WO2005023368A1 - Prophylaxis of and treatment for infections from the family chlamydiaceae using amino acids as leucine or methionine - Google Patents

Abstract

The present invention relates to a method for treatment of infections caused by the intracellular bacteria Chlamydia and Chiamydophiia using supplements of certain naturally occurring substances (nutrients), particularly amino acids.

Description

Prophylaxis of and treatment for infections with microorganisms from the family Chlamydiaceae

The present invention relates to a method for treatment of infections caused by the intracellular bacteria Chlamydia and Chlamydophila using supplements of certain naturally occurring substances (nutrients), 5 particularly amino acids.

The invention concerns specific amino acids in the L-form, namely, L- leucine, L-isoleucine, L-methionine and L-phenylalanine which, when present in amounts exceeding the amounts normally present in body fluids,o in organs, tissues or/and at target cells could lead to very appreciable adverse effects on chlamydial growth.

The invention concerns a possible adequate and safe antichlamydial therapy with no side effects using amino acids as medicaments. Amino acids fall into5 the category of foods and not drugs and are produced in a safe form.

The invention relates to the nutritional manipulation by increasing the availability of amino acids to the body which may be sufficient and beneficial in eliminating chlamydial infections. The invention relates to combinations ofo conventional antibiotic regimens, usually not sufficiently successful to treat in vivo chlamydial infections, especially chronic ones, with the increase in the body amino acid concentrations, which may be more efficacious.

Biology of Chlamydiaceae5 Members of the family Chlamydiaceae are obligate intracellular pathogens that replicate within membrane-bound vacuoles known as inclusions. They are responsible for several major diseases either in animals or humans. Until recently, the family Chlamydiaceae was represented by only a single genuso known as Chlamydia that composed of four species: Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia pecorum (Kaltenboeck, Kousoulas et al., 1993). In 1999, the chlamydial taxonomy was revised and the Chlamydiaceae family has been split into two genera (Chlamydia and Chlamydophila) encompassing three (Chlamydia trachomatis, Chlamydia suis, Chlamydia muridarum) and six (Chlamydophila pneumoniae, Chlamydophila abortus, Chlamydophila psittaci, Chlamydophila pecorum, Chlamydophila felis, Chlamydophila cavlae) species, respectively (Everett, Bush et al., 1999). For simplicity, all members of the family Chlamydiaceae are referred, here, to as chlamydiae.

Chlamydiae possess a unique developmental cycle. This cycle consists of infectious and noninfectious stages that exhibit unique morphological, biochemical, and biological properties. The infectious elementary body (EB), which is non-replicating and metabolicaliy inactive, attaches to and enters host cells (Moulder, 1991). After host cell entry, the EB is localized to a phagosome, and the primary differentiation process is initiated. This developmental process involves the commencement of bacterial metabolism and the conversion of the EB to the intracellular replicating form of the organism, termed the reticulate body (RB). The RB multiples by binary fission for a period of 24 to 36 h. After multiple rounds of replication the RB undergoes a secondary differentiation process back to an infectious EB. At this late stage in development (40 to 72 h) the host cell lyses and releases mature EBs that then reinfect neighboring host cells (Moulder, 1991).

Persistent infections

Two distinguishing characteristics of chlamydiae are its developmental cycle and predilection for causing a persistent (chronic or latent) infections (Moulder, 1991), during which the normal developmental cycle is altered, producing aberrant RB-like forms. Persistency can be established in vitro using several methods, including treatment with cytokines or antibiotics or by deprivation of certain nutrients, such as amino acids (Beatty, Byrne et al., 1994) and iron (Al-Younes, Rudel et al., 2001). Persistent infections produced can revert to normally growing organisms when the suppressor is removed or nutrients are replaced (Allan and Pearce, 1983; Al-Younes, Rudel et al., 2001 ). In humans, acute chlamydial infections can progress to persistent infections, which may lead to a pathogenic process that leads to chronic diseases including blindness, pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, arthritis, Alzheimer's disease and atherosclerosis (Hammerschlag, 2002; Villareal, Whittum-Hudson et al., 2002; Stephens, 2003).

Diseases associated with chlamydiae

Although they have a similar unique developmental cycle, chlamydiae cause a variety of human and animal diseases. Chlamydia trachomatis, primarily a pathogen of humans, is one of the most common bacterial pathogens that primarily infects columnar epithelial cells of the ocular and genital mucosae, causing sexually transmitted and ocular diseases in humans. These diseases have a significant impact on human health worldwide, causing trachoma, the leading cause of preventable blindness, and sexually transmitted diseases (STD) that include tubal factor infertility, life-threatening ectopic pregnancy, and pelvic inflammatory disease that often result in involuntary sterility (Stephens, 2003). Chlamydial STDs are also risk factors in cervical squamous cell carcinoma (Anttila, Saikku et al., 2001) and HIV infection (Chesson and Pinkerton, 2000). Infants are at risk for chlamydial eye infection and pneumonia if they pass through an infected cervix (Stephens, 2003). Chlamydia trachomatis strains (or serovars) L1 , L2 and l_3 are the etiological agents of the sexually transmitted systemic syndrome Lymphogranuloma venereum (LGV). Serovars A to C are primarily the agents responsible for the endemic blinding trachoma, while serovars D to K are associated with STDs (Guaschino and De Seta, 2000).

Chlamydophila pneumoniae is an important cause of human respiratory tract diseases, such as pneumonia, pharyngitis, sinusitis, otitis, asthma, acute bronchitis (Grayston, Campbell et al., 1990), persistent cough, chronic obstructive pulmonary disease (COPD), flu-like syndrome (Blasi, Arosio et al., 1999) and lung carcinoma (Laurila, Anttila et al. 1997). In addition, this pathogen is correlated with other non-pulmonary diseases, such as erythema nodosum (Erntell, Ljunggren et al., 1989), Guillain-Barre syndrome (Haidl, Ivarsson et al., 1992), endocarditis (Grayston, Campbell et al., 1990), Alzheimer's disease (Balin, Gerard et al., 1998), reactive arthritis (Villareal, Whittum-Hudson et al., 2002), meningoencephalitis (Koskiniemi, Gencay et al., 1996) and the blood vessel disease atherosclerosis (Campbell and Kuo, 2003).

Other species, such as C. psittaci, C. abortus and C. pecorum, are responsible for several major diseases in animals, mainly spontaneous abortion in livestock and systemic disease in birds, and can also infect rodents and cats (Schachter, 1999).

The biological mechanisms responsible for these differences in vertebrate host, tissue tropism and spectrum of diseases are unknown.

Of all the infectious diseases reported to the U.S. state health departments and the U.S. Centers for Disease Control and Prevention, C. trachomatis genital tract infections are the most common, with an estimated 4 to 5 million cases occurring annually in the United States and 3 million cases occur in Europe (Marrazzo and Stamm, 1998; Schachter, 1999). In 1995, infections with C. trachomatis were the most commonly reported bacterial disease in the U.S. (Marrazzo and Stamm, 1998), and the World Health Organization estimated that 89 million new cases would arise worldwide (Marrazzo and Stamm, 1998).

Serological surveys have shown that virtually every human has been infected with C. pneumoniae (Grayston, 2000). This prevalent pathogen causes 6 to 25% of community-acquired pneumonia. Further, this pathogen is also associated with other respiratory diseases as well as non-respiratory diseases, such as cancer and Alzheimer's disease. Importantly, increasing evidence demonstrated that C. pneumoniae is present and persistent at sites of arterial disease and, thus, contributes to coronary artery disease (Atherosclerosis). The presence of C. pneumoniae in atheromatous plaques has been demonstrated by several methods, such as polymerase chain reaction (PCR), immunocytochemistry, in situ hybridization, electron microscopy and by recovery of bacteria in tissue cultures (Ramirez, 1996). Furthermore, respiratory inoculation of C. pneumoniae in experimental animal models induced or accelerated the formation of atherosclerotic lesions (de Boer, van der Wal et al., 2000). This disease continues to be the principal cause of death in the U.S. and in most Western countries (Braunwald, 1997). For example, it causes nearly 25% of all deaths each year in the UK, whereas it causes about 40% of annual deaths in the U.S. (Gupta and Camm, 1998).

Several antibiotics, including macrolides and quinolones, were shown to have efficacious antichlamydial activity in in vitro acute infections (Donati, Rodriguez Fermepin et al., 1999; Hammerschlag, 2000). However, several reports indicated that in vitro treatment of infected cells with antibiotics, such as the macrolide drug azithromycin and the quinolone drug gemifloxacin, failed to completely eradicate chlamydiae (Kutlin, Roblin et al., 2002). Further, addition of antibiotics, such as azithromycin, penicillin and ampicillin, was reported to induce formation of persistent chlamydial infection, leading to the formation of inclusions containing viable enlarged and morphologically abnormal chlamydiae (Beatty, Byrne et al.,1994; Bragina, Gomberg et al., 2001). In humans, therapy for acute chlamydial infections have been shown to result in satisfactory cure rates in clinical trials (Ossewarde, Plantema et al., 1992; Hammerschlag, Golden et al., 1993; Hillis, Coles et al., 1998). However, relapsing chlamydial infections are a common problem, even though patients are often treated appropriately (Katz, Caine et al., 1991; Blythe, Katz et al., 1992; Xu, Schillinger et al., 2000; Guaschino, and Ricci, 2002). Relapsing chlamydial infections after antibiotic treatment appear to be a result of the persistence of chlamydiae (Munday, Thomas et al., 1995; Dean, Suchland et al., 2000). Two recent studies confirmed the resistance of C. pneumoniae infection in monocytes (Gieffers, Fullgraf et al., 2001 ) and lymphocytes (Yamaguchi, Friedman et al., 2003) to antibiotics, thought to be very efficacious in eradicating in vitro and in vivo acute infections. The susceptibility of C. pneumoniae to antibiotics in monocytes and lymphocytes likely is critical in controlling the spread of the organism from the primary site of infection (the respiratory system) to sites of chronic infection (e.g. atheromas). This chronicity of chlamydial infection have been implicated in inflammatory processes that could be important in the pathogenesis of atherosclerosis and other diseases (Huittinen, Leinonen et al., 2003).

Treatment of chlamydial infections, state of the art

After entry into a host cell, the EB is localized to a phagosome. At the very early stage of infection (1 to 3 h), the parasite exerts profound effects on the host. Through an unknown mechanism, dependent on both bacterial transcription and translation (Scidmore, Rockey et al., 1996; Al-Younes, Rudel et al, 1999), chlamydiae modify the properties of the phagosome and prevent its entry into the lysosomal pathway (Heinzen, Scidmore et al., 1996; Al-Younes, Rudel et al., 1999). Many obligate and facultative intracellular pathogens use this approach to avoid intracellular killing by using different means to interfere with cellular trafficking (Duclos and Desjardins, 2000). This unique parasite strategy provides a continuously protected intracellular niche in which chlamydiae then replicate.

1. Treatment of chlamydial infection by antibiotics

Because of the unique biology of chlamydiae, specific requirements are imposed on antimicrobial agents employed for therapy of chlamydial infections. The extracellular EBs are metabolicaliy inactive and resistant to killing. Therefore, antichlamydial agents must efficiently penetrate tissues and then cellular and inclusion membranes in order to inhibit growth of the metabolicaliy active and dividing RBs. Chlamydiae have a relatively long developmental cycle, thus, prolonged course of therapy must be adopted or an antibiotic with a long half-life must be selected.

Several antibiotics such as doxycycline, azithromycin and rifampin, were considered as first-line choices in treatment of C. pneumoniae infections and uncomplicated human genital infections with C. trachomatis (Marrazo and Stamm, 1998; Guaschino and Ricci, 2003). These antibiotics are characterized by long half-life and good tissue and cell penetration (Marrazo and Stamm, 1998; Guaschino and Ricci, 2003). Several quinolones (ofloxacin and ciprofloxacin) are also recommended as an alternative therapy for chlamydial infections in humans (Marrazo and Stamm, 1998). Therapies with other antibiotics, such as amoxicillin, erythromycin and sulfa drugs are less effective with efficacies between 60% and 80% (Marrazo and Stamm, 1998; Guaschino and Ricci, 2003). Other antibiotics were recommended, including ceftriaxone, cefoxitin, probenecid, mitronidazole, cefotetan, gentamicin (Mazzarro and Stamm, 1998, Guaschino and Ricci, 2003), levofloxacin (Baltch, Smith et al., 2003), garenoxacin (Roblin, Reznik et al., 2003a) and rifamycin derivatives ABI-1648, ABI-1657 and ABI-1131 (Roblin, Reznik et al., 2003b).

Many disadvantages were reported on the use of antibiotics. For instance, some antibiotics should not be used by pregnant and lactating women and in individuals younger than 16 years of age. Some antimicrobial agents have been associated with an unacceptable rate of chlamydial relapse. Use of antibiotics is sometimes associated with significant side effects, such as gastrointestinal intolerance (Marrazzo and Stamm, 1998; Guaschino and Ricci, 2003) and up to 20% discontinue therapy because of these adverse effects (Guaschino and Ricci, 2003). Some antibiotics have to be given for longer than one week (2 to 3 weeks) to avoid recurrence of infection, which is common (Roblin, Montalban et al., 1994). Recently, there have been reports of multi-drug resistant chlamydial infections causing relapses or persistent infections (Hammerschlag, 2002; Guaschino and Ricci, 2003).

Another more important disadvantage on the use of antibiotics is that chronic infections are less responsive to antibiotic therapy, compared to the acute infection with chlamydiae or to the in vitro infection (Beatty, Byrne et al., 1994). In addition, chlamydial infection in certain cell types were reported not responsive to antibiotic treatment. For instance, infection of C. pneumoniae in human monocytes and lymphocytes are not responsive to treatment of antibiotics usually efficacious in treatment of infection in other cell types. Thus, the reduced antimicrobial susceptibility might probably allow circulating monocytes and lymphocytes to transfer the pathogen from the respiratory tract (primary site of infection) to the cells of the vascular wall and other sites, where reinfection is initiated and, thus, chronic disease formation is promoted (Boman, Soderberg et al., 1998; Gieffers, Fullgraf et al., 2001 ; Yamaguchi, Friedman et al., 2003). In vitro experiments showed that chlamydiae in monocytes and lymphocytes showed reduced antibiotic susceptibility in the presence of rifampin, the most effective anti-C. pneumoniae drug in vitro (Gieffers, Solbach et al., 1998), and azithromycin a macrolide widely used in current treatment trials (Grayston, 1999). Beside in vitro studies, C. pneumoniae were cultured from monocytes of coronary artery disease patients undergoing experimental azithromycin treatment for coronary sclerosis. This finding proves the presence of viable chlamydiae in the bloodstream, despite antichlamydial therapy (Gieffers, Fullgraf et al., 2001 ) and indicates the not sufficiently successful antibiotic therapy of in vivo infection, compared to more efficacious in vitro treatment trials.

Antibiotic-resistant C. pneumoniae was observed not only in blood cells but also in tissues of atheromas and infected tissues of the respiratory system and joints. Treatment failures were seen in respiratory infections with chlamydial strains that seemed susceptible in acute infections in vitro (Hammerschlag, Chirgwin et al., 1992). In addition, using standard antibiotic therapeutic approaches against chlamydiae may not be successful in alleviating clinical coronary artery disease symptoms (Meier, Derby et al., 1999; Muhlestein, Anderson et al., 2000). Reduced antibiotic susceptibility of chlamydiae in tissues to antibiotic intervention is likely due to the presence of chlamydiae in a persistent state. For instance, Hammerschlag, Chirgwin et al. (1992) described persistent nasopharyngeal infection after acute respiratory illness caused by C. pneumoniae in 5 patients for periods up to 11 months, despite treatment with multiple and prolonged courses of antibiotics. Follow-up studies of 2 of these patients documented persistence 5 for 7 to 9 years (Hammerschlag, 2002). One patient was culture-positive on 14 separate occasions over the course of 9 years. In vitro susceptibility testing of these isolates did not demonstrate development of antibiotic resistance. This in vivo multiple drug resistance was also observed in C. trachomatis infections and was associated with clinical treatment failureso (Guaschino and Ricci, 2003). Taken together, chlamydial infection in tissues cannot be completely eliminated by antibiotic trials, and thus, forming persistent multidrug-resistant infections in tissues that can be reactivated and reinfection may occur, especially when antibiotic levels in tissues declined with time, causing promotion of chronic diseases in those tissues.5 2. Treatment of chlamydial infection by naturally occurring substances (Dietary supplements)

To the best of our knowledge, only one single study reported a role ofo naturally occurring substances, namely vitamin E, in the treatment of chlamydial infection. Stephens, McChesney et al. (1979) demonstrated that supplementation of lambs with vitamin E altered the course of infection and improved recovery of lambs inoculated intratracheally with chlamydiae. CΛ/amyαYa-i nfected lambs supplemented with vitamin E had less extensive5 pneumonia, greater post-infection feed consumption and significantly heavier weight gains than non-supplemented lambs. Importantly, Chlamydia were isolated from lungs of 40% of non-supplemented lambs but were not isolated from lungs of supplemented lambs. 0 General beneficial effects of the intake of amino acid supplements in the human body

Historically, amino acids have been viewed as precursors that solely used for protein synthesis. Nowadays, it is well known that amino acids participate in the regulation of other major metabolic pathways (Meijer, 2003). Amino acids participate in the body metabolism as an energy source (Layman, 2002). Further, amino acids have anti-catabolic activity; they are very effective in inhibition of autophagic degradation of cytoplasmic constituents. As a result of their anti-catabolic activity, amino acids, especially the branched chain amino acids (L-leucine, L-isoleucine and L-valine), slow skeletal muscle degradation (Louard, Barrett et al., 1995). Additionally, they are also very effective in promoting protein synthesis (Layman, 2002; Meijer, 2003). Recent in vivo and in vitro work has revealed important new information about the mechanisms involved in these effects. A number of components of the translational machinery are regulated through signaling events elicited by amino acids. Taken together, these activated components lead to a global protein synthesis, cell differentiation, increase in cell mass and to cell proliferation (Kimball, 2002; Kimball and Jefferson, 2002).

Among the amino acids extensively studied, branched-chain amino acids (L- leucine, L-isoleucine and L-valine) were found effective in stimulating mRNA translation initiation, when administered to animals. Of the branched-chain amino acids, L-leucine was most potent; its administration stimulates global rates of protein synthesis in skeletal muscles. Effects of these amino acids were confirmed in vivo. For instance, muscle protein synthesis was stimulated if the amino acids are perfused into rats (Mosoni, Houlier et al., 1993). Further, Anthony, Anthony et al. (2000) showed that orally administered L-leucine stimulated muscle protein synthesis in rats. A sustained hyperleucinemia (leucine increased two- to threefold) stimulated muscle protein synthesis in rats intravenously infused with amino acids (Garlick and Grant, 1988; Mosoni, Houlier et al., 1993; Sinaud, Balage et al., 1999).

In humans, Volpi, Mittendorfer et al. (1999) showed that muscle protein synthesis responded positively after oral administration of L-leucine. Twofold increase in plasma L-leucine level stimulated the whole-body protein synthesis in healthy humans (Elia, Folmer et al., 1989; Giordano, Castellino et al., 1996). Traditionally, athletes consume large amounts of amino acids, especially the branched chain amino acids before exercise and strength training workouts, thereby promoting greater gains in muscle mass and strength.

Intake of amino acids and their safety

Amino acids are natural substances that are usually safe with unknown side effects (Kadowaki and Kanazawa, 2003). Supplemental amino acids are available on the market in combination with various multivitamin formulas, as protein mixture, in a wide variety of food supplements, in a number of amino acid formulas and as crystalline free-form amino acids. They can be purchased as capsules, tablets, liquids and powders. Adverse effects to the human body caused by excessive amino acid intake is not reported

(Kadowaki and Kanazawa, 2003). In the literature, amino acid administration to patients, to healthy subjects or to animal models was not harmful and had beneficial effects on the whole body protein synthesis, as described before.

Other beneficial effects are also reported. For example, parenteral administration of the amino acid L-glutamine to patients after surgery is able to correct the negative nitrogen balance (Stehle, Zander et al., 1989; Neu,

Shenoy et al., 1996).

Intakes of amino acids by body builders and athletic populations

In general, amino acids participate in the body metabolism in 4 ways: 1) they serve as precursors for protein synthesis, 2) they are considered as an energy source, 3) they act as metabolic signals involved in many cell transduction pathways, particularly, those involved in the initiation of mRNA translation (Anthony, Anthony et al., 2000; Kimball, 2002; Layman, 2002), and (4) they slow tissue degradation, e.g. skeletal muscle (Louard, Barrett et al., 1995). Based on their beneficial effects and their safety, body builders and athletes consume copious quantities of amino acids and proteins before and after strength training workouts, thereby promoting greater gains in muscle mass and strength. Amino acids can be taken from protein-rich diets or, alternatively, they can be purchased as tablets, powders, capsules or liquids in the form of individual amino acids or in combination supplements. Free amino acids can be taken orally or can be administered through injections. They are available on the market in its purest form as dietary supplements that can be sold without a prescription. The increase in muscle protein synthesis may be attributed in part to an increase in amino acid supply to the muscle, thereby augmenting substrate availability for peptide synthesis. Additionally, individual amino acids may function as nutritional signaling molecules that regulate mRNA translation (Anthony, Anthony et al., 2000; Kimball, 2002; Layman, 2002).

Autophagic proteolysis as a source of bio-precursors

Because chlamydiae are obligate intracellular pathogens, they obtain most of the metabolic precursors and nutrients from the host cell. There are at least three possible sources for the soluble essential metabolites available in the cytoplasm of the host cell. First, through the entry of a wide range of extracellular nutrients and solutes through the host cell membrane by means of diffusion and transport systems. Second, through the release of metabolites from nutrient-rich vesicles of the endosomal-lysosomal endocytic pathway that is involved in the fluid phase uptake (heterophagy). Finally, through the release of biosynthetic precursors resulted from the degradation of endogenous cellular components by a process known as autophagy, a pathway that is widely used for the maintenance of cellular homeostasis (Dunn, 1990; Hoyvik, Gordon et al., 1991; Stromhaug and Klionsky, 2001). Autophagic degradation is initiated when organelles and portions of cytoplasm are sequestered in vacuoles called nascent autophagosomes that will acquire degradative enzymes upon fusion with lysosomes and then mature to late autophagosome, in which the vacuolar content is degraded and released into the cytosol (Pillay, Elliott et al., 2002). Therefore, autophagic proteolysis is considered an important determinant of the intracellular biosynthetic precursor content (Stromhaug and Klionsky, 2001).

It is well characterized that autophagic proteolysis can be modulated and controlled in vivo and in vitro by levels of amino acids in the plasma or in the cell culture medium, respectively. Several amino acids have a direct inhibitory potential in autophagic proteolysis, such as L-leucine, L- methionine, L-phenylalanine, L-glutamine, L-tyrosine, L-proline, L-tryptophan and L-histidine (Dunn, 1990; Hoyvik, Gordon et al., 1991; Fengsrud, Roos et al., 1995).

Detailled description of the invention

The state of the art does not provide any indication that amounts of amino acids which exceed the amounts normally present would be helpful in the treatment trials of chlamydial infections either in vivo or in vitro. In addition, there is no hint that supplementation with amino acids is implicated in therapeutic courses of infections caused by other extracellular or intracellular pathogens.

The extremely high prevalence of C. pneumoniae and C. trachomatis infections in humans and the inefficacy of presently used drugs (antibiotics) to completely resolve the chronic infection and the eventual role of antibiotics in establishing persistent chlamydial infections, associated with many serious chronic diseases, necessitate a more reliable and effective therapeutic approach which could help in complete resolving of chlamydial infections. Further, an effective antichlamydial vaccine is still not present. Therefore, it is the problem underlying the present invention to provide medicaments for treatment or/and prophylaxis of chlamydial infections which overcome the disadvantages as described above. In particular, medicaments are needed which are able to completely eradicate Chlamydia in order to prevent relapsing infections.

Surprisingly, it was found in the context of the present invention that increasing concentrations of amino acids, e.g. L-leucine, L-isoleucine, L- methionine or L-phenylalanine could dramatically suppress chlamydial growth. As described in the examples, supplementation of human cell cultures infected with chlamydiae with exogenous amounts of individual amino acids markedly affected at least one of the following: the inclusion size, morphology of chlamydial forms and development of infectious progeny. More importantly, among the additives used, L-methionine, L- isoleucine and L-leucine (at concentrations of 10 mM each) completely inhibited multiplication of entered bacteria, leading to total arrest of inclusion maturation and to complete suppression (100%) of the production of infectious chlamydiae. 50% of inhibition of production of infectious progeny (EBs) was obtained in C. trachomatis at a concentration of 0.25-0.5 mM L- leucine, L-isoleucine or L-methionine. C. pneumoniae was found to be slightly more sensitive to amino acid treatment. In C. pneumoniae, 50% inhibition of production of infectious progeny was obtained at a concentration of 0.1-0.25 mM L-leucine, < 0.1 mM L-isoleucine or 0.25-0.5 mM L- methionine. 10 mM L-phenylalanine, which also markedly inhibited the growth of the bacterial vacuole, very strongly reduced the production of infectious chlamydial progeny by more than 99%, compared to the control untreated infected cell cultures.

By RT-PCR analyses, it was surprisingly observed that long-term exposure to amino acids such as L-leucine, L-isoleucine or L-methionine leads to reduction and suppression of expression of chlamydial key genes over time. After a period of 5-7 days, a significant reduction of mRNA transcripts of the chlamydial genes MOMP and HSP60 was observed upon treatment with L- leucine, L-isoleucine or L-methionine, indicating that amino acids reduce and/or eliminate chlamydial growth over time.

Ultrastructural analysis by electron microscopy of chlamydiae grown for 48 h in host cells exposed, for example, to 10 mM of either L-leucine or L- methionine demonstrated the presence of single bacteria in the form of RB that are found in small aberrant bacterial vacuoles within the host cytoplasm, indicating a growth inhibition, compared to the large inclusions filled with high numbers of developing bacteria. Further, analysis of electron microscopical data revealed abnormality and distortion of some endocytosed bacteria.

A further unexpected finding is that the chlamydial growth and infectivity cannot be restored as long as increased amounts of an amino acid are present in the cell culture medium, as revealed from long-term exposure experiments, in which infected cells were continuously exposed to one of the amino acids for up to 5 days post-infection. However, the notable adverse inhibitory effects by L-leucine, L-isoleucine, L-methionine and L- phenylalanine either on the inclusion maturation or on the bacterial replication and infectivity can be partially reversed when elevated individual amino acid concentrations were withdrawn 1 or 2 days post-treatment. More importantly, removal of elevated additive concentrations at days 3, 4 or 5 post-treatment led to a dramatic decrease in the infectivity and chlamydial inclusion growth recovery. For example, withdrawal of exogenous amino acids from infected cell cultures continuously exposed for 5 days with individual amino acids did not lead to resumption of inclusion growth and resulted in zero or negligible infectivity yield. These in vivo results suggest that long-term treatments with individual amino acids could be effective in killing of chlamydiae (Example 3).

Overall, the data obtained suggest that chlamydial development is dramatically modulated by prolonged exposures to L-leucine, L-isoleucine, L-methionine and L-phenylalanine. Chlamydia affected by long amino acid treatments were unable to grow and to be infective, indicating possible antichlamydial effects exerted by these natural substances (Examples 1-3). This conclusion is supported by: (1) the presence of aberrant inclusions with single bacteria that showed some degree of distortion, shown by electron microscopy 2 days post-treatment, and (2) the inability of bacteria to restore their growth and infectivity in cell cultures exposed for relatively longer periods (Example 3).

Two (L-leucine and L-isoleucine) out of the three branched chain amino acids were found very effective in inducing markedly adverse effects on chlamydial development in vitro.

Consuming elevated amounts of amino acids is usually not associated with any health risk for people, and treatment with amino acids increase the plasma amino acid level. Use of free amino acids is advantageous, as they need no digestion and are absorbed directly into the bloodstream. The supra-physiologic amino acid level in the plasma would increase, in turn, amino acid availability to infected tissues in humans and may initiate Chlamydial eradication process. Because of the general safety of amino acids, intakes of individual amino acids or combinations thereof elevate their plasmic and tissue concentration beyond the physiological ones in order to induce the anti-chlamydial effects as shown in the examples. Alternatively, chronic intakes of dietary supplements which are rich in amino acids could be helpful in treatment regimen for chlamydial infections and in prevention from chlamydial infections.

Importantly, proteins in animal and plant tissues are made from the L-form of amino acids. Thus, the L-forms of leucine, isoleucine, methionine and phenylalanine are compatible with human biochemistry. L-Leucine, L- isoleucine, L-methionine and L-phenylalanine are essential amino acids; they cannot be synthesized in the body and need to be supplied in the diet. Consuming elevated amounts of these amino acids is usually not associated with any health risks for people. The solution to the problem outlined above concerns the use of safe naturally occurring substances (dietary supplements), namely, amino acids for treatment or/and prophylaxis of chlamydial infections. Increasing intakes of amino acids might have two long-term benefits in the human body: (1) eradication of chlamydial infection, and (2) upregulation of protein synthesis, as amino acids upregulate the capacity of tissues to synthesize proteins. Intake of exogenous amino acids to increase plasma and tissue levels of these substances could initiate and/or accelerate successful treatment of chlamydial diseases, and could improve recovery of infected persons by regular supplementation with amino acids or a mixture of them.

A first aspect of the invention is the use of at least one amino acid or/and and analogue or/and a derivative thereof as an active ingredient for the manufacture of a medicament for the prophylaxis or treatment of infecitions with microorganisms from the family Chlamydiaceae.

In a preferred embodiment, the medicament of the invention comprises at least one amino acid selected from naturally occurring L-amino acids, analogues and derivatives thereof. It is more preferred that the medicament of the invention comprises at least one amino acid selected from essential amino acids, analogues and derivatives thereof. It is most preferred that the amino acid is selected from the group consisting of L-leucine, L-isoleucine, L-methionine, L-phenylalanine, analogues and derivatives thereof.

In chlamydial treatment courses, supplementary diet to be taken could contain one of L-leucine, L-isoleucine, L-methionine or L-phenylalanine or a mixture thereof. A combinatory use of L-leucine, L-isoleucine, L- phenylalanine and L-methionine may be more efficacious and therapy using these amino acids together may require lower doses due to their synergistic antichlamydial effects. In another embodiment of the invention, the medicament is for administering elevated amounts of amino acids to target cells, tissues, organs or/and body fluids. In the in vitro experiments, exogenous additions of L-leucine, L- isoleucine, L-methionine or L-phenylalanine at concentrations of 1 mM each have shown notably adverse effects on chlamydial growth. The endogenous concentrations of these amino acids in commercially available host cell growth media range from 0.09 mM for L-phenylalanine to 0.38 mM for either L-leucine or L-isoleucine (see example 1). The composition of such host cell growth medium is approximated to the composition of blood plasma. Thus, elevated amounts of amino acids at the target cells, tissues, organs, or/and the body fluids are concentrations increased by a factor of preferably 2 to 100, more preferably for 2 to 10, and most preferably 2 to 5. In absolute concentrations, it is preferred to reach a concentration at the target cells, tissues, organs or/and body fluids of the respective amino acid of at least 1 mM and up to 10 mM, more preferably up to 5 mM, most preferably up to 2,5 mM. Analogues or/and derivatives of amino acids are preferably administered to reach a concentration of at least 1 mM and up to 10 mM, more preferably up to 5 mM, most preferably up to 2,5 mM.

A person skilled in the art, in particular a physician, is able to determine the dosage of amino acids or/and analogues or/and derivatives thereof, which have to be administered to reach plasma concentrations sufficient for effective treatment or/and prophylaxis. Suitable amounts of an amino acid or/and an analogue or/and a derivative thereof are preferably at least 1 mmol/kg body weight up to 10 mmol/kg body weight, more preferably up to 5 mmol/kg body weight, most preferably up to 2,5 mmol/kg body weight. To sustain such elevated concentrations in the body, amino acids may be taken, for example, orally as tablets, capsules or as a drink three times daily for preferably at least 2 days, more preferably at least 1 week. However, the adequate dosages and the intake periods of elevated amounts of individual amino acids, analogues, derivatives thereof, or a mixture thereof, which selectively resolve infections with obligate intracellular microorganisms of the order Chlamydiales in particular Chlamydiaceae can be identified by a method comprising

(a) establishing an animal model susceptible to chlamydial infections, and

(b) testing the efficacy of increasing intakes by infected animals in step (a) of individual amino acids selected from Table 1, analogues or derivatives thereof, or combination supplements thereof, in the treatment of chlamydial infections.

The animal model can be a susceptible mouse which shows pathological events similar to those in humans infected with Chlamydiales species, in particular with Chlamydiaceae species. A preferred mouse strain is NIH/S (Kaukoranta-Tovanen, Lauriia et al.,1993) or Swiss Webster (Yang, Kuo et al., 1995).

Further, doses which are prophylactic for chlamydial infections can be identified by a method comprising: (a) regular supplementation of animals susceptible to chlamydial infections with doses as identified by the method as described above before the experimental infection with chlamydial pathogens, and (b) infection of the amino acid-supplemented animals with Chlamydiales species, in particular Chlamydiaceae species, and

(c) testing the efficacy of the supplementation with said doses of amino acids, analogues or derivatives thereof, or a combination mixture thereof in the prevention of infections with the obligate intracellular bacteria of the order Chlamydiales, in particular of the family Chlamydiaceae. An elevated level of at least one amino acid or/and an analogue or/and derivative thereof may be a dosage increasing the amounts normally present in body fluids, in organs, tissues or/and at target cells, in particular a dosage of at least 1 mmol/kg body weight up to 10 mmol/kg body weight, more preferably up to 5 mmol/kg body weight, most preferably up to 2,5 mmol/kg body weight.

The regular intake of the amino acids by humans could have prophylactic effects leading to the prevention of chlamydial infection. In addition, regular supplementation of healthy persons with the amino acids as single amino acids or a mixture of them could alter the course of future infection and could improve recovery of persons who will be infected with chlamydiae.

The medicament of the invention can be used for treatment or/and prophylaxis of microorganisms of the genus Chlamydia or Chlamydophila. It is preferred that the microorganism is Chlamydia trachomatis or Chlamydophila pneumoniae.

The medicament may contain amino acids as such. Alternatively, analogues and derivatives of amino acids are preferably selected from the group consisting of D-leucine, D-isoleucine, D-methionine, D-phenylalanine, and derivatives thereof, and derivatives of L-leucine, L-isoleucine, L-methionine, and L-phenylalanine. More preferably, the analogues are selected from the compounds given in Table 5.

To increase the blood and the tissue level of amino acids in the body of Chlamydia patients, intake of amino acids, analogues or/and derivatives thereof, in particular increased amounts, can be via various routes. Amino acids, analogues or/and derivatives thereof can be infused intravenously into individuals, or can be given orally as tablets or capsules or as a drink, or can be administered by injection; e.g. subcutaneously. In a preferred embodiment, the medicament is for the treatment or prophylaxis of a human subject in need thereof. The medicament of the invention may not comprise a further active ingredient. However, in yet another embodiment of the invention the medicament may comprise a further active ingredient to overcome the in vivo resistance to antibiotics and, thus, accelerating complete resolving of chronic diseases associated with chlamydiae. Increasing intake of amino acids, analogues or/and derivatives thereof during antibiotic intervention might have synergistic effect on the eradication of pathogens from the bloodstream and infected tissues. Thus, increasing acids plasma and tissue levels during conventional therapeutic courses with at least one antibiotic may have long-term benefits, preventing reactivation of antibiotic-resistant pathogens that may be in persistent state.

The further active ingredient may be a substance already known for treatment and/or prophylaxis of chlamydial infections. A combination of an antibiotic and at least one amino acid or/and at least one amino acid analogue or at least one amino acid derivative has the advantageous effect that a complete eradication can be achieved. It is preferred that the antibiotic comprised in the medicament of the invention is an antibiotic which is not sufficiently successful when taken alone to prevent or treat infection with Chlamydiaceae. It is more preferred to select the antibiotics from macrolides, quinolones or combinations thereof.

As pointed out above, eradication is a major problem in patients with chronic chlamydial infections. It is thus preferred that the medicament of the invention is for treatment of patients with chronic infections with Chlamydiaceae, especially those that are associated with chronic respiratory system and heart diseases. The use of amino acids alone or together with antibiotic trials could improve the clinical condition of patients with coronary heart disease by eradication of chlamydiae from lesions in the blood vessel wall (atheromas), where antimicrobial resistance is common.

A further embodiment of the invention is a nutritional or pharmaceutical composition including an elevated level of at least one amino acid or/and an analogue or/and derivative thereof, in a therapeutically sufficient amount in combination with an antibiotic for use in the prevention or treatment of infections with microorganisms from the order Chlamydiales, in particular from the family Chlamydiaceae, especially those that are associated with chronic respiratory system and heart diseases.

Preferably, the amino acids in the pharmaceutical compositions are selected from the group consisting of L-leucine, L-isoleucine, L-methionine and L- phenylalanine. The antibiotic may be an antibiotic which is not sufficiently successful when taken alone to treat in vivo chlamydial infections. Preferably, the antibiotic is selected from macrolides or quinolones.

A further embodiment is the use of at least one amino acid, analogue or derivative thereof in a sufficient dose optimally in combination with further active ingredients for the manufacture of a nutritional or pharmaceutical composition for the prevention of infections with microorganisms from the order Chlamydiales, in particular from the family Chlamydiceae. The further active ingredient may be an antibiotic, which is preferably selected from macrolides or quinolones.

A further embodiment is a method for the treatment of infections with microorganisms from the order Chlamydiales, in particular Chlamydiaceae comprising administering a subject in need thereof a therapeutically effective dose of a medicament comprising at least one amino acid, analogue, or derivative, thereof.

Yet another embodiment is a method for the prevention of infections with microorganisms from the order Chlamydiales comprising administering a subject in need thereof a prophylactically effective dose of a medicament comprising at least one amino acid or an analogue or derivative thereof.

The invention is further illustrated by the following figure and examples: Figure 1 describes reduction and suppression of chlamydial key gene expression upon application of amino acids.

EXAMPLE 1

Effects of L-leucine, L-isoleucine, L-methionine and L-phenylalanine on the production of infectious progeny (EBs) of chlamydiae.

The goal of this experiment was to assess the production of infectious EBs in the presence of either L-leucine, L-isoleucine, L-methionine or L- phenylalanine.

The human laryngeal epithelial HEp-2 cells (ATCC-CCL23) were grown in 6- well plates in CGM (cell growth medium), which composes of RPMI 1640 supplemented with 25 mM HEPES, 10% FBS (fetal bovine serum) and 2 mM glutamine, and incubated overnight at 5% CO₂ and 37°C to allow adherence. To examine effects of elevated amounts of free amino acids on the production of infectious EBs, host cells were infected and chemicals were administered as follows: C. trachomatis serovar L2 EBs were suspended in IM (infection medium), which composes of RPMI supplemented with 5% FBS, 25 mM HEPES and 2 mM glutamine, and added directly to the cells at a multiplicity of infection (MOI) ~ 1. The cultures were incubated at 5% CO₂ and 35°C for 2 h. At the end of the incubation, infected cells were washed twice with IM and further incubated at similar conditions. Nineteen hours post-infection, old medium was aspirated from the plates and was replaced with 2 ml/well IM containing one of the additives at 10 mM concentrations. As controls, infected cells were exposed to fresh IM without additives. Treated and untreated cells were allowed to incubate at conditions mentioned before until the end of the experiment (44 h post-infection). In another number of experiments, host cells seeded in 6-well plates were pretreated with IM plus one of the additives (10 mM concentration) for 30 min before infection. EBs (MOI-1) diluted in IM containing the respective chemicals were added to the cells and allowed to adsorb for 2 h at 5% CO₂ and 35°C. The cells were rinsed 2 times and subsequently loaded with IM plus the respective chemicals and further incubated until 44 h post-infection. Control untreated cells were similarly infected and maintained in IM without extra amino acids.

Exogenous amounts of these amino acids corresponding to 10 mM concentrations were dissolved directly in the host cell growth medium (RPMI 1640 medium, which is originally supplied with these amino acids to allow host cell growth and proliferation. The growth medium usually contains the following concentrations: 0.38 mM for either L-leucine or L-isoleucine, 0.1 mM for L-methionine and 0.09 mM for L-phenylalanine. Thus, the actual concentration of each amino acid that showed the antichlamydial activity is the sum of both concentrations: the originally present in the medium and the exogenously added.

At the end of the experiment (44 h post-infection), treated and untreated control cells were removed from the wells of 6-well plates using sterile glass beads and the cells were lysed to release chlamydiae by vortexing with glass beads. The lysates were freshly utilized for the infectivity titer determination. In brief, 10-fold dilutions of lysates made in IM were inoculated onto monolayers of HEp-2 cells cultured in coverslips-containing 12-well plates. The bacteria were allowed to adsorb to cells for 2 h at 5% CO₂ and 35°C. Cells were then washed twice with IM and then supplemented with IM and incubated again under similar conditions. One day later, inclusions resulted from each dilution were visualized by immunostaining using Rabbit polyclonal antibodies specific against the genus Chlamydia that was supplied from Milan Analytica AG (La Roche, Switzerland), counted and the infectivity titer, expressed as number of IFU/ml, was estimated (see Al- Younes, Rudel et al., 2001).

L-leucine, L-isoleucine, L-methionine or L-phenylalanine added 19 h until 44 h post-infection very strongly diminished the production of infectious chlamydial forms (infectivity was reduced by more than 99%) (Table 1). Importantly, continuous treatments with L-leucine, L-isoleucine or L- methionine 30 min before the infection and during the infection completely diminished the infectivity of chlamydiae. In contrast, continuous presence of L-phenylalanine did not completely prevent infectious forms being produced but reduced the infectivity by more than 99% (Table 2).

EXAMPLE 2:

Effect of different doses of L-leucine, L-isoleucine, L-methionine and L- phenylalanine on the inclusion development and infectivity of Chlamydia in HEp-2 cells (larynx carcinoma cell line).

The goal of this experiment was to examine the effect of various concentrations of L-leucine, L-isoleucine, L-methionine or L-phenylalanine on EB production in HEp-2 cells infected with C. trachomatis L2.

The human laryngeal epithelial HEp-2 cells (ATCC-CCL23) were grown in 6- well plates in CGM (cell growth medium), which composes of RPMI 1640 supplemented with 25 mM HEPES, 10% FBS (fetal bovine serum) and 2 mM glutamine, and incubated overnight at 5% CO₂ and 37°C to allow adherence. Host cells were pretreated for 30 min with IM (infection medium), which composes of RPMI supplemented with 5% FBS, 25 mM HEPES and 2 mM glutamine, containing various concentrations of either L-leucine, L- isoleucine, L-methionine or L-phenylalanine (1 mM, 2.5 mM or 5 mM). Chlamydia trachomatis serovar L2 EBs diluted in IM containing the respective amino acid concentration were added to the cells at a multiplicity of infection (MOI) ~ 1 and allowed to adsorb for 2 h at 5% CO₂ and 35°C. The cells were rinsed 2 times and subsequently loaded with IM plus the respective amino acid concentration and further incubated until 44 h post- infection. Control untreated cells were similarly infected and maintained in IM without extra amino acid. Next, the development of infectious C. trachomatis progeny was assayed in cell cultures exposed to varying concentrations of the amino acids by routinely used infectivity titration assays. Briefly, infected cells were homogenized by vortexing using glass beads to release formed bacteria. Ten-fold dilutions of lysates made in IM were inoculated onto monolayers of HEp-2 cells cultured in coverslips- containing 12-well plates. The bacteria were allowed to adsorb to cells for 2 h at 5% CO₂ and 35°C. Cells were then washed twice with IM and then supplemented with IM and incubated again under similar conditions. One day later, inclusions resulted from each dilution were visualized by immunostaining using Rabbit polyclonal antibodies specific against the genus Chlamydia that was supplied from Milan Analytica AG (La Roche, Switzerland), counted and the infectivity titer, expressed as number of IFU/ml, was estimated (see Al-Younes, Rudel et al., 2001). Results are shown in Table 3a.

In a further experiment employing the same protocol, production of infectious progeny was tested in C. trachomatis and C. pneumoniae at concentrations of L-leucine, L-isoleucine or L-methionine ranging from 0,1 mM to 5 mM. Results are shown in Table 3b.

As shown in Table 3a and 3b, treatments at concentrations used reduced the number of infectious EBs in a dose-dependent fashion. 50% inhibition was achieved at a concentration of 0.25 mM-0.5 mM L-leucine, about 0.25 mM L-isoleucine, 0.25-0.5 mM L-methionine (C. trachomatis, Table 3b) or 0.1-0.25 mM L-leucine, < 0.1 mM L-isoleucine, 0.25-0.5 mM L-methionine (C. pneumoniae, Table 3b). Concentrations as low as 1 mM of L-leucine, L- isoleucine and L-methionine reduced the infectivity yield by more than 92% (C. trachomatis) or 76% (C. pneumoniae), whereas 1 mM of L-phenylalanine had a smaller effect on the formation of infectious progeny. When chemicals used at higher concentrations (2.5 or 5 mM), development of infectious EBs was reduced by more than 93% or totally abolished (Table 3a and b).

EXAMPLE 3:

Influence of prolonged exposures to either L-leucine, L-isoleucine, L- methionine or L-phenylalanine on the developmental cycle of chlamydiae (the production of infectious EBs). The goal of this experiment was to see if infectious progeny can be formed in continuously treated HEp-2 cells over time. Further, this experiment was designed to examine whether the infectivity suppressed by the amino acids can be reverted by the removal of elevated amounts of amino acids.

The human lar ngeal epithelial HEp-2 cells (ATCC-CCL23) were grown in 6- well plates in CGM (cell growth medium), which composes of RPMI 1640 supplemented with 25 mM HEPES, 10% FBS (fetal bovine serum) and 2 mM glutamine, and incubated overnight at 5% CO₂ and 37°C to allow adherence. Host cells were pretreated for 30 min with IM (infection medium), which composes of RPMI supplemented with 5% FBS, 25 mM HEPES and 2 mM glutamine and containing 10 mM concentrations of either L-leucine, L- isoleucine, L-methionine or L-phenylalanine. Chlamydia trachomatis serovar L2 EBs diluted in IM containing the respective amino acid concentration were added to the cells at a multiplicity of infection (MOI) ~ 1 and allowed to adsorb for 2 h at 5% CO₂ and 35°C. The cells were rinsed 2 times and subsequently loaded with IM plus the respective amino acid and further incubated for several intervals of times ranging from 1 to 5 days post- infection (specimens 4-8 in Table 4). Control untreated cells were similarly infected and maintained in IM without extra amino acid for either 1 , 2 or 3 days post-infection (specimens 1-3 in Table 4). Infectivity assessments indicated no recovery of chlamydial EB in infected cells incubated with either L-leucine, L-isoleucine or L-methionine for 1 , 2, 3, 4 or 5 days (specimens 4- 8). Similarly, long exposure of infected cells to L-phenylalanine depressed infectivity, which remained negligible and decreased over time. Collectively, these findings suggest that Chlamydia cannot complete its developmental cycle when these amino acids are continuously present in the infected cell cultures.

To test if the infectivity suppressed by these amino acids can be reverted by removal of amino acids added, cell cultures were infected in the presence of these amino acids for either 1 , 2, 3, 4 or 5 days as described above (specimens 9-13; Table 4). At these time points, culture medium containing the respective amino acids was replaced with fresh IM without elevated levels of additives and the cell cultures were incubated for additional 2 days to allow the rescue of chlamydiae. The cells were then examined microscopically (phase contrast) for inclusion detection, lysed and inoculated onto fresh HEp-2 cells for infectivity yield assessments. Table 4 confirms that early replacement (at 1 or 2 days post-treatment) of IM containing exogenous amino acids with IM without elevated amino acids led to a partial recovery of bacterial growth (specimens 9 and 10). Exposure of infected host cells to elevated amounts of individual amino acids for periods longer than 2 days led to a dramatic decrease in the number of inclusions rescued by removal of elevated levels of amino acids, as shown by phase contrast microscopy. Further, exposure to amino acids for either 3, 4 or 5 days appreciably reduced bacterial infectivity (specimens 11-13; Table 4).

Collectively, these findings suggest that Chlamydia cannot complete its developmental cycle when these amino acids are continuously present in the infected cell cultures (specimens 4-8; Table 4). Further, the data obtained here suggest that the inhibition of the chlamydial growth and infectivity induced by L-leucine, L-isoleucine, L-methionine and L-phenylalanine can be partially reversed by the removal of elevated amounts of amino acids and that the degree of rescue of chlamydial development is dramatically affected by prolonged exposures to amino acids (specimens 9-13).

EXAMPLE 4:

RT-PCR analyses of mRNA transcripts prepared from Chlamydia trachomatis infected cells for periods up to 15 d in the absence (A; control) or in the presence of 10 mM of either L-leucine (B), L-isoleucine (C) or L- methionine (D). Hep-2 cells seeded in 6-well plates were infected with C. trachomatis at MOI 0.5 and the medium with or without excess amino acids were replaced each second day. At each time point indicated, cells were harvested and total RNA was prepared for RT-PCR analysis. (A) Despite complete detachment of control untreated infected cells within 5 to 7 d postinfection, chlamydial mRNA transcripts can be detected throughout the experiment period. (B-D) long-term exposure to individual amino acids did not detach infected cells and led to reduction and suppression of the expression of key chlamydial genes over time, indicating capacity of these amino acids to reduce and/or to eliminate chlamydial growth over time. Results are shown in Fig. 1. MOMP: major outer membrane protein. HSP60: heat shock protein 60. EBs: harvested elementary bodies.

Table 1. Exogenous amino acids added at 19 h post-infection until the end of the infection (44 h) onto infected HEp-2 cells adversely affected the development of infectious C. trachomatis L ^~ 2^a.

Exogenous AA Cone. (mM) % Infectivity ± SD^b

None (control) 100 ± 2,23

L-leucine 10 0.043 ± 0.05

L-isoleucine 10 0.077 ± 0.09

L-methionine 10 0.16 ± 0.11

L-phenylalanine 10 0.44 ± 0.03

a. The experiment was performed at 2 different occasions. b. The percent infectivity of C. trachomatis L2 resulted in each treatment was determined by dividing IFU/ml estimated for that treatment by IFU/ml of the control cell monolayers unexposed to exogenous amino acids. The percent infectivity was expressed as means of the percent infectivity for 2 different experiments ± standard deviations from the means.

Table 2. Exogenous amino acids added 30 min before the infection until the end of the infection (44 h) onto infected HEp-2 cells adversely affected the development of infectious C. trachomatis L2 .

Exogenous AA Cone. (mM) % Infectivity ± SD

None (control) 100 ± 3,41

L-leucine 10 0

L-isoleucine 10 0

L-methionine 10 0

L-phenylalanine 10 0.015 ± 0.001

a. The experiment was performed at 2 different occasions. b. The percent infectivity of C. trachomatis UI resulted in each treatment was determined by dividing IFU/ml estimated for that treatment by IFU/ml of the control cell monolayers unexposed to exogenous amino acids. The percent infectivity was expressed as means of the percent infectivity for 2 different experiments ± standard deviations from the means. Table 3a. Reduction in the production of infectious C. trachomatis L2 by exogenous amino acids is dose-dependen .

Chemical Cone. (mM)¹³ % Infectivity ± SD^C

None (control) 100 ± 20.96 L-leucine 1 2.54 ± 0.12 2.5 0.01 ± 0.005

L-isoleucine 1 2.13 ± 0.2 2.5 0.01 ± 0.003 5 0

L-methionine 1 7.74 ± 0.7 2.5 0.04 ± 0.03 5 0

L-phenylalanine 1 45.04 ± 11.52 2.5 3.24 ± 0.12 5 0.23 ± 0.06

a. The experiment was performed at 2 different occasions. b. Host cells were pre-incubated for 30 min with exogenous ajnino acids at concentrations ranging from 1 to 5 mM. Cells were subsequently infected with C. trachomatis UI and incubated for an additional 48 h in the presence of the respective amounts of the additives. c. The percent infectivity of C. trachomatis UI resulted in each treatment was determined by dividing IFU/ml estimated for that treatment by IFU/ml of the control cell monolayers unexposed to exogenous amino acids. The percent infectivity was expressed as means of the percent infectivity for 2 different experiments ± standard deviations from the means. Table 3b. Exogenously added leucine, isoleucine and methionine reduce the production of infectious progeny of C. trachomatis and C. pneumoniae in a dose-dependent fashion.

% Infectivity ± SD

Chemical Cone. (mM) C. trachomatis C. pneumoniae

None (control) 100 100 0.1 88.3 ± 4.52 85.65 ± 3.32

L-Leucine 0.25 66 ± 5.94 40.7 ± 12.45 0.5 22.05 ± 7.71 35.05 ± 5.44 1 2.7 ± 2.4 13.35 ± 6.01 2.5 n n 5 0 0

L-lsoleucine 0.1 60.5 ±4.38 21.6 ± 8.2 0.25 51 ± 4.95 2.9 ± 1. 98 0.5 19.05 ± 4.3 n 1 2.05 ± 1.91 n 2.5 n 0 5 0 0

L-Methionine 0.1 89.35 ± 1.34 78.8 ± 7.21 0.25 68.1 ± 5.37 71.65 ± 6.7 0.5 32.75 ± 5.16 41.5 ± 7.79 1 6.85 ± 2.76 23.7 ± 0.71 2.5 n 6.95 ± 4.17 5 0 0.65 ± 0.35 Table 4. Influence of prolonged exposures to eitήer leucine (Leu), isoleucine (lie), metrnonine (Met) or Phenylalani e (Phe) on the production of infectious EB and rescue of infectivity of C. trachomatis UI by withdrawal of elevated levels of amino acids^a.

IFU/ml harvested from infected cell cultures exposed to Infection period nothing Specimen and treatment (controls) leucine isoleucine methionine phenylalanine 1 ld-AA 4.16 xlO³ 2 2d-AA 2.61 x 10⁸ 3 3d-AA 3.98 xlO⁷ 4 ld + AA 0 0 0 0 5 2d + AA 0 0 0 3.22 xlO⁴ 6 3d + AA 0 0 0 4.51 x 10³ 7 4d + AA 0 0 0 1.37 xlO³ 8 5d + AA 0 0 0 1.15 xlO³ 9 ld + AA 5.88 xlO⁷ 6 l0⁷ 6xl0⁷ 4.22 xlO⁷ 2d-AA 10 2d + AA 3.15x10° 1.84 10° 1.07x10° 3.56x10° 2d-AA 11 3d + AA 2.38 lO⁵ 3.86 xlO⁴ 2.14 xlO⁴ 5.64x10' 2d-AA 12 4d + AA 1.78 xl0^j 2.38 x 10^j 5.94x10^ 2.97 xlO³ 2d-AA 13 5d + AA n n n 4.16 x10^s 2d-AA

a. Host cells were pre-incubated for 30 min with HVI containing 10 mM concentrations of either leucine, isoleucine, methionine or phenylalanine (specimens 4-13) and were then infected for various time intervals in the presence of respective amounts of amino acids. For some specimens (9-13), elevated levels of AA-containing medium was replaced with IM without exogenous amino acids and then cells were incubated for additional two days in an attempt to rescue the bacterial infectivity. As controls, host cells were infected and incubated in IM without any exogenous amino acid for either 1 d, 2 d or 3 d (specimens 1, 2 and 3, respectively). Each specimen was represented by 2 wells of 6-well plates. At the end of indicated time points, all specimens were harvested, disrupted, serially diluted and inoculated Table 5

Analogues and derivatives of L-leucine, L-isoleucine, L-methionine and L- phenylalanine.

L-leucine (L-isoleucine) L-methionine L-phenylalanine

D-leucine D-methionine D-phenylalanine

D-isoleucine ethionine N-acetyl-L-phenylalanine

N-acetyl-L-leucine seleno-L-methionine L-phenylalanine methylester

L-histidyl-L-leucine L-methionine ethylester L-phenylalanine hydroxamate

L-leucinamide L-methioninamide L-phenylalaninamide

L-leucinol L-methionine hydroxamate L-pehnylalaninol

H-alpha-DL-leucine L-methionine sulfone L-phenylalanyl-L-leucine

N-methyl-L-leucine formyl-L-methionine

N-acetyl-L-leucinamide alpha-methyl-L-methionine

L-Norleucine cycloleucine

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Claims

Claims

1. The use of at least one amino acid or/and analogue or/and derivative thereof as an active ingredient for the manufacture of a medicament for the prophylaxis or treatment of infections with microorganisms from the family Chlamydiaceae.

2. The use of claim 1 , wherein the amino acid is selected from naturally occurring L-amino acids or analogues or derivatives thereof.

3. The use of claim 1 or 2, wherein the amino acid is selected from essential amino acids, analogues and derivatives thereof.

4. The use of any one of claims 1 to 3, wherein the amino acid is selected from the group consisting of L-leucine, L-isoleucine, L- methionine, L-phenylaline and analogues and derivatives thereof.

5. The use of any one of claims 1 to 4, wherein the medicament is for administering elevated amounts of amino acids to target cells, tissues, organs, or/and body fluids.

6. The use of any one of claims 1 to 6, wherein the concentrations of amino acids at the target cells, tissues, organs, or/and body fluids is at least 1 mM.

7. The use as claimed in any one of claims 1 to 6, wherein the microorganisms are from the genus Chlamydia.

8. The use as claimed in claim 7, wherein the microorganisms are Chlamydia trachomatis.

9. The use as claimed in any one of claims 1 to 6, wherein the microorganisms are from the genus Chlamydophila.

10. The use as claimed in claim 9, wherein the microorganisms are Chlamydophila pneumoniae.

11. The use of any one of claims 1 to 10, wherein the medicament contains amino acids as such.

12. The use of any one of claims 1 to 10, wherein the medicament contains at least one amino acid analogue or derivative selected from Table 5.

13. The use of any one of claims 1 to 12, wherein the medicament is for administering by parenteral, subcutaneous or intravenous routes.

14. The use of any one of claims 1 to 13, wherein the medicament is for the treatment of humans.

15. The use of any one of claims 1 to 14, wherein the medicament comprises a further active ingredient.

16. The use of claim 15, wherein the further active ingredient is selected from antibiotics.

17. The use of claim 16, wherein the antibiotics are not sufficiently successful when taken alone to prevent or treat infections with Chlamydiaceae.

18. The use of claim 15 or 16, wherein the antibiotics are selected from macrolides, quinolones and combinations thereof.

19. The use of any one of claims 1 to 18, wherein the medicament is for the treatment of patients with chronic infections with Chlamydiaceae.

20. A pharmaceutical composition including an elevated level of amino acids or/and analogues or/and derivatives thereof, in a therapeutically sufficient amount in combination with an antibiotic for use in the prevention of treatment of infections with microorganisms from the order Chlamydiales, in particular from the family Chlamydiaceae.

21. The composition of claim 20, wherein the amino acids are selected from the group consisting of L-leucine, L-isoleucine, L-methionine, L-phenylalaine and analogues and derivatives thereof.

22. The composition of claim 20 or 21 , wherein the antibiotic is not sufficiently successful when taken alone to treat in vivo chlamydial infections.

23. The composition of any of claims 20 to 22, wherein the antibiotic is selected from macrolides or quinolones.

24. The use of at least one amino acid, analogue, or derivative thereof, in a sufficient dose optionally in combination with further active ingredients, for the manufacture of a nutritional or pharmaceutical composition for the prevention of infections with microorganisms from the order Chlamydiales, in particular from the family Chlamydiaceae.

25. A method for the treatment of infections with microorganisms from the order Chlamydiales, in particular from the family Chlamydiaceae comprising administering a subject in need thereof, a therapeutically effective dose of a medicament comprising at least one amino acid, analogue or derivative thereof.

6. A method for the prevention of infections with microorganisms from the order Chlamydiales, in particular from the family Chlamydiaceae, comprising administering a subject in need thereof a prophylactically effective dose of a medicament comprising at least one amino acid, analogue or derivative thereof.