Described are ortho carboxy phenol derived acetals and compositions containing ortho carboxy phenol derived acetals which are useful for delivering biologically active compounds to cells. The acetals can be used to reversibly link up to three different molecules and have rapid hydrolysis kinetics in conditions which are present in a cell as well as in vivo. Cleavage of the acetal enhances delivery of the biologically active compound.

Description of the Invention

SUMMARY OF THE INVENTION

Compounds and methods are described for enhancing the delivery of a biologically active compound to a cell. In a preferred embodiment, the compounds comprise acid labile ortho carboxy phenol derived acetals. The acetals can be used to reversibly link up to three different molecules which are rapidly cleaved from each other upon exposure to an acidic pH environment.

In a preferred embodiment, the described ortho carboxy phenol derived acetals may be used to form acid cleavable transfection agents. The transfection agent can be a compound which is non-covalently associated with a biologically active compound to be delivered to a cell. Alternatively, the transfection agent can be a compound which is covalently linked to a biologically active compound. Cleavage of the transfection agent can release either a non-covalently associated or covalently linked biologically active compound from the transfection agent. The transfection agent may be designed such that cleavage of the transfection agent increases membrane activity of the agent.

In a preferred embodiment, we describe a composition for delivering a biologically active compound to a cell comprising: the biologically active compound electrostatically associated with a pH sensitive ortho carboxy acetal containing delivery agent to form a complex. For delivery of a polynucleotide, a preferred delivery agent is a polycation or a lipid. The ortho carboxy acetal may be present in a polymer or lipid prior to association of the polymer or lipid with a polynucleotide. Alternatively, the ortho carboxy acetal may be used to crosslink a polymer or lipid after association of the polymer or lipid with a polynucleotide. The ortho carboxy acetal may also be used to attach a functional group to a polynucleotide/delivery agent complex.

A variety of groups can be attached to an ortho carboxy phenol derived acetal. These groups may be selected from the group comprising: polynucleotide, biologically active compound, targeting moiety, ligand, interaction modifier, polycation, polymer, polymer monomer, membrane active compound, hydrophobic group, detergent, and lipid.

In a preferred embodiment, we describe labile crosslinking agents comprising: ortho carboxy acetal dialdehydes. In one embodiment, the dialdehydes may be used to link amines via a pH sensitive linkage. In this way, the dialdehydes may be used to reversibly crosslink amines present in polynucleotide/polyamine complexes, thus stabilizing the complexes. In another embodiment, the dialdehydes may be used as an acid-labile building block to synthesize lipids, polymers, and/or crosslinking reagents that may be useful in the delivery of biologically active compounds.

DETAILED DESCRIPTION

The present invention relates to the delivery of biologically active compounds to cells using pH-labile linkages and compounds incorporating these pH-labile linkages. The present invention provides compositions and methods for delivery and release of a compound of interest to a cell.

It has been shown that acid groups near an acetal group can facilitate cleavage of the acetal. In particular, ortho carboxy substituted acetals derived from ortho carboxy phenols hydrolyze 10.sup.5-10.sup.6 times faster than the corresponding acetals without ortho carboxy substitution (Fife et al. 1971). The protonated carboxylate accelerates the hydrolysis of the acetal and the carboxylate group is key to rapid hydrolysis kinetics. The corresponding acetals with ortho-substituted ester groups are approximately 22-fold slower in their hydrolysis kinetics (Dunn et al. 1970) The acid cleavage of an ortho carboxy substituted acetal derived from ortho carboxy phenol, is shown in FIG. 1 (see Original Patent). In an ortho carboxy substituted acetal, R, R.sub.1-4 can be hydrogen, any carbon-containing group (including, but not limited to any alkyl, aryl, or acyl group) or a heteroatom and R' may be any carbon-containing group (including, but not limited to any alkyl, aryl, or acyl group) or a heteroatom but not hydrogen.

Therefore, to covalently link two compounds via a rapidly hydrolyzed bond, one of the compounds is R or R' and the other is R or R.sub.1-4. For example, if one compound is R, the other compound may be R' or R.sub.1, R.sub.2, R.sub.3 or R.sub.4. If one compound is R', the other compound may be R or R.sub.1, R.sub.2, R.sub.3 or R.sub.4. If one compound is R.sub.1, R.sub.2, R.sub.3 or R.sub.4, the other compound may be R R'. Similarly, to link three compounds by rapidly hydrolyzed bonds, one of the compounds is R, a second compound is R' and a third compound is R.sub.1, R.sub.2, R.sub.3 or R.sub.4. The compounds attached to the acetal may be selected from the group comprising: biologically active compounds, polynucleotides, pharmaceutical agents, peptides, proteins, membrane active compounds, polymers, polymer monomers, transfection agents, lipids, detergents, targeting moieties, and interaction modifiers.

To illustrate the rate of cleavage of several example ortho carboxy phenol derived acetals, we synthesized the molecules 1-3 (shown in FIG. 2 (see Original Patent)) and measured the rates of acetal hydrolysis for each at pH 5.2-7.3. Acetal 1. R', R=alkyl; and R.sub.1, R.sub.2, R.sub.3, R.sub.4=hydrogen Acetal 2. R', R=alkyl; R.sub.1, R.sub.4=hydrogen; R.sub.2=carboxyl (carbon-containing group); R.sub.3=hydroxyl (heteroatom) Acetal 3. R'=alkyl; R, R.sub.2, R.sub.3, R.sub.4=hydrogen; and R.sub.1=carboxyl (carbon-containing group)

The rate of acetal hydrolysis is dependent upon several critical characteristics of the ortho-substituted phenol-derived acetal structures including the aldehyde and the phenol from which the acetal is derived. In particular, acetals derived from formaldehyde (acetal 3) hydrolyze more slowly than acetals derived from alkyl-substituted aldehydes such as acetaldehyde (acetals 1 and 2). Also, substitution of the phenol with another ortho carboxy groups increases the rate above that observed for the monocarboxylate (Dunn et al. 1970).

As can be seen by half-lives of the ortho carboxy phenol derived acetals, the rate of cleavage is rapid at pH 4-7.5. The lability of these acetals allows their use in the construction of agents that disassemble under physiological conditions to aid in drug delivery.

Saccharides are a well-known class of acetals which have established routes of synthesis. In particular, reaction of 1-bromo protected sugars with ortho carboxy derived phenolates, followed by deprotection, results in a salicylic galactoside, an ortho carboxy phenol derived acetal (FIG. 3 (see Original Patent); Capon 1963). For this ortho carboxy acetal, R and R' are linked to make the sugar.

Compounds containing multiple aldehyde groups, e.g. glutaraldehyde groups (Adami et al. 1999), are capable of efficient crosslinking. A simple method for synthesizing dialdehydes is the oxidation of cyclic compounds containing vicinal alcohol groups, such as on sugars, by sodium periodate. In particular, sugars with ortho carboxy derived phenolates may be oxidized to produce dialdehydes (see FIG. 4 (see Original Patent)). The dialdehyde may be added to a polyamine-containing particle to crosslink (i.e., cage) the polyamine, thereby stabilizing the particle. Alternatively, the dialdehyde may be used as an acid-labile building block to synthesize lipids, polymers, and/or crosslinking reagents that may be useful in delivery of biologically active compounds.

Ortho carboxy phenol derived acetals may be incorporated into polynucleotide (or other biologically active compound) delivery complexes. Many different molecules can be attached to ortho carboxy phenol derived acetals, at positions R, R', and R.sub.1-4. Biologically active compounds and a variety of functional groups may be attached to the acetal. The acetal may also be used in the construction on polymers useful for biologically active compound delivery to cells. A plurality of ortho carboxy phenol derived acetals can be incorporated into a polymer to facilitate release of side groups from the polymer or to facilitate cleavage of the polymer backbone.

A polymer can also be designed such that its presence in an endosome prevents acidification of the endosome or facilitates disruption of the endosomal membrane. For example, the polymer can contain endosomolytic properties or have endosomolytic agents or membrane fusion agents attached to it.

The labile acetal bonds described herein may be incorporated into systems that are amphipathic and increase in hydrophobicity and membrane activity upon bond cleavage. For example, the acetal may contain acetals derived from ortho carboxylate phenols having a hydrophilic, negative charge. Cleavage of the acetal separates R.sub.1-4 from R and R', which removes the link between R and R' and the carboxylate group of the ortho-substituted carboxy phenol. This loss of associated charge may make R and/or R' more hydrophobic, and therefore more likely to interact with and lyse a membrane. Using this strategy one may use acetals derived from ortho carboxylate phenol to construct lipids (where R and R' are long chain, C>10, alkyl groups), or detergents (where R or R' are long chain, C>10, alkyl groups) that become membrane active upon hydrolysis.

Functional groups include cell targeting signals, nuclear localization signals, compounds that enhance release of contents from endosomes or other intracellular vesicles (releasing signals), membrane active compounds, lipids, charged groups, polymers and polymer monomers, transfection enhancing agents, and other compounds that alter the behavior or interactions of the compound or complex to which they are attached. Charged groups include cationic groups which may be used to ionically interact with nucleic acid.

The present invention provides compositions of matter and methods for facilitating the delivery of biologically active compounds to the cells. For the purposes of this invention, the term biologically active compound is intended to encompass all naturally-occurring or synthetic compounds capable of eliciting a biological response or having an effect on biological systems, particularly cells and cellular organelles. A biologically active compound typically has some specific and intended pharmaceutical or biological action. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in a cell or tissue. The cell may be in vivo or in vitro. Biologically active compounds include, but are not limited to: pharmaceuticals, proteins, peptides, polypeptides, proteins, enzymes, enzyme inhibitors, hormones, cytokines, antigens, viruses, and polynucleotides. The term biologically active compound includes therapeutic agents that provide a therapeutically desirable effect when administered to an animal (e.g., a mammal, such as a human, see Physicians' Desk Reference, 58 ed., 2004, Medical Economics Company, Inc., Montvale, N.J., pages 201-202).

For polynucleotide delivery, it is desirable for the polynucleotide to be dissociated from components of the complex in the cell in order for the polynucleotide to be active. This dissociation may occur outside the cell, within cytoplasmic vesicles or organelles (i.e. endosomes), in the cytoplasm, or in the nucleus. The disclosed acetal linkages can be utilized in forming cleavable components of polynucleotide delivery complexes to facilitate this dissociation of the polynucleotide.

The described acetals and acetal-containing compounds can be used with a variety of delivery routes, including: intravascular (intravenous, intra-arterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal, intralymphatic, transdermal, oral, nasal, respiratory, and mucosal routes of administration.

Targeting moieties are used for targeting a compound or composition to cells, to specific cells, to tissues or to specific locations in a cell. Targeting moieties enhance the association of compounds or compositions with a cell. The moiety may increase binding of the compound to the cell surface and/or its association with an intracellular compartment. By modifying the cell or tissue localization of a compound, the function of the compound can be enhanced. The targeting moieties can be, but is not limited to, a protein, peptide, lipid, steroid, sugar, carbohydrate, or synthetic compound. Targeting moieties such as ligands enhance binding to cellular receptors. A variety of ligands have been used to target drugs and genes to cells and to specific cellular receptors. The ligand may have affinity for a target within the cell membrane, on the cell membrane or near a cell. Binding of ligands to receptors typically initiates endocytosis. Ligands include agents that target the asialoglycoprotein receptor by using asialoglycoprotein or galactose residues. Other moieties such as insulin, EGF, RGD-containing peptides, folate and other vitamins, and transferrin are other examples of cell receptor targeting ligands. Chemical groups that react with thiols or disulfide groups on cells can also be used to target many types of cells. Other targeting groups include molecules that interact with membranes such as lipids, fatty acids, cholesterol, dansyl compounds, and amphotericin derivatives. In addition viral proteins could be used to bind cells.

After interaction of a compound or complex with the cell, other targeting groups can be used to increase the delivery of the biologically active compound to certain parts of the cell. For example, nuclear localizing signals enhance delivery into proximity of the nucleus and/or entry into the nucleus. Nuclear transport signals can be proteins or peptides, such as the SV40 large T antigen NLS or the nucleoplasmin NLS, that interact with the nuclear transport machinery in the cell. Nuclear transport signals can also be proteins that make up the nuclear transport machinery. For example, karyopherin beta can be used to target compounds the nuclear pore complex.

Membrane active polymers or compounds are molecules that are able to alter membrane structure. This change in structure can be shown by the compound inducing one or more of the following effects upon a membrane: an alteration that allows small molecule permeability, pore formation in the membrane, a fusion and/or fission of membranes, an alteration that allows large molecule permeability, or a dissolving of the membrane. This alteration can be functionally defined by the compound's activity in at least one the following assays: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis and endosomal release. More specifically membrane active compounds allow for the transport of molecules with molecular weight greater than 50 atomic mass units to cross a membrane. This transport may be accomplished by either the total loss of membrane structure, the formation of holes (or pores) in the membrane structure, or the assisted transport of compound through the membrane. Membrane active compounds can enhance the release of endocytosed material from intracellular membrane enclosed vesicles. Release includes movement out of an intracellular compartment into the cytoplasm or into an organelle such as the nucleus. Chemicals such as chloroquine, bafilomycin or Brefeldin A1, viruses and viral components such as influenza virus hemagglutinin subunit HA-2 peptides, and other types of amphipathic peptides such as melittin are examples of molecules which have been shown to enhance release of endosomal contents.

An interaction modifier changes the way that a molecule interacts with itself or other molecules relative to molecule containing no interaction modifier. The result of this modification is that self-interactions or interactions with other molecules are either increased or decreased. Steric stabilizers are hydrophilic polymers that decrease electrostatic interactions between molecules and themselves and with other molecules. Steric stabilizers such as polyethylene glycol have been used to reduce interactions with blood components to increase circulatory time of a compound or composition to which they are attached by preventing opsonization, phagocytosis and uptake by the reticuloendothelial system. Other steric stabilizers include: alkyl groups, and polysaccharides.

A transfection agent, or transfection reagent or delivery vehicle, is a compound or compounds that bind(s) to or complex(es) with oligonucleotides and polynucleotides, and mediates their entry into cells. Examples of transfection reagents include, but are not limited to, cationic liposomes and lipids, polyamines, calcium phosphate precipitates, histone proteins, polyethylenimine, polylysine, and polyampholyte complexes. It has been shown that cationic proteins like histones and protamines, or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents. Typically, the transfection reagent has a component with a net positive charge that binds to the oligonucleotide's or polynucleotide's negative charge. The transfection reagent mediates binding of oligonucleotides and polynucleotides to cells via its positive charge (that binds to the cell membrane's negative charge) or via ligands that bind to receptors in the cell. For example, cationic liposomes or polylysine complexes have net positive charges that enable them to bind to DNA or RNA. For non-viral delivery, polynucleotides can be incorporated into lipid vesicles (liposomes), complexed with polymers (polyplexes) or a combination of lipids and polymers (lipopolyplexes).

Amphiphilic, or amphipathic, compounds have both hydrophilic (water-soluble) and hydrophobic (water-insoluble) parts. Hydrophilic groups indicate in qualitative terms that the chemical moiety is water-preferring. Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water. Examples of hydrophilic groups include compounds with the following chemical moieties; carbohydrates, polyoxyethylene, peptides, oligonucleotides and groups containing amines, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls. Hydrophobic groups indicate in qualitative terms that the chemical moiety is water-avoiding. Typically, such chemical groups are not water soluble, and tend not to hydrogen bonds. Hydrocarbons are hydrophobic groups.

Detergents or surfactants are water-soluble molecules containing a hydrophobic portion (tail) and a hydrophilic portion (head), which upon addition to water decrease water's surface tension. The hydrophobic portion can be alkyl, alkenyl, alkynyl or aromatic. The hydrophilic portion can be charged with either net positive (cationic detergents), negative (anionic detergents), uncharged (nonionic detergents), or charge neutral (zwitterionic detergent). Examples of anionic detergents are sodium dodecyl sulfate, glycolic acid ethoxylate (4 units) 4-tert-butylphenylether, palmitic acid, and oleic acid. Examples of cationic detergents are cetyltrimethylammonium bromide and oleylamine. Examples of nonionic detergents include, laurylmaltoside, Triton X-100, and Tween. Examples of zwitterionic detergents include 3-[(3-cholamidopropyl)dimthylammonio]1-propane-sulfonate (CHAPS), and N-tetradecyl-N,N-dimethyl-3-ammoniu-1-propanesulfonate.

A polymer is a molecule built up by repetitive bonding together of smaller units called monomers. A polymer can be linear, branched network, star, comb, or ladder types of polymer. A polymer can be a homopolymer in which a single monomer is used or can be copolymer in which two or more monomers are used.

The main chain of a polymer is composed of the atoms whose bonds are required for propagation of polymer length. For example in poly-L-lysine, the carbonyl carbon, .alpha.-carbon, and .alpha.-amine groups are required for the length of the polymer and are therefore main chain atoms. The side chain of a polymer is composed of the atoms whose bonds are not required for propagation of polymer length. For example in poly-L-lysine, the .beta., .gamma., .delta. and .epsilon.-carbons, and .epsilon.-nitrogen are not required for the propagation of the polymer and are therefore side chain atoms.

Other Components of the Monomers and Polymers: Polymers may have functional groups that enhance their utility. These groups can be incorporated into monomers prior to polymer formation or attached to the polymer after its formation. Functional groups may be selected from the list consisting of: targeting groups, interaction modifiers, steric stabilizers, and membrane active compounds, affinity groups and reactive groups.

A polyion (or polyelectrolyte), is a polymer possessing charge, i.e. the polymer contains a group (or groups) that has either gained or lost one or more electrons. The term polyion includes polycations, polyanions, zwitterionic polymers, and neutral polymers. The term zwitterionic refers to the product (salt) of the reaction between an acidic group and a basic group that are part of the same molecule. Salts are ionic compounds that dissociate into cations and anions when dissolved in solution. Salts increase the ionic strength of a solution, and consequently decrease interactions between nucleic acids with other cations. A charged polymer is a polymer that contains residues, monomers, groups, or parts with a positive or negative charge and whose net charge can be neutral, positive, or negative.

A polycation can be a polymer possessing net positive charge, for example poly-L-lysine hydrobromide or a histone. The polymeric polycation can contain monomer units that are charge positive, charge neutral, or charge negative, however, the net charge of the polymer must be positive. A polycation also can be a non-polymeric molecule that contains two or more positive charges.

A polyanion can be a polymer containing a net negative charge, for example polyglutamic acid. The polymeric polyanion can contain monomer units that are charge negative, charge neutral, or charge positive, however, the net charge on the polymer must be negative. A polyanion can also be a non-polymeric molecule that contains two or more negative charges.

A labile bond is a covalent bond that is capable of being selectively broken. That is, the labile bond may be broken in the presence of other covalent bonds without the breakage of the other covalent bonds. For example, a disulfide bond is capable of being broken in the presence of thiols without cleavage of any other bonds, such as carbon-carbon, carbon-oxygen, carbon-sulfur, carbon-nitrogen bonds, which may also be present in the molecule.

pH-labile refers to the selective breakage of a covalent bond under acidic conditions (pH<7). That is, the pH-labile bond may be broken under acidic conditions in the presence of other covalent bonds without their breakage.

The term polynucleotide, or nucleic acid or polynucleic acid, is a term of art that refers to a polymer containing at least two nucleotides. Nucleotides are the monomeric units of polynucleotide polymers. Polynucleotides with less than 120 monomeric units are often called oligonucleotides. Natural nucleic acids have a deoxyribose- or ribose-phosphate backbone. An artificial or synthetic polynucleotide is any polynucleotide that is polymerized in vitro or in a cell free system and contains the same or similar bases but may contain a backbone of a type other than the natural ribose-phosphate backbone. These backbones include: PNAs (peptide nucleic acids), phosphorothioates, phosphorodiamidates, morpholinos, and other variants of the phosphate backbone of native 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, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. The term base encompasses any of the known base analogs of DNA and RNA. The term polynucleotide includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinations of DNA, RNA and other natural and synthetic nucleotides.

A polynucleotide can be delivered to a cell to express an exogenous nucleotide sequence, to inhibit, eliminate, augment, or alter expression of an endogenous nucleotide sequence, or to affect a specific physiological characteristic not naturally associated with the cell. Polynucleotides can be delivered to cells to treat genetic disorders, treat acquired diseases such as cancer, induce an immune reaction (such as in vaccination or immunization), treat infectious disorders, add a new cellular function, or study gene function.

A polynucleotide-based gene expression inhibitor comprises any polynucleotide containing a sequence whose presence or expression in a cell causes the degradation of or inhibits the function, transcription, or translation of a gene in a sequence-specific manner.

Polynucleotide-based expression inhibitors may be selected from the group comprising: siRNA, microRNA (miRNA), small non-messenger RNAs (snmRNA), utRNA (untranslated), snoRNAs (24-mers, modified snmRNA that act by an anti-sense mechanism), tiny non-coding RNAs (tncRNAs), interfering RNA or RNAi, dsRNA, ribozymes, antisense polynucleotides, and DNA expression cassettes encoding the like. SiRNA comprises a double stranded structure typically containing 15-50 base pairs and preferably 19-25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure (small hairpin RNA, shRNA). MicroRNAs are small noncoding polynucleotides, about 22 nucleotides long, that direct destruction or translational repression of their mRNA targets. Antisense polynucleotides comprise sequence that is complimentary to a gene or mRNA. Antisense polynucleotides include, but are not limited to: morpholinos, 2'-O-methyl polynucleotides, DNA, RNA and the like. The polynucleotide-based expression inhibitor may be polymerized in vitro, recombinant, contain chimeric sequences, or derivatives of these groups. The polynucleotide-based expression inhibitor may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited.

The process of delivering a polynucleotide to a cell has been commonly termed transfection or the process of transfecting and also it has been termed transformation. The term transfecting as used herein refers to the introduction of a polynucleotide or other biologically active compound into cells. The polynucleotide may be used for research purposes or to produce a change in a cell that can be therapeutic. The delivery of a polynucleotide for therapeutic purposes is commonly called gene therapy. The delivery of a polynucleotide can lead to modification of the genetic material present in the target cell. The term stable transfection or stably transfected generally refers to the introduction and integration of an exogenous polynucleotide into the genome of the transfected cell. The term stable transfectant refers to a cell which has stably integrated the polynucleotide into the genomic DNA. Stable transfection can also be obtained by using episomal vectors that are replicated during the eukaryotic cell division (e.g., plasmid DNA vectors containing a papilloma virus origin of replication, artificial chromosomes). The term transient transfection or transiently transfected refers to the introduction of a polynucleotide into a cell where the polynucleotide does not integrate into the genome of the transfected cell. If the polynucleotide contains an expressible gene, then the expression cassette is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes. The term transient transfectant refers to a cell which has taken up a polynucleotide but has not integrated the polynucleotide into its genomic DNA.
 

Claim 1 of 7 Claims

1. An ortho carboxy dialdehyde consisting of a structure represented by: ##STR00001## (see Original Patent)

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