WO2004087924A2 - Method for delivery of substances to intracellular microorganisms - Google Patents

Abstract

The present invention concerns a method of delivering substances to obligate intracellular microorganisms. Further, the invention concerns a method of treatment of infections of intracellular microorganisms. Further, the invention concerns substances suitable for treatment of infections of intracellular microorganisms. In addition, the invention concerns stable transformants of obligate intracellular microorganisms.

Description

New PCT-Application

Max-Planck-Gesellschaft zur Fόrderung der Wissenschaften e.V.

Our Ref.: F 2511 PCT

Method for delivery of substances to intracellular microorganisms

The present invention concerns a method of delivering substances to obligate intracellular microorganisms.

Further, the invention concerns a method of treatment of infections of intracellular microorganisms. Further, the invention concerns substances suitable for treatment of infections of intracellular microorganisms.

In addition, the invention concerns stable transformants of obligate intracellular microorganisms.

Definitions

- Conjugation in the context of the present invention denotes the coupling of two molecular species. The product is referred to as a conjugate. Coupling includes covalent and non-covalent bonding of the two species. Coupling also includes direct bonding between the two species or indirect bonding via bifunctional reagents.

- Cross-linking is the conjugation of two molecular species which have some sort of affinity between them, e. g. the chemical bonding between a receptor and a ligand.

- A bifunctional reagent couples at least one molecule of a first molecular species to at least one molecule of a second molecular species. A bifunctional reagent contains at least two reactive groups for coupling with the two molecular species. A heterobifunctional reagent contains reactive groups of different specificity and is able to couple two molecular species with different functional groups. The reactive groups are dissimilar. A homobifunctional reagent contains reactive groups which are able to couple molecular species with identical functional groups. The reactive groups on the homobifunctional reagent have not to be identical.

- An antibiotic is a chemical compound which has inhibitory activity against microorganisms, viruses and eukaryotic cells. The antibiotic might be derived from the secondary metabolism of living organisms (Grafe, 2002), but may also be any compound suitable for inhibition of microorganisms, viruses and eukaryotic ceils. Inhibitory activity includes suppressing of growth, propagation, and/or killing of microorganism, viruses and eukaryotic cells. However, although particular nucleic acids disclosed in the present invention would fall under this definition, they shall be regarded as a separate class of molecules. Particular nucleic acids are able to selectively knocking out one or more genes of the microorganisms, and thereby inhibiting microorganisms.

- The term chlamydia describes the bacteria of the order Chlamydiales including the four families: Chlamydiaceae, Simikaniaceae, Parachlamydiaceae and Waddliaceae. The family Chlamydiaceae includes two genera: Chlamydia and Chlamydophila. The genus Chlamydia contains C. muridarum, C. suis and C. trachomatis. The genus Chlamydophila contains C. abortus, C. caviae, C. felis, C. pecorum, C. pneumoniae and C. psittaci. C. trachomatis and C. pneumoniae are human pathogens.

A transformant is a transformed microorganism. Transformation is a process in which nucleic acid is delivered to a microorganism. The nucleic acid may be linked to other compounds. Stable transformation is achieved if the nucleic acid is transmitted to any progeny of the microorganism. A prerequisite for stable transformation is the integration of the nucleic acid into a replicon of this microorganism, or has to represent a separate replicon of this microorganism. Transferrin (Tf) is an 80 kDa monomeric glycosylated polypeptide consisting of two globular domains. Each domain contains a high affinity binding site for one iron molecule. The amino acid sequence of human transferrin is described in Hershberger, Larson et al., 1991 , Genbank-Accession AAB22049, Version AAB22049.1 , Genlnfo Identifier 248648). In the present invention, the transferrin definition shall include transferrin derivatives and any polypeptide (and derivatives thereof) capable of binding to the transferrin receptor, e.g. conalbumin, or HFE (hereditary hemochromatosis gene product, 11 alternative transcripts, Genbank Accession-No NM_000410.2, NM_139002.1 , NM_139003.1 , NM_139004.1 , NM_139005.1 , NM_139006.1 , NM_139007.1 , NM 39008.1 , NM_139009.1 , NM_139010.1, or NM_139011.1). The Transferrin receptor protein 2 (TfR2, HFE3, Accession-No. NM_003227.1 , is also included.

The transferrin receptor (TfR) is a protein located on -the plasma membrane of mammalian and avian cells. Insects have been shown to encode and produce transferrin that is homologous to the one of vertebrates, suggestive of presence of transferrin receptor.

A target is a protein that plays an essential role in a particular disease. The disease should be treated successfully by activation or inhibition of the target.

Biology of Chlamvdiales

The order Chlamydiales comprises Gram negative obligate intracellular bacteria and includes two human pathogens: Chlamydia trachomatis and Chlamydophila pneumoniae. C. pneumoniae derives nutrients from higher eukaryotic host cell, however, the underlying mechanism of this process is not well understood.

All Chlamydiales have a unique developmental cycle. The infectious form called elementary body (EB) invades host cell and remains separated from the cytoplasm by a vacuolar structure called inclusion. Few hours after infection, EB reorganizes within inclusion to form reticulate body (RB) that unlike EB is metabolically active. RB actively synthesizes RNA, proteins and replicates. Between day 2 and 4 after infection, RB reorganizes back to the electron dense, metabolically inactive, small infectious EB that are released from inclusion and host cell to infect neighboring cells.

Genome and gene function in Chlamvdiales

Genomes of C. pneumoniae and C. trachomatis have been recently sequenced (Kalman, Mitchell et al., 1999). About 200 genes are of unknown function while function of remaining genes is hypothetically deduced based on gene homology with other bacteria. Diseases associated with Chlamvdiales

C. trachomatis causes trachoma and urogenital infections and has also been -associated directly or indirectly with other diseases like an increased risk for cervical squamous-cell carcinoma (Koskela, Anttila et al., 2000)Paavonen, 2001 and reactive arthritis (Berlau, Junker et al., 1998).

C. pneumoniae at first infects epithelial cells of the respiratory system leading to pneumonitis and other illnesses of the upper and lower respiratory tract. Presence of C. pneumoniae organisms, DNA, antigens or antibodies against was also associated with atherosclerosis (Grayston, 2000)Saikku, 2000, lung carcinoma (Jackson, Wang et al., 2000), Alzheimer disease (Balin, Gerard et al., 1998), encephalitis (Guglielminotti, Lellouche et al., 2000)Airas, Kotilainen et al., 2001, multiple sclerosis (Sriram, Stratton et al., 1999), skin lesions in diabetic patients (King, Jr., Bushman et al., 2001) and other disorders. IgG antibodies against C. pneumoniae, an indicative of present or past infections, can already be detected in blood of young children. The IgG seropositivity increases with age and reaches majority in population above the age of 60 (70-80%) (Grayston, 2000). This strongly suggests that almost anyone can be exposed to C. pneumoniae at some point in life. At this time, it is not clear if presence of C. pneumoniae or C. trachomatis initiates or accelerates progress of the diseases listed above.

Mechanism of disease induction by Chlamvdiales

Chlamydial infections are first acute and then often become persistent and can last for several years (Hammerschlag, Chirgwin et al., 1992). It is difficult to establish criteria for eradication of this bacteria. For C. pneumoniae, majority of people are seropositive and one study demonstrated that about 20% of healthy blood donors were in fact carriers of this bacteria (Bodetti and Timms, 2000).

Pathogenic mechanism of chlamydial infections investigated to date have been associated with inflammation and autoimmunity.

Local inflammation due to infection leads to tissue damage and scaring (Stephens, 1999). The inflammation is a result of production and release of inflammatory cytokines from the infected cells. Multiple cytokines have been detected in the infected tissues including proinflammatory IL-1β, IL-6, and TNF-α (Stephens, 1999).

Pathogenic mechanism behind autoimmunity involves molecular mimicry. Some of the chlamydial proteins share homology with human proteins. If the immune response during the infection will be directed against these proteins this could lead to autoimmune reaction.

There are so far two known examples of chlamydial proteins that share epitope homology with human proteins:

1. chlamydial heat shock protein 60 (HSP60)

2. hypothetical protease encoded by gene CPn0483.

Endothelial cells exposed to stress produce HSP60. High levels of human HSP60 were demonstrated in serum of patients with atherosclerosis. T cells that recognize chlamydial HSP60 due to previous or existing infection with C. pneumoniae can damage of endothelium during the cross-reactive cytotoxic immune response (Gupta and Camm, 1999). In fact, T cell lines recognizing chlamydial and human HSP60 have been established from human atherosclerotic plaques (Mosorin, Surcel et al., 2000).

The open reading frame Cpn0483 encodes hypothetical cysteine protease that belongs to the predicted hyperfamily of cysteine proteases that comprises prokaryotic and viral homologues (Makarova, Aravind et al., 2000). No homologue from this protease family was found in genomes of other Chlamydiales and bacteria.

Recently, it has been demonstrated that rats injected with a synthetic peptide derived from the sequence of Cpn0483 develop encephalomyelitis (Lenz, Lu et al., 2001). This synthetic peptide shared homology with a specific epitope of basic myelin protein known to cause autoimmunoreactivity in multiple sclerosis.

Treatment of chlamydial infection, state of the art

Chlamydiales are obligate intracellular pathogens. Thus, for their growth to be inhibited by an antimicrobial agent, this agent must penetrate cells and the chlamydial inclusion to be able to act on bacteria. Therefore, antibiotics that are likely to be active against Chlamydiales are macrolides, tetracyclines, chloramphenicol, quinolones and rifampicin. All of the above antibiotics can enter the cell and were shown to have antimicrobial activity. However, a prolonged in vitro treatment of the infected cells with azithromycin and with gemifloxacin (an enhanced-affinity fluoroquinolone) failed to completely eliminate C. pneumoniae (Kutlin, Roblin et al., 2002a, Kutlin, Roblin et al., 2002b).

Tetracyclins are most often Used at present in clinics followed by macrolides (azithromycin, clarithomycin) and quinolones (ofloxacin and sparfloxacin). Patients have to be treated for at least 4-5 weeks, which may be an unacceptable long period due to general side effects of antibiotics and resistance development. The clinical response of C. pneumoniae respiratory infection to antibiotics was often slow, with persistance of symptoms and frequent clinical relapse requiring further treatment (Grayston, Kuo et al., 1986). Persistent organisms might act as a reservoir for spread of infection and could play a role in pathogenesis of chronic diseases associated with C. pneumoniae. Chronic C. pneumoniae infections may be less susceptible to eradication with antibiotics (Gupta and Camm, 1999).

Other infections which have persistent intracellular forms which are difficult to attack The persistent infections can be caused by bacteria other than Chlamydiales. Obligate intracellular pathogens include Ehrlichia spp. and Mycobacterium spp. of which M. tuberculosis causes one of the top three infectious disease killers.

Facultative intracellular pathogens include Salmonella enterica, a pathogen which can replicate in macrophages, Brucella abortus and Legionella pneumophila.

Object of the present invention

It is still almost impossible to access obligate intracellular microorganisms. No attempts are known to deliver substances to chlamydia harvested in eukaryotic cells. These substance has to pass the eukaryotic cell membrane and the cytoplasm. The substance has to be targeted to the inclusion in which the microorganism is located, and has to pass the inclusion membrane and the bacterial (cell wall and membrane). Thus, the object of the present invention is to provide a means to access in a simple and rapid manner intracellular microorganisms with any molecules suitable for experimental and therapeutic purposes.

The local concentration of the antibiotics is not large enough for complete eradication of the chlamydia. Thus, the object of the present invention is a method and a pharmaceutical composition for antibiotic treatment of chlamydial infections with locally increased antibiotic concentrations at the chlamydial inclusions. In addition, a pharmaceutical composition with reduced side effects for long term application of antibiotics for treatment of persistent chlamydial infections is provided. The antibiotics so far used in the therapy of chlamydial infections are not specific to Chlamydia, and have more or less inhibitory effects upon other microorganisms, pathogenic or not, which might be an undesirable side effect in long term antibiotic treatment. Thus, a further object of the present invention is a method for identification of chlamydial target proteins for specific antibiotic treatment of chlamydia and other obligate intracellular microorganisms.

Chlamydial Transformants

Genetic engineering of intracellular bacteria

In many bacteria, viruses or higher organisms, function of genes is studied by using methods that enable their modification.

For bacteria, these methods include engineering nucleic acids encoding genes of interest (or parts of them) into vectors under a control of specific promoters (e.g. plasmid DNA, phagemid DNA) and introducing these constructs into bacteria using transformation, transduction, or conjugation. Transformation involves the uptake of naked DNA by competent bacteria and is usually facilitated by physical (electroporation) or chemical (calcium) means. Transduction is an exchange of DNA between bacteria using bacterial viruses while conjugation is an exchange of DNA between bacteria.

Of these three methods, using transformation to introduce nucleic acids into Chlamydiales is the most obvious choice. In all the reports about transformation of other intracellular bacteria (e.g. Coxiella burnetti, Rickettsia sp.), electroporation was used to introduce naked DNA into isolated bacteria (Lukacova, Valkova et al., 1999)Troyer, Radulovic et al., 1999. These attempts resulted in an successful transformation of the bacteria. In Coxiella burnetti, transformation with a GFP construct resulted in the expression of GFP that could be detected during several passages. However, the fluorescence intensity was lower than in E. coli transformed with the same construct, most likely due to the replication origin of the construct. Replication of the GFP construct was under control of a C. burnetii autonomous replication sequence (Suhan, Chen et al., 1994), and protein expression was performed under control of an IPTG-inducible trp/lac fusion promoter (Lukacova, Valkova, Quevedo, Perecko, and Barak, 1999, Suhan, Chen et al., 1996) derived from E. coli. In Rickettsia typhi, a GFP construct was stably introduced into the chromosome by homologous recombination. No plasmids capable of autonomous replication within rickettsiae have been identified, nor suitable selectable markers have been found. The use of antibiotic resistance to tetracycline or chloramphenicol is not advisable, due to their importance in treatment of rickettsial diseases (Troyer, Radulovic, and Azad, 1999). The approaches of Lukacova, Valkova, Quevedo, Perecko, and Barak, 1999 and Troyer, Radulovic, and Azad, 1999 are labor intensive and time consuming. Transformation of intracellular forms of bacteria or chemical means of transformation of Legionella, Coxiella, or Rickettsia have not been attempted.

Genetic engineering of Chlamvdiales

For Chlamydiales, no genetic engineering method has been developed. The only attempt to modify chlamydial genome was applied for Chlamydia trachomatis by using electroporation of EB to introduce chloramphenicol resistance gene (Tarn,

Davis et al., 1994). These experiments had, however, at least two pitfalls:

1.) the number of EB used for transformation had to be extremely high as 90% of

EB died during electroporation and 2.) obtained transformants were unstable and were lost after four generations.

The difficulties in gene manipulation of Chlamydiales are complex. The EB are extremely temperature-sensitive. As electroporation generates high local temperature within the suspension, the majority of bacteria die during this procedure. It is also unknown how electroporation affects infectivity of Chlamydiales that have survived the procedure. Chemical means of transformation of EB have not been attempted.

In the infected cell, access to Chlamydiales is through the host cell membrane, cytoplasm, vacuolar membrane, lumen of the vacuole and finally outer and inner membrane.

Further object of the present invention

Transformants of bacteria are an important tools for research in life science. Almost all methods of genetic engereering are directly or indirectly based on bacterial transformants.

The identification and/or validation of chlamydial target proteins which might be suitable for treatment of chlamydial infections might be facilitated by chlamydial transformants. Thus, a further object of the present invention is to provide a simple and convenient method for transformation of obligate intracellular bacteria (chlamydia), including suitable reporter genes, reporter gene constructs, either on a plasmid capable of autonomous replication, or for insertions into the chromosome. In addition, stable and transient transformats are provided.

Techniques of protein conjugation: state of the art

Up to now, a large variety of applications of protein conjugates are known. An overview of applications of protein conjugation can be found in Wong, 1993. By conjugation, both of the molecular species to be coupled form a new entity which combines the particular properties of both species. Proteins to be conjugated may be targeting agents, e.g. antibodies, receptor ligands, or hormones. The second molecular species may be a protein as well, or may be a lower molecular weight compound. The second species may be used e.g. for tracing the protein species, e.g. a fluorescent molecule or a molecule containing radioactive isotopes. The second species may also be a compound inducing a specific biological response, e.g. a hormone, or a toxic compound. A comprehensive description of protein conjugation and the underlying chemical reactions can be found e.g. in Wong, 1993. If at least one of the molecular species to be conjugates is a polypeptide, conjugation can be regarded as the chemical modification of a side chain of the polypeptide. Polypeptides provide several functional groups within the side chains which can be used for conjugating them with other compounds. The most reactive functional groups are amino groups (ε-amino group of serin, N-terminal amino acids), carboxyl groups (aspartic acid, glutamic acid and C-terminal amino acids), sulfhydryl groups (cysteine), thioether (methionin), imidazolyl groups (histidine), and guadinidyl groups (arginine). Less reactive side chains are phenolic groups (tyrosine), and indolyl groups (tryptophan). The hydroxyl groups of serin and threonin, respectively, are less important, as, from the viewpoint of there reactivity in an aqueous solution, they can be regarded as water derivatives.

Group specifity of reagents

Many chemical reactions suitable for conjugation of polypeptides are known. For example, in a nucleophilic substitution reaction, the functional group at an amino acid side chain with a lone pair of electrons (e.g. the amino group and the sulfhydryl group, particularly in its thiolate form) attack the electrophilic centers of the substrate. Typical leaving groups are N-hydroxysuccinimide, methanol, iodine and thiopyridine. Electrophilic reactions have been described to attack the side chains of tyrosines and histidines, e.g. by diazonium salts, particularly aryl diazonium components.

Group specific reagents have been described in the art. An overview can be found in Wong, 1993, pp. 27-48, which is included herein by reference. However, the number of groups absolutely specific towards a particular functional group is very limited.

Specific groups which couple to sulfhydryl groups are e.g. mercurials or disulfides. Isocyanates, isothiocyanates, sulfonyl halides, and imidoesters e.g. are specific for amide groups. Diazoacetates and carbodiimides e. g. are used for coupling to carboxyl groups.

New functional groups can be introduced into proteins by the modification of protein side chains. The following reactions are described in Wong, 1993, pp. 16-25 which is included herein by reference: conversion of amines to carboxylic acids conversion of amines to sulfhydryl groups, conversions of thiols to carboxylic acids, conversions of thiols to amines, conversions of carboxylic acids to amines, conversions of hydroxyl to sulfhydryl groups, conversions of tyrosine to aminotyrosine.

Introduction of spacers

A spacer arm can be introduced in order to reduce steric hindrance between the two molecular species to be coupled. The spacer removes the functional groups from the protein surface and decreases the influence of the local environment of the functional group. In its easiest form, the spacer is a backbone with a functional group at each end. The backbone may be, in its easiest form, a simple aliphatic chain, or may contain additional functional groups increasing or decreasing hydrophobicity. Bifunctional reagents in which the at least two reactive groups are spatially separated can be regarded to contain a spacer (Wong, 1993, p. 14, pp. 49-50, p. 68).

Conjugates between proteins and nucleic acids

Covalent conjugation between proteins and nucleic acids is described in Wong,

1993, p. 319.

Conjugates between protein and antibiotics

Conjugates between polypeptides and antibiotics are known in the art. These conjugates were used for examination of the antigenic properties of antibiotics (e. g. penicillin and its derivatives), which are undesired side effects of therapeutic applications. Since compounds of molecular masses less than about 1000-10000 Da are usually not intrinsically immunogenic, it was necessary to couple the antibiotics to proteins to render them immunogenic. Preparation of antigenic conjugates of e.g. penicillins (Fernandez, Warbrick et al., 1995, Zhao, Batley et al., 2000)Cliquet, Cox et al., 2001 or sulfonamide antibiotics (Spinks, Wyatt et al., 1999) are described. A conjugate of maleylated bovine serum albumin (MBSA) and p-aminosalicylic acid (PAS) in a molar ratio of 33 mol PAS/mol MBSA is able to kill intracellular Mycobacterium tuberculosis in a cell culture model more efficient than free PAS (Majumdar and Basu, 1991).

Non-covalent conjugates

Wong, 1993, pp. 128-132: Non-covalent conjugates are formed e.g. between avidin or streptavidin and biotin. The formation of the complex is essentially irreversible. Avidin and streptavidin are tetravalent, thus, they can conjugate biotin containing compounds, including proteins. Details of the application of the avidin/steptavidin- biotin method can be found e.g. in Bayer and Wilchek, 1980; Hofstetter, Morpurgo et al., 2000 or Wilchek and Bayer, 1990.

Transferrin-based vesicular system of the endocytic pathway

Endocytosis is a cellular process in which macromolecules and particulate substances are taken up. One of the well researched examples of this process is the receptor-mediated endocytosis of transferrin (Tf), a protein that carries iron in the blood.

Cell-surface transferrin receptors (TfR) bind and deliver transferrin with its bound iron to early endosomes by receptor-mediated endocytosis. After acidification, the low pH in the endosome induces transferrin to release its bound iron, but the iron-free transferrin itself (called apotransferrin) remains bound to its receptor and is recycled back to the plasma membrane as a receptor-apotransferrin complex. When it has returned to the neutral pH of the extracellular fluid, the apotransferrin dissociates from the receptor and is thereby free to pick up more iron and begin the cycle again. Thus the transferrin protein shuttles back and forth between the extracellular fluid and the endosomal compartment, avoiding lysosomes and delivering the iron that cells need to grow.

The appearence of the transferrin receptor is not limited to proliferative ceils, and can be found on cancer cells and normal dividing cells (Munns, Yaxley et al., 1998, Ponka and Lok, 1999, p. 1116). An antibody against the transferrin receptor are able to inhibit the growth of SKBR3 cells and is a mimic of the ligand holotransferrin (Poul, Becerril et al., 2000).

Use of Transferrin receptors as a marker of early endosomes TfR is a 180 kDa glycoprotein consisting of two identical disulphide-linked 90 kDa subunits (Schneider, Owen et al., 1984). This receptor is expressed on the cell surface and on the early endosomes, which are the first compartments receiving endocytosed materials. Early endosomes usually comprise two distinct compartments: the sorting endosomes, part of the endocytic pathway, which contain internalized ligands that will be delivered to lysosomes and degraded; and the recycling endosomes, which contain molecules to be returned back to the cell surface (Mukherjee, Ghosh et al., 1997).

Al Younes, Rudel et al., 2001 demonstrated that the amount of TfR co-localized with chlamydial inclusions (C. trachomatis, C. pneumoniae) dose-dependently increases in cells where iron was depleted by addition of the iron-chelator deferoxamine mesylate (DAM, up to 30 μM). Unlike in the uninfected cells, in the infected DAM- treated cells, the punctuate pattern of TfR distribution throughout the cytoplasm disappeared. Additionally, more abundant accumulation of TfR surrounding C. pneumoniae inclusions was observed unlike in the untreated infected control. Thus, it was postulated that, iron is an indispensable element and that Chlamydia may use iron transport pathways of the host by attracting TfR to the phagosome.

Al Younes, Rudel, Brinkmann, Szczepek, and Meyer, 2001 examined the interaction of the chlamydial inclusions with vesicles of the endocytic pathway of host cells deprived of iron. Analysis of the confocal microscopy data showed that the inclusions in iron-deprived host cells did not sequester the lysosomal protein marker LAMP-1 (Fig.4B), similar to those in the control infected cells (Fig.4A). Interestingly, in the DAM-treated cells, the punctuate pattern of TfR distribution throughout the cytoplasm disappeared, and more abundant accumulation of these receptors was observed surrounding C. pneumoniae vacuoles, unlike in the untreated control. These findings were corroborated by electron microscopy using gold immunolabelling, which, in contrast to unexposed cells, showed local concentration of TfR in the vicinity of the inclusion in DAM-exposed cells. Surprisingly, gold particles were found in association with small vesicles of unknown origin present either ih the lumen of the inclusion or inside compartments located adjacent to the inclusion in the DAM-expόsed cells and the unexposed control cells. Taken together, these results indicate that limiting the availability of iron does not affect the interaction of the chlamydial inclusions with lysosomes, but increases the amount of TfR attracted to the vicinity of the inclusions.

Based on microscopical observations, TfR-enriched vesicles are smaller in size when compared with lysosomes and are abundant within the cytoplasm of HEp-2 cells. Indeed, two staining patterns of TfR were observed, particularly at 20 h and 40 h after infection. TfR-positive structures were found either as aggregates intimately associated with the parasitophorous vacuoles, staining a considerable part of its circumference, or exhibited a granular discontinuous fluorescence distribution, which was also closely adjacent to the inclusion (Fig. 4B, in Al Younes, Rudel et al., 1999a, arrowhead). The granular pattern of TfR labelling was sustained in most of the cells infected for 70 h, whereas a small population of infected cells showed a reduced accumulation of TfR-containing vesicles at the periphery of the inclusion. In general, this significant concentration of TfR adjacent to the inclusionmay reflect a degree of translocation of early endosomes within the cytoplasm to the proximity of bacterial vacuoles, suggesting a possible interaction between these vacuoles and early endosomes. Thus, TfR associates with chlamydial inclusions. TfR-enriched vesicles were observed close to Chlamydial vacuoles, indicating a specific translocation of theses organelles through the cytoplasm to the vicinity of the vacuole. In other words, early or/and recycling endosomes are attracted to close proximity of chlamydial inclusions (Al Younes, Rudel et al., 1999b).

Early endosomes do not fuse with the inclusion, but they show a punctate or aggregate pattern of distribution around the pathogen vacuole. These results correlate with and strengthen those obtained by other investigators using confocal and electron microscopy. For instance, no signs of fusion of these vesicles have been documented at 18 h (Scidmore, Fischer et al., 1996) and 24 h after infection (Taraska, Ward et al., 1996) or even at very early phases (30 min, 2 h or 4 h) of infection Hackstadt, 1998. The recruitment of these vesicles and their failure to fuse might reflect two independent steps of the maturation process, whereby the fusion rather than the docking is actively prevented by the pathogen. Inhibition of the early endosomal fusion might be crucial for the subsequent fate of the inclusion, as it prevents the delivery of signalling molecules to the inclusion membrane, which may be required for the interaction with late endosomes.

Because of the absence of confluent circumferential staining, it is concluded that TfR is very likely not a constituent of the membrane of the chlamydial vacuole, at least not at the time points examined (Al Younes, Rudel, and Meyer, 1999b). The amount of TfR co-localized with chlamydial inclusions increases in tissue culture system where iron was depleted by addition of deferoxamine, an iron chelating agent. TfR was the first time reported to be inside chlamydial inclusions (Al Younes, Rudel, Brinkmann, Szczepek, and Meyer, 2001). It is important to note that the TfR is not taken up by the chlamydia.

Structure and function of transferrin and its interaction with the transferrin receptor A human transferrin sequence is disclosed in Genbank under the accession number AAB22049, Version AAB22049.1 , Genelnfo identifier 248648.

This sequence contains about 60 lysine residues (AAB22049: 58 residues) which are almost uniformly distributed along the sequence (Tanaka, Kaneo et al., 1996, p. 777). Lysine residues can be used for conjugation of various compounds.

Transferrin conjugates

It is state of the art to use transferrin to deliver substances to eukaryotic cells. The ability of transferin to be taken up by the cells is limited by the molar ratio of the substance relative to the protein moiety. Transferrin conjugates of low molecular weight substances as well as polypeptides can be taken up. Tanaka, Kaneo, and Miyashita, 1996 describe the manufacture of a conjugate of transferrin holoenzyme and the anti-tumour drug mitomycin C (MMC). MMC has a molecular weight of 334 dalton. The conjugate is taken up by eukaryotic cells and is intended to be used in cancer treatment. The conjugates can be manufactured easily in variable molar ratios. In a ratio of about 10 mol MMC/mol Tf, the activity is reduced by about 50% compared with unconjugated Tf (Tanaka, Kaneo, and Miyashita, 1996, p. 776 and Fig. 4). A ratio of 23 mol MMC/mol Tf reduces the activity to about 10%. The active ester of glutarylated MMC will be coupled to the ε-amino groups of Tf. The binding parameters (association constant, number of binding sites) of a Tf-MMC conjugate with a molar ratio of about 4 (calculated from a MMC content of 1.8 w/w% in the conjugate and a Tf molecular weight of 80 kDal) and Tf are the same in HepG2 cells (Tanaka, Fujishima et al., 2001).

Conjugates between transferrin and antibiotics have not yet been used to treatment of bacterial infection.

A conjugate of transferrin with insulin with a molar ratio 3 mol insulin/mol Tf increases transcytosis of insulin in Caco-2 cells. Transcytosis was mediated by TfR, but not by the insulin receptor. Caco-2 cells are an in vitro model of intestinal epithelium (Shah and Shen, 1996). In streptozotocin-induced diabetic rats, it could be demonstrated that transepitheliai transport via TfR-mediated transcytosis is a feasible approach for developing the oral delivery of insulin (Xia, Wang et al., 2000).

Transferrin-based transfection system for eukaryotic cells

The US patents 5,354,844 and 5,792,645 discloses conjugates of transferrin with nucleic acids. A transferrin-polycation nucleic acid complex capable of being absorbed into a cell by transferrin receptor-mediated endocytosis is described.

US patent 5,922,859 discloses a complex of internalizing factors (e.g. transferrin) conjugated to bonding factors (e.g. polycationic compounds) and non-covalently bound substances (e.g. polycationic compounds, histones) having high affinity for nucleic acids. These complexes are used for endocytic uptake of nucleic acids into eukaryotic cells. In addition, a composition is disclosed comprising the complex wherein the nucleic acid to be transferred is a therapeutically effective nucleic acid.

Sigma offers under T0288 a transferrin-polylysin-FITC conjugate for DNA delivery.

Detained description of the invention

Inventive concept

Surprisingly, transferrin-conjugated molecules (e.g. transferrin labelled with the fluorescence dye Texas Red or with biotin) are accumulated within chlamydial inclusions.

In a chase studies, exogenous transferrin could be found as soon as 5 minutes later localized in the chlamydial inclusions. These data were confirmed by electron microscopy. Immunolabeled transferrin was detected inside chlamydial inclusions and inside chlamydial cells (Example 1 and 2). In Example 3, localization of fluorescently labeled antisense oligonucleotide directed against chlamydial gene Cpn0483 and coupled with transferrin was studied by immunofluorescence. Using confocal microscopy, fluorochrome-Iabeled oligonucleotide DNA was detected inside chlamydial inclusions.

The state of the art does not provide evidence that transferrin binding to the TfR on the cell surface would be transported to the chlamydial inclusion and would be internalized into the EBs or RBs. In addition, there is no hint that transferrin-delivered molecules are accumulated within the chlamydial inclusion, or will be taken up by the chlamydia. Although transferrin is widely used as a delivery system for nucleic acids in eukaryotic cells, transferrin has not yet been suggested for delivery of molecules to intracellular bacteria.

The present invention provides substances, methods and pharmaceutical compositions suitable for treatment of chlamydial infection. These substances, methods and pharmaceutical compositions exploit the surprising findings that molecules can be accumulated within chlamydial inclusion and can be delivered to chlamydia via a targeting moiety, which is a transferrin molecule internalized into eukaryotic cells via the transferrin receptor. Further, the invention provides tools for development of substances, methods and pharmaceutical compositions for treatment of chlamydial infections, e.g. methods of transformation of chlamydia, vectors suitable for transformation of chlamydia, and transformed chlamydia. Furthermore, the invention concerns chlamydial proteins as possible drug targets for treatment of chlamydial infections, including methods for use.

The substance is

(a) suitable for suppressing growth, propagation, and/or killing of intracellular microorganism, e.g. a nucleic acid, and/or

(b) an antibiotic to which the intracellular bacteria from the order Chlamydiales are susceptible.

(c) a conjugate comprising a targeting moiety capable of targeting chlamydial inclusions within eukaryotic cells, and at least one effector molecule selected from (a) and/or (b). Eukaryotic proteins (e.g. transferrin) can be used as a carrier to deliver substances to the intracellular chlamydia. Methods for manufacture said conjugate are disclosed.

The invention therefore relates to a method of identifying compounds which selectively inhibit growth and/or propagation of Chlamydiales, comprising

(a) determining the in vitro-interaction of the compound with a polypeptide selected from the group of polypeptides encoded by the sequences described in Table 1, and

(b) contacting the compound exhibiting in vitro interaction according to step (a) with a eukaryotic cell infected with a Chlamydiales species under condition that allow the uptake of the compound, and

(c) determining the reduction of growth and propagation of the Chlamydiales species within the eukaryotic ceils exposed to the compound according to step (b).

In a preferred embodiment, the compound identified by the method of the invention is used for manufacture of a pharmaceutical composition for treatment of a chlamydial infection. The substance may be used for treatment of infections with Chlamydiales, and may be used for manufacturing a pharmaceutical composition for treatment of infections of Chlamydiales comprising an antibiotic, and/or a nucleic acid conjugated with a protein. Further, the invention concerns a method of treatment of infections of chlamydiales by administering the conjugate formed between transferrin and a molecule to a subject in need thereof, together with pharmaceutically acceptable auxiliary substances, carriers, and diluents.

The present invention can be applied to any intracellular bacteria of the order Chlamydiales (including the genus Chlamydia and/or Chlamydophila, and the species Chlamydia trachomatis and Chlamydophila pneumoniae) which all have a lifecycles very similar to the cycle of Chlamydia trachomatis and Chlamydophila pneumoniae. All Chlamydiales form the typical inclusions and can be treated by substances, methods and pharmaceutical compositions of the present invention. All Chlamydiales can be transformed by the methods and vectors of the present invention, and target proteins of the present invention can be found in all Chlamydiales.

The present invention also relates to a recombinant vector, comprising

(a) an origin of replication suitable for replication in a bacterium from the order Chlamydiales, and/or

(b) an expression signal suitable for protein expression in a bacterium from the order Chlamydiales, and/or

(c) a reporter gene operatively linked to the expression signal of (b).

Furthermore, the invention also relates to a method of introducing an organic molecule into a bacterium from the order Chlamydiales comprising

(a) forming a conjugate between said organic molecule and a polypeptide capable of binding to the transferrin receptor, and

(b) contacting the conjugate formed in step (a) with a eukaryotic cell infected with said bacterium under condition that allow the uptake of the conjugate via the transferrin receptor.

In a preferred embodiment of the method of the invention the polypeptide is transferrin.

In a further preferred embodiment of the method of the invention the organic molecule is an antibiotic.

In a particular preferred embodiment of the method of the invention the antibiotic is selected from penicillins, cephalosporins, tetracylines, quinolones, or sulfonamides.

In a more preferred embodiment of the method of the invention the organic molecule is a nucleic acid.

In a most particular preferred embodiment of the method of the invention the bacterium is from the order Chlamydiales. The transferrin capable of being internalized into chlamydial inclusion and suitable for conjugation may be selected among vertebrate transferrins known in the art (e.g. from human, chicken, or mouse) Examples of non-transferrin proteins which could substitute transferrin are conalbumin, ferritin and HFE (hereditary hemochromatosis gene product) The function of HFE protein is unknown, but it has been proposed, that HFE may negatively regulate the uptake of iron-loaded transferrin.

Nucleic acids

Substances suitable for suppressing growth and propagation of intracellular microorganisms or for killing said microorganisms may be nucleic acids (antisense, RNA, DNA, either double stranded or single stranded) targeting for example genes coding for bacterial proteins involved in transcription regulation.

Thus, a subject matter of the present invention are nucleic acids or fragments thereof for use as a medicament. Further, the present inventions concern the use of nucleic acids for manufacture of a pharmaceutical composition for the treatment of infections with obligate intracellular microorganisms from the order Chlamydiales. The nucleotide sequence of the nucleic acid can be selected from the group of sequences described in Table 1. The invention further concerns nucleic acids comprising the sequence CGTAGTTTTTTAATCAA, its use as a medicament and its use of manufacture a pharmaceutical composition for treatment of chlamydial infections.

The present invention therefore relates to a nucleic acid or a fragment thereof for use as a medicine, characterized in that the nucleotide sequence of the nucleic acid is selected from the group of sequences described in Table 1.

The present invention also relates to a nucleic acid characterized in that the nucleotide sequence of the nucleic acid is CGTAGTTTTTTAATCAA.

In a preferred embodiment the nucleic acid of the invention is used as a medicine.

In an additionally preferred embodiment the nucleic acid or a fragment thereof is DNA.

In a particularly preferred embodiment the nucleic acid or a fragment thereof of the invention is RNA and the nucleic acid sequence T is replaced by U.

In a further particularly preferred embodiment the nucleic acid or a fragment thereof of the invention is single stranded.

In an additionally particularly preferred embodiment the nucleic acid or a fragment thereof of the invention is double stranded.

In another additionally preferred embodiment the nucleic acid or a fragment thereof of the invention is used for manufacture of a pharmaceutical composition for the treatment of infections with obligate intracellular microorganisms from the order Chlamydiales.

In a more preferred use of the invention the obligate intracellular microorganism is from the genus Chlamydia. In an even more preferred use of the invention the obligate intracellular microorganism is Chlamydia trachomatis.

In an additionally even more preferred use of the invention the obligate intracellular microorganism is from the genus Chlamydophila.

In a most preferred use of the invention the obligate intracellular microorganism is Chlamydophila pneumoniae.

A person skilled in the art knows how to design a nucleic acid suitable for complete or partial suppression (gene silencing) of the translation if either the whole or partial nucleotide sequence of the gene coding for the protein is given, may this sequence represent the sense strand or the antisense strand, or may it represent DNA or RNA, single stranded or double stranded.

In another aspect, the invention provides nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above. By "stringent hybridization conditions" is intended overnight incubation at 42° C in a solution comprising: 50% formamide, 5x SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1x SSC at about 65° C.

By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 (e.g., 35, 40, 45, 50, 55, 60, 65) nt of the reference polynucleotide.

Gene silencing is performed by inhibiting the corresponding mRNA of the gene by an antisense nucleic acid. The sequences described in Table 1 are the nucleotide sequences of the coding strand of chlamydial proteins including 5' and 3' non translated sequences.

Antisense oligodeoxyribonucleotides

Antisense oligodeoxyribonucleotides can be selected on the basis of the secondary structure of the target mRNA sequence. The secondary structure of an RNA molecule is determined by intramolecular base pairing of stretches of complementary nucleotides and regions of non-pairing sequences forming loops, bulges, joints, etc. There is no obvious link between primary sequence and efficacy of a antisense nucleic acid and biological or biochemical parameters (Patzel, Steidl et al., 1999). Patzel, Steidl, Kronenwett, Haas, and Sczakiel, 1999 describe a method for selection effective antisense oligodeoxyribonucleotides at a statistical probability of about 50%. Favorable targets for antisense oligodeoxyribonucleotides include large loops (> 10 nucleotides), bulges, joints, and free ends. At least the 3' end of the antisense oligonucleotide should be complementary to a stretch of nucleotides not be involved in intramolecular base pairing. A comprehensive review about antisense oligodeoxyribonucleotides can be found in Sczakiel and Far, 2002.

Oligoribonucleotides

Recently, a method is described for usage of small double stranded RNA molecules (e.g. Zamore, Tuschl et al., 2000). Small double stranded RNA molecules can be delivered to Chlamydia as well.

Proteins conjugates with nucleic acids

A further aspect of the present invention is a conjugate of transferrin with antibiotic substances and/or with nucleic acids which have antibiotic activity against chlamydia.

Nucleic acids can be coupled either directly to the transferrin molecules, or nucleic acids can be delivered attached to a polycation moiety which is linked to transferrin.

The state of the art teaches a strong dependence of the biological activity of the transferrin conjugate and the molar ratio (Table 2). The content of the compound (%w/w) seems not to be the critical parameter, as can be seen from the fact that successful delivery can be achieved over a large scale of this parameter. However, conjugates containing ligands as large as transferrin or larger might not be internalized.

A transferrin conjugate for successful delivery to intracellular chlamydia can be designed by selecting a molar ratio below 25 (more preferable below 10, most preferable below 5). Molecules of such different structure, size, charge and function as an oligonucleotide DNA (polyanionic compound), PEI (polycationic compound), PEI and DNA in a single complex, Texas Red (sulforhodamin 101 acid chloride, MW 625, see Sigma catalogue, No. S 3388), or biotin (MW 244, Sigma No. B 4501) can be internalized into intracellular chlamydia via transferrin (Table 3, examples 1-3). Thus, the structure of the molecule conjugated to transferrin is not limiting the ability of the conjugate to be taken up by eukaryotic cells and to be delivered to intracellular chlamydia. The most important criterion for successful delivery is the molar ratio of the compound with respect to the protein moiety. A ratio up to 10 mol/mol transferrin for a small compound (MW <1000) is slightly reducing the ability to bind to eukaryotic cell surface. A ratio up to 20 reduced the ability to about 10-30% (half a log unit). Larger molecules as e.g. insulin (MW approx. 5800) can be successfully delivered with a molar ratio of 3.

Suitable polycation compounds are PEI (polyethylene-imine), or PEG (polyethylene- glycol).

The use of spacers is preferred in order to reduce possible steric hindrance of the molecule and transferrin.

In order to increase the coupling efficiency (mol molecule/mol carrier), the number of a particular functional group suitable for conjugation of a given molecule may be increased by conversion of other functional groups. Bifunctional reagents may be used which in addition to the functional group coupled to the protein contain more than one functional group for coupling of the second molecular species.

The number of functional groups available could be largely increased on a genetically engineered transferrin fusion proteins which carries a sequence of amino acids (C- terminal or N-terminal) suitable for conjugation, e.g. a sequence of lysin residues, aspartic residues, or glutamic residues, or a sequence containing any residues suitable for conjugation. Amino acids may be selected which are not abundant upon the surface of transferrin, which would help to retain the biological activity of transferrin. Other amino acids which are not suitable for conjugation may be included as spacers, e.g. glycin, or alanin.

The biological activity of transferrin may be completely retained by leaving it unconjugated. Conjugation is performed on a second protein which is connected thereafter to transferrin.

Derivatives of transferrin and the molecular species to be conjugated may be prepared in which functional groups are activated in order to increase reactivity and specificity to the other molecular species. Amino groups might be activated with cyanogen bromide. The carboxyl group might be activated by transforming it into an ester e.g. with p-nitrophenol, N-hydroxysuccinimide, or N-hydroxybenzotriazole in the presence of a carbodiimide.

Use of nucleic acids for treatment of chlamydial infections

Conjugates of nucleic acids may be used as a medicament and may be used for manufacture a pharmaceutical composition for treatment of chlamydial infections.

As an auxiliary substance, deferoxamine which increases the uptake of transferrin may be used. In vitro, iron deprivation increases the uptake of transferrin. Iron may be deprived in vitro and in vivo by the use of an iron-chelating agent, e.g. deferoxamine in a concentration of maximal 30 μM for the in vitro conditions. For an in vivo curative dosage of deferoxamine in an acute iron toxicity the injection dosage form is as follows: for adults and children over 3 years of age, the dose is based on body weight and must be determined by your doctor. The usual dose is 90 milligrams (mg) per kilogram (kg) of body weight, followed by 45 mg per kg of body weight, injected into a muscle every four to twelve hours. If it is injected into a vein, the usual dose is 15 mg per kg of body weight per hour every eight hours. Children up to 3 years of age: The usual dose is 15 mg per kg of body weight per hour, injected into a vein. For chronic iron toxicity: The usual dose for adults and children over 3 years of age is 500 mg to 1 gram a day, injected into a muscle. Or, the medicine may be injected under the skin by an infusion pump. The usual dose is 1 to 2 grams (20 to 40 mg per kg of body weight) a day, injected under the skin, over a period of eight to twenty-four hours. In blood transfusions, the usual dose is 500 mg to 1 gram a day, injected into a muscle. An extra 2 grams of the medicine is injected into a vein with each unit of blood at a rate of 15 mg per kg of body weight per hour. For children up to 3 years of age, the usual dose is 10 mg per kg of body weight a day, injected under the skin. Antibiotic coniugates suitable for treatment of chlamydial infections, its use for manufacturing a pharmaceutical composition.

The conjugates of an antibiotic with transferrin for treatment of infections with chlamydiales, or for use as a medicament, or for manufaction of a pharmaceutical composition for treatment of chlamydial infection may be selected from the group of β-lactam antibiotics (penicillins or cephalosporins), tetracyclines, oligoglycoside antibiotics, quinolones, or sulfonamides. Suitable prodrugs (e.g. active esters) of these antibiotics may be used.

The same rules as outlined above will hold for the production of a conjugate of transferrin with an antibiotic. The antibiotic may be selected among those substances which are able to inhibit growth and propagation of Chlamydia and/or killing Chlamydia in vivo and in an in vitro system. Examples are described above and below.

The antibiotic effect against Chlamydia can be determined in vitro according to standard cell culture procedures for Chlamydia, as e.g. described in Storey and Chopra, 2001 or in Hammerschlag and Gleyzer, 1983.

The use of conjugates of transferrin with said antibiotics or substances suitable for treatment of microbial infections is not yet known in the art.

Commonly known antibiotics may be conjugated with transferrin under suitable conditions, if

(a) the antibiotic contains at least one functional group which can react with an amino acid side chain of transferrin, and/or

(b) the antibiotic contains at least one functional group which can react with a bifunctional reagent which is able to react with transferrin, and/or

(c) a derivative of the antibiotic may be used which contains at least one functional group according to (a) and/or (b).

Functional groups of the antibiotic according to (a) might be nucleophilic side chains, e.g. the amino groups, the sulfhydryl groups, or the carboxyl group. Functional groups attacked by electrophilic compounds are e.g. tyrosine and histidine.

The bifunctional reagent according to (b) may serve as a spacer to reduce steric hindrance between the antibiotic and transferrin. When homobifunctional reagents are used, functional groups of the antibiotic according to (b) might be selected from the functional groups of the amino acid side chains of proteins, including the amino group, carboxyl group, sulfhydryl groups, thioether, imidazolyl group, guadinidyl group, phenolic group, indolyl group, carbohydrate side chain. When heterobifunctional reagents are used, functional groups of the antibiotic according to (b) are not limited to functional groups of the amino acid side chains of proteins.

Methods of preparing derivatives of commonly known antibiotics according to (c) are known in the art. The functional group may be links to the antibiotic at a moiety which is not essential for proper inhibitory activity. Additionally, the functional group may linked to the antibiotic via a spacer.

For preparing the conjugate, the antibiotic or the substance suitable for suppressing growth, propagation, and/or killing of intracellular microorganism may be used in a prodrug form which releases the active form after administering it to a subject in need thereof. Prodrugs may be conjugated to transferrin either by its active moiety, or by the inactive moiety which is left after release of the active forms.

When transferrin is coupled to the inactive moiety, the active form may be released after uptake by the eukaryotic host cell, either upon catalysis by eukaryotic host enzymes or by bacterial enzymes, e.g. by cleavage of an ester bonding. β-lactam antibiotics (including penicillins and cephalosporins) are suitable for treatment of infections of intracellular microorganisms including Chlamydiales. β- lactam antibiotics act via inhibition of enzymes involved in the synthesis of peptidoglycan which is a major constituent of eubacterial cell wall. Most of them are transpeptidases (penicillin binding proteins, PBPs). However, chlamydia do not contain peptidoglycans in amounts sufficient for sacculus formation. It is suggested that the β-lactam antibiotics interfere in RB division, thus leading to large RB which are called "penicillin forms" (Storey and Chopra, 2001). A large number of derivatives of naturally occurring penicillins are known in the art. Some of them are used in clinical practice for treatment of bacterial infections (Rote Liste, 2001). Benzylpenicillin, ampicillin, meciliinam, ceftriaxone, cefotaxime, imipenem, and meropenem have a minimal bactericical concentration of 4.0 μg/ml, 2.0 μg/ml, 0.25 μg/ml, 16.0 μg/ml, 250 μg/ml, 512 μg/ml, and 64 μg/ml, respectively upon chlamydia which is defined to be the lowest concentration of antibiotic in the first cycle of infection that resulted in no inclusions in the second cycle of infection (for details see Storey and Chopra, 2001, Hammerschlag and Gleyzer, 1983).

Thus a conjugate of transferrin with a β-lactam antibiotic (including penicillin or a derivative of penicillin and cephalosporin) is suitable for treatment of chlamydial infections, can by used as a medicament, and can be used for manufacture of a pharmaceutical composition for treatment of chlamydial infections.

In order to minimize steric hindrance between transferrin and the penicillin moiety, and in order to retain the inhibitory activity of the penicillin moiety, preferred conjugates of penicillin derivatives and transferrin are coupled via the side chain R¹ (Fig. 1). Derivatives of the common penicillin structure according to (a) and (b) are ampicillin (including prodrug esters as e.g. pivampicillin, bacampicillin, or talampicillin), amoxicillin, and epicillin (each modified at R¹). They contain at R¹ an amino group which might be suitable for conjugation with transferrin. Other derivatives according to (a) and (b) are e.g. carbenicillin, and ticarcillin which contain at R¹ a carboxyl group which might be suitable for conjugation with transferrin. Functional groups according to (c) might be linked to commonly known antibiotics at R¹ of the general structure formula (Figure 2).

Further, transferrin can be conjugated with a cephalosporin. Many derivatives or the general structure (see Figure 3) are known which are used in clinical practice (see Rote Liste, 2001 , e.g. cefalexin, cefaclor, cefadroxil, or cefamandole). Derivatives of the common cephalosporin structure according to (a) and (b) are e.g. cefalexin, cefaloglycin, cefaclor, SCE 100, cefradine, cefroxadine, cefadroxil, cefatrizine (which contain an amino group or a phenolic group at the R¹ side chain), cefamandole, or cefonicid (which contain a hydroxyl group). These functional groups might be suitable for conjugation with transferrin. Functional groups according to (c) might be linked to commonly known antibiotics at R¹ of the general structure formula. Tetracyclines are first choice antibiotics for treatment of chlamydial infections. Thus, a conjugate of transferrin with a tetracycline is suitable for treatment of chlamydial infections, can by used as a medicament, and can be used for manufacture of a pharmaceutical composition for treatment of chlamydial infections.

Transferrin can be conjugated with tetracycline. Many derivatives or the general structure (see Figure 4) are known which are used in clinical practice (see Rote Liste, 2001, e.g. tetracycline, doxycycline, minocycline, oxytetracycline). Derivatives of the common tetracycline structure according to (a) and (b) are e. g. tetracycline, doxycycline, minocycline, oxytetracylcine 6-demethyltetracycline, 6- demethylchlortetracycline, or rolitetracycline which contain hydroxyl groups (either in the position R⁵ or R^6α). These functional groups might be suitable for conjugation (see Grafe, 2002, p. 134, p. 269) with transferrin.

Functional groups according to (c) might be linked to commonly known tetracyclines. Due to its affinity to the targets at the bacterial ribosome, functional groups may be preferably introduced at residue R⁵, R^6α, R^6β or R⁷ or at positions C4 to C10 with minimal inference with the inhibitory activity of tetracyclines.

Furthermore, transferrin can be conjugated with an oligoglycoside antibiotic, e.g. clindamycin or lincomycin which are used in clinical practice (Rote Liste, 2001).

In a further embodiment of the present invention, transferrin can be conjugated with quinolone antibiotics. Quinolone antibiotics are inhibitors of bacterial gyrase A subunit (=topoisomerase A). Clinical important quinolone antibiotics are e.g. ciprofloxacin, ofloxacin, moxifloxacin, norfloxacin, enoxacin, pefloxacin, fleroxacin, levofloxacin, cinoxacin, or pipemidic acid.

For inhibition of topoisomerase, the oxygen and carboxyl group of the quinolone moiety is important which binds to topoisomerase in a complex together with Mg²⁺. Thus, structural modifications for conjugation with transferrin have to be done preferably not at the oxygen and not at the carboxyl group.

In a further embodiment of the present invention, transferrin can be conjugated with sulfonamides.

The present invention therefore relates to a conjugate comprising a polypeptide and a nucleic acid or a fragment thereof which is capable of being internalized into a eukaryotic cell by receptor-meditated endocytosis, characterized in that the nucleotide sequence of the nucleic acid is selected from the group of sequences described in Table 1 or is CGTAGTTTTTTAATCAA.

In a more preferred embodiment of the invention the polypeptide is transferrin.

In a most preferred embodiment of the present invention the receptor-meditated endocytosis is mediated by the transferrin receptor.

The present invention further relates to a conjugate comprising a polypeptide, a polycation and a nucleic acid or a fragment thereof which is capable of being internalized into a eukaryotic cell by receptor-meditated endocytosis, characterized in that the nucleotide sequence of the nucleic acid is selected from the group of sequences described in Table 1 or is CGTAGTTTTTTAATCAA.

In a preferred embodiment of the invention the polycation is PEI.

The present invention additionally relates to a conjugate comprising a polypeptide and an antibiotic, characterized in that the conjugate is capable of being internalized in a eukaryotic cell by receptor-meditated endocytosis.

In a preferred embodiment of the conjugate of the present invention the polypeptide is transferrin.

In a further preferred embodiment of the conjugate of the present invention the receptor-meditated endocytosis is mediated by the transferrin receptor.

In a further more preferred embodiment of the conjugate of the present invention the antibiotic is selected from penicillins, cephalosporins, tetracylines, quinolones, or sulfonamides.

In an additional preferred embodiment of the conjugate of the present invention the conjugate is used as a medicine.

The present invention further relates to the use of the conjugate of the present invention for manufacture of a pharmaceutical composition for the treatment of infections with an obligate intracellular bacterium from the order Chlamydiales.

In a preferred embodiment of the use of the conjugate the obligate intracellular bacterium is from the genus Chlamydia.

In a more preferred embodiment of the use of the conjugate the obligate intracellular bacterium is Chlamydia trachomatis.

In an even more preferred embodiment of the use of the conjugate the obligate intracellular bacterium is from the genus Chlamydophila.

In an most preferred embodiment of the use of the conjugate the obligate intracellular bacterium is Chlamydophila pneumoniae.

Stable transformants of obligate intracellular microorganisms To be able to transform the metabolically active, actively replicating RB one has to use transport system that will transport DNA from outside of host cell to chlamydial cell. Thus, naked DNA is delivered to chlamydial cell during the infection using host vesicular system of the endocytic pathway.

A method of producing a stable transformant of an obligate intracellular bacterium comprising

(a) conjugating a DNA molecule with transferrin, and (b) contacting the conjugate formed in step (a) with a eukaryotic cell infected with said obligate intracellular bacterium under conditions that allow the uptake of the complex via the transferrin receptor.

The present invention relates to a method of stably transforming a bacterium from the order Chlamydiales comprising

(a) providing a nucleic acid suitable for transforming said bacterium, and

(b) forming a conjugate between said nucleic acid and a polypeptide capable of binding to the transferrin receptor, and

(c) contacting the conjugate formed in step (b) with a eukaryotic cell infected with said bacterium under condition that allow the uptake of the conjugate via the transferrin receptor.

The present invention further relates to a transformed bacterium from the order Chlamydiales characterized in that the transformation is stable.

In a preferred embodiment of the present invention the bacterium is from the genus Chlamydia.

In a more preferred embodiment of the present invention the bacterium is Chlamydia trachomatis.

In a further preferred embodiment of the present invention the bacterium is from the genus Chlamydophila.

In a more preferred embodiment of the present invention the bacterium is Chlamydophila pneumoniae.

The invention furthermore relates to a method of silencing a gene in a bacterium from the order Chlamydiales comprising

(a) providing a nucleic acid suitable for gene silencing, and

(b) forming a conjugate between said nucleic acid and a polypeptide capable of binding to the transferrin receptor, and

(c) contacting the conjugate formed in step (b) with a eukaryotic cell infected with said bacterium under condition that allow the uptake of the conjugate via the transferrin receptor.

The present invention also relates to a method of introducing an organic macromolecule into an obligate intracellular bacterium comprising

(a) contacting said organic macromolecule with transferrin and optionally a polycationic compound; and

(b) contacting the complex formed in step (a) with a eukaryotic cell infected with said obligate intracellular bacterium under condition that allow the uptake of the complex via the transferrin receptor. In a preferred embodiment of the method of the present invention said organic macromolecule is a (poly) peptide.

In a further preferred embodiment of the method of the present invention the organic macromolecule is a nucleic acid molecule.

In a more preferred embodiment of the method of the present invention the nucleic acid molecule is a DNA molecule.

In an even more preferred embodiment of the method of the present invention the DNA molecule comprises a recombination promoting sequence.

In a most preferred embodiment of the method of the present invention the recombination promoting sequence is a cre-lox sequence.

In a further preferred embodiment of the method of the present invention the organic macromolecule is an antibiotic.

The present invention further relates to a method of inhibiting or reducing a propagation or growth of an obligate intracellular bacterium comprising

(a) contacting an antisense nucleic acid molecule complementary to at least a part of an mRNA encoding an essential (poly) peptide of the obligate intracellular bacterium with transferrin and a polycationic compound; and

(b) contacting the complex formed in step (a) with a eukaryotic cell infected with said obligate intracellular bacterium under condition that allow the uptake of the complex via the transferrin receptor.

In a preferred embodiment of the method of the present invention the antisense nucleic acid molecule is an oligonucleotide.

The present invention also relates to a method of producing a transfected/transformed obligate intracellular bacterium comprising the steps of (a) contacting a recombinant vector that carries a selectable marker and that can be propagated in said obligate intercellular bacterium with transferrin and a polycationic compound;

(b) contacting the complex formed in step (a) with a eukaryotic cell infected with said obligate intracellular bacterium under condition that allow the uptake of the complex via the transferrin receptor; and

(c) selecting for said selectable marker.

In a preferred embodiment of the method of the present invention the vector is an expression vector.

In a preferred embodiment of the method of the present invention the obligate intracellular bacterium belongs to the order Chlamydiales.

In a more preferred embodiment of the method of the present invention the bacterium belongs to the species Chlamydia trachomatis or Chlamydia pneumoniae.

In a preferred embodiment of the method of the present invention the polycationic compound is polyethylenimine.

Furthermore, the invention relates to a transformed/transfected obligate intercellular bacterium obtainable by the method of the invention.

The method of any one of claims 44 to 46, wherein the eukaryotic cell is deprived of iron prior to or during said contact with said complex.

In a preferred embodiment of the method of the present invention, the eukaryotic cell is deprived of iron prior to or during said contact with said complex.

The invention is further illustrated by the following examples: EXAMPLE 1

Localization of transferrin labeled with fluorescent dye Texas Red in Hep-2 cells (larynx carcinoma cell line) infected with Chlamydophila pneumoniae strain TW 183 and strain AR39 - confocal microscopy studies.

The goal of this experiment was to find out if transferrin is translocated into the chlamydial inclusions.

Hep-2 (ATCC CCI23) cells were grown on glass coverslips in a 24 well plate in tissue culture medium MEM (Minimal Essential Medium) supplemented by 10% fetal bovine serum and 2mM L-glutamine. Subconfluent cells were infected with C. pneumoniae strain TW 183 (ATCC VR-2282) or strain AR39 (ATCC 53592), multiplicity of infection (MOI, the ratio of the number of bacteria and the number of cells to be infected)) of 1 with the aid of centrifugation (900g, 1 h, at 37°C) with freshly thawed C. pneumoniae EB diluted in medium. After infection, cells were incubated in MEM containing 5% fetal bovine serum, 1 micromolar cycloheximide and 30 micromoiar deferoxamine mesylate (DAM) for 16h under standard tissue culture conditions. Cycloheximide is used as a standard additive in chlamydial cultures to inhibit host but not bacterial protein synthesis. As a control, uninfected cells were grown using the same media and conditions. Sixteen hours after infection, cells were washed twice with MEM supplemented with 30 micromolar DAM followed by 1 h incubation under standard tissue culture conditions, to remove natural transferrin that was present in fetal bovine serum. Next, the medium was replaced by MEM containing 30 micromolar DAM and 25 microliters/well of [1mg/ml] solution of transferrin labeled with Texas Red and anti-chlamydial polyclonal rabbit serum (Milan Analytica AG, Switzerland, see Al Younes, Rudel, and Meyer, 1999a). The cells were fixed at different times, starting at 5 minutes up to 24h after addition of Texas Red-transferrin. Localization of transferrin and chlamydial inclusions was determined using confocal microscopy.

Unlike traditional epifluorescent microcopy, the confocal microscope technology enables "slicing" the cell to unequivocally determine localization of detected molecules. This is how it was determined that transferrin is in fact inside of the inclusion and not on top of it. The combined fluorescence images of labelled transferrin and labelled anti-chlamydial polyclonal rabbit serum demonstrates localization of transferrin inside of the chlamydial inclusions. About 50% of the inclusions showed accumulation of transferrin. Thus, this experiment demonstrated that transferrin was taken up by the iron-starved host cells and was delivered inside the chlamydial inclusion within the first 5 minutes. Longer incubation times did not increase the amount of transferrin within the inclusions.

EXAMPLE 2

Localization of transferrin labeled with biotin in Hep-2 cells (larynx carcinoma cell line) infected with Chlamydophila pneumoniae strain TW 183 - electron microscopy studies.

The goal of this experiment was to determine exact location of translocated transferrin within the chlamydial inclusions.

Hep-2 cells were grown on glass coverslips in a 24 well plate using tissue culture medium MEM (Minimal Essential Medium) supplemented by 10% fetal bovine serum and 2mM L-glutamine. Subconfluent cells were infected with C. pneumoniae strain TW 183, multiplicity of infection 1 with the aid of centrifugation (900g, 1h, at 37°C). After centrifugation, fresh media composed of MEM, 5% fetal bovine serum, 2mM L- glutamine, cycloheximide and 30 micromolar DAM was added. Sixteen hours after infection, cells were washed 3 times with MEM containing 2mM L-glutamine, cycloheximide and 30 micromolar DAM and next incubated for 1h with MEM containing only 30 micromolar DAM. Next, cells were washed twice with MEM containing 2mM L-glutamine, cycloheximide and 30 micromolar DAM followed by incubation for 23h in MEM containing 2mM L-glutamine, cycloheximide and 30 micromolar DAM supplemented with 50 micrograms/ml of transferrin labeled with biotin (Molecular Probes). Thirty hours after infection cells were fixed and processed for electron microscopy detection of biotin-labeled transferrin with avidin coupled with 12nm gold particles (Figure 1).

This experiment showed that transferrin is not only found in the chlamydial inclusions but more importantly inside the bacteria.

EXAMPLE 3

Localization of synthetic oligonucleotides labeled with fluorescent dye Cy3 in Hep-2 cells (larynx carcinoma cell line) infected with Chlamydophila pneumoniae strain TW 183 - confocal microscopy studies.

The goal of this experiment was to determine if a nucleic acid can be delivered inside chlamydial inclusion using transferrin as a carrier.

Hep-2 cells were grown on glass coverslips in a 12 well plate using tissue culture medium MEM (Minimal Essential Medium) supplemented by 10% fetal bovine serum and 2mM L-glutamine. Subconfluent cells were infected with C. pneumoniae strain TW 183, multiplicity of infection 1 with the aid of centrifugation (900g, 1h, at 37°C). After infection, cells were incubated in MEM containing 5% fetal bovine serum, 1 micromolar cycloheximide and 30 micromolar deferoxamine (DAM) for 16h under standard tissue culture conditions. As a control, uninfected cells were grown using the same media and conditions. Sixteen hours after infection, old medium was replaced by a fresh one. In the next step, transfection was carried using DuoFect, a transferrin-based transfection kit from Quantum Biogene (Heidelberg, Germany, now Q-Biogene). The double-internalizing factor conjugated with synthetic DNA was prepared as follows: 75 microliters of HBS buffer were mixed by pipetting with 0.65 microliters of PEI-transferrin and 1 microgram of synthetic single-stranded oligonucleotide cgtagttttttaatcaa (Cpn0483) labeled with fluorescent dye Cy3. After 20 minutes incubation at room temperature, the mixture was added to the cells. Four hours later (=20h post infection) cells were washed with PBS and fixed in 4% paraformaldehyde. Intracellular pathogen was visualized by immunostaining with polyclonal antibodies (anti-chlamydial polyclonal rabbit serum, see above). Confocal microscopy revealed localization of labeled synthetic DNA within the chlamydial inclusions. 40h post infection, all the labelled DNA can be found inside the inclusion.

This experiment showed that nucleic acid can be delivered into the chlamydial inclusion.

The components of the Duofect transfection kit are described in detail in US patent 5,792,645 and 5,354,844 (see also Ogris, Steinlein et al., 1998, Kircheis, Kichler et al., 1997, Boussif, Lezoualc'h et al., 1995). FIGURE LEGENDS:

Figure 1

Shown is electron microscope image demonstrating chlamydial inclusion containing reticular bodies inside. Transferrin visualized by round black immunogold particles (arrowheads) is found inside bacterial cells consistently localized just below the bacterial membrane (see the insert with the enlargement).

Figure 2

General structure of penicillin taken from Grafe (2002). R¹ and R² have the meaning as defined in Grafe (2002).

Figure 3

General structure of cephalosporins taken from Grafe (2002). R¹, R² , and R³ have the meaning as defined in Grafe (2002).

Figure 4

General structure of tetracyclines taken from Grafe (2002). R, R⁵, R^6a|Pha , R^6beta and

R⁷ have the meaning as defined in Grafe (2002).

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Claims

Claims

1. A nucleic acid or a fragment thereof for use as a medicine, characterized in that the nucleotide sequence of the nucleic acid is selected from the group of sequences described in Table 1.

2. A nucleic acid characterized in that the nucleotide sequence of the nucleic acid is CGTAGTTTTTTAATCAA.

3. The nucleic acid as claimed in claim 2, wherein the nucleic acid is used as a medicine.

4. The nucleic acid or a fragment thereof as claimed in claim 1, wherein the nucleic acid is DNA.

5. The nucleic acid or a fragment thereof as claimed in any of the claims 1 to 3, wherein the nucleic acid is RNA and wherein in the nucleic acid sequence T is replaced by U.

6. The nucleic acid or a fragment thereof as claimed in any of the claims 1 to 5, wherein the nucleic acid is single stranded.

7. The nucleic acid or a fragment thereof as claimed in any of the claims 1 to 5, wherein the nucleic acid is double stranded.

8. The use of a nucleic acid or of a fragment thereof of any one of claims 1 to 7 for manufacture of a pharmaceutical composition for the treatment of infections with obligate intracellular microorganisms from the order Chlamydiales.

9. The use as claimed in claim 8, wherein the obligate intracellular microorganism is from the genus Chlamydia.

10. The use as claimed in claim 9, wherein the obligate intracellular microorganism is Chlamydia trachomatis.

11. The use as claimed in claim 8, wherein the obligate intracellular microorganism is from the genus Chlamydophila.

12. The use as claimed in claim 11, wherein the obligate intracellular microorganism is Chlamydophila pneumoniae.

13. A conjugate comprising a polypeptide and a nucleic acid or a fragment thereof which is capable of being internalized into a eukaryotic cell by receptor-meditated endocytosis, characterized in that the nucleotide sequence of the nucleic acid is selected from the group of sequences described in Table 1 or is CGTAGTTTTTTAATCAA.

14. A conjugate as claimed in claim 13, wherein the polypeptide is transferrin.

15. A conjugate as claimed in the claims 13 or 14, wherein the receptor-meditated endocytosis is mediated by the transferrin receptor.

16. A conjugate comprising a polypeptide, a polycation and a nucleic acid or a fragment thereof which is capable of being internalized into a eukaryotic cell by receptor-meditated endocytosis, characterized in that the nucleotide sequence of the nucleic acid is selected from the group of sequences described in Table 1 or is CGTAGTTTTTTAATCAA.

17. A conjugate as claimed in claim 16, wherein the polycation is PEI.

18. A conjugate comprising a polypeptide and an antibiotic, characterized in that the conjugate is capable of being internalized in a eukaryotic cell by receptor- meditated endocytosis.

19. A conjugate as claimed in claim 17, wherein the polypeptide is transferrin.

20. A conjugate as claimed in the claims 17 or 19, wherein the receptor-meditated endocytosis is mediated by the transferrin receptor.

21. A conjugate as claimed in any of the claims 17 to 20, wherein the antibiotic is selected from penicillins, cephalosporins, tetracylines, quinolones, or sulfonamides.

22. A conjugate as claimed in any of the claims 13 to 21 for use as a medicine.

23. The use of the conjugate as claimed in any of the claims 13 to 21 for manufacture of a pharmaceutical composition for the treatment of infections with an obligate intracellular bacterium from the order Chlamydiales.

24. The use as claimed in claim 23, wherein the obligate intracellular bacterium is from the genus Chlamydia.

25. The use as claimed in claim 24, wherein the obligate intracellular bacterium is Chlamydia trachomatis.

26. The use as claimed in claim 25, wherein the obligate intracellular bacterium is from the genus Chlamydophila.

27. The use as claimed in claim 26, wherein the obligate intracellular bacterium is Chlamydophila pneumoniae.

28. A method of identifying compounds which selectively inhibit growth and/or propagation of Chlamydiales, comprising

(a) determining the in vitro-interaction of the compound with a polypeptide selected from the group of polypeptides encoded by the sequences described in Table 1, and

(b) contacting the compound exhibiting in vitro interaction according to step (a) with a eukaryotic cell infected with a Chlamydiales species under condition that allow the uptake of the compound, and

(c) determining the reduction of growth and propagation of the Chlamydiales species within the eukaryotic cells exposed to the compound according to step (b).

29. The use of the compound identified by the method as claimed in claim 28 for manufacture of a pharmaceutical composition for treatment of a chlamydial infection.

30. A method of stably transforming a bacterium from the order Chlamydiales comprising

(a) providing a nucleic acid suitable for transforming said bacterium, and

(b) forming a conjugate between said nucleic acid and a polypeptide capable of binding to the transferrin receptor, and

(c) contacting the conjugate formed in step (b) with a eukaryotic cell infected with said bacterium under condition that allow the uptake of the conjugate via the transferrin receptor.

31. A transformed bacterium from the order Chlamydiales characterized in that the transformation is stable.

32. A bacterium as claimed in claim 31 wherein the bacterium is from the genus Chlamydia.

33. A bacterium as claimed in claim 32 wherein the bacterium is Chlamydia trachomatis.

34. A bacterium as claimed in claim 31 wherein the bacterium is from the genus Chlamydophila.

35. A bacterium as claimed in claim 34 wherein the bacterium is Chlamydophila pneumoniae.

36. Recombinant vector, comprising

(a) an origin of replication suitable for replication in a bacterium from the order Chlamydiales, and/or

(b) an expression signal suitable for protein expression in a bacterium from the order Chlamydiales, and/or

(c) a reporter gene operatively linked to the expression signal of (b).

37. A method of introducing an organic molecule into a bacterium from the order Chlamydiales comprising

(a) forming a conjugate between said organic molecule and a polypeptide capable of binding to the transferrin receptor, and

(b) contacting the conjugate formed in step (a) with a eukaryotic cell infected with said bacterium under condition that allow the uptake of the conjugate via the transferrin receptor.

38. The method as claimed in the claim 37, wherein the polypeptide is transferrin.

39. The method as claimed in the claims 37 or 38, wherein the organic molecule is an antibiotic.

40. The method as claimed in the claim 39, wherein the antibiotic is selected from penicillins, cephalosporins, tetracylines, quinolones, or sulfonamides.

41. The method as claimed in the claims 37 or 38, wherein the organic molecule is a nucleic acid.

42. The method as claimed in any of the claims 37 to 41 , wherein the bacterium is from the order Chlamydiales is.

43. A method of silencing a gene in a bacterium from the order Chlamydiales comprising

(a) providing a nucleic acid suitable for gene silencing, and

(b) forming a conjugate between said nucleic acid and a polypeptide capable of binding to the transferrin receptor, and

(c) contacting the conjugate formed in step (b) with a eukaryotic cell infected with said bacterium under condition that allow the uptake of the conjugate via the transferrin receptor.

44. A method of introducing an organic macromolecule into an obligate intracellular bacterium comprising

(a) contacting said organic macromolecule with transferrin and optionally a polycationic compound; and

(b) contacting the complex formed in step (a) with a eukaryotic cell infected with said obligate intracellular bacterium under condition that allow the uptake of the complex via the transferrin receptor.

45. A method of inhibiting or reducing a propagation or growth of an obligate intracellular bacterium comprising

(a) contacting an antisense nucleic acid molecule complementary to at least a part of an mRNA encoding an essential (poly)peptide of the obligate intracellular bacterium with transferrin and a polycationic compound; and

(b) contacting the complex formed in step (a) with a eukaryotic cell infected with said obligate intracellular bacterium under condition that allow the uptake of the complex via the transferrin receptor.

46. A method of producing a transfected/transformed obligate intracellular bacterium comprising the steps of

(a) contacting a recombinant vector that carries a selectable marker and that can be propagated in said obligate intercellular bacterium with transferrin and a polycationic compound;

(b) . contacting the complex formed in step (a) with a eukaryotic cell infected with said obligate intracellular bacterium under condition that allow the uptake of the complex via the transferrin receptor; and

(c) selecting for said selectable marker.

47. A transformed/transfected obligate intracellular bacterium obtainable by the method of claim 46.

48. The method of any one of claims 44 to 46, wherein the eukaryotic cell is deprived of iron prior to or during said contact with said complex.

Table 1: target proteins of Chlamydophila pneumoniae CWL029

The sequences described by the Accession-Wo. and GenBank-identifiers are the coding sequences without regulatory sequences (e.g. promotors) . The complete sequences can be found under

AE001363 . 1

c

CD

m m m

7i c m r σ>

</>

C Tabelle 1 (continued) CD </>

m m m

TJ

C m r σ>

Table 2:

Conjugates of transferrin, state of the art, for delivery to eukaryotic cells. Based on the information of the state of the art, underlined parameters are calculated with 80 kdal as a molecular weight of transferrin.

c

CD

m m m

TJ c m r σ> Table 3.

10

Properties of compounds to be delivered to chlamydial inclusion and to chlamydia in example 1 to 3.