WO2010133975A2 - Reducible polyamino disulfides as transfectants - Google Patents

Abstract

The invention relates to polynucleotide transfection (e.g. of pDNA or siRNA), and in particular, the use of polyplexes (complexes of a polynucleotide and a reducible (disulfide) polymer) as delivery vehicles. Compositions of the invention contain a reducing agent, such as glutathione (GSH) or N-acetyl cysteine (NAC). These are able to reduce, and so break, S- S bonds in the reducible disulfide polymer formed form polymerisation of thiol-containing monomers. This can result in the release of the polynucleotide (e.g. pDNA or siRNA). The reducible (disulfide) polymer is preferably formed from constituent monomer(s) units having a thiol group at each end, and a middle repeating section or unit of a polyamine, such as a spermine molecule.

Description

REDUCIBLE POLYAMINO DISULFIDES AS TRANSFECTANTS

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to U.K. Application GB0908963.2, filed May 22, 2009, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions for use in the delivery and/or transfection of pharmaceutically active substances, e.g., a nucleic acid molecule of interest (NOI or polynucleotide, which terms are used interchangeably) such as DNA (including antisense oligodeoxynucleotides), RNA (and, in particular, sz-RNA), proteins, peptides, and other pharmacologically active substances intended to be delivered to cells. Such compositions comprise two or more of the following three components, namely:

- a reducing agent;

- a reducible (disulfide) polymer (or monomer(s) or oligomer(s) of 2 to 10 monomer units capable of forming said polymer);

- a (pharmaceutically) active substance (such as a polynucleotide).

The reducible (disulfide) polymer and active substance may together form a complex, such as a polyplex.

BACKGROUND OF THE INVENTION

In the art, some disulfide polymers are known to form polyplexes with polynucleotides. These polyplexes disintegrate into their constitutive parts in the reductive environment of the cell. For example, low molecular weight polypepetides based on poly-L- Lysine (PLL) incorporating 2-5 cysteine residues, are known. One polypeptide, Cys-Trp- Lysπ-Cys, was more effective than an analogue with alkyl-protected cysteines. It proved equal to PLL (150 kDa) in one cell line, and superior in another. Polypepetides based on Cys-(Lys)io-Cys oligomers have also been studied. They were oxidised without the presence of pDNA to form polymers of 187 kDa that were then used to condense pDNA.

Incorporation of histidine can allow shorter lysine sequences be used without affecting efficacy, such as in Cys-His-Lys₆-His-Cys. Derivatives of this molecule functionalised with polyethylene glycol (PEG) and sugar moieties allowed polymers for the delivery of pDNA to the livers of mice to be produced that ensured high transfection levels and presence of the transgene for up to 12 days. Further in vivo work used derivatised Cys- His-Lys₆-His-Cys with either a transglutaminase peptide sequence, to allow for its covalent attachment to a fibrin matrix, or a nuclear localisation signal (NLS).

Later, the incorporation of even more histidines into a monomer of the form Cys-His₆-

Lys₃-His₆-Cys resulted in 59 kDa polymers² that mediated transfection levels greater than those of PEI (branched, 25 kDa).

A bifunctional reversible addition fragmentation chain transfer initiator has been used to synthesise poly[2-(dimethylaminoethyl) methacrylate]s terminated at either end with thiols. These could then be oxidised to linear, higher molecular weight species. When tested in Bl 6F10 cells, the reducible polymer achieved a 3-4 fold increase in transfection upon nonreducible poly[2-(dimethylaminoethyl) methacrylate]s.

One strategy has been to place disulfide bonds at more regular intervals in the backbone of the polymer to produce a macromolecule that is degradable under reductive conditions, such as bioreducible poly(amido amine)s, synthesised via the polycondensation of N,N'-cystaminebisacrylamide with a variety of amines.

Other reducible poly(amido amine)s have employed ω- functionalised primary amines as bivalent monomers to ensure that the product has a linear architecture. Reaction with disulfide containing crosslinkers, such as dimethyl-3,3'-dithiobispropionimidate (DTBP) or dithiobis(succinimidylpropionate) (DSP) results in branched reducible polyamines, the most effective of which proved to be the equal of PEI (linear, jetPEl™) in terms of transfection across 4 cell lines tested. Other polymers which have been thiolated to allow for reducible lateral stabilisation include PEG-PLL conjugates, chitosan and α,β-poly[N-2-hydroxyethyl)-D,L-aspartamide. PLL based polyplexes with the crosslinker DTBP retained the transfection efficacy of PLL up to a 1 :1 ratio of cross-linker to primary amines.

A reducible version of linear PEI has been synthesised via the functionalisation of oligoethylenimines with thiol tips followed by the oxidation of the resulting dithiols to form polymers with molecular weights of between 11 and 20 kDa.¹ Their most effective vector was able to transfect the two cell lines tested to within an order of magnitude of PEI (linear, 22 kDa, ExGen 500) and cytotoxicity was greatly reduced. The reducible polymers remained non-toxic up to a concentration of 100 μg/mL, whereas PEI virtually ablated cell viability at 20 μg/mL. Reducible polymers rapidly disintegrated upon cell uptake, in contrast to PEI, as evidenced by self-quenching dyes conjugated each to both the reducible polymer and PEL These results were obtained for free polymer, rather than the relevant polyplexes, and it may be that differences exist in uptake and trafficking as well as their susceptibility to degradation that might mean that these results would not be extended to a reducible polyplex.

Transfection of anionic nucleic acids can require efficient carrier systems. Cationic polymers have proven an impressive means of carriage in vitro but their utility in vivo is limited by cytotoxicity. Nevertheless, various forms of the cationic polymer polyethyleneimine (PEI) have been used in in vivo experiments and in Phase I clinical trials. Linear polyethylenimine (commercially avialable asyetPEI™ from Polyplus) is regarded as one of the most effective synthetic vectors currently available. However, more effective vectors are still desired, with lower toxicity.

SUMMARY OF THE INVENTION

The present invention relates to formulations for the delivery of a phramaceutically active agent to a cell or a target tissue of interest, as well as to a method for delivering a pharmaceutically active agent using the inventive composition. "Composition" and "formulation" are used interchangeably to connote a solid, a solution, or a dispersion of a pharmacetically active agent, including but not limited to a nucleic acid molecule (see below), in a suitable physiologically acceptable carrier. Pharmaceutical compositions can contain other agents, such as stabilizers, colorants, salts, and other pharmaceutically acceptable excipients.

According to a first aspect of the invention there is thus provided a composition comprising:

a) a reducible (disulfide) polymer (or monomer(s) or oligomer(s) of 2 to 10 monomer units capable of forming (such as by polymerising) said polymer); and

b) a reducing agent.

A second aspect of the invention relates to a composition (or product) comprising:

a) a reducible (disulfide) polymer (or monomer(s) or oligomer(s) of 2 to 10 monomer units capable of forming (such as by polymerising) said polymer); and

b) a reducing agent;

(as a combined preparation) for simultaneous, separate or sequential therapy (of a disease or disorder or other medical condition).

Such composition(s) may optionally also comprise: a) an active (such as a therapeutic or pharmaceutical) substance, such as a polynucleotide.

In the second aspect, the reducing agent is suitably provided to a patient or individual first. In other words, it is preferred that delivery of the active substance takes place after delivery of the reducing agent, either by separate administration, or, for example, by use of a delayed release formulation. Thus, the present invention contemplates the use of a nebulizer or a pressured metered dose inhaler (MDI), for delivery to the lungs of a composition as described here. Formulations suitable for the sequential delivery of reducing agent and active pharmaceutical to the lungs can be in powder form or in the form of a solution that is aerosolized during delivery. For sequential delivery, a suitable time interval between administration of the reducing agent and administration of the active pharmaceutical is in the range of 0.1 hour to 2 hours.

The invention (in a third aspect) further relates to (the use of) a composition of the first and/or second aspect (for use) in medicine, as a delivery vehicle, or as a transfection or transportation agent, for example across a membrane, or for the treatment of a human or animal body. This aspect of the invention additionally relates to the composition for use in administration of polynucleotides, for example in gene or RNAi therapy, or for enhancing the penetration of, for example, the active substance.

The invention additionally relates, in a fourth aspect, to a method of delivering an active substance, by administering the composition(s) of the invention, or indeed administering two or more of:

a) a reducing agent;

b) a reducible (disulfide) polymer (or monomer(s) or oligomer(s) of 2 to 10 monomer units capable of forming said polymer); or

c) an active substance. The invention further relates to a composition of any previous aspect for use (in the manufacture of a medicament for):

- as a delivery vehicle, or as a transfection or transportation agent, or for use in administration of polynucleotides, for example in gene or RNAi therapy, or to enhance penetration of a therapeutic agent; or

- to treat a disease or disorder or any other medical condition.

The invention additionally relates to a composition comprising:

a) a reducing agent; and

b) a complex (for example, a polyplex) of a polynucleotide and a (reducible) polymer (or monomer(s) or oligomer(s) of 2 to 10 monomer units capable of forming said polymer).

The invention additionally provides a complex of a polynucleotide and the reducible polymer (such as a polyplex). Such a complex can be used as a delivery vehicle, as a transfection or transportation agent, and can have the same uses as described above for the compositions of the invention.

The present invention can thus also provide a (condensed and/or packaged) polymer/polynucleotide complex, comprising a polymer and polynucleotide, for use in delivering the polynucleotide to (the cytosol or nucleus of) a eukaryotic cell, wherein

(i) the polymer is a reducible (disulfide) polymer (or monomer(s) or oligomer(s) of 2 to 10 monomer units capable of forming said polymer);

(ii) the polymer is capable of binding to the polynucleotide; and

(iii) the polymer is capable of condensing and/or packaging the polynucleotide and, preferably, wherein the polynucleotide is heterologous to the polymer. The present invention also provides a process of producing a (non- viral), polynucleotide delivery vector or vehicle, comprising a condensed and/or packaged polymer/polynucleotide complex, and optionally a reducing agent, which comprises:

a) optionally, forming a reducible (disulfide) polymer with one or more (e.g. dithiol) monomers or oligomers of 2 to 10 monomer units;

b) contacting a nucleic acid sequence of interest (NOI), with said polymer (or monomer(s) or oligomer(s) of 2 to 10 monomer units capable of forming said polymer), said polymer being (i) capable of binding to the NOI; and (ii) capable of condensing or packaging the NOI; and wherein the NOI is heterologous to the polymer; and

c) optionally, contacting the polymer/polynucleotide complex thus formed with a (cationic) lipid and/or a moiety comprising one or more functional groups that can be coupled to one or more functional groups present in the reducible (disulfide) polymer.

Thus, once formed, the condensed and/or packaged polymer/polynucleotide complex may be further coupled to other moieties, such as targeting moieties. Such coupling may be via reaction between one or more functional groups present in the polymer and complementary functional groups of the additional moiety. One example of such a "post coupling" strategy is aminoxy coupling, as described in WO 02/48170.

As noted above, the present invention further provides a method for introducing a nucleic acid molecule into a eukaryotic cell. The inventive method comprises (A) providing a polyplex that is the product of contacting a polycationic reducible disulfide polymer with a polynucleotide and (B) delivering said polyplex to a target tissue in a subject such that said polyplex comes into contact with a reducing agent at said target tissue, so as to cause reductive degradation of the polyplex to release the polynucleotide in the target tissue. Preferably, the target tissue is mucosal, epithelial, lung tissue, neural or cancer cells, more preferably a lung or mucosal epithelial cell.

Thus, the present invention relates to a non-viral delivery vector (or vehicle) comprising a reducible (disulfide) polymer that is coupled, reversibly or irreversibly, to one or more polynucleotide(s). Illustrative of such polynucleotides are those in the categories of DNAs, RNAs, DNA/RNA hybrids, antisense oligonucleotides, siRNAs, and /m-RNAs.

The present invention also provides a method for delivering to a cell a polynucleotide, such as an siRNA, the method comprising the step of providing to the environment of a cell, tissue, or organ the non-viral delivery vector that includes the polyplex and a reducing agent.

The aspect of the present invention also relates to a process for preparing a non- viral delivery vector or polyplex, wherein one or more polynucleotide(s) are coupled, reversibly or irreversibly, to one or more reducible (disulfide) polymer(s), optionally first formed from one or more monomer(s) or oligomer(s) of 2 to 10 monomer units capable of forming said polymer.

The present invention also encompasses a pharmaceutical composition comprising the polymer, reducing agent and/or active substance, and a pharmaceutically acceptable carrier or diluent. The present invention is advantageous since it can inter alia provide a method for delivering siRNA using non-viral mediated methods.

According to another aspect, the invention provides an assemblage for use in non- viral delivery of polynucleotides of interest. The inventive assemblage comprises a polyplex, formed by contacting the polynucleotide with a polycationic reducible disulfide polymer, and a reducing agent. The assemblage can be such that the polyplex and reducing agent are separated, one from the other, or that they are together in formulation (see below). Formulations of the polyplex and reducing agent can be provided in particulate form, preferably as nanoparticles suitable for targeting and entering lung and mucosal tissue. The present invention additionally relates to a method, or composition substantially as described herein, and with reference to any one of the Examples or Figures.

Reducing Agent

The reducing agent may be an antioxidant (e.g. glutathione (GSH) or uric acid or N- acetylcysteine or alpha-lipoic acid or a vitamin, such as ascorbic acid (vitamin C) or vitamin E (such as alpha-tocopherol)), a radical scavenger, and/ or an agent with a protective effect.

Preferably, such an agent will have one or more reducing group(s). It may be a mucolytic agent, inflammation inhibitor and/or may be a chemoprotective agent. The reducing agent may also act to eliminate or reduce oxidative stress.

Preferably the agent will comprise one or more thiol (or, sulfhydryl, -SH, the terms are used interchangeably) groups. Suitably it will be able to reduce, for example, the (disulfide) polymer employed in the invention. Examples of thiol-containing reducing agents include dithiothreitol or precursors thereof, such as dithiothreitol tetra-acetate, ammonium thioglycolate and thioglycolic acid (TGA) and derivatives thereof. The reducing agent can be a polythiol, for example, poly(D-glucosamine)-cysteine, or a carbomer-cysteine. Further examples are provided below.

The reducing agent may be a polypeptide. Preferably the reducing agent is a mono-, di- or tri-peptide, or an analogue thereof.

Preferably the reducing agent will comprise (one or more) cysteine (Cys) group(s) or residue(s).

Preferably, the reducing agent has the following formula (I):

^(I)

wherein:

Ri represents H; acetyl; Ci_-6 alkyl; C_2-6 alkylene; Ci_-6 alkoxy; C_6-)o aryl; a 5- to 10- membered heteroaryl group; acyl or mercaptoacyl (e.g. of the formula X-R₄-C(O)-, wherein R₄ is a linear or branched lower alkyl residue, e.g. Ci_-4, e.g. C₂ or C₃, and X is H or SH; a residue of an alpha amino acid (for example a monopeptide residue), such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine; or a polyamide residue (e.g. a polypeptide residue, such as a di- or tri- peptide residue) comprising such amino acids; R₂ is OH; Ci_-6 alkyl; C_2-6 alkylene; Ci_-6 alkoxy; C_6-I0 aryl; a 5- to 10- membered heteroaryl group or a residue of an alpha amino acid such as those mentioned above, or a polyamide residue (e.g. a polypeptide residue, such as a di- or tri- peptide residue) comprising such amino acids;

R₃ is H; C_1-6 alkyl; C_2-6 alkylene; Ci_-6 alkoxy; C_6-I0 aryl; a 5- to 10- membered heteroaryl group; an optionally S-substituted cysteine residue, wherein the optional substituent may be a fatty saturated or unsaturated acid radical or a radical of an aromatic acid (e.g. benzoic acid), or their salts; optionally substituted benzyl (e.g. substituted by a lower (suitably C_2-8) alkoxycarbonyl group or a carboxy group), Ci_-6 hydroxyalkyl, allyl or propargyl; and n represents an integer of 1 to 10, preferably 1 to 5, suitably 1 or 2.

Preferably, Ri is H, acetyl or a residue of cysteine, glutamic acid, glutamine or glycine, preferably glutamic acid.

Preferably, R₂ is OH or a residue of cysteine, glutamic acid, glutamine or glycine, preferably glycine. In a preferred embodiment, R₃ is H, i.e. the reducing agent is of formula (Ia):

In formula (Ia), Ri, R₂ and n have the same definitions as above. Preferably, Ri is H, acetyl or glutamate (preferably such that it forms a peptide bond with its gamma carboxyl group). Preferably, R₂ is OH or glycine. Preferably n is 1.

In one preferred embodiment, the compound of formula (Ia) is cysteine, glutathione or

N-acetyl cysteine, more preferably N-acetyl cysteine.

The reducing agent may comprise glutathione (GSH) or glutaredoxin(s) or thioredoxin(s), or precursor or analogues thereof, cysteine or an analogue thereof, or preferably N-acetyl cysteine (which is a precursor to GSH) or a precursor or analogue thereof.

Without wishing to be bound by theory, it is thought that the reducing agent is able to reduce the disulfide polymer, thereby resulting in less tight binding or release of the polynucleotide to which it may be complexed. Surprisingly, addition of a reducing agent extracellularly can lead to an increased uptake and delivery into the cell. This can mean that the active agent, such as a polynucleotide, can be delivered more effectively. The reducing agent may therefore enhance the penetration of the complex of the disulfide polymer and active agent.

Preferably, the reducing agent is administered to an individual (or patient or (human or animal) subject - the terms are interchangeable) prior to the (disulfide) polymer. Such a polymer may be in the form of a complex (such as a polyplex) with a polynucleotide.

The reducing agent may be administered up to one hour, optionally 4, 6, 8, 10 or 12 hours, before the active agent/therapeutic substance is administered. Preferably, administration is approximately 30 minutes, such as 20 or 10 minutes, before administration of the active agent. Alternatively, the polymer and reducing agent may be administered at the same time but separately, or they may be administered with delayed release for one or the other of these components (e.g., the polymer). The administration may be to a mucosal membrane, for example, intranasally via inhalation, or sublingually. Typically, the reducing agent is formulated either as a dry powder, for use with a dry powder inhaler (DPI), or is dissolved in a suitable solvent for delivery to the lungs as aerosolized particles, using a nebulizer or a metered dose inhaler (MDI) (see below).

Reducible (disulfide) Polymer

The reducible (disulfide) polymer suitably comprises 2 (or more) sulfur atoms. In particular, the constituent monomer units may comprise 2 (or more) sulfur atoms. The polymer may comprise one or more (e.g. terminal) thiol (-SH) groups. The polymer (or indeed the monomer(s) or oligomer(s) from which the polymer is formed) may be linear, or cyclic. The polymer is suitably a non-peptide, but if it is a peptide, it preferably does not contain any disulfide bridges (-S-S-).

Preferably the polymer (or its constituent monomer units) comprises one or more amines, for example one or more secondary (and/or tertiary) amines. Suitably each monomer unit comprises between 1 and 15 (secondary and/or tertiary) amines, suitably, between 2 and 8, for example 2, 3, 4 or 5 amines. One or all of these amines may be secondary and/or tertiary amines.

Suitably a thiol group (-SH), or a salt thereof, will be present at both ends, or termini, of the polymer.

Preferably the polymer will have a middle section, such as repeating unit(s). Such repeating units can comprise a (preferably secondary or tertiary) amine group. Suitably the middle section comprises a spermine or putrescine molecule. The polymer will preferably contain one or more reducible disulfide linkages or bonds (-S-S-). One or more of these linkages may degrade in a reducing environment.

The polymer is preferably able to associate with, and/or condense, DNA or another polynucleotide.

Preferably, the constituent monomer will have 6 to 50, preferably 6 to 30, carbon atoms. In one embodiment, both polymer and monomer will have no oxygen atoms.

The polymer should preferably be GRAS (generally recognised as safe), and can therefore act as a (preferably non-toxic) transfection agent.

Suitably, the (resultant) polymer has a molecular weight from 10,000 to 30,000, such as from 11,000 to 25,000, preferably from 12,000 to 18,000.

Preferably, the polymer is able to enter a cell by endocytosis, for example through the endosome.

Preferably the polymer contains one or more disulfide bonds, usually formed between two thiols (-SH) by an oxidation reaction. Disulfide bonds can be (relatively easily) cleaved by a reducing agent, forming two thiols. Thus, it is preferable that the reducing agent, which itself can comprise a thiol, is able to reduce disulfide bonds present in the polymer.

The polymer of the invention comprises constituent monomer units. It may be formed from the monomer(s) corresponding to such units, or from oligomer(s) of 2 to 10 monomer units. Suitably, the monomer(s) or oligomer(s) comprise at least two thiol groups.

Suitably the monomer(s) or oligomer(s) have the following chemical formula II:

wherein: p is an integer of from 1 to 10, such as 1 to 5, for example 1 or 2; each n and m individually represents an integer of from 1 to 10, such as from 1 to 5, preferably 2; each y is individually an integer of from 1 to 10, preferably 2 to 8, suitably 2, 3 or 4, and each repeating unit y is the same or different, wherein in each unit y:

x is an integer, preferably of from 1 to 10, preferably 2, 3 or 4;

A is a disulfide group (-S-S-) and a is an integer chosen from 0 and 1 ;

- each B is -CHR'- and each b is an integer of from 0 to 10, preferably from 0 to 4, suitably 1 or 2;

each C and D are independently -C(=O)- and each c and d are independently an integer chosen from 0 and 1 ; each R and R' are independently chosen from H, Ci_-6 alkyl; C_2-6 alkylene; C_2-6 alkynyl, Ci_-6 alkoxy; C_6-I0 aryl; a 5- to 10- membered heteroaryl group; Ci_-6 aldehyde group, Ci_-6 ketone group, Ci_-6 carboxamide (aminoxy) group, Ci_-6 alkylamine or Ci_-6 alkyl thiol.

The repeating unity may thus comprise any of the following formulae (III) to (VI):

wherein each of R, R' and x are as defined above, and: in formula (III), a, b, c and d are all 0; in formula (IV), a is 1 , b is an integer of 1 to 4, and c and d are 0; in formula (V), a, b and d are 0 and c is 1 ; in formula (VI), a, b and c are 0 and d is 1.

In the above formulae (II) to (VI):

Preferably, R is H.

Preferably, R' is H.

In another embodiment, R and R' are H, with the proviso that at least one R or R' is Ci_-6 alkylthiol.

Preferably, a is 0.

In one embodiment, a, b, c and d are each zero.

In a preferred embodiment, a, b, c and d are zero and R is H, so that the polymer can be represented by the following formula (Ha):

wherein n, x, y, m and p are as defined above, namely: p is an integer of from 1 to 10, such as 1 to 5, for example 1 or 2; each n and m individually represents an integer of from 1 to 10, such as from 1 to 5, preferably 2; each y is individually an integer of from 1 to 10, preferably 2 to 8, suitably 2, 3 or 4, and each repeating unit y is the same or different, wherein in each unit y, x is an integer, preferably of from 1 to 10, preferably 2, 3 or 4; Preferred values of n, x, y, m and p are, independently, as follows: n is 2; x is 2, 3 or 4; y is from 1 to 8, preferably 2 or 3; p is 1 or 2.

In one embodiment, each x is 2.

In another, preferred, embodiment, y is greater than or 1, e.g. 2 or 3, and each x may be the same or different (preferably different) and is an integer chosen from 2, 3 and 4.

In the case where p is 1, the above formulae (II) and (Ha) also represent the preferred constituent monomer units of the polymer of the present invention.

The reducible (disulfide) polymer may thus comprise a polyamine, as a repeating unit, such as of the types described in WO 97 045442. These (polyamines) may comprise:

Putrescine: H₂N-(CH₂)₄-NH₂

Spermine: H₂N-(CH₂)₃-NH-(CH₂)₄-NH-(CH₂)₃-NH₂

CDAN-PA: H₂N-(CH₂)₂-NH-(CH₂)₃-NH-(CH₂)₂-NH₂

Norspermidine: H₂N-(CH₂)₃-NH-(CH₂)_3-NH₂

Norspermine: H₂N-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)_3-NH₂

Caldopentamine: H₂N-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)_3-NH₂

Caldohexamine: H₂N-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)_3-NH₂

Such polyamines (PA) may comprise, or be, the middle section of the reducible

(disulfide) polymer or the polymer may be derived therefrom. Putrescine, spermine and CDAN-PA (3,7-Diazanonane-l,9-diamine) are preferred polyamine moieties. Polyamines entirely composed of aminopropyl moieties are typically produced under stress conditions (hot springs, desert plants).

Spermine, normally found in millimolar concentrations in the nucleus, can act as a free radical scavenger and is capable of protecting intracellular DNA from free radical attack.

Formation of Reducible (Disulfide) Polymer

The polymer may be formed from a single type of monomer (or oligomer of 2 to 10 monomer units). Alternatively, a copolymer of two or more different monomers (or oligomers) may be formed. Each monomer or oligomer may suitably be of formula (II) or (Ha) defined above. The monomers or oligomers may be cyclic or linear.

Where two or more monomers (or oligomers) are used, at least one of these could comprise three or more thiol groups, which may introduce branching into the polymer. For example, the polymer could be formed from a mixture comprising a first kind of monomer (or oligomer) comprising two thiol groups and a second kind of monomer (or oligomer) comprising three (or more) thiol groups, for example in proportions of 90% di-thiol and 10% tri-thiol.

When forming the polymer, in one embodiment "chain stoppers" may be added in order to terminate the polymer chain, for example to control or regulate the molecular weight of the polymer. Such chain stoppers may be a molecule (e.g. a monomer, oligomer or polymer) having a single thiol group, or even a reducing agent as described above comprising a thiol group (for example added in low concentration).

Design of Suitable Monomers for reversible oxidative Polymerisation

The present invention encompasses nucleic acids of interest (NOI), peptides/proteins or other pharmacologically active substances (referred to hereafter as 'payload') encapsulating polymers that are oxidised and stable upon storage and during the act of delivery, but that are reduced and disintegrating at the point of delivery to the cells of interest. Both the cytosol and the nucleus of a cell are strongly reducing environments due to the high concentrations (about 5mM) of glutathione (GSH) and GSH-dependent enzyme systems maintained therein. In contrast, the extra-cellular milieu is non-reducing.

Reduction, oxidation, and isomerization of protein disulfide bonds in the cytosol and endoplasmic reticulum (ER) of eukaryotic cells are carried out by enzymes of the thioredoxin family. Thioredoxin and glutaredoxin catalyze reduction of disulfide bonds in the cytosol and nucleus. These enzymes use reduced co factors (e.g., glutathione) as electron donors for the reduction of protein or other disulfide bonds. Therefore, disulfide bonds should remain relatively stable, until exposed to the reducing environment of the cytosol.

The expectation was that reductive degradation of the payload-encapsulating agent would lead to the controlled release of the payload to the cytosol or nucleus, thereby potentially improving the efficacy of transfection or pharmacological effect of the delivered substance(s), but also leading to the formation of polymer fragments that should be easier to metabolise than their polymeric analogues, and hence be potentially less damaging to cells. In particular, the basic polymer- forming repeat units may be based on naturally occurring polyamines (PAs), polycations capable of neutralising the charge on NOIs and negatively charged (anionic) pharmaceutically activate substances that, owing to their charge, would be unable to cross membranes.

The polymer may be pre-formed by oxidation of free thiol groups before bringing it into contact with the payload, or (oxidative) polymer formation may occur in the presence of the payload. Typically, for formation of the polymer-payload complexes, lyophilised mercaptoethyl polyamine derivatives will be dissolved in adequate aqueous buffer and polymerisation will start by molecular oxygen naturally dissolved in that buffer. In general, polymerisation can be accelerated by leading a stream of oxygen through the aqueous buffer or by using DMSO. Monomers with two (terminal) thiol groups can form linear polymers only. Addition of monomers with three (or more) thiol groups can laterally connect linear polymers. Addition of a thiol-containing reducing agent can act as chain stopper. Combination of these elements allows highly individual, tailor-made polymer networks to be assembled, such as by use of different types of linear or branching monomers, addition of chain stoppers, addition of oxygen, control of time and temperature, addition of counter-ions, pre- formation vs. formation in the presence of payload, etc.

Condensation and packaging of anionic payload, such as NOI, through interaction with the polymer is an entropy driven process, as electrostatic interaction between these two types of (macro)molecules allows counter-ions to be released into solution; and the larger the molecules involved, the stronger the effect is expected to be.

As an example, eight disulfide monomers, which then can polymerise to disulfide polymers, are shown below. All can be used to make efficacious nucleic acid therapy vectors and/or can be used to prepare a reducible polymer (by a suitable reducing agent).

(a) HS-(CH₂)₂-NH -(CH₂)₄-NH-(CH₂)₂-SH

(di-mercaptoethyl putrescine, 1)

(b) HS-(CH₂)₂-NH -(CH₂)₂-NH-(CH₂)₃-NH-(CH₂)₂-NH-(CH₂)₂-SH

(di-mercaptoethyl 3,7-Diazanonane-l,9-diamine, 2)

(c) HS-(CH₂)₂-NH -(CH₂)₃-NH-(CH₂)₄-NH-(CH₂)₃-NH-(CH₂)₂-SH

(di-mercaptoethyl spermine, 3)

(d) HS-(CH₂)₂-NH-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₂-SH

(di-mercaptoethyl norspermidine, 4)

(e) HS-(CH₂)₂-NH -(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₂-SH

(di-mercaptoethyl norspermine, 5) (f) HS-(CH₂)₂-NH -(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₂- SH

(di-mercaptoethyl caldopentamine, 6)

(g) HS-(CH₂)₂-NH -(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₃-NH-(CH₂)₃- NH-(CH₂)₂-SH

(di-mercaptoethyl caldohexamine, 7)

(h) HS-(CH₂)₂-NH-CH₂)₃-NH-CH₂)₃-NH-CH₂)₃-NH-CH₂)4-NH-(CH₂)₂-SH

(di-mercaptoethyl homocaldopentamine, 8)

(i) HS-(CH₂)₂-NH- CH₂)₃-NH- CH₂)₃-NH- CH₂)₄-NH- CH₂)₃-NH-(CH₂)₂-SH

(di-mercaptoethyl thermopentamine, 9)

One synthesis of disulfides can be based on commercially available polyamines.

Briefly, [(4-methoxybenzyl)sulfanyl]acetaldehyde can be obtained in 2 steps from the reaction of mercaptoethanol with-methoxy-benzylmercaptan. [(4-methoxybenzyl)sulfanyl] acetaldehyde can subsequently be used to selectively functionalise the terminal primary amines with protected thiol tips via reductive amination (Gordon et al., J. Med. Chen. 1988, 31, 2199-2211). Removal of the 4-methoxybenzyl protecting groups can be carried out in refluxing trifluoroacetic acid (TFA) to yield the monomer(s) with free thiol groups.

The monomers can subsequently be re-dissolved in adequate buffer and oxidised in a stream of oxygen and/or with DMSO to start the polymerisation process. Oxidation may further be accelerated in the presence of a solid state interface with a high oxygen binding capacity, such as charcoal. Depending on the reaction conditions, the concentration of the monomers, and their size, formation of oligomers (n=2-10) and/or intramolecular cyclisation may be favoured. Oxidation may be carried out in the presence or absence of the poly-anionic payload. It is known in the art that the presence of polynucleotides can serve as a template to assist the formation of a polymer network. This polymer formation may start with preformed oligomer units.

Thiol-disulfide exchange (disulfide reshuffling) is known to occur at rapid speed but requires the free thiol group to be deprotonated to a thiolate anion. Typical thiol pKa values are of the order 8.3, so that this reaction would require enzymatic assistance to occur at neutral pH. However, the high polyamine density (protonated amine pKa values, such as for spermine, are in the range 8-10) in the forming polymer and, when present, reducing agents may help such reshuffling to also occur at neutral pH. Thus disulfide reshuffling may be a process that can assist formation of polymers with induced fit to the template.

Uptake and mechanisms of action

It is thought that there are a number of different effects in the uptake of a polynucleotide, for example in a mucosa, especially if they are condensed or encapsulated by a disulfide polymer. Reduced polymers with free thiol groups may facilitate mucosal attachment and penetration of the polymer, albeit possibly with the loss of less tightly condensed or enclosed polynucleotides. Oxidised polymers, on the other hand, are likely to ensure (maximum) polynucleotide condensation or encapsulation, but possibly with a lower degree of either membrane attachment, or membrane transmigration of the polymer. The present invention concerns polymers which may have bioadhesive and biodegradable properties.

It is thought that the reducing agent, for example a thiol group-containing agent, administered at the same time, before or after the polymer administration, could thus be used to optimise uptake and transfection

The composition may be a delayed release formulation. Alternatively or additionally, the polymer (or constituent monomers) and or polyplex may be kept separate from, or not in contact with, the reducing agent (but in the same composition). This is so that the reducing agent may come into contact with the polymer or polyplex later, such as after administration. This may prevent the reducing agent contacting (and so reducing) the polymer, for example, prior to administration.

It is thought that the thiol groups may facilitate attachment, for example to mucous membranes. They may react with disulfide or thiol groups, open epithial cell tight junctions, or cause redox changes in surface proteins (which may result in associated conformational changes). For example, the inhibition of efflux pumps, that remove substances to be delivered out of the cell and thus impede their uptake, is known. Efflux pump mechanisms can operate at the blood brain barrier to remove substances that have leaked through said barrier.

The oxidation of thiol groups may lead to the formation of disulfide bonds, which can be an important element of stabilising the three-dimensional structure of a protein (as they may link different parts of the protein backbone on the same or separate chains). The formation of disulfide bonds can also be used in the assembly of polymers, from thiol containing monomers. Thus, the disulfide polymers of the invention can provide a useful means for the delivery of polynucleotides to the cytosol (such as siRNA, antisense oligonucleotides, micro RNA or nucleus DNA).

The polynucleotide may be encapsulated in the reducible (disulfide) polymer, which may provide a stabilised network. The polymer may provide for condensation and/or delivery of the polynucleotide, for example to the cell. It is thought that the polymer (which may form a network) may be broken down by the reducing environment in the cytosol of the target cell. This may result in the release of the polynucleotide.

The disulfide polymer may therefore be used as a non-viral carrier, because the presence of disulfide linkages may favourably influence gene delivery properties for example increasing polynucleotide binding ability, enabling removal of shielding of "stealth" (PEG) groups, fine tuning of the buffer capacity for enhanced endosomal escape and/or improving carrier-unpacking, and furthermore decreasing cytotoxicity. For example, deshielding and DNA unpacking may be effected by the reducing environment, for example in the cytosol and/or nucleus. This reducing capacity is often provided by glutathione (GSH).

One of the preferred reducing agents of the invention is N-acetyl cysteine (NAC). This can be provided in both oral or inhaled formulations. By providing oral NAC, for example in advance of the polymer, it may help to replenish or boost the internal GSH pool, such as in the epithelial lining fluid (ELF) of the lung or other mucosa, by increasing the intracellular amount of GSH and thus increasing its secretion into the ELF, particularly in disease (e.g., sickle cell anaemia) and intoxication states. By providing inhaled NAC, for example in advance of the polymer, it may help to directly support the reductive action of GSH.

It is preferred that the reducing agents of the invention, such as NAC, may allow for rapid membrane penetration, for example from the gastric fluid by passive diffusion. NAC may therefore act as a substitute, for example for cysteine, and may increase the levels of cysteine available for the formation of cysteine disulfide dimers (cystine), which can then be taken up into cells by a cystine/glutamate transporter system for the production of intracellular glutathione.

The presence of the reducing agent, such as GSH, or NAC as mentioned, in the ELF may lead to a reduction in the number of disulfide bonds. This may itself result in a loosening of the polymer network, and thus support uptake of the polynucleotide and subsequent escape from early endsomes (as the low pH in the late endosome may impede and uncatalysed disulfide reduction by GSH).

The reducible polymer may therefore be first complexed with the polynucleotide, to form a polyplex. This may be taken up by the cell, for example by endosomal uptake. Once in the cell, reductive disintegration of the polymer can take place that can result in release of the polynucleotide. The polynucleotide can then pass into the cytosol or into the nucleus.

Thus, preferably the polymer is bioreducible, in other words it can be reduced once inside the cell. The polymer can be triggered to release of the polynucleotide, such as siRNA, from the complex or polyplex, for example in the reducing environment of the cytosol. In this way, siRNA can be directed towards the RNA-induced silencing complex (RISC), which occurs mainly in the cytoplasmic space. This can allow the delivery of the siRNA (or other polynucleotide) to the RISC, preferably to achieve the desired RNAi activity. Thus, a timely and efficient unpacking of the polymer/polynucleotide complex, and the release of the polynucleotide from the complex or polyplex, can be achieved.

Condensing of polynucleotide

At first glance, siRNA and double-stranded DNA (dsDNA) share many common properties. They are both double-stranded polynucleotides. They each have anionic phosphodiester backbones with the same negative charge to nucleotide ratio, and both can interact electrostatically with cationic agents. Upon a deeper look at the fundamental aspects of siRNA and plasmid DNA delivery, however, it is clear that there are several key differences which make the two phenomena distinct.

The pDNAs used in gene therapy, are often several kilobase pairs long. They may possess a molecular topography which allows them to be condensed into (small, nanometric) particles e.g. when complexed with a cationic agent. This condensation, driven by electrostatic attractions, can ensure that pDNA can be entirely encapsulated and may protect against enzymatic degradation The persistence length (the length scale over which the chains behave as rigid rods) of dsDNA is about 50 nm, and that of double-stranded RNA is about 70 nm, making RNA a stiffer molecule. At a rise per base pair value of 2.1 K, one persistence length for RNA is about 260 bp. RNA segments shorter than this value may essentially behave as rigid rods, suggesting that 21 bp siRNAs are not likely to further condense. Thus instead these types of polynucleotide may simply be "packaged" with the polymer of the invention. The flexibility of the polymer system of the present invention allows the use of these different types of polynucleotide to be accommodated, by tailoring for the condensation or packaging required when the polynucleotide is DNA or siRNA, respectively.

Polynucleotides and nucleic acid sequences of interest (NOI)

Nucleic acid sequences of interest (NOIs, or polynucleotides) intended to be delivered to cells using the composition or complex of the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. Thus, NOIs may comprise DNA, for example plasmid DNA (pDNA) or antisense oligodinucleotides (ODNs), or RNA, for example siRNA or miRNA, or DNA/RNA hybrids, or chemical modifications thereof.

Thus, they may be polynucleotides which include within them synthetic or modified nucleotides, number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3 'and/or 5'ends of the molecule. The polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of the NOIs.

Preferably the polynucleotide is other than a plasmid, for example other than a luciferase expression plasmid, or a plasmid comprising a luciferase gene, or part thereof.

The NOI may comprise a heterologous gene. The term "heterologous gene" encompasses any gene. The heterologous gene may be any allelic variant of a wild-type gene, or it may be a mutant gene. The term "gene" is intended to cover polynucleotide sequences which are capable of being at least transcribed. Thus, sequences encoding mRNA, tRNA and rRNA, as well as antisense constructs, are included within this definition.

Polynucleotides may, for example, comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogues thereof. Sequences encoding mRNA may optionally include some or all of 5' and/or 3' transcribed (but untranslated) flanking sequences naturally, or otherwise, associated with the translated coding sequence. It may optionally further include the associated transcriptional control sequences (normally associated with the transcribed sequences), for example transcriptional stop signals, polyadenylation sites and/or downstream enhancer elements.

The transcribed sequence of the heterologous gene is preferably operably linked to a control sequence permitting expression of the heterologous gene in mammalian cells, preferably neuronal cells, such as cells of the central and peripheral nervous system, cancer or epithelial cells. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence can be ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.

The control sequence comprises a promoter allowing expression of the heterologous gene and a signal for termination of transcription. The promoter can be selected from promoters which are functional in mammalian, preferably human cells. The promoter may be derived from promoter sequences of eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression of the heterologous gene is to occur, preferably a cell of the mammalian central or peripheral nervous system. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of β-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter or promoters of herpes virus genes.

It may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell.

Inducible means that the levels of expression obtained using the promoter can be regulated. In addition, any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above. Furthermore, the use of locus control regions (LCRs) may be desirable.

The heterologous gene will typically encode a polypeptide of therapeutic use. In accordance with the present invention, suitable NOI or polynucleotide sequences include those that are of therapeutic and/or diagnostic application such as, but are not limited to: sequences encoding cytokines, chemokines, hormones, antibodies, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumour suppressor protein, growth factor, membrane protein, vasoactive protein, anti- viral 1 protein, and derivatives therof (such as with an associated reporter group).

Examples of polypeptides for therapeutic use include neurotrophic factors such as nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BNTF) and neurotrophins (such as NT-3, NT-4/5) which have potential as therapeutic agents for the treatment of neurological disorders such as Parkinson's disease.

Suitable polynucleotides for use in the present invention in the treatment or prophylaxis of cancer include NOIs encoding proteins which: destroy the target cell (for example a ribosomal toxin), act as: tumour suppressor (such as wild-type p53) ; activator of an anti-tumour immune mechanism (such as a cytokine, co-stimulatory molecule and/or immunoglobulin) ; inhibitor of angiogenesis ; or which provide enhanced drug sensitivity (such as a pro-drug activation enzyme) ; indirectly stimulate destruction of target cell by natural effector cells (for example, strong antigen to stimulate the immune system or convert a precursor substance to a toxic substance which destroys the target cell (for example a prodrug activating enzyme). An encoded protein could also destroy bystander tumour cells (for example with secreted antitumour antibody-ribosomal toxin fusion protein), indirectly stimulated destruction of bystander tumour cells (for example cytokines to stimulate the immune system or procoagulant proteins causing local vascular occlusion) or convert a precursor substance to a toxic substance which destroys bystander tumour cells (eg an enzyme which activates a prodrug to a diffusible drug).

Polynucleotide may be used which encode an antisense transcript or ribozyme (which can interfere with the expression of a cellular or pathogen genes), for example, with expression of cellular genes for tumour persistence (for example against aberrant myc transcripts in Burkitts lymphom or against bcr-abl transcripts in chronic myeloid leukemia. The use of combinations of polynucleotides is also envisaged.

Instead of, or as well as, being selectively expressed in target tissues, the polynucleotide may encode a pro-drug activation enzyme, which may have no significant effect or no deleterious effect until the individual is treated with one or more pro-drugs upon which the enzyme or enzymes act. In the presence of the active polynucleotide, treatment of an individual with the appropriate pro-drug can lead to enhanced reduction in tumour growth or survival.

The polynucleotide may also encode an antigenic polypeptide for use as a vaccines.

Preferably such antigenic polypeptides are derived from pathogenic organism, for example bacteria or a virus. Examples of such antigenic polypeptides include a hepatitis C virus antigen, hepatitis B surface or core antigen, HIV antigen, pertussis toxin, cholera toxin or diphtheria toxin.

The polynucleotide may also include (or, indeed, exclude) a marker gene (for example encoding β-galactosidase or green fluorescent protein) or genes whose products regulate the expression of other genes (for, example, transcriptional regulatory factors).

Where a disease is caused by a defective gene, a polynucleotide may be admistered that encodes a fully functional allele of the gene, such as in the case of cystic fibrosis. Toxicity of the monomers

In general, the preferred polyamines for use as monomer units in the reducible (disulfide) polymers of the present invention are non- toxic (at the concentrations suitably employed). It may be possible, however, to use monomers/polymers even under conditions where they would be expected to show some toxicity, through the choice of an appropriate reducing agent.

For example, spermine is unique among the natural polyamines in that, although it does occur at high concentrations in the nucleus and is essential for nucleotide synthesis, it is believed that it can be toxic through modulation of cellular NMDA receptors and enhancement of the glutamate-induced influx of Ca²⁺ into the cell. Normally, the Ca²⁺ concentration inside of cells is kept several orders of magnitude lower as compared to the extracellular space through active transport mechanisms. If the Ca²⁺ concentration remains above a certain threshold for prolonged periods of time, apoptotic events may be triggered that lead to the death of the cell. As a result, glutamate (and other substances) is released from the dying cells, which compounds the effect of NMDA receptor activation in the affected tissue.

Recent studies show that the glutamate receptor agonist N-methyl-D-aspartate (NMDA) can trigger acute lung injury (ALI), manifested by high-permeability pulmonary edema, and that NMDA receptor subtypes are expressed in normal rat lung. As GSH can, to some extent, counteract oxidative damage during pre-apoptotic events, such as resulting from polyamine oxidase activity, there is a rationale for co-administering GSH or GSH precursors or molecules with similar effects when spermine-based polyplexes are administered to lung or other tissue.

Furthermore, in unstressed tissue (low concentrations of Ca²⁺), spermine may act synergistically with GSH by acting as a free radical scavenger, thus inhibiting H₂O₂ production and maintaining protein sulfhydryl and GSH level at reduced levels. Thus, in some circumstances, the choice of GSH or a GSH precursor, in particular N- acetyl cysteine, as the reducing agent may help prevent or reduce toxicity of formulations, particularly in situations where oxidative stress is likely to occur.

Pharmaceutical Formulations

The polynucleotide-reducible polymer complex (polyplex) and reducing agents of the invention can be suitably formulated as a dry powder or as a solution capable of aerosolization. In accordance with the invention, therefore, suitable means for delivering compounds to the mucosal membrane of the lungs include a dry powder inhaler (DPI), a nebulizer, and a metered dose inhaler (MDI).

1. Dry Powder Inhaler (DPI)

DPIs are an alternative to the aerosol-based inhalers. The dry powder formulation can include one or more active pharmaceutical constituents as well as biocompatible additives that form the desired formulation or blend. As used herein, the term "dry powder" is used interchangeably with "dry powder formulation" and means a dry powder that comprises one or a plurality of constituents or ingredients having one or a plurality of particles that are of the same size or have different sizes. Dry powder formulations may contain particles that have the same density or particles that have different densities. For example, a low density dry powder means dry powders having a density of about 0.8 g/cm or less.

Individual dispensable quantities of dry powder formulations include both active and inactive ingredients. The inactive ingredients can include additives added to enhance flowability or to facilitate aeorolization delivery to the desired systemic target. The typical size of active ingredient particles in a dry powder formulation range from between about 0.5- 50 μm, typically in the range of between about 0.5 μm-20.0 μm, and more typically in the range of between about 0.5 μm-8.0 μm.

The dry powder formulation can also include flow-enhancing ingredients, which typically have particles whose size may be larger than the particle size of the active ingredient. In certain embodiments, the flow-enhancing ingredients can include excipients having particulate sizes on the order of about 50-100 μm. Examples of excipients include lactose and trehalose. Other types of excipients also can be employed, such as, but not limited to, sugars which are approved by the United States Food and Drug Administration ("FDA") as cryoprotectants (e.g., mannitol) or as solubility enhancers (e.g., cyclodextrine) or other compounds that are generally recognized as safe.

Delivery using the DPI route is often preferred when a low dose of an active agent is to be delivered to the lungs. Generally, the dose delivered is in the range from about 0.1 mg to about 50 mg, preferably about 1 mg to 40 mg. That is, DPIs can be used to deliver a dose that contains about 0.5, 1, 2, 3, 4, 5, 10, 15, 20 or 25 mg of the pharmaceutical agent of interest.

DPIs are breath- activated and do not use a chemical propellant to eject medication. Each device contains medication in the form of powder which is dispersed into particles by inspiration. DPIs require the patient to place the lips around a mouthpiece and inhale rapidly to disperse the powder into nanoparticles suitable to reach the lung. Typically, aerosolization upon inhalation results in the production of nanoparticles whose size is in the range from about 40 nm to about 300 nm. Some DPI's use foil pouches or disks to hold the medication (diskhalers) and require the patient to load the diskhaler with a medication disk or foil pouches. Activation of the device punctures a pouch or disk to release powdered pharmaceutical agent of interest that is inhaled through a mouthpiece.

2. Nebulizer And Metered Dose Inhalers (MDI)

Compounds in accordance with the invention, can be formulated as a solution for aerosolization and delivery to the mucosal tissue of lungs. Liquid formulations according to this aspect of the invention are sterile and are generally filled in vials or canisters including unit dose vials for use with a nebulizer or a MDI device. In one embodiment, the polymer- nucleotide complex (polyplex) and reducing agent are filled in separate vials for sequential delivery to the lungs. Typical concentration for the polyplex in the formulation is in the range from about 0.01 μg/l .Oμl to about 100 μg/lOOμl, while the concentration of the reducing agent is in the range of 100 μM to about 200 mM.

In one embodiment, therefore, a subject in need of therapy is provided a formulation capable of delivering between 10 mg/kg to 50 mg/kg of reducing agent per unit dose. Preferably, the device delivers 15 mg/kg to 40 mg/kg of reducing agent, such as a dose of 20 mg/kg, 25 mg/kg, 30 mg/kg, 32 mg/kg, 34 mg/kg, 36 mg/kg, or 38 mg/kg per unit dose. Similarly, each dose of the polyplex comprises between 0.5 mg/kg to about 2.0 mg/kg of nucleotide that is complexed with 0.6 mg/kg to about 4.0 mg/kg of reducible polymer. Using the above mentioned dose ranges, polyplexes that have a nitrogen/phosphate (N/P) ratio in the range from about 0.5 to about 20 can be obtained, preferably an N/P ratio in a range from about 2 to about 8. In one aspect of the inventive formulation, therefore, 1 mg/kg of nucleotide is complexed with 1.2 mg/kg of polymer to give a polyplex that has a N/P ratio of 5.0. Other suitable formulations can contain a polyplex having an N/P ratio of about 8.0.

The volume of solution packaged for use with a nebulizer or MDI is typically in the range from about 0.1 ml to about 5 ml, preferably in the range from about 0.5 ml to 3.0 ml. In one embodiment, therefore, the volume of solution packaged for use with the nebulizer is about 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, or about 3.0 ml Pharmacologically suitable solvents include, but are not limited to, polar solvents, such as, compounds that contain hydroxyl groups or other polar groups. Such solvents include, but are not limited to, water or alcohols, such as ethanol, isopropanol, and glycols including propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether, glycerol and polyoxyethylene alcohols. Preferably the liquid formulation is buffered in a pH range that is physiologically acceptable and does not degrade or otherwise inactivate or precipitate the pharmaceutical agent. A suitable pH range is between 3.0 to about 8.0, preferably a pH of about 4.0, 5.0, 6. 0, 6.5, 7.0, or 7.5. Suitable buffers include without limitation citrate, phosphate, malate, borate, TRIS, MES 2-(N- morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), and formate. The stability of liquid formulations is particularly susceptible to the ionic strength of the solution. Ionic strengths of the compositions provided herein are from about 0 to about 0.4, or 0 to 0.4. For instance, the ionic strength of the compositions is about 0.05 to about 0.16. Compositions having a lower ionic strength exhibit improved stability over formulations having higher ionic strength. The particular ionic strength of a given composition for long term storage provided herein may be determined using standard stability assays well known to those of skill in the art.

The inventive formulations may further include pharmaceutically acceptable organic or inorganic salts to maintain the tonicity of the formulation. Exemplars of such salts without limitation include ammonium carbonate, ammonium chloride, ammonium lactate, ammonium nitrate, ammonium phosphate, ammonium sulfate, ascorbic acid, bismuth sodium tartrate, boric acid, calcium chloride, calcium disodium acetate, calcium gluconate, calcium lactate, citric acid, dextrose, diethanolamine, dimethylsulfoxide, edetate disodium, edetate trisodium monohydrate, fluorescein sodium, fructose, galactose, glycerin, lactic acid, lactose, magnesium chloride, magnesium sulfate, mannitol, polyethylene glycol, potassium acetate, potassium chlorate, potassium chloride, potassium iodide, potassium nitrate, potassium phosphate, potassium sulfate, propylene glycol, silver nitrate, sodium acetate, sodium bicarbonate, sodium biphosphate, sodium bisulfite, sodium borate, sodium bromide, sodium cacodylate, sodium carbonate, sodium chloride, sodium citrate, sodium iodide, sodium lactate, sodium metabisulfite, sodium nitrate, sodium nitrite, sodium phosphate, sodium propionate, sodium succinate, sodium sulfate, sodium sulfite, sodium tartrate, sodium thiosulfate, sorbitol, sucrose, tartaric acid, triethanolamine, urea, urethan, uridine and zinc sulfate. In certain embodiments, the tonicity adjusting agent can be sodium chloride.

Formulations suitable for drug delivery using a MDI device, are provided in canisters under pressure and may contain a propellant or are propellant free formulations. Typically, a propellant is a pharmaceutically acceptable liquefied gas, such as a hydrofluoroalkane.

In another aspect, the polyplex and reducing agents of the invention can be formulated for delivery through the mucous membrane of the eye. For example, the polyplex and reducing agent may be formulated as separate sterile eye drop solutions. Suitable formulations include combining the polyplex and reducing agent with sterile water or a pharmaceutically acceptable buffer. For aqueous solutions, the isotonicity of the formulation is adjusted by adding the appropriate amount of sodium chloride and the solution is sterile filtered prior to use. Such formulations are suitable to treat disease conditions of the eye, for example, to deliver a sz-RNA polyplex to treat macular degeneration. Treatment can be effected by administering the reducing agent first, followed by administration of the polyplex.

Administration

Pursuant to the invention, a polyplex preferably is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition, which may be for human or animal use. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline, as well as other pharmaceutically acceptable solvents exemplified above. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration or inhalation. Delivery may be to or through a mucosal layer (e.g., in the lung, mouth, nose, GI tract, rectum or vagina). Typically, each NOI or polynucleotide may be administered at a dose of from 10 ng to 10 μg per, e.g., kg body weight, preferably from 0.1 to 10 μg (e.g., per kg), more preferably from 0.1 to 2 μg (e.g., per kg body weight).

Alternatively, transfection of patient cells may be carried out ex vivo e.g., by removing tissue and isolating cells, transfecting the isolated cells using the inventive formulations comprising polyplex and reducing agent followed by reimplantation of the transfected cells into the patient. The term "patient" and "subject" are being used interchangeably throughout the specification, and refer to a human or animal in need of therapy using the inventive composition.

The compositions may be suitable for targeting and entering the lung or nasal mucosal tissue or the CNS and/or peripheral circulation, for example via the nasal and/or olfactory epithelium. Thus, the composition may be administered intranasally or orally. Apart from i.v. administration, the composition may also be applied to cells of the oral epithelium and epidermis that are capable of absorption by endocytosis. The oral cavity is lined with a mucous membrane that is covered with squamous epithelium and embedded mucous glands. The buccal mucosa is similar to the sublingual mucosal tissue and both achieve high absorption rates.

In a preferred embodiment, the compositions of the present invention are especially suitable for mucosal delivery. In particular, the compositions may be delivered into the lung, through nasal mucosa (e.g., to facilitate entry directly into the CNS), through inhalation, or sublingually (thus avoiding, for example, degradation and loss of active pharmaceutical agent in the GI tract and liver, and also avoiding the need for injection).

As mentioned above, delivery to the lungs is facilitated by using a dry powder inhaler, a nebulizer or a metered dose inhaler, or any device of similar function. Use of any one of these devices would permit the sequential delivery of reducing agent and polyplex. In one embodiment, therefore, a dry powder formulation of the reducing agent is first delivered to the lungs using a disc or foil pouch, followed by administration of the polyplex. Typically, the polyplex is administered after a time interval in the range of about 0.1 hour to about 2 hours. The actual interval of time between delivery of the reducing agent and polyplex depends on several factors, which include without limitation the concentration of reducing agent and polyplex in the dry powder formulation, the particle size of the powder, and the ability of the patient to inhale and aerosolize the powdered formulation to nanoparticles suitable for entry in to the lungs.

The amount of reducing agent delivered can be equal to the amount of disulfide linkages of the polyplex, but it is preferably in excess of the moles of disulfide bonds available for reduction. For instance, the mole ratio of reducing agent to polyplex may be between about 1 and 50. Thus, the amount of reducing agent delivered to the mucosal tissue can be at least 5-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, or 45-fold greater than the amount of polyplex delivered. In another aspect, dry powder formulations of the reducing agent and polyplex are appropriately mixed to form a composition suitable for use. Such a composition is then packaged within a foil pouch or disk for use with a DPI device. Sequential delivery and mucosal absorption of reducing agent and polyplex from such a mixture can be effected by using dry powder formulations having particles of different sizes. For example, the particle size for a dry powder formulation of the reducing agent can be smaller than the particle size of the powdered formulation of the polyplex.

Formulations of the reducing agent and polyplex for use with a nebulizer are prepared by dissolving a dry powder with a sterile pharmaceutically acceptable diluent. Dissolution of the dry powder in the diluent can be carried out at the site of care, or by the patient just prior to use. For MDI delivery, separate canisters containing reducing agent or polyplex dissolved in a suitable propellant are given to a patient. Depending on the condition to be treated, the inventive formulations are administered once a day, or may be administered at regular intervals several times during the day, such as administration at time intervals of every 4, 6, 8, or 12 hours. Preferably, the polyplex and reducing agent are formulated for once-a-day delivery.

The routes of administration, dosages and disease conditions that can be treated are intended only as a guide. It should be understood that a skilled practitioner will readily be able to determine the optimum route of administration and dosage for any particular patient and disease condition.

Uses and medical conditions

The compositions or complexes in the present invention may be used to efficiently transfect eukaryotic cells, in particular mammalian cells, with polynucleotides. The compositions may be particularly efficient compared with prior art compositions in transfecting cells in mucous and other membranes, such as in the lung, for lung related illnesses, such as asthma, COPD (chronic obstructive pulmonary disease), cystic fibrosis, viral or bacterial infections, particularly to the lungs, influenza, allergies, and other respiratory ailments as well as non-respiratory tract related conditions, for example, diabetes and other related insulin resistance disorders.

The compositions or complexes in the present invention may also be used to transfect neuronal cells via the nasal mucosa. The olfactory neural pathway provides both intraneuronal and extra neuronal pathways in to the brain. The intraneuronal pathway involves axonal transport and may require hours to days for drugs to reach different brain regions. The extra neuronal pathway through perineural channels can allow drugs to be delivered directly to the brain parenchymal tissue, to the cerebro spinal fluid (CSF), or to both. This can allows therapeutic agents to reach the CNS with in minutes. Intranasal delivery of agents to the CSF is not surprising as CSF normally drains along the olfactory axon bundles as they traverse the cribriform plate of the skull and approach the olfactory submucosa in the roof of the nasal cavity where the CSF is then diverted in to the nasal lymphatics

This has specific implications for clinical applications where it is desired to introduce polynucleotides into such cells, such as of the central (or peripheral) nervous system of a human or animal. More generally, the compositions complexes in the present invention may be used in a variety of delivery applications such as gene therapy, DNA vaccine delivery and in vitro transfection studies.

Examples of diseases that may be treated and/or alleviated include those of the central (or peripheral) nervous system, such as neurodegenerative diseases and damage to nervous tissue as a result of injury/trauma (including strokes). In particular, neurodegenerative diseases include motor neurone disease, an inherited disease, such as familial dysautonomia and infantile spinal muscular atrophy, and late onset neurodegenerative diseases such as Parkinson's and Alzheimer's disease.

The composition complexes of the invention may also be used to administer therapeutic genes systemically to a patient suffering from a malignancy, for example cancer. Other diseases of interest include diseases caused by mutations, inherited or somatic, in normal cellular genes, such as cystic fibrosis, thalessemias and the like.

Further areas of interest include the treatment of immune-related disorders such as organ transplant rejection and autoimmune diseases by siRNA therapy.

Preferred features and characteristics of one aspect of the invention are equally applicable, mutatatis mutandis, to one or more other aspects of the invention. In addition, any preferred or optional characteristics of one aspect, presented positively, could also be taken (in the appropriate context) as the opposite, i.e. a negative feature, limitation or exclusion. Thus, when the specification refers to a positive preference, that could also be regarded as an equivalent negative statement or limitation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows cell death, as measured by LDH release, at different amine molar concentrations for compound (3) (small diamonds) and the reference compound jetPEI™ (large squares).

Figure 2 shows the β-Gal Expression from murine lung following intranasal installation of polyplexes constructed with compound (3) and jetPEI™. 1 : non-treated control; 2: polyplex formed from (3) at N/P ratio of 5; 3: polyplex formed from (3) at N/P ratio of 8; 4: polyplex formed from (3) at N/P ratio of 5 with prior administration of 5μmol NAC; 5: polyplex formed with jetPEI™; 6: polyplex formed with jetPEI™ and prior administration of 5μmol NAC.

Figure 3 shows 5,5'-dibromo-4,4'-dichloro-indigo stained lung lobes resulting from successful transfection of pDN A encoding for /3-galactosidase. A: untreated mice; B: mice treated with polyplex formed from (3); C: mice treated with polyplex formed from (3) and prior administration of 5μmol NAC; D: mice treated with polyplex formed with jetPEI™.

Figure 4 shows sections of the lung lobe in Figure 3, C at the medium magnifications indicated. Dark dots indicate 5,5'-dibromo-4,4'-dichloro-indigo staining. Figure 5 shows sections of the lung lobe in Figure 3, A (left) and Figure 3, C (right) at larger magnifications. Dark dots indicate 5,5'-dibromo-4,4'-dichloro-indigo staining.

The present invention will now be illustrated by means of the following Examples which are illustrative only and are not to be construed as limiting.

Example 1 - Toxicity of compound (3)

Uncomplexed compound (3) (HS-(CH₂)₂-NH -(CH₂)₃-NH-(CH₂)₄-NH-(CH₂)₃-NH- (CH₂)₂-SH) was diluted with a mixture of 4 mM HEPES and Opti-MEM (150 μl, 1 :1, v:v) to the concentrations indicated and then incubated with CHO-Kl cells for 24 h prior to the measurement of LDH content of both media and cell lysate (n = 3, +/- standard deviation, Figure 1). Results are represented as a function of the amine groups present in the solution (mMol). Over the range 1-3 μg/lOOμl, corresponding to 0.13-0.4mM, the toxicity of compound (3) is negligible when applied to the extracellular medium, in contrast to jetPEI™.

Example 2 - Lung transfection capacity of the reducible (disulfide) polymer formed from compound (3)

Polynucleotide therapy can require the use of discrete polyplexes of a few hundred nanometres in diameter or less (of the order 70nm to 250nm). The physical characterisation of the polyplexes formed from the disulfides of the present invention demonstrated that they were able to form such discrete polyplexes at low N/P ratios, making them strong candidates for polynucleotide therapy, and hence they were tested in an in vivo setting versus the current gold standard jetPEI™.

Their ability to condense pDNA at low N/P ratios, similar to those observed with linear PEI (25 kDa) implied the formation of higher molecular weight species in the presence of pDNA providing evidence for polymerisation.

Polyplexes, containing 25 μg of pDNA in 100 μl_ of 5 % glucose, were delivered intranasally to BALB/c mice. After 24 hours the mice were sacrificed and their lungs analysed for β-galactoside expression, with the results obtained summarised in Figure 2. In non-treated mice, the level of β-gal expression was virtually zero. In mice treated with polyplex formed from (3), significant β-gal expression was found at both the N/P ratios of 5 and 8 (1.66 ± 0.40, n=10 and 1.61 ± 0.32 n=5 [ng of β-gal protein per 100 mg of lung protein], respectively). Instillation of NAC (5OmM in lOOμl 5% glucose) 30 minutes prior to the polyplex instillation led to expression levels similar tojetPEI™ (3,66 ± 0,32, n=5 for the polypex versus 3.06 ± 0.26, n=15 for jetPEI™ [ng of β-gal protein per lOOmg of lung protein]), while addition of NAC to jetPEl™ remained without effect, indicating that the cause of the observed synergy was not the mucolytic nature of NAC, but rather its ability to raise the local reductive environment and, it is thought, to reduce (at least) part of the disulfide bonds in the disulfide polymer.

Example 3 - Lung tissue distribution of the reducible (disulfide) polymer formed from compound (3)

Histological analysis of the murine lung tissue was performed by OCT cryosectioning. and paraffin embedding treatment, hi both cases, Eosin counterstaining was used to better identify those areas expressing /3-gal in blue. Successful transfection is illustrated by the dark staining in Figure 3-5. Major sites of transfection are the airway epithelia in the bronchioles and bronchioli. Transfection may also occur in the parenchyme.

Experimental details

Formation of Polyplexes

Disulfide oligomers were pre- formed at concentrations of 5mg/ml in cone HCL + 1 % DMS overnight, precipated, washed, lyophilised, and redissolved at concentrations of lμg/μl (3.125mM) in 4 raM HEPES (pH=7.4). A solution in 5% glucose, was added at a weight : weight ration of 1.2: 1 polymer to pDNA and 1.92: 1 polymer to pDNA, corresponding to a N/P ration of 5 and 8, respectively. The resulting solution was shaken, centrifuged at 13,200 rpm for 5s and then allowed to stand for 30 mins at room temperature before use. The solution was subsequently adjusted to contain 25μg pDNA in a volume of 1 OOμl.

Agarose Gel Electrophoresis

Standard Conditions: Polyplex solutions of various N/P ratios containing 500 ng of pDNA in lOμL of buffer were made-up using the procedure described previously. Each sample was diluted with 10 μL of buffer and then incubated for a further 30 mins after which they for 1 h at 110 V through a 0.8 % agarose gel plate containing 20 ng μL ^"' of ethidium bromide. The resulting plate was visualised under UV light.

Reductive Conditions: Polyplex solutions of various N/P ratios containing 500 ng of pDNA in 10 μL of buffer were made-up. Each sample was diluted with 10 μL of 200 mM DTT and then incubated for a further 30 mins after which they for 1 h at 110 V through a 0.8 % agarose gel plate containing 20 ng μL ^"' of ethidium bromide. The resulting plate was visualised under UV light.

Transfection

Polyplexes were prepared in 5% glucose using (3) and jetPEI™ as polymer to encapsulate pDNA encoding for a /3-galactosidase (|S-gal). Polyplexes at a concentration of 25μg/100μl and N/P ratios of 5 and 8 were delivered intranasally to the lungs of female BALB/c mice. 24 hours later, the mice were sacrificed and their lungs removed. The amount of /3-gal expression was determined using standard β-Gal ELISA assays and expressed in ng of β-gal protein per 100 mg of lung protein (Figure 1). NAC (5OmM in 100 μl 5% glucose) was instilled in some groups of mice 30 minutes prior to the polyplex instillation

Histology

Mouse lung tissue originating from the left lobe was fixed by placing it into a 0.5% glutaraldehyde solution for 10 minutes. It was then washed repeatedly with PBS prior to incubation in an X-gal solution for 24 h at 37 °C. The tissue was then washed with PBS and dehydrated by sequential immersion in 70%, 95% and absolute ethanol. The tissue was subsequently soaked in a mixture of benzyl benzoate and benzyl alcohol (2:1, v:v) for examination by light microscopy.

Lung tissue successfully transfected with pDNA encoding for the /3-galactosidase enzyme is capable of cleaving X-gal to galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue dye, which allows transfected tissue to be easily identified.

OCT sections were prepared by sequential immersion in 100% MeOH, MeOH in PBS and finally PBS. The tissue samples were then soaked with 20% sucrose in PBS before being embedded in a mixture of Tissue-Tek OCT and 20% sucrose in PBS and frozen via immersion in liquid N₂. 9 μm thick sections of the frozen samples were counterstained with Eosin and analysed. Other samples were embedded in paraffin and then cut into 4 μm sections which were then counterstained with Eosin. Both types of samples were then analysed for /3-gal staining by light microscopy.

References

1. Lee, Y.; Mo, H.; Koo, H.; Park, J.-Y.; Cho, M. Y.; Jin, G.-W.; Park, J.-S. Bioconj. Chem. 2007, 18 (1), 13-18.

2. Read, M. L.; Singh, S.; Ahmed, Z.; Stevenson, M.; Briggs, S. S.; Oupicky, D.; Barrett, L. B.; Spice, R.; Kendall, M.; Berry, M.; Preece, J. A.; Logan, A.; Seymour, L. W. Nucleic Acids Res. 2005, 33 (9).

Claims

1. An assemblage for use in non- viral delivery of a polynucleotide of interest, comprising (A) a polyplex that is the product of contacting a polycationic reducible disulfide polymer with said polynucleotide with (B) a reducing agent, wherein, the polyplex and reducing agent are provided in a form suitable for nasal or oral delivery.

2. The assemblage of claim 1, comprising a mixture of particulate polyplex and particulate reducing agent , wherein the latter has release characteristics that are faster than those of the former.

3. The assemblage of claim 2, wherein the polyplex and reducing agent are in the form of nanoparticles.

4. A polyplex that is the product of contacting a polycationic reducible disulfide polymer with said polynucleotide, wherein said polyplex is in a form suitable for targeting and entering lung or nasal mucosal tissue.

5. A method for non- viral delivery of a polynucleotide of interest, comprising

(A) providing a polyplex that is the product of contacting a polycationic reducible disulfide polymer with said polynucleotide and

(B) delivering said polyplex to a target tissue in subject such that said polyplex comes into contact with a reducing agent at said target tissue, whereby said reducing agent causes reductive degradation of said polyplex to release said polynucleotide in said target tissue.

6. The method of claim 5 , wherein said reducing agent is selected from the group consisting of reduced glutathione, N-acetyl cysteine, and cysteine.

7. The method of claim 6, wherein said reducing agent is N-acetyl cysteine.

8. The method of claim 5, wherein said polynucleotide of interest is selected from the group consisting of DNA, RNA, si- RNA, mi-RNA, antisense oligonucleotides and DNA/RNA hybrids.

9. The method of claim 8, wherein said polynucleotide of interest is si-KNA.

10. The method of any of claims 5 through 8, wherein said delivering is effected by entering lung or nasal mucosal tissue.