WO2003077953A2

WO2003077953A2 - Bacteriophage composition

Info

Publication number: WO2003077953A2
Application number: PCT/GB2003/001035
Authority: WO
Inventors: David West; Vladimir Pasechnik
Original assignee: Phage Genomics Inc.; POLYANSKAYA, Natasha
Priority date: 2002-03-12
Filing date: 2003-03-12
Publication date: 2003-09-25
Also published as: AU2003209495A1; GB0205786D0; WO2003077953A3

Abstract

The present invention provides a composition which comprises a complex of a bacteriophage and a cationic delivery moiety, such as a cationic polymer, in particular a cationic peptoid or a cationic peptide. More specifically, the present invention provides a composition comprising a complex of a bacteriophage and a delivery peptide or a protective or stabilising peptide. The present invention further provides a method for the delivery of bacteriophage across a membrane, such as a cell membrane; and a method for the treatment of bacterial infection, in particular intracellular bacterial infection, using a complex according to the invention.

Description

BACTERIOPHAGE COMPOSITION

The present invention relates to a composition which comprises a complex of a bacteriophage and a cationic delivery moiety, such as a cationic polymer, in particular a cationic peptoid or a cationic peptide. More specifically, the present invention relates to a composition comprising a complex of a bacteriophage and a delivery peptide or a protective or stabilising peptide. The present invention further concerns a method for the delivery of a bacteriophage across a membrane, such as a cell membrane; and a method for the treatment of bacterial infection, in particular intracellular bacterial infection, using a complex according to the invention.

A wide variety of diseases and pathogenic conditions are caused or characterised by bacterial infection of cells and/or cellular compartments. The early stages of tuberculosis, for example, involve the infection of lung macrophages by inhaled bacilli, followed by intracellular multiplication of the bacilli within infected macrophages and transmission to uninfected macrophages and monocytes, which subsequently transport the bacilli to regional lymph nodes to initiate the next stages in TB disease progression. For this reason, the development of agents capable of inactivating or eliminating intracellular bacteria is a crucial objective in attempts to combat disease. Considerable effort has been dedicated in the art towards the development of antibiotics, which are capable of entering into infected cells and/or cellular compartments and eliminating bacteria therein. Treatment with antibiotics can constitute an extremely effective approach towards the elimination of intracellular bacteria. Nevertheless, this approach is hindered by the ability of bacterial strains, on exposure to a particular antibiotic, rapidly to "acquire" antibiotic resistance, primarily by means of mutation.

Another approach towards the treatment of bacterial infection involves the deployment of bacteriophage. Bacteriophage, otherwise known as bacterial viruses, are capable of infecting specific bacteria and replicating therein. On induction of the lytic phase within an infected bacterium, the bacteriophage produces enzymes which lyse the infected bacterium, resulting in bacterial death and the release of further infective bacteriophage. Bacteriophage have obvious potential as tools for eliminating infective bacteria. Nevertheless, whilst phage have been successfully employed for eliminating extracellular bacteria, problems have been encountered in achieving the entry of bacteriophage into eukaryotic cells and cellular compartments, and hence attempts to use phage for the treatment of intracellular infection have met with limited success. Accordingly, the development of an anti-bacterial composition including a bacteriophage as active agent, which composition is capable of being entered into cells or cellular compartments, so as to enable the elimination of bacteria within said cells or cellular compartments by the action of the bacteriophage, remains an important objective. According to one aspect of the present invention therefore, there is provided a composition comprising a complex of a bacteriophage and a cationic delivery moiety, characterised in that said composition is buffered to a pH above the pi of said bacteriophage such that said bacteriophage and the delivery moiety are held together electrostatically. The present inventors have found that some bacteriophage, in particular mycobacteriophage, possess an isoelectric point (pi) which is below neutral. That is to say, at a neutral or approximately neutral pH, for example at or near the pH of blood which is buffered to pH 7.4, such bacteriophage are negatively charged. It has been found that at neutral or approximately neutral pH, such bacteriophage are capable of forming stable complexes with cationic delivery moieties, in particular cationic polymers such as peptides, which have an isoelectric point above neutral; such as polyarginine.

Suitably, said composition may be buffered using any suitable buffering agent. In preferred embodiments, said composition may be buffered to approximately pH 7.5 using 50mM Tris buffer. Preferably, said cationic delivery moiety when held together electrostatically with said bacteriophage does not substantially impair the activity, in particular the lytic activity and/or the replication, of said bacteriophage. Thus, a lytic bacteriophage in a complex according to the present invention preferably remains capable of replicating and/or lysing bacteria. Alternatively, said cationic delivery moiety may be adapted to become dissociated from said bacteriophage on or following delivery of said bacteriophage into a cell or cellular compartment, such that the bacteriophage can effectively lyse susceptible bacteria within said cell or cellular compartment.

In one aspect of the invention, said cationic delivery moiety is arranged when held together electrostatically with said bacteriophage to stabilise or protect said bacteriophage. Thus, said delivery moiety may be configured to surround or envelop the whole of or a part of said bacteriophage, so as to protect the bacteriophage, for example from enzymatic attack. In some embodiments, therefore, said delivery moiety may comprise a polypeptide which includes a plurality of cationic amino acid residues, such as arginine or lysine, and one or more amino acid residues which serve to introduce a particular three-dimensional conformation to said polypeptide, such as proline which is known to induce a beta-conformation in polypeptides.

In another aspect of the invention, said cationic delivery moiety is arranged when held together electrostatically with said bacteriophage to enable said bacteriophage to cross a cellular membrane, such as an outer eukaryotic cell membrane or a membrane defining a compartment of a eukaryotic cell. The present inventors have surprisingly found that a complex in accordance with the present invention, wherein said cationic delivery moiety is a delivery macromolecule which consists of a polymer backbone comprising between 6 and 100 cationic sidechains each individually selected from -(CH₂)_a-(NH)b- C(=NH)-NH₂ where a is an integer between 2 and 12, preferably 2, 3, 4, 5, 6, 7, 8 or 9, and b is 0 or 1, is capable of being internalised into cells and cellular compartments. In many embodiments, said macromolecule is not a ligand to any cell-surface receptor, in particular any transmembrane receptor. Moreover, the present inventors have found that the anti-bacterial properties of the bacteriophage are preserved on entry of such a complex into a cell or cellular compartment, notwithstanding the association of the bacteriophage with said cationic delivery macromolecule.

According to yet another aspect of the present invention therefore, there is provided a method for delivery of a bacteriophage into a cell or a cellular compartment, comprising the steps of providing a composition comprising a complex in accordance with the present invention of said bacteriophage with said cationic delivery macromolecule, and introducing said complex to said cell or cellular compartment, such as to permit the delivery of said bacteriophage into said cell or cellular compartment.

Suitably, said bacteriophage may be lytic to at least one strain of bacteria, advantageously a strain of virulent bacteria. Said strain of bacteria may be a strain which may infect said cell or cellular compartment. Thus, said composition may be effectively used in the elimination of intracellular and/or intracompartmental bacteria, following the delivery of said complex into a cell or compartment infected with said bacteria. According to yet another aspect of the invention therefore, there is provided a method for the treatment of a disease or condition in a patient that is characterised by the infection of the patient's cells by bacteria; comprising the steps of providing a composition comprising a complex in accordance with the present invention wherein said bacteriophage is lytic to said bacteria, and admininstenng said composition to said patient such that the complex can be transported to said cells for delivery of said bacteriophage therein. Said composition may, for example, be administered to said patient orally or parenterally, for example by injection into the bloodstream. Many diseases and conditions are characterised by intracellular bacterial infection and would be susceptible to treatment in accordance with the invention; examples of such diseases and conditions include tuberculosis (M. tuberculosis), zoonotic disease of cattle (Mycobacteria bovis), leprosy (Mycobacteria leprae), typhoid (Salmonella typhi), paratyphoid (Salmonella paratyphϊ), brucellosis (Brucella spp), Legionnaire's disease (Legionella pneumophila), listeriosis (Listeria monocytogenes), tuleraemia (Francisella tularensis), Rocky Mountain fever (Rickettsia rickettsii), epidemic typhus (Rickettsia prowazekiϊ), endemic typhus (Rickettsia typhi), Q fever (Coxiella burnetii), chlamydial STD (Chlamydia trachomatis), psitticosis (Chlamydia psittaci), pneumonia (Chlamydia pneumoniae), granuloctic ehrlichiosis (Ehrlichia), shigellosis (Shigella), whooping cough (Bordetella), toxoplasmosis (Toxoplasma gondii), and anthrax (Bacillus anthracis).

According to yet another aspect of the invention, there is provided a medicament comprising a composition in accordance with the invention and, optionally, one or more pharmaceutically acceptable excipients or carriers. Such a medicament may advantageously be adapted for administration to a patient for the treatment of a disease or condition characterised by intracellular bacterial infection, in accordance with the invention.

According to yet a further aspect of the invention, there is provided a method for the use of a composition in accordance with the present invention, in the manufacture of a medicament in accordance with the present invention for use in treating a disease or condition in a patient which is characterised by intracellular bacterial infection.

In some embodiments, said polymer backbone may comprise a non- peptide backbone, such as an alkyl, alkenyl, amide, thioether, sulfonyl, ester, ether, ketone or peptoid backbone. Cationic delivery macromolecules comprising a non-peptide backbone may show advantageously increased resistance to intracellular and/or extracellular degradation.

In other embodiments, however, said polymer backbone may comprise a peptide backbone. Thus, said cationic delivery macromolecule may comprise a cationic delivery polypeptide which is formed from a plurality of amino acids, each amino acid optionally including at least one sidechain. Advantageously, at least one, and preferably all, of said cationic sidechains has a structure as defined above wherein a is 3 and b is 1, and forms part of an arginine residue within said polypeptide. Said polypeptide may additionally include one or more cationic amino acids, such as lysine, histidine, ornithine, citrulline, or isomers or analogues thereof. Each amino acid in said polypeptide may comprise a D or an L isomer; preferably an L isomer.

Suitably, the total molecular weight of said cationic delivery polypeptide may not exceed 20kDa, and may more preferably be in the range 5- 15kDa. Suitably, therefore, said cationic delivery polypeptide may comprise no more than 100, preferably no more than 50 amino acids. Advantageously, said cationic delivery polypeptide may comprise at least 5 amino acids. In most preferred embodiments, said cationic delivery polypeptide may consist of between 6 and 100, such as between 6 and 15, or between 25 and 85, amino acids.

In some especially preferred embodiments, said cationic delivery polypeptide may comprise a polyarginine homopolymer, preferably a homopolymer comprising 6-15 arginine residues, such as 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 arginine residues; or a homopolymer comprising between 28 and 84 arginine residues.

In other alternative embodiments, said cationic delivery polypeptide may comprise a naturally-occurring delivery protein or a fragment thereof; such as, for example, the Tat peptide of fflV-1 (WO 91/09958, US-A-5804604) or a fragment thereof, particularly a fragment which comprises or consists of residues 49-57 thereof having the sequence RKKRRQRRR (WO 94/04686 and

Fawell et al (1994) Proc. Nat'l Acad. Sci USA 91:664-668); or a fragment of the Antennapedia protein comprising residues 43-58 thereof having the sequence RQIKIWFQNRRMKWKK (Brugidou et al (1995) Biochem. Biophys. Res. Commun. 214:685-93); or fϊbroblast growth factor or a fragment thereof (Lin et al, 1995 J. Biol. Chem. 270:14255-14258); or Galparan (transportan) or a fragment thereof (Pooga et al, 1998 FASEB J. 12:67-77); or HSN-1 structural protein NP22 or a fragment thereof (Elliott et al, 1997 Cell 88:223-233); or a translocation peptide derived from Drosophila melanogaster homeodomain protein (Derossi D et al, J. Biol. Chem. 1994; 269:10444-10450); or a KALA/GALA-type peptide (Wyman et al, Biochemistry 1997; 36: 3008-3017). Anti-bacterial bacteriophage of various types suitable for use in the present invention are available in the art, some examples being described and characterised in US-A-5688501 and US-A-6121036. In preferred embodiments, said bacteriophage may comprise mycobacteriophage, such as mycobacteriophage R51, D29, TM4, L5, or Bxbl. Mycobacteriophage, specifically mycobacteriophage TM4, is lytic to strains of mycobacteria such M. paratuberculosis avium (MAP), which is implicated in tuberculosis and has also been implicated in Crohn's disease. Mycobacteriophage, in particular R51 and D29, have been found to have a pi of approximately 4-5, and consequently a negative charge at an approximately neutral physiological pH, such as the pH of blood.

Said bacteriophage may be selected at the panel of variants of a microbial pathogen such as M. tuberculosis strain H37R, Mycobacterium bovis (var BCG), and multi-drug-resistant variants of a target pathogen, such as M. tuberculosis strains W and H. A deposit of mycobacteriophage R51 , satisfying the requirements of the Budapest Treaty, is held in the name of the applicant at the ΝCIMB, 23 St Machar Drive, Aberdeen AB243RY, Scotland, UK under deposit number 41119, and was deposited on 31 October 2001. A deposit of mycobacteriophage BG1, satisfying the requirements of the Budapest Treaty, is held in the name of the applicant at the ΝCIMB, 23 St Machar Drive, Aberdeen

AB24 3RY, Scotland, UK under deposit number 41124, and was deposited on 30 November 2001.

Said bacteriophage and said delivery moiety are held together in a complex in accordance with the present invention by virtue of the electrostatic attraction therebetween. Preferably, said bacteriophage and said delivery moiety are not held together by any covalent bond and/or by any other type of bond (apart from electrostatic attraction) therebetween.

Suitably, said cell or cellular compartment may comprise a eukaryotic cell or compartment, such as a lymphocyte, or macrophage, or a dendritic, neuronal, kidney, vascular smooth muscle, dermal, epithelial or endothelial cell or compartment thereof.

Said medicament of the present invention may be suitable for use in the treatment of diseases or conditions characterised by intracellular infection by bacteria; including tuberculosis. Said medicament may be suitable for administration to a patient in need thereof by way of oral, sublingual, transdermal or parenteral administration. In especially advantageous embodiments, said medicament may be suitable for administration by intranasal spray, or by injection into peripheral blood vessels.

For oral or parenteral administration, it is greatly preferred that the medicament is administered in the form of a unit-dose preparation, such as a unit dose oral or parenteral preparation.

Such unit-dose preparations are prepared by admixture and are suitably adapted for oral or parenteral administration, and as such may be in the form of capsules, oral preparations, lozenges, injectable and liquid infusible solutions or suspensions or suppositories.

Capsules for oral administration are usually presented in a unit dose, and contain conventional excipients such as binding agents, fillers, diluents, tabletting agents, lubricants, disintegrants, colourants, flavourings, and wetting agents. The capsules may be coated according to well known methods in the art.

Said medicament may optionally include one or more additives, such as fillers, disintegrants, lubricants, wetting agents, and/or preservatives. Suitable fillers for use include cellulose, mannitol, lactose, trehalose and other similar agents. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate. Suitable pharmaceutically acceptable preservatives include propyl p-hydroxybenzoate and sorbic acid.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as esters of glycerine,. propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

For parenteral administration, fluid unit dose forms may be prepared comprising a sterile vehicle. The components of the preparation, depending on the vehicle and the concentration, can be either suspended or dissolved. Parenteral solutions are normally prepared by dissolving the components of the preparation in a vehicle and filter sterilising before filling into a suitable vial or ampoule and sealing. Advantageously, adjuvants such as a local anaesthetic, preservatives and buffering agents are also dissolved in the vehicle. To enhance the stability, the preparation may be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner except that the complex may be suspended in the vehicle instead of being dissolved and sterilised by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent may be included in the preparation to facilitate uniform distribution of the complex of the invention. As is common practice, the preparations will usually be accompanied by written or printed directions for use in the treatment concerned.

Following is a description, by way of example only, of embodiments of the invention; with reference to the accompanying figures, in which: Figure 1 shows the results of isoelectric focusing of mycobacteriophage

D29, indicating a peak at the pi value of D29 (pi = 4.1).

Figure 2 shows the results of isoelectric focusing of mycobacteriophage R51 , indicating a peak at the pi value of R51 (pi = 3.9).

Figure 3 shows the results of isoelectric focusing of mycobacteriophage R51 mixed with poly-arginine, indicating a peak at pH 4.0 (unbound phage) and a peak at pH 6.3, corresponding to the pi value of the poly-arginine/phage complex.

Figure 4 shows the results of live/dead bacterial counts on lysis of infected U937 (immortalised culture) cells, without prior incubation ("con no ph"), or following incubation of the cells with phage alone ("ph only" - control), phage/polyarginine mix ("ph + R300"), phage/transferrin mix ("ph + Tf ' - comparative), phage/polylysine mix ("ph + polyLys" - comparative), or phage/polyarginine-cholesterol mix ("ph + R300C" - comparative).

Figure 5 shows the results of live/dead bacterial counts on lysis of infected mouse macrophage cells, following incubation of the cells with phage alone ("ph" - control), phage/polyarginine mix ("ph+300"), phage/cationic peptide mix ("ph+Nl" - comparative), and BCG strain of M. bovis (control).

Figure 6 shows the results of a gel filtration performed on a mix of phage R51 and polyarginine labelled with Alexafluor 488. Figure 7 shows the results of in vivo experiments on groups of mice infected with M. tuberculosis strain W4, demonstrating the efficacy of phage complexed with poly-Arg in treating infection. Description 1 : Bacteriophage

The overall charge on the surface of candidate mycobacteriophage, including mycobacteriophage D29 and mycobacteriophage R51 which has been deposited at the NCIMB under deposit number 41119, has been established at physiological pH by prediction in silico and practically using isoelectric focusing (IEF) to define the isoelectric point of the phage.

Computer analyses were carried out using the computer package Nector ΝTI Suite, Version 6, in order to predict the pi values for the head and tail proteins of mycobacteriophage D29 on the basis of their physical properties. The results of these analyses are set out below in Tables 1-6.

Table 1 : D29 gp 6 minor tail protein analysis

Table 2 : D29 gp 17 major head protein analysis

Table 3 : D29 gp 23 major tail protein analysis

Table 4 : D29 gp 26 minor tail protein analysis

Table 5 : D29 gp 27 minor tail protein analysis

Table 6 : D29 gp 28 minor tail protein analysis

The computer-generated analysis of these predictions indicates that the head of phage D29 has a pi of 4.95, and the tail proteins have pi values between 4.5 and 5.5. The overall pi value is therefore be predicted to be around 5.0, indicating that the phage will have a net negative charge at neutral pH.

This prediction is borne out by isoelectric focusing (IEF). IEF is a known technique for determining the pi of proteins, and involves the electrophoresis of proteins across a pH gradient established in a non-denaturing gel. On initiation of electrophoresis, each protein will move across the gel to the point where the pH matches the pi of the protein so that the protein is uncharged. The pi of the protein can therefore be measured by reference to the position of the protein in the pH gradient following electrophoresis. The results of IEF on phage D29 are illustrated in Figure 1. As seen in this figure, a peak is observed at approximately pH 4.1, which indicates that the pi of phage D29 is approximately 4.1 and that the phage will be negatively charged at neutral pH. Meanwhile, the results of IEF on phage R51 are illustrated in Figure 2, where a peak is observed at approximately pH 3.9, indicating that the pi of phage R51 is approximately 3.9. Accordingly, phage R51 will also be negatively charged at neutral pH.

Description 2 : Delivery macromolecules Preferred delivery agents for use in the invention are peptides R300 and Nl .

R300 is a cationic poly-arginine homopolymer having a molecular weight between 5-15kDa (28-84 Arg residues). Arginine has a pi of 11.15, and is therefore positively charged at physiological pH. Nl is a cationic peptide having the amino acid sequence RQI-KIWFQRRRMKWKKC. Each of R300 and Nl may be synthesised according to methods well known in the art.

Example 1 : Phage/delivery agent complex formation

Based on the respective pi values of the delivery agents and the mycobacteriophage, it is predicted that on mixing of the two species, mutual electrostatic attraction between each delivery agent and the phage will result in the formation of a stable phage-delivery agent complex.

This prediction has been confirmed using gel filtration studies. Phage R51 and polyarginine polypeptide (labelled with Alexafluor 488) were mixed together to allow the formation of an electrostatic complex. Thereafter, the phage/polypeptide mix was passed down a Sephacryl HP S20026/60 column (commercially available from Pharmacia., Amersham). 1ml samples were analysed for fluorescence and titre. Correlation of these two, as illustrated in figure 6, indicates the formation of a stable complex between polyarginine delivery agent and phage. The stability of the phage/polyarginine complex was also demonstrated by

IEF of the phage/polypeptide mix. As seen in Figure 3, IEF reveals a peak at around pH 4 (corresponding to unbound R51 phage) and a peak at pH 6.3, which corresponds to the phage/polyarginine complex, and indicates that this complex is not separated in the course of electrophoresis across a pH gradient. A peak corresponding to polyarginine (pi > 14) is outside the range shown on this figure.

Example 2 : Cellular uptake and anti-bacterial activity of complex

To test the cellular uptake and anti-bacterial activity of the R51/polyarginine complex, the phage/polypeptide mix was added to U937 (immortalised culture) cells infected with mycobacteria. After incubation, the cells were washed and lysed, and a count of live and dead bacteria released from the cells was performed, using the Baclight bacterial viability kit (Molecular Probes, cat# L13152). Comparisons were made with the live/dead bacterial count from non-incubated infected U937 cells; infected U937 cells incubated with R51 phage only; infected U937 cells incubated with an R51/transferrin mix; infected U937 cells incubated with an R51/polylysine mix; and infected U937 cells incubated with a phage/R300C mix. R300C is a variant of R300, bearing cholesterol residues. The mean results of several experiments are shown in Figure 4. As seen in this figure, the proportion of dead bacteria released from cells incubated with phage/polyarginine is significantly higher than that released from any of the comparative or control runs, indicating that active phage has been successfully delivered to the interior of these cells. A similar experiment was performed to test the cellular uptake of active phage into primary mouse macrophage cells. In this case, primary mouse macrophage cells were infected with mycobacteria, and thereafter incubated with R51 phage; R51/polyarginine mix; R51/N1 mix; and BCG M. bovis strain. After incubation, the cells were washed and lysed and a count was taken of dead versus total bacterial count. The mean results of several runs are illustrated in Figure 5. As seen in this figure, incubation of infected macrophages with R51/polyarginine produces a marked increase in the proportion of dead bacteria released following lysis, indicating that active phage has successfully been delivered to the interior of the cells during incubation. Example 3

In vivo experiments were carried out in order to demonstrate the efficacy of the present invention in treating intracellular infection. Groups of 6 mice (C57B1/6 females) were infected with Mycobacterium tuberculosis strain W4 (dose of 10³ bacterium delivered intranasally), and left for 24 hours. Each group of mice was then treated with buffer (control), or phage, or phage + poly- Arg, in accordance with the following protocol :

Phage : Cocktail of R51 and D29 (titre of approx. 7 x 10⁸ on W4; titre approx 1 x 10¹¹ on M. smegmatis)

Poly-Arg : peptide of molecular weight 5-15kDa Sigma; mixed with phage to 4ug/ml final concentration, such as to form an electrostatic complex)

Each treatment was delivered intranasally. Treatments were repeated a further 6 times at 24 hour intervals thereafter. The mice were then sacrificed, and lungs and spleen harvested.

Tissue samples from lungs and spleen of each group were ground in saline containing 0.1% Tween and equal volumes of tissue were plated on appropriate media. Cfu counts were taken after a month's incubation at 37°C. The results of this experiment are shown in Figure 7. As seen in the Figure, phage complexed with poly-Arg is approximately twice as effective at eliminating infection as phage on its own, indicating that the phage complexed with poly-Arg is capable of accessing intracellular infective bacteria, including infective bacteria located in macrophage cells.

Claims

1 A composition comprising a complex of a bacteriophage and a cationic delivery moiety, characterised in that said composition is buffered to a pH above the pi of said bacteriophage such that said bacteriophage and the delivery moiety are held together electrostatically.

2 A composition as claimed in claim 1, which is buffered to approximately pH 7.5.

3 A composition as claimed in claim 1 or claim 2, wherein said cationic delivery moiety is arranged when held together electrostatically with said bacteriophage to stabilise or protect said bacteriophage.

4 A composition as claimed in any preceding claim, wherein said cationic delivery moiety is arranged when held together electrostatically with said bacteriophage to enable said bacteriophage to cross a cellular membrane, such as an outer eukaryotic cell membrane or a membrane defining a compartment of a eukaryotic cell, whereby said bacteriophage can be delivered into said cell or cellular compartment.

5 A composition as claimed in claim 4, wherein said cell or cellular compartment comprises a eukaryotic cell or compartment, such as a lymphocyte, or macrophage, or a dendritic, neuronal, kidney, vascular smooth muscle, dermal, epithelial or endothelial cell or compartment thereof.

6 A composition as claimed in any preceding claim, wherein said cationic delivery moiety is a delivery macromolecule which consists of a polymer backbone comprising between 6 and 100 cationic sidechains each individually selected from -(CH2)a-(NH)b-C(=NH)-NH₂ where a is an integer between 2 and 12, preferably 2, 3, 4, 5, 6, 7, 8 or 9, and b is 0 or 1.

7 A composition as claimed in claim 6, wherein said polymer backbone comprises a non-peptide backbone, such as an alkyl, alkenyl, amide, thioether, sulfonyl, ester, ether, ketone or peptoid backbone.

8 A composition as claimed in claim 6, wherein said polymer backbone comprises a peptide backbone which is formed from a plurality of amino acids, each amino acid optionally including at least one sidechain.

9 A composition as claimed in claim 8, wherein said peptide backbone includes one or more cationic amino acids selected from arginine, lysine, histidine, ornithine, citrulline, or isomers or analogues thereof.

10 A composition as claimed in claim 8 or claim 9, wherein the total molecular weight of said cationic delivery macromolecule does not exceed 20kDa.

11 A composition as claimed in any of claims 8-10, wherein said cationic delivery macromolecule consists of between 25 and 85 amino acids.

12 A composition as claimed in any of claims 8-10, wherein said cationic delivery macromolecule comprises a polyarginine homopolymer consisting of between 28 and 84 arginine residues.

13 A composition as claimed in claim 8 or claim 9, wherein said cationic delivery macromolecule comprises a naturally-occurring delivery protein or a fragment thereof; such as, for example, the Tat peptide of HTV-1 or a fragment thereof, particularly a fragment which comprises or consists of residues 49-57 thereof having the sequence RKKRRQRRR; or a fragment of the Antennapedia protein comprising residues 43-58 thereof having the sequence RQKIWFQNRRMKWKK; or fibroblast growth factor or a fragment thereof; or Galparan (transportan) or a fragment thereof; or HSN-1 structural protein NP22 or a fragment thereof; or a translocation peptide derived from Drosophila melanogaster homeodomain protein; or a KALA GALA-type peptide.

14 A composition as claimed in any preceding claim, wherein said bacteriophage and said delivery moiety are not held together by any covalent bond therebetween.

15 A composition as claimed in claim 5 or any claim dependent thereon, wherein said bacteriophage is lytic to at least one strain of bacteria which may infect said eukaryotic cell or compartment, said composition being arranged such that following delivery of said bacteriophage into said cell or said cellular compartment, said bacteriophage is capable of effecting lysis of bacteria within said cell or compartment.

16 A composition as claimed in claim 15, wherein said bacteriophage comprises mycobacteriophage, such as mycobacteriophage R51, D29, TM4, L5, or Bxbl.

17 A method for delivery of a bacteriophage into a cell or a cellular compartment, comprising the steps of providing a composition in accordance with any of claims 4-16 comprising a complex of said bacteriophage with a cationic delivery macromolecule, and introducing said complex to said cell or cellular compartment, such as to permit the delivery of said bacteriophage into said cell or cellular compartment.

18 A method for the treatment of a disease or condition in a patient that is characterised by the infection of the patient's cells by bacteria; comprising the steps of providing a composition as claimed in claim 15 or claim 16 and admininstering said composition to said patient such that the complex can be transported to said cells for delivery of the bacteriophage therein.

19 A method as claimed in claim 18, wherein said composition is administered to said patient orally or parenterally, for example by injection into the bloodstream.

20 A method for the use of a composition as claimed in claim 15 or claim 16 in the manufacture of a medicament for use in treating a disease or condition in a patient which is characterised by intracellular bacterial infection.

21 A method as claimed in any of claims 18-20, wherein said disease or condition is selected from tuberculosis, zoonotic disease of cattle, leprosy, typhoid, paratyphoid, brucellosis, Legionnaire's disease, listeriosis, tuleraemia, Rocky Mountain fever, epidemic typhus, endemic typhus, Q fever, chlamydial STD, psitticosis, pneumonia, granuloctic ehrlichiosis, shigellosis, whooping cough, toxoplasmosis, and anthrax.

22 A medicament comprising a composition as claimed in claim 15 or claim 16 and, optionally, one or more pharmaceutically acceptable excipients or carriers.