independent of the L or D form, the melittin peptide sequence can be inverted (retro).

Claims (21)

A reversible modifier for amphiphilic membrane active polyamines comprising a compound having a structure represented by the following comprising a targeting ligand covalently linked to a dipeptide-amidobenzyl-carbonate:

(Where
X is -NH-
Y is —NH—
R1 is -CH _3,
R2 is _{- (CH} 2) 3 -NH-C (O) -NH 2,
R4 consists of a targeting ligand that is uncharged and has a targeting ligand or linker ,
R5 is —CH ₂ —O—C (O) —Z in the 2, 4, or 6 position, where Z is

And
R6 is hydrogen) ;
Here, the targeting ligand is selected from the group consisting of a hapten, a vitamin, and a cell surface receptor ligand . Targeting ligand is the asialoglycoprotein receptor (ASGPr) ligand agent according to claim 1. The ASGPr ligand is selected from the list consisting of lactose, galactose, N-acetylgalactosamine, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, Nn-butanoylgalactosamine, and N-iso-butanoyl-galactosamine. Item 3. The agent according to Item 2. Z is

The agent according to any one of claims 1 to 3, wherein R-A ¹ A ² -amidobenzyl-carbonate (wherein
R consists steric stabilizer,
A ¹ is a hydrophobic amino acid, and A ² is a hydrophilic uncharged amino acid linked to A ¹ via an amide bond, where the hydrophilic uncharged amino acid is uncharged at neutral pH)
A reversible modifier for amphiphilic membrane active polyamines comprising :
Here, the steric stabilizer is polyethylene glycol (PEG),
The hydrophobic amino acid is selected from the group consisting of phenylalanine, valine, isoleucine, leucine, alanine, and tryptophan;
The agent, wherein the hydrophilic uncharged amino acid is selected from the group consisting of asparagine, glutamine, and citrulline . The agent according to claim 5, wherein the amidobenzyl is a p-amidobenzyl group. The agent according to claim 5 or 6, wherein the carbonate is an activated amine-reactive carbonate. The agent according to any one of claims 5 to 7, wherein R is neutral. A reversible modifier for amphiphilic membrane active polyamines comprising a compound having a structure represented by:

(Where
R consists steric stabilizer,
R ₁ is a hydrophobic amino acid side chain;
R ₂ is a hydrophilic uncharged amino acid side chain, wherein the hydrophilic uncharged amino acid is uncharged at neutral pH;
Y is —NH— or —O—;
R ₅ is in the 2, 4, or 6 position and is —CH ₂ —O—C (O) —Z, wherein Z is

And R ₆ is independently hydrogen, alkyl, — (CH ₂ ) _m —CH ₃ , — at each of the 2, 3, 4, 5, or 6 positions except for the position occupied by R _5. (CH ₂ ) — (CH ₃ ) ₂ , or a halide, where n and m are each independently an integer of 0 to 4)
Here, the steric stabilizer is polyethylene glycol (PEG),
The hydrophobic amino acid is selected from the group consisting of phenylalanine, valine, isoleucine, leucine, alanine, and tryptophan;
The hydrophilic uncharged amino acid is selected from the group consisting of asparagine, glutamine, and citrulline . Delivery polymers for delivering polynucleotides to cells in vivo, including:
M ¹ _y -P-M ² _z
Where
P is an amphiphilic membrane active polyamine,
M ¹ consists of a targeting ligand with a targeting ligand or linker linked to P via a dipeptide-amidobenzyl-carbamate linkage,
M ² is a dipeptide - amide benzyl - consists steric stabilizer which is connected to P through a carbamate connecting,
y and z are each integers greater than or equal to zero,
y + z has a value greater than 50% of the primary amine on P, as determined by the amount of amine on polyamine P in the absence of masking agent, and the dipeptide-amidobenzyl-carbamate linkage is :
^R-A 1 A ² - amido benzyl - carbamate -P
(Where
R consists steric stabilizer targeting ligand or M ^2, having a targeting ligand or linker M ^1,
A ¹ is a hydrophobic amino acid, and A ² is a hydrophilic uncharged amino acid linked to A ¹ via an amide bond, wherein the hydrophilic uncharged amino acid is uncharged at neutral pH; and P is have a structure represented by the amphiphilic membrane active polymer)
Wherein the targeting ligand is selected from the group consisting of a hapten, a vitamin, and a cell surface receptor ligand;
The steric stabilizer is polyethylene glycol (PEG),
The hydrophobic amino acid is selected from the group consisting of phenylalanine, valine, isoleucine, leucine, alanine, and tryptophan;
The hydrophilic uncharged amino acid is selected from the group consisting of asparagine, glutamine, and citrulline . 11. A delivery polymer according to claim 10 , wherein the amidobenzyl is a p-amidobenzyl group. M ¹ and ^{M 2} are uncharged or is charge neutrality independently claim 10 or 11 delivery polymer according. Targeting ligand is a cell receptor ligand, delivery polymer of any one of claims 10-12. Cell receptor ligand is ASGPr ligand claim 13 delivery polymer according. The ASGPr ligand is selected from the list consisting of lactose, galactose, N-acetylgalactosamine, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, Nn-butanoylgalactosamine, and N-iso-butanoyl-galactosamine. Item 15. The delivery polymer according to Item 14 . R—A ¹ A ² -amidobenzyl-carbamate-P is:

(Where
Y is —NH— or —O—;
R ₁ is the side chain of the hydrophobic amino acid A ¹ and R ₂ is the side chain of the hydrophilic uncharged amino acid A ^{2 where} the hydrophilic uncharged amino acid is uncharged at neutral pH) 16. A delivery polymer according to any one of claims 10 to 15 having the structure represented. The delivery polymer according to any one of claims 10 to 16 , wherein the amphiphilic membrane active polyamine is selected from the list consisting of random, block, and alternating synthetic polymers. The delivery polymer according to any one of claims 10 to 16 , wherein the amphiphilic membrane active polyamine is a melittin peptide. The delivery polymer according to any one of claims 10 to 18 , wherein the amphiphilic membrane active polyamine is further covalently bound to the polynucleotide. The delivery polymer of claim 19 , wherein the polynucleotide comprises an RNA interference polynucleotide. 21. The delivery polymer of claim 20 , wherein the RNA interference polynucleotide is selected from the group consisting of DNA, RNA, dsRNA, siRNA, and miRNA.

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