Pentamidine for use in the treatment or prevention of sexually transmitted infections caused by chlamydia SPP. or neisseria SPP.

Abstract

The present invention is in the field of treatment and / or prevention of sexually transmitted infections. The present invention relates to pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use in treatment and / or prevention of a sexually transmitted infection (STI) caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. The present invention further relates to agents, compositions, and / or pharmaceutical compositions for use in treatment and / or prevention of a STI, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the agents, compositions, and / or pharmaceutical compositions comprises at least pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these. The present invention further relates to methods of treatment and / or prevention of a STI in a subject in need thereof, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. The present invention is particularly useful for both pre- and post-exposure prophylaxis (PrEP / PEP) of STIs caused by Chlamydia spp. and / or Neisseria spp. and may even replace existing treatment options. Further, the present invention is particularly relevant for individuals who are at high risk of sexually transmitted infections (STIs), such as sex workers.

Description

[0001] Medizinische Universitat Wien

[0002] CeMM - Forschungszentrum fur Molekulare Medizin GmbH

[0003] PENTAMIDINE FOR USE IN THE TREATMENT OR PREVENTION OF SEXUALLY TRANSMITTED INFECTIONS CAUSED BY

[0004] CHLAMYDIA SPP. OR NEISSERIA SPP.

[0005] The present invention is in the field of treatment and / or prevention of sexually transmitted infections. The present invention relates to pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use in treatment and / or prevention of a sexually transmitted infection (STI) caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. The present invention further relates to agents, compositions, and / or pharmaceutical compositions for use in treatment and / or prevention of a STI, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the agents, compositions, and / or pharmaceutical compositions comprises at least pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these. The present invention further relates to methods of treatment and / or prevention of a STI in a subject in need thereof, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. The present invention is particularly useful for both pre- and post-exposure prophylaxis (PrEP / PEP) of STIs caused by Chlamydia spp. and / or Neisseria spp. and may even replace existing treatment options. Further, the present invention is particularly relevant for individuals who are at high risk of STIs, such as sex workers.

[0006] The risk of death or severe health restraints from infectious diseases has decreased in developed countries based on the availability of efficient antimicrobial measures, including prophylaxis and treatment (el Bcheraoui, C. et al. (2018), JAMA 319(12): 1248-1260). However, many sexually transmitted infections (STIs) are currently increasing in North America, Asia and Europe (see, e.g., Gokengin, D. et al. (2023), Lancet Reg Health Eur. 34: 100738), especially among individuals engaging in high-risk sex practices, including unprotected sex (Bosetti, D. et al. (2022), Open Forum Infect Dis 9(12):ofac592). This trend even intensified during the COVID-19 pandemic due to reduced screening and access to healthcare facilities (Wright, S.S. et al. (2022), Sex Transm Dis 49(4):E61-E63; Pagaoa, M. et al. (2021), Sex Transm Dis 48(10)798-804).

[0007] The most common bacterial STIs in humans are Chlamydia trachomatis Ct infections with an estimated prevalence of around 4% in the Americas and around 2% in Europe with highest infection rates in the population between 15 to 24 years (Huai, P. et al. (2020), BMC Infect Dis 20(10):798-804; Newman, L. et al. (2015), PLoS One 10(12):e0143304). There are different Ct serovars characterized by their major outer membrane proteins (MOMP): Serovars A-C primarily cause ocular infection, while serovars D-K preferentially infect the urogenital tract and serovars L1-L3 can lead to lymphogranuloma venereum (Stevens, M.P. et al. (2010), J Clin Microbiol 48(6):2060-2065). Ct is a Gram-negative obligate intracellular biphasic pathogen utilizing the host cell machinery for replication (Gitsels, A. et al. (2019), Front. Microbiol. 10:2329). Briefly, the infectious form of Ct, the Ct elementary bodies (EBs) can interact with various surface receptors on epithelial cells, which facilitate uptake of the pathogen (Gitsels, A. et al. (2019), Front. Microbiol. 10:2329). Using cell cytoskeletal proteins, EBs form an inclusion within the endocytic vacuoles where they transform into the non-infectious replicative form of Ct, the Ct reticulate bodies (RBs), replicating by utilizing nutrients provided by the host cell. After finishing their replication cycle, RBs transform back into EBs and leave the cell by host cell lysis or extrusion (Gitsels, A. et al. (2019), Front. Microbiol. 10(2329): 1-27; Witkin, S.S. et al. (2017), Clinical and Vaccine Immunology 24(l):e00203-17). Since the Ct developmental cycle strictly depends on the host cell, treatment approaches do not need to rely solely on antibiotics inhibiting, e.g., bacterial protein synthesis. Other exemplary direct approaches, may be the combinatorial treatment with minor groove binders (MGBs) and antiviral agents, thereby targeting the pathogen translation and / or transcription (WO 2005 / 025565) or directly interfering with the integrity of the outer membrane of the pathogen (e.g., WO 2023 / 077235). Still, they may also consider alternative mechanisms by interfering with cell entry or modulating host cell structures (Bommana, S. and Polkinghorne, A. (2019), Front Microbiol 10(113):l-9). Alternative strategies involve, amongst others, inhibiting type III secretion systems of Ct, blocking chlamydial attachment by destroying the bacterial membrane or binding certain membrane structures, and enhancing host cell defense mechanisms by cytokines or blocking of metabolic processes utilized by Chlamydia spp. (Bommana, S. and Polkinghorne, A. (2019), Front Microbiol 10(113):l-9; Hou, C. et al. (2022), Front Microbiol 13(987662): 1-13). Even though human chlamydial infections can be treated with antibiotics, there are studies reporting tetracycline resistance by the presence of a Tet(C)-island in the genome in the species-specific strain Chlamydia suis {C suis) infecting pigs (Bommana, S. and Polkinghorne, A. (2019), Front Microbiol 10(113): 1-9; Dugan, J. et al. (2004), Antimicrob Agents Chemother 48(10)3989-3995).

[0008] Another STI of concern is Neisseria gonorrhoeae Ng , inter alia, because of its high rates of antibiotic resistance. For example, a large portion of clinical Ng isolates are resistant to early-developed antibiotics like penicillin, sulfonamides, tetracyclines, and additional resistance to extended -spectrum cephalosporins and macrolides, which are currently recommended for treatment, is on the rise (Unemo, M. and Shafer, W.M. (2014), Clin Microbiol Rev 27(3)587-613; Unemo, M. et al. (2021), Lancet Microbe 2(ll)e627-e636). Moreover, co-infections of / ^ with various STIs are common (Althaus, C.L. et al. (2014), Health Technol Assess (Rockv) 18(2)1-100), as there exists an increased susceptibility of already infected individuals to other STIs (Leonard, C.A. et al. (2019), Curr Clin Microbiol Rep 6:182-191; Su, X. et al. (2022), J Infect Dis 226(12)2192-2203).

[0009] A significant problem with both Ct and Ng infections is posed by the high rates of asymptomatic cases that can still cause severe long-term sequelae in females like pelvic inflammatory disease (PID) resulting in (tubal) infertility or ectopic pregnancy (Davies, B. et al. (2016), Lancet Infect Dis 16(9)1057-1064). Preventive measures include screening programs for individuals at high risk of STIs. Nevertheless, screening is expensive for the health care system, and a cost-effectiveness assessment is often not available (Low, N. et al. (2021), The Lancet 398(10309)1386-1388; US Preventive Services Task Force, Davidson, K.W. et al. (2021), JAMA 326(10)949-956). The ultimate goal would be to develop a vaccine as preventive measure, which was not successful so far despite 60 years of research with only one vaccine candidate currently being in a clinical trial (Phillips, S.et al. (2019), Front Microbiol 10(70): 1-18; Abraham, S. et al. (2019), Lancet Infect Dis 19(10)1091-1100.; de la Maza, L.M.et al. (2017), Clin Vaccine Immunol. 24(4):e00543-16; de la Maza, L.M.et al. (2021), Expert Rev Vaccines 20, 421-435). Other preventive strategies involve pre- and post-exposure prophylaxis (PrEP / PEP) with doxycycline (also herein referred to as doxy) which has shown high efficacy in patient groups with high-risk behavior for the acquisition of chlamydial infections and syphilis that are frequently using HIV-PrEP (Molina, J.M. et al. (2018), Lancet Infect Dis 18(17):308-317; Bolan, R.K. et al. (2015), Sex Transm Dis 42(2)98-103; Luetkemeyer, A.F. et al. (2023), New England Journal of Medicine 388(14): 1296-1306). HIV-Prep may inter alia comprise the use of tenofovir alafenamide / emtricitabine (Descovy), tenofovir disoproxil fumarate / emtricitabine (Truvada), or cabotegravir (Apretude). The concern of antimicrobial resistance of / and other sexually transmitted pathogens, as well as commensals, greatly limits the doxy-PEP. In a recent study from Luetkemeyer et al., the development of resistance in A^and Staphylococcus aureus isolates was increased in the doxy-PEP group (Luetkemeyer, A.F. et al. (2023), New England Journal of Medicine 388(14): 1296- 1306).

[0010] As laid out above, the main challenges of the current treatment options for sexually transmitted infections (STIs) caused by Chlamydia spp. and / or Neisseria spp., such as Chlamydia trachomatis Ct) and Neisseria gonorrhoeae {Ng), are the increasing antibiotic resistance rates and the high rates of asymptomatic cases.

[0011] Accordingly, there is still a need for improved means and methods for the treatment and / or prevention of sexually transmitted infection (STI) caused by Chlamydia spp. and / or Neisseria spp..

[0012] The above technical problem is solved by the provisions of the embodiments as characterized in the claims and as described herein below.

[0013] Specifically, the present invention provides pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use in treatment and / or prevention of a sexually transmitted infection (STI) caused by Gramnegative bacteria selected from the group of Chlamydia spp. and Neisseria spp..

[0014] The present invention can, in particular, be effectively used for both pre- and post-exposure prophylaxis (PrEP / PEP) of STIs caused by Chlamydia spp. and / or Neisseria spp. Especially asymptomatic cases pose a significant risk, as infected individuals may not realize they have an STI and can still spread the infection to others or experience severe long-term sequelae. The present invention is particularly relevant for individuals who are at high risk of sexually transmitted infections (STIs), such as (but not limited to) sex workers, and / or individuals that tend to engage or engage in sexual activities, including high risk sexual encounters or sexual activities with unknown persons and / or multiple partners.

[0015] Accordingly, one of the aspects of the present invention is the prophylactic (i.e., preventive) use of pentamidine or an analogue of pentamidine in STI management. This is for example illustrated in the appended examples wherein it is documented that, for example, formation of Ct inclusions and Ct replication are significantly impaired by pentamidine and / or analogues thereof. Even after removal of pentamidine from exposed tissues or cells the inhibitory effect persists; see also discussion herein below and in the appended examples.

[0016] A further gist of the present invention is that pentamidine and / or an analogue of pentamidine can conveniently be employed by the individual to be treated. As also documented in the appended examples, it could convincingly be shown by the inventors that in in vitro and in vivo experiments (topical) administration of pentamidine can prevent infection with STI causing agents like Ct and Ng with high efficacy; see also discussion herein below and in the appended examples. Accordingly, topical application is one of the preferred administration modes of the present invention.

[0017] The present invention also provides an agent, a composition, and / or a pharmaceutical composition for use in treatment and / or prevention of a STI, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the agent, the composition, and / or the pharmaceutical composition comprise at least pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these.

[0018] Furthermore, the present invention relates to a method of treatment and / or prevention of a sexually transmitted infection (STI) in a subject in need of such treatment and / or prevention, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. Said method comprising administering to the subject an effective amount of pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these.

[0019] The present invention further provides a method of treatment and / or prevention of a sexually transmitted infection (STI) in a subject in need thereof, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., comprising the steps of (i) providing a composition comprising at least pentamidine (or an analogue thereof) and (ii) administering said composition of (i) to the vagina, rectum, eye, pharynx, or onto a primary sex organ of the subject. As documented in the appended examples provided herein, the inventors have surprisingly found that pentamidine (or an analogue thereof) is effective in the treatment and / or prevention of STI caused by Gram-negative bacteria selected from the group of Chlamydia ssp. or Neisseria ssp.. In particular, the present invention relates to pentamidine (or an analogue thereof) for the use in treating and / or preventing of STI, preferably caused by Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng).

[0020] Upon screening an extensive set of compounds (more than 2,000), pentamidine was the only candidate compound that significantly reduced Ct burden comparable to antibiotic treatment upon systemic and topical treatment; see illustratively in appended Figures 5B and 50. The minimal inhibitory or minimal bactericidal concentrations of pentamidine against Klebsiella pneumoniae and Escherichia coii was found to be between 2000 pM and 250 pM (Stokes, IM. et al. (2017), Nat. Microbiol. 2(17028): 1-8; Cebrero- Cangueiro, T. et al. (2018), Front. Cell. Infect. Microbiol. 8(363):l-7). However, the inventors unexpectedly found that already pentamidine at concentrations as low as between about 1 to about 50 pM modulates the host metabolism and thereby inhibits the growth of Ct and Ng, see illustratively in appended Figures 2B, 4B, or 11B. This unexpected finding makes pentamidine (or an analogue thereof) an ideal agent for pre- or post-exposure prophylaxis of STIs.

[0021] Chlamydial infections are often asymptomatic in both women and men. Asymptomatic gonorrhoeae has also been recognized for decades to occur in both women and men. Therefore, the treatment and, in particular, the prevention of infection with Chlamydia spp. and Neisseria spp. is of need. With pentamidine (or an analogue thereof) the present invention provides novel means and methods for the treatment and, in particular, the prevention of an STI, in particular of an infection both by Chlamydia spp. and Neisseria spp.. Its ease of administration, in particular, by topical and thereby highly convenient (self-)administration such as by cream, ointment, lubricant, or other means, coupled with low effective concentrations, make the present invention a convenient and effective solution for the management of STIs. It might be advantageous and is feasible that concentrations of pentamidine (or analogs thereof, as defined herein) higher than between about 1 to about 50 pM are to be used in accordance with the present invention. It is one of the gists of the present invention that pentamidine (or analogs thereof, as defined herein) is used in the herein provided preventions and treatments of STIs, since pentamidine (or analogs thereof, as defined herein) is shown herein to have (if at all) minor negative side effects, in particular in low concentrations as shown and proposed herein. The term "low concentration" means, in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof to be used in vitro and / or in vivo. It is evident for the skilled person that also higher concentrations than between about 1 to about 50 pM can be employed without deviating from the gist of the present invention. However, it has been shown herein and is illustrated in the non-limiting appended examples that pentamidine, as well as its analogues, can successfully be employed in the treatment and / or prevention, preferably also in prophylactic uses, of STIs, like Cfand Ng infections. The use of the present invention can increase treatment adherence, reduce transmission rates, and promote more effective disease management. Therefore, the present invention is also of particular use for individuals who are at risk or high risk of sexually transmitted infections (STIs), such as sex workers.

[0022] Pentamidine is proposed in the art for the treatment of protozoan diseases like leishmaniasis, trypanosomiasis and to prevent pneumocystis pneumonia in immune compromised people (Hafiz, S., and Kyriakopoulos, C. (2023), Pentamidine (StatPearls Publishing)). It is usually administered intravenously or by inhalation in the case of pneumocystis pneumonia prophylaxis every few weeks. Accordingly, resistance is almost exclusively known in Protozoans (Basseli, M. et al. (1997), Parasitol Res, 83: 413^418; Bray, P.G. et al. (2003), Trends Parasitol, 19(5):232-9; Capela, R. et al. (2019), Int. J. Mol. Sci., 20(5748): 1-30). This means that no relevant resistances have developed in bacteria causing STIs.

[0023] As will be further detailed herein below, one aspect of the present invention is that it was surprisingly found that, although the (exact) mechanism of action remains unknown, pentamidine inhibits infection of bacteria like Chlamydia spp. and / or Neisseria spp. although it does not have a direct impact on Ct. Pentamidine is also not being bactericidal for the commensal microbiome. However, pentamidine reduces the metabolic activity of host cells to a degree that impairs the pathogen already at the development of inclusions, see illustratively in appended Figure 7. This indirect effect is particularly advantageous for the present invention, as resistances against pentamidine (or an analogue) are not expected to develop due to reduced mutation pressure. Because pentamidine is effective via the host cell and not directly on the pathogen, pentamidine (or an analogue thereof) can also be used against pathogen strains that have developed resistance against other antibacterial agents.

[0024] As documented in the appended non-limiting examples, bacteria like Chlamydia spp. and / or Neisseria spp. are used herein as exemplified bacteria in order to support the gist of the present invention. As in particular documented herein Chlamydia spp., e.g., Ct, is taken up into pentamidine treated cells and early steps of inclusion formation occurs. In context of the present invention, it was surprisingly found that replication of Ct is significantly impaired by pentamidine and even after washout of pentamidine, the inhibitory effect persists. As further illustrated in the appended non-limiting Example 6, pentamidine permanently inhibits replication of Ct at an early timepoint, rather than merely suppressing bacterial translation. It has been surprisingly shown that the unexpected inhibitory effect on bacterial replication observed in pentamidinetreated cells is likely due to metabolic changes within the host cells (see appended non-limiting Example 7). In other words, pentamidine (or analogues thereof) unexpectantly acts indirectly via the host cell / host tissue rather than directly onto the pathogen (see illustratively in appended Figure 7J). As used herein, "host cell(s)" refers to individual living cells within an organism, that may be invaded by pathogens. Host tissue refers to the organized collection of (host) cells within an organism that can be colonized, invaded, or otherwise affected by foreign organisms, such as pathogens, during infection. As used herein, "host tissue(s)" may be composed of diverse cell types whose composition and function may change dynamically in response to infection or environmental stimuli. Host tissues in context of the present invention are not particularly limited but may include bodily surfaces, such as, but not-limited to, epidermis and mucous(al) membranes.

[0025] The indirect inhibition of pathogen infection via host tissue / cell modulation as provided herein for pentamidine and / or its analogue enables early and effective suppression of infection. This stands in clear contrast to previously described treatment methods which are based on directly targeting bacterial pathogens. WO 2005 / 025565 discloses combinatorial treatments with the minor groove binder (MGB) Distamycin A, and an analogue of Distamycin A (namely Suradista, defined in WO 2005 / 025565 as an antiviral agent) for use against various pathogens including viruses, fungus, and protozoans. However, such a combinatorial treatment approach relies on the direct inhibition of the pathogen's translation and / or transcription machinery. WO 2023 / 077235 proposes the use of specific pentamidine analogues to specifically target the outer membrane (OM) of in particular Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa. The disruption of the OM is intended to primarily enhance the susceptibility of bacteria, particularly antibiotic-resistant strains, to antibacterial agents.

[0026] Neither WO 2005 / 025565 nor WO 2023 / 07723, point to the direct protective effect of pentamidine or its analogues on host cells and host tissue as illustrated in the herein appended examples. This surprising finding enables a novel and distinctive use of pentamidine (and / or an analogue thereof) in the treatment of STIs, in particular in the prevention of infection. Accordingly, the present invention provides for use of pentamidine (and / or an analogue thereof) as a protective agent to inhibit STI -causing bacteria (in particular Chlamydia spp. and / or Neisseria spp.) from infecting the individuals' or subject's cells or tissues. These findings prompted the present inventors to propose the successful use of pentamidine (or an analogue thereof), in particular also in medium- to long-term prevention of an infection with a sexually transmitted disease (like Chlamydia spp. and / or Neisseria spp. infections). This renders pentamidine (or an analogue thereof) in particular useful to be administered on tissues / cells before sexual encounters / intercourse. As also documented in the appended examples, pentamidine (or an analogue thereof) has, even up to 48 hours after administration, this particular protective effect of arresting the pathogens in the non-pathogenic inclusion phase. In other words, in context of the present invention, it was also surprisingly found that the protective effect of pentamidine (and / or its analogue) can be achieved immediately when the drug is applied to corresponding tissues, like mucosal membranes or epidermal parts of the anogenital tract, like vagina and rectum, but also other parts of the body like the mouth and eyes. Yet, it is also surprising that the drug shows its protective effects over mid- and long-term periods before the potential exposure to STI- causing bacteria, in particular Chlamydia spp. and Neisseria spp.. Such a mid- to long-term prevention may comprise the administration of the drug even at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, or at least about 48 hours before a possible contact or infection with the respective Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. As shown in the appended non-limiting examples, the protective effect of pentamidine, is maintained even when administration was up to 48 hours prior to an infection, even at 37°C (i.e., body temperature). As explained above, the present invention provides a treatment strategy based on a mechanism divergent from those described in the art. Accordingly, the present invention provides for a truly novel and inventive med ica l / cli n ica I intervention of STI. This is the case because pentamidine (or an analogue thereof) exegete its protective effect (but also a treatment effect) over a rather long time period and can therefore be applied to the host cells (e.g., in mucosal membranes or epidermal parts) before these sides of potential infection are exposed to the pathogen, e.g. Chlamydia spp. and Neisseria spp.. Therefore, and in accordance with the present invention, by targeting the host cell's metabolic pathways essential for pathogen infection and replication with pentamidine (and / or an analogue thereof), inter alia, protects these host cell from infection and enables selective inhibition of STI-causing Gram-negative bacteria, such as Chlamydia spp. and Neisseria spp..

[0027] It has further been demonstrated by the inventors that pentamidine is stable and retains its effective properties at body temperature (about 37 °C) for at least 48 hours (see illustratively in appended Figure 17). Furthermore, the administration of pentamidine prior to infection (i.e., in a preventive / prophylactic manner) and subsequent removal (e.g., washed out) as early as 24 hours post-infection already results in significant impairment of Ct growth (see illustratively in appended Figure 7G-7I). It has furthermore been demonstrated that formulations in accordance with the present invention are stable under storage and application conditions. The data presented in the appended non-limiting examples demonstrates that pentamidine (or an analogue of pentamidine) may, inter alia, be advantageously administered to the relevant body cavities, such as the anogenital tract, mouth, and eyes. Examples of the form of administration are provided herein. Due to these surprising advantages of maintained stability and effectiveness at body temperature and the fact that pentamidine (or an analogue of pentamidine) can easily be applied to, for example, the anogenital tract but also other body parts and cavities it is envisaged that pentamidine (or an analogue of pentamidine) or an agent, composition, and / or pharmaceutical composition comprising at least pentamidine (or an analogue of pentamidine) may be conveniently selfadministered by a subject in need thereof.

[0028] Since pentamidine has been shown herein to be effective indirectly via the host cell, it may be advantageously employed in the prevention (i.e. prophylaxis) of STIs caused by, for example, Chlamydia spp. and / or Neisseria spp., while also retaining efficacy in the treatment of established infection with these pathogens. Accordingly, pentamidine (or an analogue thereof), in particular when applied topical (i.e., local) in low effective concentrations as described herein (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof), allows for efficient and selective targeting, and thus treatment and / or especially prevention, of STIs caused by Gramnegative bacteria, such as for example Chlamydia spp. and / or Neisseria spp., without having bactericidal effects on the commensal microbiome. Pentamidine (or an analogue thereof) can be used to supplement or replace existing treatment options for bacterial STIs. Replacement of current treatment options is particularly useful when the individual being treated is unable to tolerate the respective treatment, such as in cases of severe side effects or allergic reactions. For instance, a person who is allergic to tetracyclines would be incompatible with doxy PrEP / PEP.

[0029] Accordingly, the means and methods provided herein in particular in context of the treatment and / or prevention as described herein of an STI like a Chlamydia spp. and / or Neisseria spp. infection may lead to either additional treatment or prevention options, supplementation of current treatment options, as well as, to replacement of treatment / prevention options. For example, it is envisaged that current antibiotic treatments, like but not limiting to doxy PrEP / PEP, may be replaced within the herein described PrEP or PEP employing pentamidine (or an analogue thereof). It is therefore envisaged that subjects who currently receive or have received such an antibiotic treatment, in particular doxy PrEP / PEP and / or HIV-PrEP / PEP, may benefit from the herein described treatment / prevention options comprising pentamidine and or its (functional) analogues.

[0030] Accordingly, the present invention provides pentamidine (or an analogue thereof) for use in treatment and / or prevention of a sexually transmitted infection (STI) caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. In particular, the present invention is also useful for individuals who are undergoing (or are prone to and / or willing to undergo) PrEP and / or PEP treatments and may even replace an existing treatment option. Moreover, the means and methods of the present invention for use in treatment and / or prevention of the respective STI as described herein are easy to administer and have no observed side effects, unlike doxycycline-PrEP and / or -PEP. This may be, inter alia, relevant for individuals with a high risk of contracting STI, for example sex worker (female and male) or individuals with incompatibilities against the respective treatment option, for example doxy allergy.

[0031] It is of note that the present invention is not limited to the medical and / or therapeutic uses described herein. Encompassed is also the non-medical prophylactic use of pentamidine and / or (an) functional analogue(s) thereof; corresponding non-medical uses may comprise the use in creams, lotions, foams (e.g., lathers), lubricants, etc., to be employed before and / or during intended sexual intercourse. Envisaged are also further ways of administration like suppositories (vaginal or anal) or other administration means, in particular to the genital regions but also, if desired, to the eye, the nose, or the oral region, including the mouth (including lips and tongue), pharynx, and oesophagus. In context of the present invention, non- medical (prophylactic) means and methods refer to any action, method, or measure for the purpose of preventing, reducing the risk of, or controlling the transmission of infections with Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., without exerting a therapeutic or curative effect on an established disease in the subject. Like for medical uses, the administration of the herein described non-medical compositions, comprising at least pentamidine and / or (an) analogue(s) thereof, may comprise a non-medical use of an ointment, gel, clyster, pessary, vaginal ring, mechanical contraceptive, such as diaphragm or condom, liquid, irrigation, eye lotion, eye gel, eye drops, lozenge, troche, chewing gum, spray, mouthwash, gargle, or a powder for resuspension in liquid, provided these do not require a medical professional for their implementation and do not exert a direct therapeutic effect on the body.

[0032] As has been show and is discussed herein, pre-administered pentamidine maintains its protective effect against STIs, in particular against Ct, even when removed after 24 hours after infection for at least about 48 hours. As the inventors have also credibly shown in the appended non-limiting Example 11, pentamidine is stable and maintains its protective effect for at least about 48 hours at 37 °C. Accordingly, pentamidine (or an analogue thereof) may be advantageously employed in the prevention (i.e. prophylaxis) of infection with Chlamydia spp. and Neisseria spp., while also being effective in the treatment of said infection with Chlamydia spp. and Neisseria spp.. Thus, in context of the present invention, pentamidine (or an analogue thereof) may be prophylactically administered (e.g., as a PrEP treatment) at least about 0 hours, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, or at least about 48 hours before a possible contact or infection with the respective Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. Preferably, the possible contact or infection may be with Ct and / or Ng.

[0033] As indicated herein above, in one aspect, the present invention provides pentamidine (or an analogue thereof) for use in treatment and / or prevention of a STI, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. In context of the present invention, the STI may, for example, be caused by Chlamydia trachomatis Ct or Neisseria gonorrhoeae {Ng}.

[0034] In context of the present invention, the term "prevention" (and grammatical variations thereof such as "prevent" or "preventing") relates to obtaining a prophylactic benefit in a subject. With respect to achieving a prophylactic benefit, the object is to delay or prevent the symptoms or underlying cause (i.e., bacterial infection) associated with STI caused by a respective Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. Preferably, the object is to prevent bacterial infection without causing physiologically negative consequences for the subject. Desirable effects of prevention as described herein include but are not limited to, prophylaxis, preventing occurrence or recurrence of disease or symptoms associated with disease. Specific preventive measures, in context of the present invention may include, but are not limited to, pre-exposure prophylaxis (PrEP) and / or post-exposure prophylaxis (PEP).

[0035] Pre-exposure prophylaxis (PrEP) refers to the administration of an active compound or a combination of active compounds to a subject in need thereof prior to potential exposure to, for example, an STI, with the aim of preventing infection. In context of the present invention, PrEP comprises the prophylactic use of pentamidine (or an analogue thereof) by a subject at risk, administered before sexual contact or other potential exposure events (e.g., directly before or within about 1 hour, about 6 hours, about 12 hours, about 18 hour, about 24 hours, or about 48 hours before potential exposure), to reduce the likelihood of infection with Chlamydia spp. and / or Neisseria spp.. PrEP is primarily intended for use by individuals at risk of acquiring an STI, such as through (unprotected) sexual contact with a person of unknown, suspected, or confirmed exposure or profile of STIs or other situations where there is a risk of acquiring an STL

[0036] Post-exposure prophylaxis (PEP) refers to the administration of an active compound or a combination of active compounds to a subject after a potential exposure to, for example, an STI but before the onset of symptoms or confirmed infection, with the aim of preventing the establishment of infection. In context of the present invention, PEP comprises the timely (e.g., directly after or within about 1 hour, about 6 hours, about 12 hours, about 18 hour, or about 24 hours, or about 48 hours after exposure) administration of pentamidine (or an analogue thereof) following suspected or confirmed exposure to Chlamydia spp. and / or Neisseria spp.. PEP is intended for (emergency) use following events such as (unprotected) sexual contact with a person of unknown, suspected, or confirmed exposure or profile of STIs or other situations where there is a risk of acquiring an STI.

[0037] In context of the present invention, the term "treatment" (and grammatical variations thereof such as "treat" or "treating") relates to obtaining a therapeutic benefit in a subject. With respect to achieving a therapeutic benefit, the object is to eliminate, lessen, decrease the severity of, ameliorate, or slow the progression of the symptoms or underlying cause (i.e., bacterial infection) associated with STI caused by a respective Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. Desirable effects of treatment include but are not limited to, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, improved prognosis, and cure.

[0038] In context of the present invention, the subject may be any mammal susceptible to infection by Gramnegative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. In particular, the subject may be a mammal such as a human, pet (e.g., dog or cat), farm animal (e.g., cow, horse, pig), zoo animal, etc.. Preferably herein, the subject is a human. The state of health of the subject is not particularly limited, and the subject may or may not have a prior infection with the respective Gram-negative bacteria selected from the group of Chlamydia ssp. or Neisseria ssp..

[0039] Besides further advantages, the present invention provides for a treatment and / or prevention option that may replace or supplement current antibacterial STI treatment options, allowing for a reduced usage of available antibiotics. As pentamidine (or analogues thereof) is generally used in the treatment of protozoan disease and not in the treatment of bacterial STIs, there are no resistances known for bacteria causing STIs. In contrast, due to its binding to lipopolysaccharide, pentamidine is able to perturb the outer membrane of Gram-negative bacteria and, thus, possess potential as adjuvants for conventional antibiotics (e.g., Stokes, IM. et al. (2017), Nat. Microbiol. 2(17028): 1-8). Furthermore, as the inventors have shown that pentamidine does not directly act on the pathogen strain (e.g., Cf) but via the host cell, by using pentamidine (or analogues thereof) in the treatment and / or prevention of STIs further resistances in the pathogen strains are less likely to occur. Furthermore, by inhibiting the formation of Ct EBs and the formation of Ct inclusion bodies pentamidine (or analogues thereof) prevents the spreading and progression of the infection within the host's body.

[0040] Accordingly, in some embodiments of the present invention, pentamidine (or an analogue thereof) inhibits growth of STI-causing Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., in particular STI caused by C?or Ng.

[0041] In context of the present invention, "pentamidine" may also be referred herein as "Pent" or"4,4'-[Pentane- l,5-diylbis(oxy)]bis-[benzenecarboximidamide]" or "l,5-bis(4-amidinophenoxy)pentane". Pentamidine is represented by Formula I, below. Pentamidine may have a molecular weight of about 340 g / mol; see also further calculations below. The term "pentamidine" also includes, but is not limited to, stereoisomers of pentamidine and / or pharmaceutically acceptable salt of pentamidine and of the respective stereoisomers of pentamidine. An example of a pentamidine salt may be pentamidine isethionate salt. Pentamidine isethionate salt may have a molecular weight of about 592 g / mol; see also further calculations below.

[0042] The present invention is furthermore not particularly limited to pentamidine, but also relates to any functional analogue of pentamidine (also referred to herein as "analogue of pentamidine" or "pentamidine analogue"). Examples of functional pentamidine analogues are known in the art (see, e.g. WO 2023 / 077235). In particular, in context of the present invention, functional pentamidine analogues may be selected from the group of 4,4'-(((phenylazanediyl)bis(ethane-2,l-diyl))bis(oxy))dibenzimidamide, 3,3'-(heptane-l,7-diyl)dibenzimidamide,

[0043] 3-(7-(3-(pyrrolidine-3-yl)phenyl)heptyl)benzimidamide, 4,4'-(2,2'-((lR,3S)-cyclohexane-l,3-diyl)bis(ethane-2,l-diyl))dibenzimidamide, 4,4'-(2,2'-((lR,3R)-cyclohexane-l,3-diyl)bis(ethane-2,l-diyl))dibenzimidamide,

[0044] 4-(2-((lS,3R)-3-(4-(pyrrolidine-3-yl)phenethyl)cyclohexyl)ethyl)benzimidamide, 3,3'-(((lR,3S)-cyclohexane-l,3-diyl)bis(ethane-2,l-diyl))dibenzimidamide, 7,7'-(heptane-l,7-diyl)bis(isoquinolin-l-amine), and 4-(7-(4-(pyrrolidine-3-yl)phenyl)heptyl)benzimidamide. The term" analogue of pentamidine" also includes stereoisomers of pentamidine analogues and pharmaceutically acceptable salt of pentamidine analogues and of the respective stereoisomers of pentamidine analogues. In the context of the present invention, a functional analogue is an analogue which has at least 50%, 60%, 70%, 80%, 90%, 100%, or even more activity in comparison to pentamidine in means and methods like in vitro and / or in wk? assays provided herein. Testing for the activity of functional analogues is simple and can be conducted as per the means and methods provided herein and as illustrated in the appended non- limiting examples.

[0045] In the context of the present invention, the term "pentamidine (or an analogue thereof)" comprises pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any of any one these. Non-limiting suitable analogues of pentamidine are, inter alia, described herein above. Accordingly, the present invention provides for medical and non-medical uses of pentamidine, (an) analogue(s) of pentamidine, and / or (a) pharmaceutically acceptable salt(s) of said pentamidine or of said analogue(s) of pentamidine in treatment but also prevention, including non-medical preventions, of STIs, in particular of C?and Ng infections.

[0046] The transformation back from the non-infectious reticulate bodies (RBs) to infectious elementary bodies (EBs) represents a crucial stage in the cycle of chlamydial infection. As illustrated in the appended nonlimiting Example 6, pentamidine (or an analogue thereof) inhibits the formation of Ct EBs. Therefore, the present invention is particularly useful for the prevention of further spreading of the infection within an infected host. The inhibition of EB formation makes pentamidine (or an analogue thereof) suitable for the prevention and especially ideal for treatment of STI caused by Ct, as the STI cannot proliferate in the host. Accordingly, in some embodiments of the present invention, pentamidine (or an analogue thereof) may inhibit formation of Ct elementary bodies.

[0047] Additionally, the inventors have found, that pentamidine also inhibits formation of Ct inclusions, as illustrated in the appended non-limiting Example 6. Therefore, the present invention is particularly useful for the prevention of the progression of the infection within an infected host cell into the replicative stage of the Ct life cycle. The inhibition of inclusion formation makes pentamidine (or an analogue thereof) suitable for the treatment and especially ideal for the prevention of STI caused by Ct, as the associated STI cannot become manifest in the host. This further corroborates, that pentamidine (or an analogue thereof) is particularly useful in treatment and / or prevention of a STI caused by Gram-negative bacteria, in particular, selected from the group of Chlamydia spp., preferably, in treatment and / or prevention of a STI caused by Ct. Accordingly, in some embodiments of the present invention, pentamidine (or an analogue thereof) may inhibit formation of Ct inclusions.

[0048] It has been found that pentamidine shows rapid and long-lasting therapeutic and prophylactic effects, without any macroscopic or histopathologic side effects, when administered topically. The absence of macroscopic or histopathologic side effects makes it clear that the present invention allows the repeated use and / or use for extended periods of time. The repeated use and / or use for extended periods of time without side effects further enable the effective use of pentamidine (or an analogue thereof) in PrEP and / or PEP treatment, in particular for individuals that are particularly at risk of STI infection. As described herein, pentamidine (or an analogue thereof) may be used to supplement or replace the treatment doxy-PrEP and / or -PEP, wherein supplementation would lead, for example, to reduced side effects. The replacement of a PrEP and / or PEP treatment option may be especially desired for individuals that have incompatibilities with such medication, for example, individuals that have developed a doxycycline allergy.

[0049] Pentamidine (or an analogue thereof) has a long-lasting bactericidal effect on Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., in particular, on C?and Ng see illustratively in the appended Figures 7G to 71 and 11A to 11C). However, pentamidine (or an analogue thereof) is much less cumbersome for the natural bacterial flora and / or the microbiome. In fact, the inventors have shown, as illustrated in non-limiting Example 8, that pentamidine, even after 24 hours incubation at concentrations up to 50 pM, at best has bacteriostatic effects on Lactobacillus acidophilus (L. acidophilus but shows no bactericidal effects. Thus, although the mechanism of action is not yet known, pentamidine exhibits specific bactericidal and protective effects against Ct and Ng without being bactericidal for the microbiome. Consequently, in some embodiments of the present invention, pentamidine (or an analogue thereof) has no bactericidal effect on natural bacterial flora and / or the microbiome. In context of the present invention, the location of the natural bacterial flora and / or the microbiome is not particularly limited and relates to the intended region of administration. Examples of the location of the natural bacterial flora and / or the microbiome may include but are not limited to the skin, mouth (including lips and tongue), nose, eyes, pharynx, oesophagus, cervix, vagina, vulva, penis, anus, rectum, and the tissues directly adjacent to these regions (i.e., tissues in direct anatomical contact with the exemplary listed sites). In context of the present invention, the organism of the natural bacterial flora and / or the microbiome is not particularly limited, and examples thereof include Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus delbrueckii, and Lactobacillus case! In further embodiments of the present invention, pentamidine (or an analogue thereof) has no bactericidal effect on the natural genital flora and / or the natural skin flora, in particular the natural vaginal flora. In some further embodiments of the present invention, pentamidine (or an analogue thereof) has no bactericidal effect on bacteria selected from the group of Lactobacillus spp., and / or Bifidobaterium spp., preferably Lactobacillus acidophilus.

[0050] In the context of the present invention, bacteriostatic effect refers to the inhibition or reduction of bacterial growth while in contact with an agent, but regrowth of the bacteria is possible once the agent is removed. In context of the present invention, bactericidal effect refers to the inhibition of bacterial growth while in contact with an agent and / or the inhibition of regrowth of the bacteria after removal of the agent. In particular, the combination of both effects is preferred in context of the present invention, i.e., the inhibition of bacterial growth while in contact with an agent and the inhibition of regrowth of the bacteria after removal of the agent in vivo(\.e., in or on the subject), but also in vitro. In context of the present invention, having no bactericidal effect refers to the absence of inhibition of bacterial growth while in contact with said agent and / or the absence of inhibition of regrowth after removal of said agent, i.e., an agent having no bactericidal effect may allow bacterial growth while in contact with said agent and / or the regrowth after removal of said agent. The person skilled in the art is aware that bacteriostatic and bactericidal effects can be measured, for example in vitro or ex vivo, illustrative examples are provided herein in the appended non-limiting examples.

[0051] Methods for testing the bacteriostatic and / or bactericidal effect of an agent are known in the art. For example, an in vitro or ex vivo method for measuring the bacteriostatic effect of pentamidine (or an analogue thereof) may be measuring the growth rate of a strain with and without the presence of pentamidine (or an analogue thereof). An exemplary method may comprise the following steps: (a.i) providing a strain on which the effect of pentamidine (or an analogue thereof) is to be tested,

[0052] (b.i) preparing at least two samples of said strain of (a), preferably originating from a single colony of said strain of (a), at least one of said samples will be a control sample (i.e., untreated)

[0053] (c.i) adding pentamidine (or an analogue thereof) to the samples that are not the control sample(s), preferably in various concentrations,

[0054] (d.i) measuring the optical density of all samples after a reasonable incubation to allow for bacterial growth (at least several rounds of replication under growth conditions),

[0055] (e.i) calculating growth rates, comparing growth of the treated samples with the control sample (e.g., as % growth compared to control)

[0056] (h.i) determining bacteriostatic effect of pentamidine (or an analogue thereof) on the tested strain.

[0057] The bacteriostatic effect is given when bacterial growth of the treated sample is inhibited or reduced compared to the control (untreated) sample.

[0058] A specific example of the above method for measuring the bacteriostatic effect of pentamidine (or an analogue thereof) may comprise the following steps

[0059] (a.ii) providing a strain (for example Lactobacillus acidophilus, in particular strain ATCC 4356) on which the effect of pentamidine (or an analogue thereof) is to be tested,

[0060] (b.ii) preparing at least two samples of said strain of (a), preferably originating from a single colony of said strain of (a), at least one of said samples will be a control sample (i.e., untreated), for example, in 0.9 % NaCI at a McFarland standard of 0.5,

[0061] (c.ii) adding pentamidine (or an analogue thereof) to the samples that are not the control sample(s), preferably in various concentrations (for example in 1:2 dilutions from 50 pM),

[0062] (d.ii) measuring the optical density (for example at 600 nm wavelength) of all samples after, for example, 24 hours incubation,

[0063] (e.ii) calculating growth rates, comparing growth of the treated samples with the control sample (e.g., as % growth compared to control)

[0064] (h.ii) determining bacteriostatic effect (for example % growth rate less than 100 %) of pentamidine (or an analogue thereof) on the tested strain. Wherein the bacteriostatic effect is determined as described above.

[0065] For example, an in vitro or ex vivo method for measuring the bactericidal effect of pentamidine (or an analogue thereof) may be measuring the growth rate of a strain with and without the presence of pentamidine (or an analogue thereof) and determining regrowth of the strain after treatment. An exemplary method may comprise the above steps (a.i) to (h.i) and may comprise the following steps: (g.i) spreading the liquid cultures of step (d.i) on fresh growth medium (for example agar) plates, (h.i) incubating plates for a reasonable incubation to allow for bacterial growth (at least several rounds of replication under growth conditions),

[0066] (i.i) evaluating regrowth (e.g., as CFU / ml).

[0067] The bactericidal effect is given when bacterial growth of the treated sample is inhibited or reduced compared to the control (untreated) sample and / or the regrowth of the bacteria is inhibited.

[0068] Optionally, before step (g.i) a step comprising spinning down the culture, removing supernatant, adding the same volume (as the removed supernatant volume) of fresh medium, and resuspending the culture, may be added.

[0069] A specific example of the above method for measuring the bactericidal effect of pentamidine (or an analogue thereof) may comprise the above steps (a.ii) to (h.ii) and may comprise the following steps (g.ii) spreading 5pl of the liquid cultures of step (d.ii) on fresh agar plates, (h.ii) incubating plates for 48 hours,

[0070] (i.ii) evaluating regrowth (e.g., as CFU / ml).

[0071] Wherein the bactericidal effect is determined as described above.

[0072] It is evident for the skilled person that tests and measurements may be subject to slight variations / inconsistencies. In context of the present invention the term "inhibition of regrowth" encompasses bacterial regrowth of about 1%, about 2%, about 3%, about 4%, about 5%, and up to about 10%, these variations may be for example due to potential (for example, spontaneous) mutations occurring within the tested strains and leading, for example, to certain resistances. This should be a rare event.

[0073] The present invention provides a treatment and / or prevention option for STIs that does not have a bactericidal effect on the natural flora and / or microbiome, in particular, on the natural genital flora. Therefore, the present invention is especially useful as or for prophylaxis. The use of pentamidine (or an analogue thereof), according to the present invention, is particularly beneficial for individuals that are at high risk of STI, for example, individuals that are members of marginalized communities (e.g., people of color, indigenous / first nation people, members of the LGBTQ+ community, homeless people, prisoners), individuals that engage in high-risk sexual behavior, (e.g., individuals that practice unprotected sex, in particular those individuals that practice unprotected sex with a person unknown to them, individuals with changing and / or multiple sexual partners, in particular sex workers (female and male) or a person having sex with a sex worker, individuals in the chemsex scene), victims of sexual assault, individuals who experience application errors when using mechanical contraceptives (e.g., condoms), or individuals that have a reduced / weakened immune system (e.g., individuals with autoimmune diseases, individuals having or having had cancer, individuals that are undergoing or having undergone chemotherapy, individuals that have another STI (e.g., co-infection), or individuals that have human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS)). The use of pentamidine (or an analogue thereof), according to the present invention, may further be beneficial for individuals that have incompatibility (e.g., allergic reaction) to an existing treatment option (e.g., antibiotics). An example of an incompatibility of an individual to an existing treatment would be a tetracycline or penicillin allergy.

[0074] In context of the present invention, the administration frequency is not particularly limited, but an example thereof is once or multiple times. Examples for multiple administrations may be two, three, four, or five times. Administration frequency can also naturally depend on the form of administration. The administration frequency can be, for example, three times a day, twice a day, once a day, once in two days, once in three days, once in four days, once in five days, once in six days, once in a week, once in ten days, or once in two weeks. The administration frequency can also include dosing holidays, for example, from about 1 day to about 7 days between administration. However, as illustrated in the appended examples a single administration may be enough to treat and / or prevent infection, see illustratively in appended Figures 7 and 11.

[0075] During systemic treatment, pentamidine can have side effects like ventricular arrhythmia, hypotension, nephrotoxicity, or hepatic dysfunction (Hafiz, S. and Kyriakopoulos, C. (2023); Pentamidine (StatPearls Publishing)). As illustrated in the appended non-limiting examples, pentamidine (or an analogue thereof), when administered topically (i.e., locally), shows no macroscopic side effects and no histopathological alterations that indicate inflammation or tissue destruction neither at the site of administration (uterus) nor at distant organs such as the liver or kidney, see illustratively in appended Figures 5D and 5E. Even in the mouse model with transcervical inoculation of the pathogen (see, for example, appended non-limiting Example 4) pentamidine is shown to be protective against the respective pathogen. Accordingly, in some embodiments of the present invention, pentamidine (or an analogue thereof) is intended to be administered topically, i.e., locally. In particular, pentamidine (or an analogue thereof) for the use in treatment and / or prevention of a sexually transmitted infection (STI) as described herein, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., may be administered topically, i.e., locally. Thus, pentamidine (or an analogue thereof) is, preferably, formulated such that it can be administered topically, i.e., locally. As mentioned herein, one of the advantageous aspects of the present invention is that pentamidine (or an analogue of pentamidine) can conveniently be self-employed by the individual to be treated. The administration may be assisted, for example, by the use of an applicator. The applicator may be comprised in a kit that also further comprises at least pentamidine (or an analogue thereof). Topical administration minimizes systemic side effects, minimizes drug interaction and contraindications, and preserves the integrity of the natural flora and / or microbiome. This makes the present invention especially useful for the prolonged and / or repeated application, for example, onto organs and / or tissues with high risk of infection.

[0076] An infection, in context of the present invention, is an infection of a bodily surface. Bodily surfaces include but are not limited to epidermis and mucous membranes. Suitable examples of mucosal membranes include the mucosa of the mouth (including lips and tongue), nose, eyes, pharynx, oesophagus, cervix, vagina, penis, and rectum. Therefore, in context of the present invention, the region of administration is not particularly limited, and examples thereof include the epidermis and mucous membrane, such as the mouth (including lips and tongue), nose, eyes, pharynx, oesophagus, cervix, vagina, vulva, penis, anus, and rectum. Accordingly, in some embodiments of the present invention, topical administration is intravaginal, intrarectal, ocular, pharyngal, or onto a primary sex organ.

[0077] The form of administration of pentamidine (or an analogue thereof), in context of the present invention, for intravaginal administration is not particularly limited, and examples thereof include the administration by ointment, gel, cream, lubricant, foam (e.g., lather), clyster, suppository, pessary, diaphragm, vaginal ring, or irrigation. It is envisaged, that the intravaginal administration also reaches the cervix and thus treatment and / or prevention of STI caused by a respective Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., in particular Cfand Ng, is thereby extended to the cervix.

[0078] In context of the present invention, the form of administration of pentamidine (or an analogue thereof) for intrarectal administration is not particularly limited, and examples thereof include the administration by ointment, gel, cream, foam (e.g., lather), lubricant, clyster, suppository, or irrigation.

[0079] In context of the present invention, the form of administration of pentamidine (or an analogue thereof) for ocular administration is not particularly limited, and examples thereof include the administration by eye ointment, eye gel, or eye drops.

[0080] In context of the present invention, the form of administration of pentamidine (or an analogue thereof) for pharyngal administration is not particularly limited, and examples thereof include the administration by lozenge, troche, chewing gum, spray, mouthwash, gargle, or powder for resuspension in liquid.

[0081] In context of the present invention, the form of administration of pentamidine (or an analogue thereof) for administration onto a primary sex organ is not particularly limited, and examples thereof include the administration by ointment, cream, or lubricant. In context of the present invention the ointment, cream, or lubricant may preferably be (pre-)administered to a mechanical contraceptive, such as a condom (for male and female), diaphragm, cervical cap, and contraceptive sponge. As used herein, the term "mechanical contraceptive" refers to any device primarily intended to physically block or impede the passage of sperm into the uterus, thereby preventing fertilization. This includes, but is not limited to, (male and female) condoms, diaphragms, cervical caps, and contraceptive sponges, regardless of their material composition or specific design.

[0082] As illustrated in the non-limiting appended examples, in mice, pentamidine was inoculated locally and the infection was performed transcervical (i.e., intrauterine), in accordance with the established mouse model for human Ct. Accordingly, topical treatment and / or preventative administration is feasible with, for example, creams and suppositories for application at the penis and anal region, respectively. Application to the female lower genital tract and especially the cervix may, for example, include vaginal application by cream, suppository, sprays, or foam (e.g., lather) that might allow dissemination of pentamidine to the cervix.

[0083] As illustrated in the non-limiting appended examples, the inventors have, inter alia, provided exemplary gel formulations, illustratively using poloxamer (POX) or hydroxyethyl cellulose (HEC) as gelling agents. The inventors also provided exemplary methods for characterization of such formulations (see illustratively in appended non-limiting Example 10). Accordingly, the person skilled in the art is able to adjust the formulation of the present invention to the respective requirements of the intended region of administration and / or the corresponding form of administration. The person skilled in the art is also able to find suitable alternative compounds for the respective formulation of the present invention.

[0084] In context of the present invention, the formulation is preferably selected so as to be compatible with contraceptive means. In particular, compatibility with mechanical contraceptives, such as condoms (for example, latex condoms), diaphragms, cervical caps, and contraceptive sponges, is preferred.

[0085] In context of the present invention, the inventors have found that a single administration of pentamidine is enough to treat and prevent infection, see illustratively in appended non-limiting Examples and, e.g., Figures 7 and 11. Having pentamidine (or an analogue thereof) at hand that, already after a single administration, allows treatment and / or prevention of STI caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. greatly increases the convenience and ease of administration by reducing the time and effort required for the administration. Furthermore, by providing a streamlined treatment regimen, in particular a reduced treatment duration, the availability of a single effective dose regimen also enhances treatment adherence, while ensuring the full therapeutic benefit of the medication. Timely and effective treatment and / or the prevention of infection with a single dose, furthermore, helps prevent the development of complications associated with untreated STIs, in particular, if asymptomatic.

[0086] Accordingly, in context of the present invention, pentamidine (or an analogue thereof) may inhibit growth of the respective Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. after a single treatment (i.e., administration). Preferably, pentamidine (or an analogue thereof) inhibits growth of C?and Ng after a single treatment (i.e., administration).

[0087] In context of the present invention, the administration time is not particularly limited, and pentamidine (or an analogue thereof) can be administered before and / or after a possible contact or infection with the respective Gram-negative bacteria for the purpose of treatment and / or prevention as described herein. However, in some preferred embodiments of the present invention, pentamidine (or an analogue thereof) is to be administered at least about 0 hours, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, or at least about 48 hours before (e.g., as a PrEP treatment) and / or after (e.g., as a PEP treatment) a possible contact or infection with the respective Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. In preferred embodiments of the present invention, the possible contact or infection is with Cfand Ng. In context of the present invention, 0 hours refers to administration of pentamidine (and / or an analogue thereof) immediately before and / or directly after the potential exposure to the pathogen, in particular STI-causing Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp..

[0088] As used herein, the term "about" indicates and encompasses an indicated value and a range above and below that value. For example, the term "about" may indicate the designated value ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% without substantial loss of activity or effect.

[0089] In context of the present invention, pentamidine (or an analogue thereof) is used at concentrations that are bactericidal for the respective Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. Pentamidine has already been tested against some limited non-STI related Gram-negative bacteria, such as Klebsiella pneumoniae, Escherichia coii, and Enterobacter cloacae (Stokes, IM. et al. (2017), Nat. Microbiol. 2(17028): 1-8; Cebrero-Cangueiro, T. et al. (2018), Front. Cell. Infect. Microbiol. 8(363):l-7), where the minimal inhibitory or minimal bactericidal concentrations were found to be between about 250 pM and about 2000 pM (i.e., between about 85 and about 680 pg / ml (as calculated for pentamidine) or between 147.5 and about 1180 pg / ml (as calculated for a salt like pentamidine isethionate)). The inventors, however, have surprisingly found that pentamidine may also be used in much lower concentrations (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof, i.e., at least between about 0.34 and about 17 pg / ml (as calculated for pentamidine) or at least between about 0.59 and about 29.4 pg / ml (as calculated for a salt like pentamidine isethionate)) against STI-causing Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. Advantageously, such minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of pentamidine (or an analogue thereof) against the respective Gram-negative bacteria may be at least between about 1 and about 50 pM, at least between about 1 and about 25 pM, at least between about 1 and about 10 pM, or at least between about 1 and about 5 pM. In particular, the minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of pentamidine (or an analogue thereof) for use in treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., may be at least between about 1 and about 50 pM, preferably at least between about 1 and about 25 pM, more preferably at least between about 1 and about 10 pM, even more preferably at least between about 1 and about 5 pM. As supported by the appended non-limiting examples, these concentrations do not affect cell viability and proliferation of HeLa and primary cervical epithelial cells, see illustratively in appended Figures 7K-7M and 10. The inventors have found that the exact MIC and / or MBC may vary dependent on the specific organism. For example, the preferred concentration range for the treatment and / or prevention of a Ct infection is at least about 1 to about 10 pM (i.e., at least between about 0.34 and about 3.4 pg / ml (as calculated for pentamidine) or at least between about 0.59 and about 5.9 pg / ml (as calculated for a salt like pentamidine isethionate)), more preferred is a concentration of at least about 1 to about 5 pM (i.e., at least between about 0.34 and about 1.7 pg / ml (as calculated for pentamidine) or at least between about 0.59 and about 2.95 pg / ml (as calculated for a salt like pentamidine isethionate)). The inventors have further found that a preferable concentration for the treatment and / or prevention of a Ng infection is at least between about 3 and about 25 pM (i.e., at least between about 1.02 and about 8.5 pg / ml (as calculated for pentamidine) or at least between about 1.77 and about 14.75 pg / ml (as calculated for a salt like pentamidine isethionate)). Similarly, it is further envisaged that for some analogues of pentamidine the concentration might also vary in comparison to pentamidine concentrations. Being able to effectively use pentamidine (or an analogue thereof) at low concentrations (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof) not only signifies cost-effectiveness, but also reduced risk of adverse effects (e.g., toxicity or development of resistances), thereby enhancing accessibility to treatment, particularly in resource-constrained settings where affordability and availability of medication are crucial determinants of healthcare access. Affordability and availability of medication may be especially relevant in terms of patient compliance where these factors, amongst other advantages, promotes more effective disease management and reduced transmission rates within the communities.

[0090] Without being bound by theory, examples of routinely used concentration and total dose of pentamidine (or an analogue thereof) may be higher than the above stated minimal concentration. In particular, said higher concentrations and total doses may be used as no side effects have been observed when administration was topically; even when half of the highest still tolerable systemic dose (at about 45.2 mM) was applied in vivo to mice, see appended non-limiting examples.

[0091] In context of the present invention, examples of routinely used concentrations and amounts may include the administration to areas such as the vagina, cervix, anus, or penis by ointment, gel, cream, lotion, or foam (e.g., lather) with a pentamidine (or an analogue thereof) concentration between about 0.01 % and about 10 %, equalling to about 0.1 mg / g and about 100 mg / g. A more preferred concentration may be between about 0.1 % and about 5 %, equalling to about 1 mg / g and about 50 mg / g. A typical volume for a single application of the ointment, gel, cream, lotion, or foam (e.g., lather) with a pentamidine (or an analogue thereof) may be between about 1 g to about 5 g and thus may contains a total pentamidine dose between about 0.1 mg and about 500 mg, preferably between about 1 mg and about 250 mg.

[0092] Furthermore, in context of the present invention, examples of routinely used concentrations and amounts may include the administration to areas such as the vaginal or rectal tract by suppository with a pentamidine (or an analogue thereof) concentration between about 0.1 mg / g and about 100 mg / g (equalling to about 0.01 % to about 10 %). A more preferred concentration may be between about 1 mg / g and about 50 mg / g (equalling to about 0.1 % to about 5 %). A typical volume of a suppository may be about 1 g and thus may contains a total pentamidine dose between about 0.1 mg and about 100 mg.

[0093] Furthermore, in context of the present invention, examples of routinely used concentrations and amount may include the administration as lubricant, spray, by rinsing solution applied as a vaginal douching, anal clyster, vaginal or cervical foam and / or lather, or gel suppository with a pentamidine (or an analogue thereof) concentration between about 0.1 mg / ml and 30 mg / ml (equalling to about 0.01 % to about 3 %). A typical volume for application is between 1-10 ml, therefore the final drug dose of a single application may be between about 0.1 mg and about 300 mg, preferably between about 1 mg and about 100 mg.

[0094] Furthermore, in context of the present invention, examples of routinely used concentrations and amount may include a total drug dose may be between 0.1 mg and 300 mg for a coating of surfaces such as condoms, diaphragms, pessaries, cervical cap, contraceptive sponge, or vaginal rings.

[0095] Accordingly, without being bound by theory, examples of a routinely used total dose of pentamidine (or an analogue thereof) may be between about 0.1 mg and about 300 mg, preferably between about lmg and about 100 mg. However, as laid out herein, for protection against an STI caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., in particular C?or Ng, lower concentrations (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof) will be sufficient.

[0096] As illustrated in the non-limiting appended examples, the inventors have shown that pentamidine concentrations of 2.6 to 0.32 % (corresponding to total doses of 0.4 to 0.05 mg) can effectively reduce chlamydial burden in mice. In particular, doses between about 1.3 and about 0.32 % are shown to be particularly effective in reducing chlamydial burden (see illustratively in appended Figure 12).

[0097] Accordingly, the concentration of pentamidine (or an analogue thereof), in accordance with the present invention may be less than about 10 %, more preferably less than about 9.5 %, more preferably less than about 9 %, more preferably less than about 8.5 %, more preferably less than about 8 %, more preferably less than about 7.5 %, more preferably less than about 7 %, more preferably less than about 6.5 %, more preferably less than about 6 %, more preferably less than about 5.5 %, more preferably less than about 5 %, more preferably less than about 4.5 %, more preferably less than about 4 %, more preferably less than about 3.5 %, more preferably less than about 3 %, more preferably less than about 2.5 %, more preferably less than about 2 %, more preferably less than about 1.5 %, even more preferably less than about 1.3 %.

[0098] Accordingly, in context of the present invention, the concentration of pentamidine (or an analogue thereof) may be between about 10 % and about 0.01 %, preferably between about 9 % and about 0.02 %, more preferably between about 8.5 % and about 0.03 %, more preferably between about 8 % and about 0.04 %, more preferably between about 7.5 % and about 0.05 %, more preferably between about 7 % and about 0.06 %, more preferably between about 6.5 % and about 0.07 %, more preferably between about 6 % and about 0.08 %, more preferably between about 5.5 % and about 0.09 %, more preferably between about 5 % and about 0.1 %, more preferably between about 4.5 % and about 0.12 %, more preferably between about 4 % and about 0.14 %, more preferably between about 4 % and about 0.16 %, more preferably between about 3.5 % and about 0.18 %, more preferably between about 3 % and about 0.22 %, more preferably between about 2.6 % and about 0.24 %, more preferably between about 2.5 % and about 0.26 %, more preferably between about 2 % and about 0.28 %, more preferably between about 1.5 % and about 0.3 %, even more preferably between about 1.3 % and about 0.32 %, even more preferably between about 1.3 % and about 0.65 %. In context of the present invention, preferred concentration of pentamidine (or an analogue thereof) may be about 2.6 %, about 1.3 %, about 1 %, about 0.65 %, or about 0.32 %, particularly preferred therein may be the concentration of about 1 %.

[0099] In context of the present invention, the concentration of pentamidine (or an analogue thereof) given as percent (%) may be weight percent (% w / w), preferably for solid or semi-solid formulations, or weight-to- volume percent (% w / v), preferably for liquid formulation, unless another reference value is expressly indicated. Gels, creams, or ointments, for example, are considered semi-solid formulations in the context of this application. The person skilled in the art is able to convert the concentration of pentamidine (or an analogue thereof) given as relative concentration in percent (e.g., % w / w or % w / v) into absolute concentrations given as, for example mg / g or mg / ml, and vice-versa. An example thereof may be multiplying % (w / w) by 10 to get mg / g. As illustrated in the non-limiting appended examples, the inventors have, inter alia, provided exemplary gel formulations, illustratively using a pentamidine concentration of 1 % (w / w) (see illustratively in appended non-limiting Example 10). Accordingly, the person skilled in the art is able to adjust the concentration of pentamidine (or an analogue thereof) to the respective requirements of the intended region of administration (and / or the corresponding form of administration) in accordance with the present invention.

[0100] As indicated above, most infections with Chlamydia ssp. and Neisseria ssp. remain largely subclinical in women and men and consequently are often not detected. Therefore, an effective PrEP and / or PEP treatment option is essential. Current PrEP and / or PEP treatment options, however, usually involve systemic administration and the administration of high dosages. This is often associated with the development of resistances, a variety of systemic side effects, and the disruption of the natural microbiome. For example, often the local microbiomes of areas not affected by the infection to be treated and / or prevented (e.g., the gut microbiome) may be affected by said treatment options. The present invention may be used in much lower concentrations (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof), may be administered topically, and does not have a bactericidal effect on the (local) microbiome. Therefore, the present invention may advantageously be used as or for a PrEP and / or PEP treatment option. Accordingly, in some embodiments of the present invention, pentamidine (or an analogue thereof) may be used as or for a pre- and / or post-exposure prophylaxis (PrEP and / or PEP) treatment. In particular, in some embodiments of the present invention, pentamidine (or an analogue thereof) is to be used in combination with one or more further PrEP and / or PEP treatment(s), e.g., in combination with doxycycline-PrEP, doxycycline-PEP, HIV- PrEP and / or HIV-PEP.

[0101] STI PrEP and / or PEP treatment options often are used in patient groups with high-risk for the acquisition of STI as a co-infection. Such an STI-PrEP / PEP is often also used and / or recommended for use by those subjects who are also using HIV-PrEP and / or PEP. Accordingly, in some embodiments of the present invention, pentamidine (or an analogue thereof) is used in the treatment and / or prevention of a STI in a human immunodeficiency virus (HlV)-positive patient or a subject having acquired immunodeficiency syndrome (AIDS). In some preferred embodiments of the present invention, said HIV-positive patient is receiving doxycycline PrEP and / or PEP.

[0102] HIV pre-exposure prophylaxis (PrEP) is used to protect against HIV infection. HIV-negative individuals take an antiretroviral medication regularly or as needed, prior to potential exposure to the human immunodeficiency virus (HIV), with the aim of prevent HIV infection. HIV-PrEP is particularly recommended for people at high risk of HIV, such as but not limited to men who have sex with men (MSM) or people who frequently change sexual partners without using condoms. Examples of approved and regularly used HIV PrEP medications include but are not limited to tenofovir alafenamide / emtricitabine (Descovy), tenofovir disoproxil fumarate / emtricitabine (Truvada), or cabotegravir (Apretude). Accordingly, in some embodiments of the present invention, pentamidine (or an analogue thereof) is used in accordance with the pesent invention the treatment and / or prevention of a STI in a human immunodeficiency virus (HIV)- negative subjects that receive HIV- PrEP.

[0103] HIV-post-exposure prophylaxis (PEP) is used to protect against HIV infection. HIV-PEP refers to the administration of a short course of antiretroviral medication to individuals who are HIV-negative or of unknown HIV status, after a suspected or confirmed exposure to HIV, with the aim of preventing HIV infection. HIV-PEP is initiated as soon as possible and preferably within 72 hours after the suspected or confirmed exposure to HIV. HIV-PEP is primarily intended for emergency use following events such as unprotected sexual contact, sharing of injection equipment, occupational exposures (e.g., needlestick injuries), or sexual assault. HIV-PEP regimen usually consists of a combination of three antiretroviral agents taken daily for 28 days. Examples of recommended regimens include but are not limited to tenofovir disoproxil fumarate / emtricitabine combined with either raltegravir or dolutegravir. Alternative regimens may be used depending on individual patient factors, drug resistance, or tolerability. Accordingly, in some embodiments of the present invention, pentamidine (or an analogue thereof) is used in the treatment and / or prevention of a STI in a human immunodeficiency virus (HlV)-negative subjects that receive HIV- PEP.

[0104] All embodiments for the pentamidine (and / or its analogues) use in prevention and / or treatment provided herein also apply for the combinational use with HIV-PrEP and / or PEP.

[0105] In another aspect, the present invention relates to an agent for the treatment and / or prevention of a STI, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the agent comprises at least pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these as an active ingredient, preferably wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct) or Neisseria gonorrhoeae (Ng).

[0106] In context of the present invention an agent may also refer to exploratory and research agents. In context of the present invention, an agent may also refer to a composition or a pharmaceutical composition. A pharmaceutical composition, in context of the present invention is a composition for pharmaceutical use.

[0107] Accordingly, the present invention also relates to a composition and / or to a pharmaceutical composition for the treatment and / or prevention of a STI, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the composition comprises at least pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these as an active ingredient, preferably wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis (Ct) or Neisseria gonorrhoeae (Ng).

[0108] The pharmaceutical composition may include, in addition to pentamidine (or an analogue thereof) pharmaceutically acceptable carriers, excipients, auxiliary substances, preservatives, solvents, pH adjusting agents, and / or viscosity modulating agents. The person skilled in the art knows suitable formulations for respective pharmaceutical compositions and will readily be able to choose suitable compounds, such as pharmaceutically acceptable carriers or excipients, depending, e.g., on the formulation and administration route of the pharmaceutical composition of the invention. Examples of the pharmaceutically acceptable carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, a physiological saline solution and the like. Further examples of the pharmaceutically acceptable carrier may be hydroxyethyl cellulose (e.g., hydroxyethyl cellulose 250G), polyoxypropylene glycol, and a combination of polyoxypropylene glycol and polyethylene glycol, such as a poloxamer (e.g., poloxamer 407). According to the need, a generally used additive such as a stabilizer, a wetting agent, an emulsifier, a binder, a tonicity agent, a permeant, and / or an excipient can be suitably added. Examples of solvent include water, saline solution or any other physiological solution, ethanol, DMSO, glycerol, oil such as vegetable oil, or a mixture thereof. Accordingly, in context of the present invention, pentamidine (or an analogue thereof) may be preferably formulated in a pharmaceutically acceptable carrier.

[0109] As illustrated in the non-limiting appended examples, the inventors have, inter alia, provided exemplary gel formulations, illustratively using poloxamer (POX) or hydroxyethyl cellulose (HEC) as gelling agents. In particular, the inventors provided an exemplary poloxamer (POX)-based gel formulation comprising pentamidine isethionate, poloxamer 407 (POX), propylene glycol, and water, wherein concentration of said pentamidine isethionate is 1 % (w / w), wherein the concentration of said poloxamer 407 (POX) is 15 % (w / w), wherein the concentration of said propylene glycol is 20 % (w / w), and wherein the concentration of said water is 64 % (w / w). The inventors also provided an exemplary hydroxyethyl cellulose (HEC)- based gel formulation comprising pentamidine isethionate, hydroxyethyl cellulose 250G, propylene glycol, and water, wherein concentration of said pentamidine isethionate is 1 % (w / w), wherein the concentration of said hydroxyethyl cellulose (HEC) 250G is 3 % (w / w), wherein the concentration of said propylene glycol is 20 % (w / w), and wherein the concentration of said water is 76 % (w / w). Furthermore, the inventors provided exemplary methods for production and characterization of such formulations (see illustratively in appended non-limiting Example 10). Accordingly, the person skilled in the art is able to adjust the formulation of the agent, composition, and / or pharmaceutical composition of the present invention to the respective requirements of the intended application (and / or the corresponding application form) and is also able to find suitable alternative compounds for the respective formulation of the agent, composition, and / or pharmaceutical composition of the present invention.

[0110] The administration frequency of the agent, the composition, and / or the pharmaceutical composition of the present invention is not particularly limited, but an example thereof is once or multiple times. Examples for multiple administrations may be two, three, four, or five times. Administration frequency can also naturally depend on the mode of administration. The administration can be, for example, three times a day, twice a day, once a day, once in two days, once in three days, once in four days, once in five days, once in six days, once in a week, once in ten days, or once in two weeks. The administration can also include dosing holidays, for example, from about 1 day to about 7 days between administration. However, as illustrated in the appended examples a single administration may be enough to prevent infection, see illustratively in appended non-limiting Examples 6 to 8.

[0111] The present invention, besides further advantages, is associated with no macroscopic or histopathologic side effects when applied topically. Therefore, in context of the present invention the agent, the composition, and / or the pharmaceutical composition may be administered topically. In particular, the agent, the composition, and / or the pharmaceutical composition of the present invention comprising at least pentamidine (or an analogue thereof) for the use in treatment and / or prevention of a sexually transmitted infection (STI) as described herein, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., may be administered topically. Preferably, topical administration of the agent, the composition, and / or the pharmaceutical composition of the present invention is intravaginal, intrarectal, ocular, pharyngal, or onto a primary sex organ. Thus, the agent, the composition, and / or the pharmaceutical composition of the present invention is, preferably, formulated such that it can be administered topically, i.e., locally. The administration of the agent, the composition, or the pharmaceutical composition of the present invention may be assisted by the use of an applicator. As illustrated in the non-limiting appended examples, the inventors have, inter alia, provided illustratively exemplary gel formulations with neutral pH. In context of the present invention, the pH of the formulation is to be adjusted to the corresponding pH of the tissue and / or organ to which said formulation is intended to be administered. Non-limiting examples are formulations of neutral pH for administration to the anus and / or rectum or formulations of acidic pH for administration to the vaginal tract. Accordingly, the person skilled in the art is aware that the pH of the agent, composition, and / or pharmaceutical composition of the present invention may need adjustment according to the pH of the tissue and / or organ it is intended to be administered. Also, the person skilled in the art knows suitable pH adjusting agents and will readily be able to choose same and adjust the pH appropriately.

[0112] In context of the present invention, the form of administration of the agent, the composition, and / or the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include an ointment, gel, cream, lotion, foam (e.g., lather), lubricant, clyster, suppository, liquid, irrigation, eye lotion, eye gel, eye drops, lozenge, troche, spray, mouthwash, or a gargle. Administration may also be via a mechanical contraceptive, such as diaphragm, condom, cervical cap, or contraceptive sponge, or via a vaginal ring, pessary, chewing gum, or a powder for resuspension in liquid.

[0113] In context of the present invention, the administration time of the agent, the composition, and / or the pharmaceutical composition of the present invention is not particularly limited, and pentamidine (or an analogue thereof) can be administered before and / or after a possible contact or infection with the respective Gram-negative bacteria for the use of treatment and / or prevention. However, according to the present invention, the agent, the composition, or the pharmaceutical composition may be preferably administered at least about 0 hours, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least about 6 hours, at least about 24 hours, or at least about 48 hours before (e.g., as a PrEP treatment) and / or after (e.g., as a PEP treatment) a possible contact or infection with the respective Gram-negative bacteria. In context of the present invention, 0 hours refers to administration of pentamidine (and / or an analogue thereof) immediately before and / or directly after the potential exposure to the pathogen, in particular STI-causing Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. In context of the present invention, the storage conditions of pentamidine (or analogues thereof), the agent, the composition, and the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include but are not limited to storage at room temperature (i.e., at a temperature in the range of about 15 °C to about 25 °C, preferably between about 21 °C and 25 °C) or storage under refrigeration (i.e., at a temperature in the range of 2°C to 8°C). Storage may be carried out in sealed containers, protected from light, moisture, and / or oxygen as appropriate, depending on the physical and chemical stability requirements of the respective formulation. Other storage conditions may be selected as required to maintain the stability, potency, and usability of the agent, compound, or composition of the present invention.

[0114] As laid out herein, the inventors have found that, in context of the present invention, pentamidine (or analogues thereof) may be used at much lower concentrations (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof) against STI -causing Gram negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. Therefore, according to the present invention, the concentration of pentamidine (or an analogue thereof) at least comprised in the agent, the composition, and / or the pharmaceutical composition of the present invention may be at least between about 1 and about 50 pM, preferably at least between about 1 and about 25 pM, more preferably at least between about 1 and about 10 pM, even more preferably at least between about 1 and about 5 pM. In particular, the concentration of pentamidine (or an analogue thereof) at least comprised in the agent, the composition, and / or the pharmaceutical composition of the present invention for use in treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., may be at least between about 1 and about 50 pM, preferably at least between about 1 and about 25 pM, more preferably at least between about 1 and about 10 pM, even more preferably at least between about 1 and about 5 pM.

[0115] As laid out above and without being bound by theory, examples of routinely used concentration and total dose of pentamidine (or an analogue thereof) may be higher than the above stated minimal concentration. In particular, said higher concentrations and total doses may be used as no side effects have been observed when administration was topically. Accordingly, examples of a routinely used total dose of pentamidine (or an analogue thereof) may be between about O.lmg and about 300mg, preferably between about lmg and about 100 mg. However, as laid out herein, for protection against an STI caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., in particular Ct or Ng, lower concentrations (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof) will be sufficient.

[0116] The present invention also relates to a method of treatment and / or prevention of a STI, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., preferably wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct) or Neisseria gonorrhoeae (Ng). In particular, the present invention relates to a method of treatment and / or prevention of a sexually transmitted infection (STI) in a subject in need thereof, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., the method comprising administering to the subject an effective amount of pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these as described herein. Preferably wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis (Ct) or Neisseria gonorrhoeae (Ng). Said method may also be comprising the steps of

[0117] (i) providing a composition that comprises at least pentamidine (or an analogue thereof); and

[0118] (ii) administering said composition of (i) to the vagina, rectum, eye, pharynx, or onto a primary sex organ.

[0119] In context of the present invention, the method may be a method of treatment and / or prevention of a sexually transmitted infection (STI) in a subject in need thereof, wherein the STI is caused by Gramnegative bacteria selected from the group of Chlamydia spp. and Neisseria spp., the method comprising administering to the subject an effective amount of pentamidine (or an analogue thereof), wherein the minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of said pentamidine (or an analogue thereof) is at least between about 1 and about 50 pM. Preferably wherein said Gramnegative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis (Ct) or Neisseria gonorrhoeae (Ng). Accordingly, in said composition of (i) comprising at least pentamidine (or an analogue thereof) the concentration of said pentamidine (or an analogue thereof) corresponds to the MIC and / or MBC of at least between about 1 and about 50 pM, more preferably at least between about 1 and about 25 pM, more preferably at least between about 1 and about 10 pM, even more preferably at least between about 1 and about 5 pM. As indicated herein above, the inventors have found that the exact MIC and / or MBC of pentamidine (or an analogue thereof) may vary dependent on the specific organism. For example, the particularly preferred concentration range for the treatment and / or prevention of a Ct infection is at least about 1 to about 10 pM, even more preferred is a concentration of at least about 1 to about 5 pM. The inventors have further found that a preferable concentration for the treatment and / or prevention of a Ng infection is at least between about 3 and about 25 pM. It is further envisaged that for some analogues of pentamidine the concentration might also vary in comparison to pentamidine concentrations.

[0120] In accordance with the present invention, the administering to the subject an effective amount of pentamidine (or an analogue thereof) (e.g., the administration of said composition of (i) comprising at least pentamidine (or an analogue thereof)) is performed at least about 0 hours, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, or at least about 48 hours before (e.g., as a PrEP treatment) and / or after (e.g., as a PEP treatment) a possible contact or infection with the respective Gramnegative bacteria. The administration frequency for the administering to the subject an effective amount of pentamidine (or an analogue thereof) (e.g., the herein described composition of (i)) of the methods of the present invention is not particularly limited, but an example thereof is once or multiple times. Examples for multiple administrations may be two, three, four, or five times. Administration frequency can also naturally depend on the form of administration. The administration frequency can be, for example, three times a day, twice a day, once a day, once in two days, once in three days, once in four days, once in five days, once in six days, once in a week, once in ten days, or once in two weeks. The administration frequency can also include dosing holidays, for example, from about 1 day to about 7 days between administration. However, a single administration may be enough to prevent infection.

[0121] As described herein above and as evident from the appended non-limiting examples, pentamidine, when administered topically, i.e., locally, showed no macroscopic side effects and no histopathological alterations that indicate inflammation or tissue destruction neither at the site of administration (uterus) nor at distant organs such as the liver or kidney, see illustratively in appended Figures 5D and 5E. Accordingly, an effective amount of pentamidine (or an analogue thereof) (e.g., said composition of (i) comprising at least pentamidine (or an analogue thereof)) is intended to be preferably administered to a subject in need thereof topically, i.e., locally. In particular, the composition of (i) comprising at least pentamidine (or an analogue thereof) for the use in treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., may be administered topically. Thus, said composition of (i) is, preferably, formulated such that it can be administered topically, i.e., locally. The administration of said composition of (i) may be assisted, for example, by the use of an applicator. In particular, topical administration may be intravaginal, intrarectal, ocular, pharyngal, or onto a primary sex organ.

[0122] According to the present invention, the administration of an effective amount of pentamidine (or an analogue thereof) (e.g., by the administration of said composition of (i) comprising at least pentamidine (or an analogue thereof)) to a subject in need thereof is preferably by ointment, gel, cream, lotion, foam (e.g., lather), lubricant, clyster, suppository, , liquid, irrigation, eye lotion, eye gel, eye drops, lozenge, troche, spray, mouthwash, or a gargle. Administration may also be via a mechanical contraceptive, such as diaphragm, condom, cervical cap, or contraceptive sponge, or via a vaginal ring, pessary, chewing gum, or a powder for resuspension in liquid.

[0123] As described herein above, the present invention is, besides further advantages (and non-limiting), beneficial as pentamidine (or analogues thereof) has (have) so far never been used against bacteria causing STI. Accordingly, no prior resistances to pentamidine (or an analogue thereof) of STI-causing bacteria, in particular, no Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., are known to exist. Furthermore, the inventors have found that, although the mechanism of action remains unknown, pentamidine (or analogues thereof) does not directly act on the pathogen strain, in particular Ct, but via the host cell. The indirect mode of action of pentamidine (or an analogue thereof), combined with its low effective concentrations (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof), reduces the mutation pressure and effectively treats and prevents STIs caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. Therefore, the development of resistance in these pathogens is not expected to be a concern in the future.

[0124] The present invention is furthermore beneficial as it is associated with fewer side effects, for example, compared to a current treatment option, such as doxycycline. This is due to the low concentrations of pentamidine (or an analogue thereof) (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof) in combination with a preferably topical (self-)administration regimen. The topical administration regimen and the finding that already a single administration of pentamidine (or an analogue thereof) is effective in the treatment and / or prevention of STI greatly improves the convenience and ease of application and leads to increased treatment adherence, making the disease management more effective and reducing transmission rates within communities.

[0125] After topical administration of pentamidine (or an analogue thereof), neither the administration site (e.g., uterus) showed macroscopic or histopathologic side effects nor did distant organs, such as the liver or kidney, show pathological changes. Furthermore, pentamidine (or an analogue thereof) has no bactericidal effect on the natural bacterial flora and / or the microbiome. Therefore, the present invention is particularly advantageous for prolonged and / or repeated administration regimens, for example, a prolonged preexposure or post-exposure prophylaxis regimen, consecutive treatments after repeated infections, and / or repeated prophylactic treatment. An advantageous prophylaxis regimen is further facilitated as only low concentrations of pentamidine (or an analogue thereof) (in particular in context of the present invention, a range of at least between about 1 to about 50 pM of pentamidine or the analogue thereof) are required for effective treatment and / or prevention of STI caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. This also ensures that the natural bacterial flora and / or microbiome are not affected. Additionally, the present invention is particularly useful for treating and preventing STI caused by Ct, as it prevents the formation of Ct inclusions and EBs in the host cell.

[0126] Therefore, the present invention is useful for the treatment and / or prevention as described herein of STI caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp.. In particular, the present invention may be used to supplement or replace current treatment options. This may be, inter alia, relevant for individuals with a risk or high risk of contracting STI, for example, sex workers (female and male) or individuals with incompatibilities against a respective antibacterial treatment option, for example, doxy allergy. Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[0127] As used herein, the singular forms "a," "an," and "the" include the plural referents unless the context clearly indicates otherwise. The terms "include", "such as" and "and the like" are intended to convey inclusion without limitation, unless otherwise specifically indicated.

[0128] As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0129] As used herein, the term "comprising" also specifically includes "consisting of" and "consisting essentially of" the recited elements, unless specifically indicated otherwise.

[0130] As used herein, the terms "comprising", "including", "having" or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. The terms "comprising" / "including" / "having" encompass the terms "consisting of" and "consisting essentially of". Thus, whenever the terms "comprising" / "including" / "having" are used herein, they can be replaced by "consisting essentially of" or, preferably, by "consisting of".

[0131] The terms "comprising" / "including" / "having" mean that any further component (or likewise features, integers, steps and the like) can be present. The term "consisting of" means that no further component (or likewise features, integers, steps and the like) can be present. The term "consisting essentially of" or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed product, composition, use or method and the like. The term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

[0132] Furthermore, the present invention relates to the following items:

[0133] 1. Pentamidine (or an analogue thereof) for use in treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp..

[0134] 2. Pentamidine (or an analogue thereof) for use of item 1, wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct) or Neisseria gonorrhoeae Ng).

[0135] 3. Pentamidine or an analogue thereof for use of items 1 or 2, wherein said analogue of pentamidine is selected from the group consisting of 4,4'-(((phenylazanediyl)bis(ethane-2,l-diyl))bis(oxy))dibenzimidamide, 3,3'-(heptane-l,7-diyl)dibenzimidamide,

[0136] 3-(7-(3-(pyrrolidin-3-yl)phenyl)heptyl)benzimidamide, 4,4'-(2,2'-((lR,3S)-cyclohexane-l,3-diyl)bis(ethane-2,l-diyl))dibenzimidamide, 4,4'-(2,2'-((lR,3R)-cyclohexane-l,3-diyl)bis(ethane-2,l-diyl))dibenzimidamide,

[0137] 4-(2-((lS,3R)-3-(4-(pyrrolidin-3-yl)phenethyl)cyclohexyl)ethyl)benzimidamide, 3,3'-(((lR,3S)-cyclohexane-l,3-diyl)bis(ethane-2,l-diyl))dibenzimidamide, 7,7'-(heptane-l,7-diyl)bis(isoquinolin-l-amine), and 4-(7-(4-(pyrrolidine-3-yl)phenyl)heptyl)benzimidamide.

[0138] 4. Pentamidine (or an analogue thereof) for use of items 1 to 3, wherein said pentamidine (or an analogue thereof) inhibits growth of the respective Gram-negative bacteria.

[0139] 5. Pentamidine (or an analogue thereof) for use of items 1 to 4, wherein said pentamidine (or an analogue thereof) inhibits formation of Ct elementary bodies.

[0140] 6. Pentamidine (or an analogue thereof) for use of items 1 to 5, wherein said pentamidine (or an analogue thereof) inhibits formation of Ct inclusions.

[0141] 7. Pentamidine (or an analogue thereof) for use of any one of items 1 to 6, said pentamidine (or an analogue thereof) has no bactericidal effect on the natural bacterial flora and / or the microbiome. 8. Pentamidine (or an analogue thereof) for use of any one of items 1 to 7, said pentamidine (or an analogue thereof) has no bactericidal effect on the natural genital flora and / or the natural skin flora, in particular the natural vaginal flora.

[0142] 9. Pentamidine (or an analogue thereof) for use of any one of items 1 to 8, said pentamidine (or an analogue thereof ) has no bactericidal effect on bacteria selected from the group of Lactobacillus spp., and / or Bifidobaterium spp., preferably Lactobacillus acidophilus.

[0143] 10. Pentamidine (or an analogue thereof) for use of any one of items 7 to 9, wherein having no bactericidal effect comprises the absence of inhibition of bacterial growth while contacting said pentamidine (or an analogue thereof) and / or the absence of inhibition of regrowth after removal of said pentamidine (or an analogue thereof).

[0144] 11. Pentamidine (or an analogue thereof) for use of any one of items 1 to 10, wherein pentamidine (or an analogue thereof) is to be administered once or multiple times.

[0145] 12. Pentamidine (or an analogue thereof) for use of items 1 to 11, wherein pentamidine (or an analogue thereof) is to be administered topically.

[0146] 13. Pentamidine (or an analogue thereof) for use of item 12, wherein the topical administration is intravaginal, intrarectal, ocular, pharyngal, or onto a primary sex organ.

[0147] 14. Pentamidine (or an analogue thereof) for use of item 13, wherein the intravaginal administration is by ointment, gel, cream, foam, lubricant, suppository, clyster, pessary, diaphragm, vaginal ring, or irrigation.

[0148] 15. Pentamidine (or an analogue thereof) for use of item 13, wherein the intrarectal administration is by ointment, gel, cream, lubricant, suppository, clyster, or irrigation.

[0149] 16. Pentamidine (or an analogue thereof) for use of item 13, wherein the ocular administration is by eye ointment, eye gel, or eye drops.

[0150] 17. Pentamidine (or an analogue thereof) for use of item 13, wherein the pharyngal administration is by lozenge, troche, chewing gum, spray, mouthwash, gargle, or powder for resuspension in liquid.

[0151] 18. Pentamidine (or an analogue thereof) for use of item 13, wherein the administration onto a primary sex organ is by ointment, cream, or lubricant. 19. Pentamidine (or an analogue thereof) for use of items 14, 15 or 18, wherein said ointment, cream, or lubricant is (pre-)administered to a mechanical contraceptive.

[0152] 20. Pentamidine (or an analogue thereof) for use of any one of items 1 to 19, wherein said pentamidine (or an analogue thereof) inhibits growth of the respective Gram-negative bacteria after a single treatment (i.e., administration).

[0153] 21. Pentamidine (or an analogue thereof) for use of items 1 to 20, wherein said pentamidine (or an analogue thereof) is to be administered at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours before a possible contact or infection with the respective Gram-negative bacteria.

[0154] 22. Pentamidine (or an analogue thereof) for use of claims 1 to 21, wherein said pentamidine (or an analogue thereof) is to be administered at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours after a possible contact or infection with the respective Gram-negative bacteria.

[0155] 23. Pentamidine (or an analogue thereof) for use of any one of items 1 to 22, wherein the minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of said pentamidine (or an analogue thereof) against the respective Gram-negative bacteria is at least between about 1 and about 50 pM, preferably at least between about 1 and about 25 pM, more preferably at least between about 1 and about 10 pM, even more preferably at least between about 1 and about 5 pM.

[0156] 24. Pentamidine (or an analogue thereof) for use of any one of items 1 to 23, wherein said pentamidine (or an analogue thereof) is to be used as a pre- and / or post-exposure prophylaxis (PrEP and / or PEP) treatment.

[0157] 25. Pentamidine (or an analogue thereof) for use of items 24, wherein said pentamidine (or an analogue thereof) is to be used in combination with one or more further PrEP and / or PEP treatment(s) (e.g., doxycycline).

[0158] 26. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claim 25, wherein the one or more further PrEP and / or PEP treatment(s) is / are selected from from the group consisting of doxycycline PrEP, doxycycline PEP, HIV-PrEP, and HIV-PEP. Pentamidine (or an analogue thereof) for use of any one of items of 1 to 26, wherein the treatment and / or prevention of a STI is in a HIV-positive patient or a subject having AIDS. Pentamidine (or an analogue thereof) for use item 27, wherein the HIV-positive patient is receiving doxycycline PrEP and / or PEP. Pentamidine (or an analogue thereof) for use of any one of claims of 1 to 28, wherein said pentamidine (or an analogue thereof) is formulated in a pharmaceutically acceptable carrier. An agent for the treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the agent comprises at least pentamidine (or an analogue thereof), preferably wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct or Neisseria gonorrhoeae {Ng). A composition for the treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the composition comprises at least pentamidine (or an analogue thereof), preferably said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis {Cf} or Neisseria gonorrhoeae {Ng). A pharmaceutical composition for the treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the pharmaceutical composition comprises at least pentamidine (or an analogue thereof), preferably wherein said Gram-negative bacteria selected from the group gonorrhoeae {N The agent for the use of item 30, the composition for use of item 31, or the pharmaceutical composition for the use of item 32, wherein said agent, said composition, or said pharmaceutical composition is administered once or multiple times. The agent for the use of any one of items 30 or 33, the composition for use of any one of items 31 or 33, or the pharmaceutical composition for the use of any one of items 32 or 33, wherein said agent, said composition, or said pharmaceutical composition is to be administered topically. 35. The agent for the use of item 34, the composition for use of item 34, or the pharmaceutical composition for the use of item 34, wherein the topical administration is intravaginal, intrarectal, ocular, or pharyngal.

[0159] 36. The agent for the use of any one of items 30, or 33 to 35, the composition for use of any one of items 31 or 33 to 35, or the pharmaceutical composition for the use of any one of items 31 to 35, wherein said agent, said composition, or said pharmaceutical composition is an ointment, gel, cream, lotion, foam, lubricant, clyster, suppository, pessary, diaphragm, vaginal ring, condom, liquid, irrigation, eye lotion, eye gel, eye drops, lozenge, troche, chewing gum, spray, mouthwash, gargle, or a powder for resuspension in liquid.

[0160] 37. The agent for the use of any one of items 30, or 33 to 36, the composition for use of any one of items 31 or 33 to 36, or the pharmaceutical composition for the use of any one of items 32 to 36, wherein said agent, said composition, or said pharmaceutical composition is to be administered at least 0 hours, at least 6 hours, at least 24 hours, or at least 48 hours before a possible contact or infection with the respective Gram-negative bacteria.

[0161] 38. The agent for the use of any one of claims 30, or 33 to 37, the composition for use of any one of claims 31 or 33 to 37, or the pharmaceutical composition for the use of any one of claims 32 to 37, wherein said agent, said composition, or said pharmaceutical composition is to be administered at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours after a possible contact or infection with the respective Gram-negative bacteria.

[0162] 39. The agent for the use of any one of items 30, or 33 to 38, the composition for use of any one of items 31 or 33 to 38, or the pharmaceutical composition for the use of any one of items 31 to 38, wherein the concentration of said pentamidine (or an analogue thereof) is at least between about 1 and about 50 pM, preferably at least between about 1 and about 25 pM, more preferably at least between about 1 and about 10 pM, even more preferably at least between about 1 and about 5 pM.

[0163] 40. The agent for the use of any one of claims 30, or 33 to 39, the composition for use of any one of claims 31 or 33 to 39, or the pharmaceutical composition for the use of any one of claims 31 to

[0164] 39, wherein the concentration of the pentamidine (or an analogue thereof) within said agent, composition or pharmaceutical composition is between about 10 % and about 0.01 %.

[0165] 41. The agent for the use of any one of claims 30, or 33 to 40, the composition for use of any one of claims 31 or 33 to 40, or the pharmaceutical composition for the use of any one of claims 32 to

[0166] 40, wherein the concentration of the pentamidine (or an analogue thereof) within said agent, composition or pharmaceutical composition is between about 5 % and about 0.1 %, 42. The agent for the use of any one of claims 30, or 33 to 41, the composition for use of any one of claims 31 or 33 to 41, or the pharmaceutical composition for the use of any one of claims 32 to 40, wherein the concentration of the pentamidine (or an analogue thereof) within said agent, composition or pharmaceutical composition is between about 1.3 % and about 0.32 %.

[0167] 43. The agent for the use of any one of claims 30, or 33 to 42, the composition for use of any one of claims 31 or 33 to 42, or the pharmaceutical composition for the use of any one of claims 32 to

[0168] 42, wherein the concentration of the pentamidine (or an analogue thereof) within said agent, composition or pharmaceutical composition is between about 1.3 % (w / w) and about 0.32 % (w / w).

[0169] 44. The agent for the use of any one of claims 30, or 33 to 43, the composition for use of any one of claims 31 or 33 to 43, or the pharmaceutical composition for the use of any one of claims 32 to

[0170] 43, wherein the agent, composition, or pharmaceutical composition further comprises poloxamer 407 (POX), propylene glycol, and water.

[0171] 45. The agent for the use of claim 44, the composition for use of claim 44, or the pharmaceutical composition for the use of claim 44, wherein concentration of the pentamidine (or an analogue thereof) is 1 % (w / w), wherein concentration of the poloxamer 407 (POX) is 15 % (w / w), wherein concentration of the propylene glycol is 20 % (w / w), and wherein the concentration of the water is 64 % (w / w).

[0172] 46. The agent for the use of any one of claims 30, or 33 to 43, the composition for use of any one of claims 31 or 33 to 43, or the pharmaceutical composition for the use of any one of claims 32 to 43, wherein the agent, composition, or pharmaceutical composition further comprises hydroxyethyl cellulose 250G (HEC), propylene glycol, and water.

[0173] 47. The agent for the use of claim 46, the composition for use of claim 46, or the pharmaceutical composition for the use of claim 46, wherein concentration of the pentamidine (or an analogue thereof) is 1 % (w / w), wherein concentration of the hydroxyethyl cellulose 250G (HEC) is 3 % (w / w), wherein concentration of the propylene glycol is 20 % (w / w), and wherein the concentration of the water is 76 % (w / w).

[0174] 48. The agent for the use of any one of claims 30, or 33 to 47, the composition for use of any one of claims 31 or 33 to 47, or the pharmaceutical composition for the use of any one of claims 32 to 47, wherein said agent, said composition, or said pharmaceutical composition is to be used as a pre- and / or post-exposure prophylaxis (PrEP and / or PEP) treatment. The agent for the use of claim 48, the composition for the use of claim 48, or the pharmaceutical composition for the use of claim 48, wherein said agent, said composition, or said pharmaceutical composition is to be used in combination with one or more further PrEP and / or PEP treatment(s) (e.g., doxycycline). The agent for the use of claim 49, the composition for the use of claim 49, or the pharmaceutical composition for the use of claim 49, wherein the one or more further PrEP and / or PEP treatment(s) is / are selected from the group consisting of doxycycline PrEP, doxycycline PEP, HIV-PrEP, and HIV- PEP. The agent for the use of any one of claims 30, or 33 to 50, the composition for use of any one of claims 31 or 33 to 50, or the pharmaceutical composition for the use of any one of claims 32 to 50, wherein the treatment and / or prevention of a STI is in a HIV-positive patient or a subject having AIDS. The agent for the use of claim 51, the composition for the use of claim 51, or the pharmaceutical composition for the use of claim 51, wherein the HIV-positive patient is receiving doxycycline PrEP and / or PEP. A method of treatment and / or prevention of a sexually transmitted infection (STI) in a subject in need thereof, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., preferably wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct) or Neisseria gonorrhoeae (Ng), the method comprising the steps of

[0175] (i) providing a composition, wherein said composition comprises at least pentamidine (or an analogue thereof), preferably wherein the concentration of said pentamidine (or an analogue thereof) is at least between about 1 and about 50 pM, more preferably at least between about 1 and about 25 pM, more preferably at least between about 1 and about 10 pM, even more preferably at least between about 1 and about 5 pM; and

[0176] (ii) administering said composition of (i) to the vagina, rectum, eye, pharynx, or onto a primary sex organ, preferably, wherein the administration is performed at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours before a possible contact or infection with the respective Gram-negative bacteria. 59. The method of item 58, wherein said composition of (i) is to be administered once or multiple times.

[0177] 60. The method of any one of items 58 to 59, wherein said composition of (i) is to be administered topically.

[0178] 61. The method of any one of items 58 to 60, wherein the administration of said composition of (i) is by ointment, gel, cream, lotion, foam, lubricant, clyster, suppository, pessary, diaphragm, vaginal ring, condom, liquid, irrigation, eye lotion, eye gel, eye drops, lozenge, troche, chewing gum, spray, mouthwash, gargle, or by a powder for resuspension in liquid.

[0179] 62. A method of treatment and / or prevention of a sexually transmitted infection (STI) in a subject in need thereof, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., the method comprising administering to the subject an effective amount of pentamidine (or an analogue thereof).

[0180] 63. The method of claims 62, wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct) or Neisseria gonorrhoeae Ng .

[0181] 64. The method of any one of claims 62 or 63, wherein the minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of said pentamidine (or an analogue thereof) is at least between about 1 and about 50 pM.

[0182] 65. The method of any one of claims 62 to 64, wherein the administration is to the vagina, rectum, eye, pharynx, or onto a primary sex organ.

[0183] 66. The method of any one of claims 62 to 65, wherein the administration is performed as pre- and / or post-exposure prophylaxis (PrEP and / or PEP).

[0184] 67. The method of any one of claims 62 to 66, wherein the administration is performed at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours before a possible contact or infection with the respective Gram-negative bacteria.

[0185] 68. The method of any one of claims 62 to 67, wherein the administration is performed at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours after a possible contact or infection with the respective Gram-negative bacteria. 69. The method of any one of claims 62 to 68, wherein said effective amount of pentamidine (or an analogue thereof) is administered once or multiple times.

[0186] 70. The method of any one of claims 62 to 69, wherein said effective amount of pentamidine (or an analogue thereof) is administered topically.

[0187] 71. The method of claim 70, wherein the topical administration is intravaginal, intrarectal, ocular, pharyngal, or onto a primary sex organ.

[0188] 72. The method of claim 71, wherein the intravaginal administration is by ointment, gel, cream, foam, lubricant, clyster, suppository, or irrigation.

[0189] 73. The method of claim 71, wherein the intravaginal administration is via a pessary, diaphragm, or vaginal ring.

[0190] 74. The method of claim 70, wherein the intrarectal administration is by ointment, gel, cream, lubricant, suppository, clyster, or irrigation.

[0191] 75. The method of claim 70, wherein the ocular administration is by eye ointment, eye gel, or eye drops.

[0192] 76. The method of claim 70, wherein the pharyngal administration is by lozenge, troche, chewing gum, spray, mouthwash, gargle, or powder for resuspension in liquid.

[0193] 77. The method of claim 70, wherein the administration onto a primary sex organ is by ointment, cream, or lubricant.

[0194] 78. The method of any one of claims 72, 74, or 77, wherein said ointment, cream, or lubricant is (pre-)administered to a mechanical contraceptive.

[0195] 79. The method of any one of claims 62 to 78, wherein the subject is at high risk of an STI caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp..

[0196] 80. The method of any one of claims 62 to 79, wherein the subject is engaging in high-risk sex practices, such as unprotected sex with changing and / or multiple partners or unprotected sex with an unknown person. 81. The method of any one of claims 62 to 80, wherein the subject has an incompatibility to an existing treatment option for an STI caused by said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp..

[0197] 82. The method of any one of claims 62 to 81, wherein the subject is receiving one or more further pre- and / or post-exposure prophylaxis (PrEP and / or PEP) treatment(s) (e.g., doxycycline).

[0198] 83. The method of claim 82, wherein the one or more further PrEP and / or PEP treatment is selected from doxycycline PrEP, doxycycline PEP, HIV-PrEP, and HIV-PEP.

[0199] 84. The method of any one of claims 62 to 83, wherein the subject has a reduced / weakened immune system.

[0200] 85. The method of any one of claims 62 to 84, wherein the subject is HIV-positive or has AIDS.

[0201] 86. The method of any one of claims 85, wherein the subject is receiving a doxycycline pre- and / or post-exposure prophylaxis (PrEP and / or PEP) treatment.

[0202] Sequence Listing qPCR forward primer (5' to 3'):

[0203] GGA GGC TGC AGT CGA GAA TCT (SEQ ID NO.: 1) qPCR reverse primer (5' to 3'):

[0204] TTA CAA CCC TAG AGC CTT CAT CAC A (SEQ ID NO.: 2) qPCR labeled probe (5' to 3'):

[0205] [6-FAM]-TCG TCA GAC TTC CGT CCA TTG CGA-[TAMRA] (SEQ ID NO.: 3)

[0206] The invention is also characterized by the following figures, figure legends, and the following non-limiting examples.

[0207] Brief Description of Drawings

[0208] Fig. 1 - Medium-throughput screen reveals compounds inhibiting chlamydia growth.

[0209] A, A schematic of the experimental setup of the medium-throughput screen. HeLa cells were seeded in 384-well-plates containing lOpM of compounds. Ct containing media was added after 6h on top. Plates were incubated for 40h. B-C, Overview of test conditions. B, Overlay of fluorescent images of DAPI (blue), Evans blue (red), and C?-inclusions (green) infected with different MOI (Perkin Elmer Operetta high-content automated confocal microscope, 20X long field WD); representative images; scale bars, 100 pm. C, One representative field of view per well is depicted after fixation with either 100% methanol (MetOH), 4% paraformaldehyde (PFA) or 4% PFA containing 1% methanol. D, Plot showing number of nuclei vs number of inclusions of initial screen. Each dot represents one test compound (Cpd). DMSO treated wells as negative controls (DMSO), empty wells (Empty), and azithromycin treated wells as positive controls (AZT lOnM, AZT 2nM, AZT 50nM). Cut-off for cytotoxic drugs was 100 nuclei per FOV. E, Percentage of control vs compound ID for compounds being not cytotoxic. Hits are compounds which reduce chlamydia growth to less than 50 percent of control. F, Tree map plot of the drug classes and subclasses identified to inhibit Ct growth.

[0210] Fig. 2 - Validation experiments reveal inhibition of Ct growth across various serovars. A, Schematic of the compound sets used herein and the sequential screening steps applied to obtain a selection of a final set of 3 drugs for in vivo experiments. B, Dose response curves for the 20 compounds remaining after the validation conditions . 3-fold dilutions in duplicates starting from 13.5pM for Cfserovars E, F, and L2 were assessed (POC, percentage of DMSO treated control). C, Representative images of indicated compounds in 13.5pM concentration (Perkin Elmer Operetta high-content automated confocal microscope, 20X long field WD); scale bars, 100 pm.

[0211] Fig. 3 - Cytotoxicity assay with CellTiter-Glo.

[0212] HeLa cells were treated with compounds in 3-fold serial dilutions starting at 13.5pM for 48h to assess their cytotoxicity in the absence of Ct in duplicates. Cell viability was determined by measuring metabolic activity in each well using CellTiter-Glo. Percentage of control (POC) calculation was based on 0.1 % DMSO-treated cells (100 % viability anticipated).

[0213] Fig. 4 - Addition of compounds before or after Ct infection.

[0214] To assess the effect of compounds if administered after infection, the top 20 compounds were added in 3- fold serial dilutions starting at 13.5pM to HeLa cells 1 h after infection with CM2-GFP. The cells were incubated for 42 h and POC was compared with results from the previous dose-response validation screen with compounds being present already before Confection. A, Scheme of experimental setup. Created with BioRender. B, Dose response curves in duplicates.

[0215] Fig. 5 - Pentamidine prevents Coinfection in vivo

[0216] A., Schematic of setup of animal experiments. Created with BioRender. B, Bacterial burden in mouse uteri at the end of experiment in different groups under systemic plus loca l / topical treatment: mock treated mice (Ctr), pentamidine (Pent), Doxy, dolutegravir (DTG), or metergoline (Meterg) treated mice. Bacterial burden is indicated as pg Ct DNA per pg host DNA. One-way ANOVA and Tukey's multiple comparisons tests were performed to find differences between treatment groups (*p < 0.05, **p < 0.01). Data are presented as the mean of biological replicates ± s.d. C, Bacterial burden in mouse uteri after local pentamidine or mock (Ctr) treatment was analyzed by unpaired t-test (**p < 0.01). Data are presented as the mean of biological replicates ± s.d. D, Histopathological grading of uteri. Uteri from mice receiving 5x local treatment and Ct infection (same mice as shown in 3C, n=7-10) and mice treated once locally (n=3) were stained with H&E and assessed by a pathologist 24h after last treatment. E, Pentamidine absorption 24h after lx local treatment in different organs (fmol / mg) and serum (ng / pl).

[0217] Fig. 6 - Chlamydia incidence in HIV+ and HIV- patients with and without dolutegravir (DTG) treatment

[0218] Kaplan-Meier curves for the probability for Ct infection (upper chart) and the individual risk for Ct infection (lower chart) are presented.

[0219] Fig. 7 - Pentamidine acts more rapidly against chlamydia than antibiotics by an indirect effect via host cells.

[0220] A-D, HeLa cells pre-treated with lpM pentamidine (Pent) for 6h were infected with CM2 (no GFP) at MOI 100. Ct uptake was quantified after washing 2x with PBS to remove unbound Ct, PFA fixation, and antibody staining for CMPS in 10 FOV per condition (Olympus 1X53, LUCPIanFL N, 40x). Significant differences were tested by unpaired t-tests (*p < 0.05, ****p < 0.0001). Data are presented as the mean of 10 FOV ± s.d. A, Representative images of chlamydia uptake and inclusion formation 2h and 24h after infection. Scale bars, 20pm; CMPS (green), Evans blue (red), DAPI (blue). B, Ratio of chlamydia particles (size=10- 500 pxA2) and nuclei per FOV 2h after infection. C, Ratio of chlamydia particles (size=10-500 pxA2) and nuclei per FOV 24h after infection. D, Ratio of chlamydia inclusions (size>2000 pxA2) and nuclei per FOV 24 h after infection. E, Quantification of Ct uptake with qPCR. HeLa cells were treated with 1 or 5pM pentamidine and infected with CM2 (no GFP, MOI 100). HeLa cells were harvested 2h or 24h after infection and ratios of Ct DNA / host DNA were determined (n=3 independent experiments). F-I, Medium containing lOnM azithromycin (AZT), lOOnM Doxy, lpM or 5pM pentamidine was replaced every 24h supplemented with freshly prepared compounds or just medium. Significant differences were tested by two-way ANOVA with to Sidak's multiple comparisons test (*p < 0.05, **p < 0.01). F, Representative images of washout experiments 72h after infection (Olympus 1X53, Cplan N, lOx). Scale bars, 100pm. G, HeLa cells treated with indicated compounds, analysis 48h after infection (n=4 independent experiments, 10 FOV per experiment were analyzed, Olympus 1X53, LCAch N, 20x). H, HeLa cells treated with indicated compounds, analysis 72h after infection (n=3 independent experiments, 10 FOV per experiment were analyzed, Olympus 1X53, LCAch N, 20x). I, Quantification of Ct burden with qPCR. Hela cells were harvested 48h after infection and ratios of Ct DNA / host DNA were determined (n=4 independent experiments). J, Ct pretreated with pentamidine before infection. HeLa cells were infected with pre-treated Ct (for 30min or 2h; indicated pentamidine concentration during Cf-pretreatment) or HeLa cells were pre-treated with pentamidine 6h before infection (final concentration of 10 or 1 pM concentration). Quantification of inclusions per FOV (Olympus 1X53, LCAch N, 20x) normalized to inclusions in untreated control samples (analyzed 10 FOV per experiment). K-M, Seahorse analysis using the mitotic stress test kit, performed on 8,000HeLa cells treated with compounds (Ctr = DMSO, 3-fold dilutions of pentamidine starting at 13.5 pM or lOOnM Doxy) and / or CM2-GFP (MOI 2.5) for 48h. Data are pooled replicates from two independent runs (n=14-16), mean ± s.d. (normalized to cell number). K, Oxygen consumption rate (OCR) in pmol / min. L, Extracellular acidification rate (ECAR) in mPH / min. M, Basal respiration of all treatment conditions (OCR).

[0221] Fig. 8 - C6-NBD-sphingomyelin is recruited to inclusions in pentamidine treated cells, but inclusions do not develop further.

[0222] HeLa cells were treated with lpM pentamidine (right panel) or DMSO (Control; left panel) for 6h before infection with CM2 MOI 2.5 (no GFP-expressing). 2h, 6h, or 18h after infection, cells were harvested and stained with C6-NBD-Ceramide, anti-CMPS, phalloidin (F-actin), and DAPI. Representative images are shown.

[0223] Fig. 9 - Pentamidine, in contrast to methotrexate, does not inhibit Ct growth via SLC19A1.

[0224] HCT116 Renilla- / - and SLC19A1- / - (SLC) cells treated with 5pM pentamidine (Pent) or 5pM methotrexate (MTX) 6h before infection were analyzed 48h after infection. A, Representative images, scale bar = 100pm.

[0225] B, Percentage of control (POC; n=2 independent experiments).

[0226] Fig. 10 - Viability of Hela cells and primary EC is not impacted by pentamidine.

[0227] A-C, HeLa cells were treated with various concentrations of pentamidine (13.5pM - 0.16pM) and DMSO for 48h. A, Flow-cytometric analysis of 7-AAD negative cells, 3 independent experiments. B, Analysis of LDH-activity in pentamidine treated samples in comparison to untreated cells, 2 independent experiments.

[0228] C, Flow-cytometric analysis of proliferating cells by assessing dilution of CellTrace Violet (CTV), 3 independent experiments. D-E, primary cervical epithelial cells were treated with various concentrations of pentamidine and DMSO for 48h. D, Flow-cytometric analysis of 7-AAD negative cells, 2 independent experiments. E, Analysis of LDH-activity in pentamidine treated samples in comparison to untreated cells, 2 independent experiments.

[0229] Fig. 11 - Pentamidine inhibits Neisseria gonorrhoeae growth in vitro bu does not stop growth of Lactobacilli.

[0230] A, Liquid 24h cultures of Ng in the presence of 2-fold serial dilutions of pentamidine (starting at 50pM). B, Quantification of growth of Ng in pentamidine treated cultures in comparison to DMSO control cultures. Data are presented as the means of 3 independent experiments. C, Representative image of Ng bacterial smears derived from pentamidine-treated 24h liquid cultures. D, Quantification of growth of L. acidophilus in pentamidine treated cultures in comparison to control cultures. Data are presented as the means of 4 independent experiments. E, Representative image of L. acidophilus bacterial smears derived from pentamidine-treated 24h liquid cultures. F, Quantification of L. acidophilus bacterial smears derived from pentamidine-treated 24h liquid cultures. Fig. 12 - Dose titration of pentamidine reveals lower concentrations are more beneficial.

[0231] The values presented show the bacterial load in the uterus after dose titration of pentamidine (Pent) with uterine administration of pentamidine 4 days after infection (dissolved in NaCI). All pentamidine doses tested reduced the bacterial burden compared to untreated mice, lower concentrations (0.2 mg - 0.1 mg) seemed to be particularly beneficial. LOD= Limit of detection

[0232] Fig. 13 - Rheology of POX gel formulation indicates good physical stability.

[0233] Comparison of the dynamic viscosity of exemplary hydrogels with 15 % (w / w) poloxamer 407 (POX) from week 0 to 4 shown as flow curve (A) and viscosity curve (B). The values shown represent the mean of n=3 samples; the standard deviation is not shown graphically for reasons of clarity. Poloxamer gels maintained similar flow curves and viscosity over the course of 4 weeks, indicating good physical stability.

[0234] Fig. 14 - Rheology of HEC gel formulation indicates good physical stability.

[0235] Comparison of the dynamic viscosity of exemplary hydrogels with 3 % (w / w) hydroxyethyl cellulose (HEC) from week 0 to 4 shown as flow curve (A) and viscosity curve (B). The values shown represent the mean of n = 3 samples; the standard deviation is not shown graphically for reasons of clarity. HEC gels maintained similar flow curves and viscosity over the course of 4 weeks, indicating good physical stability.

[0236] Fig. 15 - POX and HEC gels remain pH stable over at least 4 weeks.

[0237] Comparison of the pH value of hydrogels with A) 15 % (w / w) POX and B) 3% (w / w) HEC with and without active substance (Pent) from week 0 to 4. The values shown are the mean and standard deviation of n=3 samples. While Pent as additive increases the pH value slightly in comparison to pure gels, the pH value remained stable over 4 weeks in neutral range suitable for body sites with neutral pH such as rectal mucosa.

[0238] Fig. 16 - HPLC analysis of POX and HEC gels indicates pentamidine content stability for at least 1, respectively 2 weeks.

[0239] Results of HPLC analysis revealed no significant differences in pentamidine (Pent) content in formulations over 1-2 weeks in POX and HEC gels indicating sufficient stability of the formulations for immediate treatment.

[0240] Fig. 17 - Pentamidine activity after storage in gel formulation.

[0241] Results of Ct inclusions per field of view (FOV) 24h after infection in presence of exemplary pentamidine gel-based formulations stored at different conditions. The gel formulations were either stored at room temperature (RT) or at 37°C for 48h before treatment of cells with a concentration of lOpM. Examples

[0242] To provide a beter understanding of the present invention and of its many advantages certain embodiments of the invention will now be described with reference to the following examples, which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0243] Example 1 - General Methods

[0244] A) Bacterial strains and cell culture

[0245] Cfserovar CTL2P-pGFP: :pSW2 (referred CM2-GFP) was purchased from the Chlamydia Biobank (University of Southampton) (Wang, Y. et al. (2011), PloS Pathog 7, el002258.). Cfserovars L2 (DSM 19102), E (DSM 19131), and F (DSM 19410) were purchased from the DSMZ-German Collection of Microorganisms and Cell Culture GmBH. Serovar CM2-GFP was propagated in McCoy cell monolayers in DMEM supplemented with 10% FBS (Biowest, Nuaille, France), lx non-essential amino acids (MEM NEAA 100X, Gibco™, Thermo Fisher Scientific) in the presence of 10 U / ml penicillin (Sigma, St. Louis, USA) to select for GFP expressing bacteria. Strains L2, E, and F were propagated in HeLa cells and 1 pg / ml cycloheximide (Sigma, Burlington, USA) was added to the medium as described previously. All strains were purified using gastrografin gradient centrifugation and titers were determined as described previously (Scidmore, M.A. (2005), Curr Protoc Microbiol Chapter 11, Unit 11A.1; Faris, R. and Weber, M (2019), Bio Protoc 9(24):e3459; Klasinc, R. et al. (2021), Pathogens 10(12):1617). Purified stocks were stored in sucrose-phosphate-glutamate buffer at -80°C and thawed immediately before use (220 mM sucrose, 8.6 mM Na2HPO4, 3.8 mM KH2PO4 and 5 mM L-glutamic acid).

[0246] HeLa cells (ATCC® CCL-2TM) and McCoy cells (ATCC® CRL-1696TM) were maintained in DMEM with 10% FBS. During infection, all cells were cultured in DMEM with 10% FBS (supplemented with 10 U / ml penicillin if OL2-GFP was used).

[0247] CRISPR / Cas9 knock out lines of HCT116 cells transduced with sgRNA targeting either SLC19A1 (CE0540- U) or Renilla luciferase (CE04CG-T) cDNA were obtained from RESOLUTE 38 and cultured in RPMI containing 10 % FBS. During infection, all cells were cultured in DMEM with 10 % FBS (supplemented with

[0248] 10 U / ml penicillin).

[0249] Primary cervical epithelial cells were derived from hysterectomy samples from premenopausal healthy donors which were recruited at the University Hospital, Medical University of Vienna, Austria after obtaining appropriate fully informed writen consent. Cervical biopsies were digested overnight at 4°C with dispase

[0250] 11 (2U / ml). Epithelial layer is scraped off with tweezers and cultivated in Keratinocyte Growth Medium-2 (Lonza) and CnT-IsoBoost until the first split (CnT-ISO-50, CELLnTEC). The study was approved by the Local Ethics Commitee of the Medical University of Vienna (ECS 1503 / 2020). For Neisseria gonorrhoeae (Ng) experiments, the fully antibiotics susceptible strain ATCC 49226 was used. / Vp' were cultured on homemade agar plates with Gonococcal base medium supplemented with Kellogg's supplement I and II (Ragland, S.A. and Criss, A.K. (2019), Methods in Molecular Biology 1997:319-345).

[0251] B) Medium-throughput compound screening

[0252] The compound screening was performed in collaboration with the CeMM Molecular Discovery Platform using a customized library of 2,167 compounds. The compounds derived from the NIH clinical collection, CeMM library of unique drugs CLOUD, and collections of anti-cancer agents, natural products, epigenetic compounds, metabolites, and kinase inhibitors were spotted on 384-well assay plates at concentrations of typically 10 pM (ranging from 10 - 50 pM) in 0.1% DMSO. As positive control, azithromycin at concentrations of 2, 10, and 50 nM was used. 2,000 HeLa cells were seeded per well in 25 pl and incubated for 6 hours to ensure adherence of cells. Then, Ct serovar L2 (CM2-GFP) at MOI 2.5 were added on top in 25 pl suspension and incubated for 42 h. Cells were fixed with 3.7% paraformaldehyde with 1% methanol and stained with lpg / ml DAPI and 0.002% Evans blue. Number of nuclei and chlamydia inclusions were counted in one FOV for each well. Z-factors were calculated using negative controls (DMSO) and positive controls (azithromycin 50 nM) from each plate individually (Zhang, 1-H.et al. (1999), J Biomol Screen 4(2):67-73). All plates passed the quality control with Z'>0. By linear regression, the percentage of control (POC) was calculated, setting the mean of negative controls to 100% and the mean signal of positive controls to 0% separately for each plate. Hits were defined as compounds with POC <50 and number of nuclei >100. Compounds with less than 100 nuclei were considered toxic (306 in total).

[0253] For the dose response validation, all hits except known antibiotics were tested in serovar L2 in an 8-point dose-response in duplicates in 3-fold dilutions, typically starting at 13.5 pM. Azithromycin was used at an assay concentration of 67.5 nM. Number of nuclei and inclusions were determined in one FOV per well. Top hits must fulfil the criteria of POC <60 and the number of nuclei must be >150.

[0254] The 20 top candidates from the validation screen were tested also with serovar E and F in a 4-point doseresponse in duplicates in 3-fold dilutions. As for these serovars, a centrifugation step is crucial to obtain sufficient infectivity, the plates were centrifuged for 30 min at 600 g before incubation for 46 h. In experiments with serovars not expressing GFP, cells were permeabilized with a 0.1% saponin / PBS solution for 20 min before immunofluorescence staining with FITC-conjugated anti-C? LPS monoclonal antibody (B410F, Invitrogen, 1:100) in 0.1% Saponin in PBS + 2% BSA for 30 min.

[0255] C) Compounds for in vivo and in vitro tests

[0256] For other assays than medium-throughput screening, the following compounds and suppliers were used: azithromycin (Synovo, Tubingen, Germany), doxycycline (Vibravenos, Pfizer, Poce-sur-Cisse, France), dolutegravir (MedChemExpress, Monmouth Junction, USA), metergoline (Sigma, St. Louis, USA), pentamidine isethionate (Thermo Fisher Scientific, Germany). Compounds were dissolved in DMSO and diluted in 0.9% NaCI to final concentrations indicated in the respective experiments (final DMSO concentration in the cultures <1%).

[0257] D) In vitro experiments with cell lines

[0258] For in vitro uptake and washout experiments, HeLa cells were seeded in DMEM + 10% FCS in the presence of pentamidine and infected with CM2 (MOI 100 for uptake, MOI 2.5 for washout) after 6 hours. After infection, cells were incubated at 37°C in a 5% CO2 atmosphere for indicated time points (2 hours, 6 hours, 18 hours, 24 hours, or 48 hours). For washout experiments, the culture media was removed, cells were washed twice with 200-500pl pre-warmed PBS and culture medium was replaced with fresh, pre-warmed media supplemented either with fresh compounds or empty vehicle (DMSO only). After washout, cells were incubated for another 24 to 48 hours before assessment of number of inclusions counting the number of inclusions per field of view by microscopy at a 20x resolution. A total of 10 fields of view were analyzed per replicate per experimental run. For read-out by qPCR, wells were washed 2x with 200 - 500pl PBS before harvest, trypsinized with 200pl Trypsin-EDTA (0.05%) for 5 minutes at 37°C in the incubator before washing the cells of with 1 ml DMEM + 10% FCS and collecting them in a 1.5ml tube. Cells were spun down for 5 minutes at 300g, supernatant was sucked off and cell pellets were resuspended in 200pl PBS. DNA isolation was performed according to the manual of the Qiagen DNA mini kit.

[0259] For microscopy, cells were fixed with 4% paraformaldehyde and stained as described for the Ct screen. Acquisition was performed with an Olympus 1X53 microscope (LUCPIanFL N, 40x).

[0260] For qPCR, DNA was isolated with Qiagen DNA mini kit (see Example l.F, infra}.

[0261] For viability assays with HeLa cells and primary epithelial cells either calorimetric assays (CellTiter-Glo and LDH-assays) or flow cytometry-based assays (staining with CellTrace Violet and 7-AAD) were used. Cells were cultured in the presence of pentamidine for 48 hours before viability was assessed.

[0262] For CellTrace Violet 49abelling, 1 million HeLa cells were incubated in 1ml PBS containing lpM CellTrace Violet for 15min. Cells were washed with 5ml medium before plating. After harvest, cells were stained with 2.5 pg / ml 7-AAD in PBS for lOmin and directly acquired with a Cytek Aurora flow cytometer. FACS data was analyzed using FlowJo.

[0263] For C6-NBD-sphingomyelin staining and confocal microscopy, HeLa cells were treated with lpM pentamidine or DMSO in controls for 6 hours before infection with CM2 MOI 2.5 (no GFP-expressing). At indicated time points (2 hours, 6 hours or 18 hours after infection), C6-NBD-Ceramide was added to the medium (final concentration 5pM C6-NBD-Ceramide in 0.05% BSA) and incubated for 30min at 37°C. Cells were washed 2x with PBS and cells were incubated with fresh DMEM + 10% FCS for 60min at 37°C to allow back-exchange. Cells are fixed with 4% PFA solution for 20min at 37°C. Subsequently, cells are stained with anti-Cf-LPS antibody (Clone 512F, Invitrogen, 1.3|jg / ml in 0.1% saponin in 2% BSA / PBS) for 30min. Secondary antibody staining with anti-mouse-IgG (AF680, Invitrogen, 5pg / ml in 0.1% saponin in 2% BSA / PBS) was combined with phalloidin staining (AF594, Invitrogen, 1:100) for 30min at RT. After washing with PBS, counterstaining with DAPI for 5min, coverslips were mounted onto slides. Samples were acquired at a confocal laser scanning microscope (Olympus, FLUOVIEW-FV 3000, equipped with OBIS lasers: 405, 488, 561, 640 nm and x60 UplanXApo objectives and Olympus FV31S-SW software).

[0264] For assessment of cellular metabolic activity, a Seahorse XF Cell Mito Stress Test (Agilent) was performed. 8,000 HeLa cells were seeded per well in 80pl DMEM supplemented with 10% FCS in the presence of pentamidine. Cells were incubated for 6 h before adding 80pl medium containing CM2-GFP (MOI 2.5). Cells were incubated for 40 hours at 37°C, 5% CO2 before exchanging the culture medium with assay medium (Agilent Seahorse DMEM, ImM pyruvate, 2mM glutamine and lOmM glucose). Plates were incubated for 1 hour at 37°C without CO2 before loading them into the Agilent Xfe96 Extracellular Flux Analyzer. The assay was performed using lpM oligomycin, lpM FCCP and 0.5pM rotenone / antimycin A.

[0265] E) Mouse model of Ct infection

[0266] All mouse experiments were approved by the Institutional Review Board of the Austrian Ministry of Sciences (BMBWF 2020-0.380.439). Female 6-8-week-old C57BL / 6JRj mice were purchased from Janvier labs / Charles River and maintained under specific pathogen free conditions in Biosafety Level 2 (BSL-2) facilities at the Medical University of Vienna. Mice were housed in standard cages in a temperature and humidity-controlled room with a 12 h light / dark cycle.

[0267] Mice were treated with 2.5 mg medroxyprogesterone acetate (MPA; TCA, Tokyo, Japan) in 100 pl PBS subcutaneously 7 days before infection to normalize the estrous cycle. Prophylactic treatment with compounds was started one day before infection and was repeated every 24 h until the end of experiment. The doses chosen for in vivo treatment were according to the highest still tolerated systemic doses reported in the literature (Warf, M.B. et al. (2009), Proc Natl Acad Sci U S A 106(44)18551-18556; Heredia, A. et al. (2017), Journal of Antimicrobial Chemotherapy 72(9):2570-2573; Ellis, M.J. et al. (2019), Nat Commun 10(1): 197) and suitable for dilution in small volume for transcervical application. Doses were calculated for an average mouse weight of 20g. For systemic treatment, doses were 0.8 mg pentamidine, 0.45 mg dolutegravir, 0.25 mg doxy or 0.1 mg metergoline in 100 pl 0.9% NaCI containing 10% DMSO administered intraperitoneally. For topical (i.e., local) treatment, mice received half of the systemic dosage in 15 pl 0.9% NaCI with 10% DMSO alone in control conditions or containing 0.4 mg pentamidine, 0.22 mg dolutegravir, 0.125 mg doxy or 0.05 mg metergoline by intrauterine application using a NSET device. For intrauterine infection, lx 106IFU of CM2-GFP in sucrose-phosphate-glutamate buffer were added to the local treatment dose and administered to the uterus using a NSET device as previously described (Gondek D.C. et al. (2012), J Immunol., 189(5):2441-2449). On day 4 after infection, uteri were minced and snap frozen.

[0268] For absorption studies, mice were treated once transcervically with 15 pl 0.9% NaCI with 0.4 mg pentamidine and organs and serum were harvested 24 h after treatment. Organs were either snap frozen in liquid nitrogen for targeted MS / MS or paraffin embedded for histopathological assessment.

[0269] F) qPCR to assess Ct burden

[0270] To assess the chlamydia burden in the murine uteri or in HeLa cells, DNA was isolated using the QIAamp DNA mini kit (Qiagen) and host GAPDH DNA and chlamydia 16S DNA were quantified by qPCR on a StepOnePlus™ Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific) in a multiplexed manner as previously described (Gondek D.C. et al. (2012), J Immunol., 189(5):2441-2449; Stary, G. et al. (2015), Science 348(6241):aaa8205-l-14). Using standard curves from known amounts of Ct and host DNA, the amount of chlamydia DNA (in pg) per unit weight of host DNA (in pg) allowed to calculate the ratio of pathogen DNA / host DNA. Primers are listed in Table 1.

[0271] Table 1 - Primers for qPCR

[0272] G) Antimicrobial susceptibility testing

[0273] For susceptibility testing of Ng, liquid medium containing Kellogg's supplement I and II and NaHCCh was prepared as previously described (Ragland, S.A. and Criss, A.K. (2019), Methods in Molecular Biology 1997:319-345). Pentamidine was added at a concentration of 3.12, 6.25, 12.5, 25, and 50 pM and samples were inoculated with a 0.5 McFarland bacterial suspension in NaCI 1:100.

[0274] For susceptibility testing of Lactobacillus acidophilus, the ATCC 4356 strain was cultured on MRS plates. Inocula were prepared by dissolving single colonies in 0.9% NaCI solution at a McFarland standard of 0.5 and using bacterial solution 1:500 in MRS broth containing various pentamidine concentrations. OD600 was measured after 24 h and growth rate was calculated comparing to optical density of medium only and untreated cultures. To assess bactericidal effect of pentamidine, 5pl of 24 h liquid cultures treated with various drug concentrations were spread out on fresh agar plates and incubated for 48h.

[0275] H) Chlamydia incidence in HIV+ and HIV- patients with and without dolutegravir (DTG) treatment

[0276] For this analysis, all 67-positive results from 04 / 2014-11 / 2020 from males visiting the HIV- and STI-clinic of the Medical University of Vienna were retrospectively evaluated, and patients' characteristics (HIV status and DTG exposure) were retrieved from the medical records. GraphPad Prism 8 was used to perform the statistical analyses. Nominal variables were plotted as number and percentage of patients with a specific feature. To calculate the incidence rate of infections and the respective 95 % confidence interval (95 % CI), the person-time method was used. Reinfections during the observational period were analyzed using a Kaplan-Meier curve and a log-rank test was used to compare the incidence of (^-reinfections by HIV- status. The presented analysis complies with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. The Local Ethics Committee of the Medical University of Vienna provided the ethical approval (2175 / 2020). Due to the retrospective design, the need for an informed consent had been waived.

[0277] I) Statistical analysis

[0278] Statistical analysis of medium-throughput screen was done in R calculating Z-factors and linear regression models. All other statistical analyses were done in Graphpad Prism version 9.5.0. For analysis of mouse experiments, outliers were excluded using Grubbs' method with alpha = 0.05. One-way ANOVA and Tukey's multiple comparisons were performed to find differences between treatment groups. For comparison between only two groups, unpaired t-tests were performed. For statistical analysis of in vitro experiments, two-way ANOVA with matching across row and multiple comparison testing according to Sidak's (comparing 2 means) or Tukey's (more than 2 means) was used.

[0279] Example 2 - Identification of reagents inhibiting chlamydia growth in a medium -th rough put compound screen

[0280] To identify novel classes of pharmacological agents preventing STI infection, in particular wherein the STI is caused by gram-negative bacteria selected from the group of Chlamydia spp., by inhibiting chlamydia growth, the inventors chose a library of ~2,200 compounds, which include approved drugs and well described experimental molecules. As Oinfected cells are not dividing, the inventors defined the optimal ratio of 2,000 HeLa cells per 384-well infected with C7-L2-GFP at a MOI of 2.5 (Figures 1A and IB). To determine the number of nuclei and inclusions in each condition, the cells were fixed with 4% paraformaldehyde / 1% methanol that allowed for detection of endogenous GFP signal of Ct inclusions and nuclear staining with DAPI and cell staining with Evans blue for HeLa cells (Figure 1C). A decrease in the number of cell nuclei per field of view (FOV) to less than 100 was considered as cytotoxic effect of a given compound and excluded from analysis (Figure ID). To test the efficiency of the screening compounds to reduce Ct growth, the cutoff was set to 50% in relation to the negative control (DMSO) (percentage of control, POC < 50) and the positive control (azithromycin 50nM) (Figure IE). In the initial screening approach, 88 compounds were identified having the potential to control chlamydia infection. Only 26 identified compounds were known antibiotics. These compounds were excluded from subsequent analysis as the aim was to identify novel inhibitors. The screen revealed the following main substance classes as effective: i) anti-neoplastics, ii) anti-virals, and iii) psychoactive drugs (Figure IF).

[0281] In an initial dose-responses validation experiment (see Example IB, supra} of the remaining 62 reagents in 3-fold dilutions revealed 28 reagents that showed a reduction of chlamydia growth by more than 40% (POC < 60) in two or more concentrations (Figure 2A Step 3). These 28 compounds were further evaluated in various conditions to select the 20 most effective candidates for chlamydia inhibition (data not shown).

[0282] Example 3 - Validation reveals candidate drugs being effective against different genital chlamydia serovars

[0283] CM2 causing lymphogranuloma venereum is more invasive and grows faster in vitroVnan genital serovars CM and CM (Faris, R.et al. (2019), Front Cell Infect Microbiol 9(399): 1-14). Therefore, the inventors further investigated if the remaining 20 compounds validated from the primary screen, see Example 2, supra, are reactive against different genital Ct serovars in various concentrations (Figure 2A Step 4).

[0284] The most efficient compounds identified are avapritinib, CAY10571, CAY10574, dolutegravir, EKI-785, metergoline, methotrexate, pentamidine and pixantrone. Most drug candidates showed a similar trend in all three Ct serovars (Figure 2B). By using the CellTiter-Glo it was demonstrated that compounds used in the validation screen are well tolerated by the cells as viability was above 90% for all compounds (Figure 3). In addition to the experimental layout with the compound treatment before Ct infection (Figure 1A), the effectivity of drugs if added 1 hour after infection was assessed (Figure 2A Step 4, and Figure4). This timepoint of infection was chosen to identify drugs that have an effect during early chlamydia life cycle.

[0285] A slight decrease for dolutegravir and pentamidine was observed if compounds were added after infection (Figure 4). This suggests that those compounds might play a role during early events in chlamydia replication or might take some time until they are fully active within the cells. Hence, those compounds might not only be suitable for treatment but also prevention of chlamydial infection. Pentamidine, dolutegravir, and metergoline were selected to be tested in a mouse model for genital chlamydia infection (Figures 2A Step 5 and Figure 2C) as i) these compounds significantly block Cfgrowth in various conditions, ii) their targets in the chlamydia life cycle have not yet been described. As will be illustrated below, pentamidine has been surprisingly shown in the following examples as being the only active tested compound that can also be employed in vivo in sensible concentrations. Dolutegravir and metergoline did not protect, e.g., mice from infections like chlamydial infections.

[0286] Example 4 - Pentamidine is effective in a mouse model for genital chlamydia infection

[0287] To investigate if the compounds inhibiting C? growth in HeLa cells also work in vivo during female genital tract infection, the inventors established a mouse model of Ct infection for prophylactic compound treatment. Mice received drug doses every 24 hours starting one day before infection, resembling continuous schemata for pre-exposure prophylaxis (Bolan, R.K. et al. (2015), Sex Transm Dis 42(2)98- 103). At the peak of chlamydia burden 4 days after genital Ct infection (Gondek D.C. et al. (2012), J Immunol., 189(5):2441-2449), uteri were harvested to quantify chlamydia burden (Figure 5A). Mice receiving a combination of systemic and local treatment with doxy or pentamidine had a significantly lower chlamydia burden compared with DMSO-treated mice (Figure 5B). Treatment with either dolutegravir or metergoline, however, did not protect mice from chlamydia replication within their uteri (Figure 5B).

[0288] Next, the inventors explored the effect of local application of pentamidine in the mouse model for genital Ct infection starting one day prior infection. Advantageously, local pentamidine treatment significantly reduces the chlamydia burden in mice (Figure 50). In comparison to the systemic pentamidine treatment where all mice presented with bradykinesia, these therapy-related adverse events in mice were not observed with local treatment regime. Histopathological assessment of uteri of locally treated mice revealed similar grading of inflammation between pentamidine-treated and -untreated CZ-infected mice (Figure 5D). Additionally, three mice received only one local dose of pentamidine to assess pathology (Figure 5D) and absorption (Figure 5E) of pentamidine upon transcervical drug inoculation. Singular drug doses do not cause any pathological changes in the uterus, liver or kidney of these mice and the highest drug levels could be detected within the uterus (Figure 5E). Therefore, pentamidine represents an effective compound for prophylaxis against Ct infection in a mouse model having potential as local microbicide preventing human Ct infection.

[0289] Example 5 - Lack of effectiveness of dolutegravir against Ct infection in people with HIV

[0290] For dolutegravir, a cohort of people living with HIV (PLWH) receiving dolutegravir as part of their antiretroviral therapy was analyzed. Individuals receiving dolutegravir are as likely to acquire a Ct infection as patients without dolutegravir intake (Figure 6, Tables 2, and 3). Therefore, the results suggest that in the mouse model for genital chlamydia infection and in humans dolutegravir does not reach inhibitory levels in tissue to prevent Ct infection. Table 2 - Chlamydia incidence in HIV+ and HIV- patients with and without dolutegravir (DTG) treatment. Table shows the chlamydia infections per year in HIV negative and positive men. HIV positive patients with and without DTG treatment were assessed separately.

[0291] Table 3 - Chlamydia incidence in HIV+ and HIV- patients with and without dolutegravir (DTG) treatment. Ct serovar typing of HIV+ patients with and without DTG treatment. (Fishers Exact test: p=0.815)

[0292] Example 6 - Pentamidine impairs Cf rapidly and permanently

[0293] The mode of action for pentamidine in protozoan infection is not well described. Pentamidine is reported as an inhibitor of i) DNA, RIMA, or protein biosynthesis (Sun, T. and Zhang, Y. (2008), Nucleic Acids Res 36(5)1654-1664) and topoisomerase (Singh, G. and Dey, C.S. (2007), Acta Trap 103(3)172-185; Shapiro, T.A. and Englund, P.T. (1990), Proc Natl Acad Sci U S A 87(3)950-954), ii) polyamine synthesis (Basselin, M.et al. (1997), Exp Parasitol 85(3)274-282), iii) folate metabolism (Girardi, E. et al. (2020), Nat Chem Biol 16(4)469-478; Waalkes, T. and Makulu, D. (1976), Natl Cancer Inst Monogr 43:171-177.), and iv) membrane integrity (Stokes, IM. et al. (2017), Nat Microbiol 2(2)1-21).

[0294] To learn if pentamidine has a direct effect on Ct or an indirect effect by inhibiting host cell metabolic processes, the inventors first tested if pentamidine acts directly on Ct before they enter host cells rather than acting indirectly via affecting the host cell metabolism. Upon treatment of Ct with different concentrations of pentamidine 30 minutes or 2 hours before adding Ct to the untreated cells, a Ct growth reduction in HeLa cells was not observe (Figure 7J). Next, the inventors investigated if pentamidine interferes with Ct uptake or with later steps during replication. 2 hours after infection, the number of bacterial particles taken up in both conditions were comparable (Figure 7A, 7B and 7E). However, after 24 hours Ct inclusions only occur in control cells, whereas single Ct particles are present in pentamidine treated cells (Figures 7A - 7E). To determine if Ct particles taken up in pentamidine treated cells are showing hallmarks of bacterial growth, the inventors used C6-NBD-ceramide labeling. Upon uptake in the cells, C6- NBD-ceramide is modified to sphingomyelin at the Golgi apparatus, resulting in transfer and retainment of sphingomyelin in Ct inclusions upon bacterial growth (Mital, let al. (2013), PloS One 8(17):e63426; Hackstadt, T.et al. (1995), Proc. Natl. Acad. Sci. USA 92(11)4877^4881). Confocal imaging of infected cells treated with C6-NBD-ceramide was performed to assess the recruitment of C6-NBD-sphingomyelin to the inclusions. C6-NBD-sphingomyelin accumulates in the Golgi apparatus, and the findings demonstrate a clear colocalization of C6-NBD-sphingomyelin with Cf-LPS positive vesicles and inclusions in both controls and pentamidine treated samples. Notably, in the pentamidine treated cells there was a halt in the development of the inclusions (Figure 8). This suggests that the uptake and early steps in the Ct life cycle are not impaired by pentamidine but only later stages, including bacterial replication, are affected by pentamidine. To elaborate on the function of pentamidine in Ct infection, the inventors investigated the kinetics of clearance of chlamydia upon in vitro treatment. Therefore, HeLa cells were pre-treated with compounds in a similar set-up as in the initial screen (see Examples IB and 2, supra}. Next, compounds were washed out 24 hours or 48 hours after infection, respectively (Figures 7F - 71). While Ct persists and still proliferates in azithromycin- and doxy-treated samples if washout occurs already 24 hours after infection, Ct growth is significantly impaired after the 24-hour washout in pentamidine-treated samples (Figure 7F, 7G and 71). A comparable effect to pentamidine is achieved for azithromycin and doxy if they are present for at least 48 hours (Figure 7H). This suggests that pentamidine permanently inhibits Ct growth at an early timepoint and not only suppresses bacterial translation as it occurs in the presence of macrolides and tetracyclines.

[0295] In summary, the results show that Ct is taken up into pentamidine treated cells and early steps of inclusion formation occur. However, replication of Ct is significantly impaired and after washout of pentamidine, the inhibitory effect persists. With this, pentamidine is surprisingly found to be suitable for use in treatment and / or prevention of chlamydial infection / STI caused, e.g., by Ct.

[0296] Example 7 - Impairment of Ct growth by modulation of host cell metabolism

[0297] As direct disruption of Ct by pentamidine could not be observed, the inventors assessed other mechanisms of pentamidine inhibiting Ct growth indirectly via host cells. Solute carriers (SLC) might be essential for Ct growth by providing necessary factors of the bacterial life cycle. The reduced folate carrier SLC19A1 was shown to be transporting anti metabolites like methotrexate and pentamidine (Girardi, E. et al. (2020), Nat Chem Biol 16(4)469-478). Treatment of SLC19A1 - / -cells with methotrexate resulted in loss of antichlamydia effects, whereas treatment with pentamidine leads to a similar reduction of Ct growth as observed for Renilla- / - control cells (Figure 9). It can therefore be concluded that pentamidine and the antifolate methotrexate have different modes of action in their activity against Ct.

[0298] The inventors next tested the possibility that the host metabolism might be influenced by pentamidine in a way that prevents further Ct growth in infected cells. For that Seahorse analysis was performed, using the mitotic stress test kits on HeLa cells treated with pentamidine and infected with Ct. When assessing the oxygen consumption rate (OCR), pentamidine treated cells not only have a reduced basal respiration, but also the spare capacity for mitochondrial respiration is reduced (Figure 7K). Interestingly, infected control cells perform more oxidative phosphorylation at baseline. This difference is not observed in pentamidine treated samples. Similarly, pentamidine treated cells perform less glycolysis as shown by the extracellular acidification rate (ECAR) and increased glycolysis in infected control cells, indicating that Ct proliferation is reflected by high energy demand by the host cells (Figure 7L). This effect of reduced metabolic activity in pentamidine treated cells is dose dependent, while doxy-treated cells show similar basal respiration as control cells (Figure 7M). This data shows that, by reducing the basal metabolism in host cells via pentamidine, growth of Ct within the host cells is inhibited. The inventors next investigated if the metabolic effect of pentamidine results in reduced viability and proliferation of the host cells. By performing LDH assays and FACS-based viability assays (7-AAD staining and CellTrace Violet staining), HeLa cell viability and proliferation was observed to be not affected by pentamidine (Figure 10A - 10C). As HeLa cells are robust cells, the viability of primary cervical epithelial cells upon pentamidine treatment was additionally assessed, which validated the observations from HeLa cells in this more physiologically relevant cell type (Figure 10D and 10E). These data show that the unexpected inhibitory effect on bacterial replication observed in pentamidine-treated cells is probably due to metabolic changes within the host cells. However, the drug doses used herein do not impact viability and proliferation of HeLa cells and primary cervical epithelial cells as host cells of Ct.

[0299] Example 8 - Pentamidine inhibits growth of Ng while commensals of the physiologic vaginal flora retain replication capacity

[0300] As C?and Ng infections often coincide, PrEP or PEP strategies are usually designed to be effective against C?and Ng infection (Molina, IM. et al. (2018), Lancet Infect Dis 18(17):308-317; Luetkemeyer, A.F. et al. (2023), New England Journal of Medicine 388(14): 1296-1306). Therefore, the antimicrobial potential of pentamidine against A^was assessed (see Example l.G, supra) as it was reported to have some inhibitory effect in other gram-negative bacteria such as E. coli (Bichowsky-Slomnitzki, L. (1948), J Bacteriol 55(1):27-31). By treating liquid cultures of Ng with pentamidine, the inventors identified the minimal inhibitory concentration between 3.12 and 6.25 pM (Figures 11A and 11B). By spreading the liquid cultures after 24 hours of pentamidine treatment on untreated plates, the minimal inhibitory concentration was determined to correspond to the minimal bactericidal concentration as re-growth of / ^was absent (Figure 11C).

[0301] Vaginal dysbiosis is a common side effect of anti biotic treatment (Coudray, M.S. and Madhivanan, P. (2020), Eur J Obstet Gynecol Reprod Biol., 245:143-148). As prophylactic treatment of STIs should not permanently affect the urogenital microbiome, the effect of pentamidine on Lactobacillus acidophilus as a representative species of the female vaginal flora was assessed next. Reduced growth of the bacteria in the presence of pentamidine could be observed, however, the drug did not have a bactericidal effect on L. acidophilus 'm any concentration tested as bacteria grew back after withdrawal of the drug (Figure 11D and 11E). The data indicate that the bactericidal pentamidine concentrations do not have a lasting detrimental effect on the human female genital tract microbiome. Unexpectedly, pentamidine demonstrated efficacy against, e.g., Ct in vivo and inhibiting / V^ growth in vitro, all while preserving L. acidophilus, a key component of the natural vaginal flora. Furthermore, when applied topically, pentamidine successfully prevented Ct infection. However, (without being bound by theory) its mode of action appears to be mediated through the host cells, because direct incubation with pentamidine does not affect Ct. Instead, pentamidine appears to modify the metabolic activity within the host cells. In comparison to azithromycin and doxy, the effect of pentamidine is rapid and long-lasting, making it ideal as topical agent against bacterial STIs in a therapeutic and / or prophylactic setting.

[0302] Example 9 - Pentamidine dose finding

[0303] Pentamidine dissolved in 0.9 % NaCI solution has been shown to successfully prevents infection with Ct when applied locally in the uterus (see Example 4, supra). Here, a concentration of 0.4 mg in 15 pl per treatment was used. This corresponds to a concentration of 2.6 % (or 45.2 mM). Further, lower dosages were therefore tested in mouse experiments in order to determine a optimal concentration for the formulations.

[0304] For Pentamidine dose titration the same animal model was used as described before (see Example IE and Example 4). Briefly, 6-8 week old female C57BL / 6JRj mice (Janvier labs / Charles River; RRID: IMSR_RJ:C57BL-6JRJ) were treated subcutaneously with 2.5 mg medroxyprogesterone acetate (TCA; Cat# M1964) in 100 ml PBS 7 days prior to infection to normalize the estrous cycle. Prophylactic uterine treatment with pentamidine (dissolved in 15 pl 0.9 % NaCI containing 10 % DSMO) started one day before infection and repeated every 24 hours until the end of the experiment. The doses selected were 0.4 mg (2.6 % w / v), 0.2 mg (1.3 % w / v), 0.1 mg (0.65 % w / v) and 0.05 mg (0.32 % w / v). For intrauterine infection, lxlO6IFU of CM2-GFP (CTL2P-pGFP: :pSW2; Chlamydia Biobank; CT401) in sucrose-phosphate- glutamate buffer was added to the local treatment dose and administered into the uterus using an NSET device. On day 4 after infection, uteri were minced and snap frozen. To assess the chlamydia burden in the murine uteri, DNA was isolated and analysed as described in Example IF, supra. The primers are listed in Table 1.

[0305] All doses tested showed the desired effect of reducing the bacteria detected, indicating good protection against Ct. However, a concentration between 0.32 - 1.3 % performed best (Figure 12). Accordingly, an active substance concentration of 1 % w / v pentamidine was chosen for the subsequent exemplary formulations (see Example 10, infra).

[0306] In vitro, the inventors have identified the effective concentrations of pentamidine against Ct in the range of 1 - 50 pM. In vivo, the inventors have been able to determine an effective range of 0.4 - 0.05 mg total dose in mice. Based on the body weight of a mouse, this would correspond to a dose of 20 - 2.5 mg / kg. In patients, the clinically applied and approved dose of pentamidine is 4 mg / kg against infections with Pneumocystis pneumonia, trypanosomes and Leishmania. Accordingly, the specified total doses are in a comparable range. However, even lower effective concentrations are conceivable as the presence of the active ingredient in the formulation could also have an effect on efficacy of Pentamidine (or analogs thereof, as defined herein).

[0307] With an average application amount of 1 - 5 g cream, 1 % (w / w) pentamidine isethionate would, for example in a 75 kg person, correspond to a total dose of 0.13 - 0.66 mg / kg body weight, which is considerably lower than the systemic dose approved for humans (4 mg / kg). Accordingly, it is envisaged that the pentamidine concentration will preferably be between 0.01 - 10 %, more preferably between 0.1 - 5 %, in line with the mouse and patient doses.

[0308] It might be advantageous and is feasible that concentrations of Pentamidine (or analogs thereof, as defined herein) higher than between about 1 to about 50 pM are to be used in accordance with the present invention. It is one of the gists of the present invention that Pentamidine (or analogs thereof, as defined herein) is used in the herein provided preventions and treatments of STIs, since Pentamidine (or analogs thereof, as defined herein) is shown herein to have (if at all) minor negative side effects, in particular in low concentrations as shown and proposed herein.

[0309] Example 10 - Exemplary pentamidine formulations

[0310] Based on the dose titration in mice (see Example 9, supra) exemplary formulations of gels containing pentamidine isethionate (Pent) for application such as to the anogenital tract were manufactured and characterized as described herein below. The key materials are listed in Table 4.

[0311] Table 4 - Key materials for exemplary pentamidine formulations

[0312] Described herein are exemplary formulations for neutral pH, which can be preferably administered onto tissues and / or organs that have neutral pH. Formulations with acidic pH, for example for the application to tissues and / or organs that have acidic pH, can be produced correspondingly, e.g., by adjusting the pH with citric acid. Characterization and stability testing of such acidic formulations can also be performed as described herein below.

[0313] Poloxamer 407 (POX') based ael formulation

[0314] To prepare poloxamer (POX) gel, poloxamer 407 (15 % w / w), propylene glycol (20 % w / w), and injection grade water (64 % w / w) was combined and stirred at room temperature until fully dissolved (intermitted cooling was used to speed up the process). Pentamidine isethionate (1 % w / w) was mixed in until completely dissolved. The gel was protected from light and oxygen to ensure uniform distribution. The vials containing the ready-made formulation were stored at room temperature (23 °C ± 2 °C) until characterization.

[0315] Hvdroxyethyl cellulose (HEO based ael formulation

[0316] For preparation of hydroxyethyl cellulose (HEC) gels, HEC 250G (3 % w / w) was mixed with propylene glycol (20 % w / w) in a sealed container before gradually adding preheated water for injection (76 % w / w). The gel was allowed to swell for at least 30 minutes while stirring and until fully dissolved. Pentamidine isethionate (1 % w / w) was mixed in until completely dissolved whilst being protected from light and oxygen. The gel was protected from light and oxygen to ensure uniform distribution. The vials containing the ready-made formulation were stored at room temperature (23 °C ± 2 °C) until characterization.

[0317] The exemplary gel formulations were manufactured in triplicates in 10 g units and tested for several parameters to characterize their potential to be used, for example, in human anogenital region. The visual inspection revealed well-dissolved, clear gel solutions after manufacturing as described herein above. The gels were stored at room temperature (23 °C ± 2 °C) to determine the shelf life in a real-life situation. Storage in the fridge, however, is thought to increase stability even more. The first timepoint of analysis of formulations was chosen to be 12 h after manufacturing followed by time points 1, 2, and 4 weeks after manufacturing using the following methods.

[0318] Rheology: Flow curve, viscosity, and physical stability

[0319] The formulations were measured using an air-bearing rheometer (modular compact rheometer MCR 302 equipped with a Viscotherm VT2 thermostatic control system and RheoPlus® software, Anton Paar GmbH, Graz, Austria). Flow curves were recorded at 37 °C (corresponding to body temperature on mucosa) with a cone / plate system (cone / plate measuring tool CP-25, diameter 25 mm, angle 2°) in the shear rate range of 1-100 / s. Approximately 0.5 g of the gels were applied to the plate with a pipette without air bubbles. The gels were then given a regeneration time of 3 minutes at 37 °C before the measurement was started.

[0320] The dynamic viscosity of the poloxamer hydrogel (Figure 13) is high at the beginning of the measurement but decreases rapidly during the measurement as the shear rate increases. The dynamic viscosity at a shear rate of 1 / s is greater by a factor of 10 than the viscosity at a shear rate of 10 / s. The dynamic viscosity of the hydrogel with HEC as a gelling agent (Figure 14) is lower by a factor of 85 at a shear rate of 1 / s compared to the gel with poloxamer as a gelling agent and only decreases by a factor of 1.4 at 10 / s. The shear thinning properties of the gels at 37 °C indicate good spreadability and adherence, for example, to the anogenital mucosal region after application. Further fine-tuning of overall gel viscosity is possible through variation of the polymer content. Both hydrogels maintained their properties when re-evaluated after storage (Figures 13 and 14; week 1-4).

[0321] PH stability

[0322] The formulations were analyzed using a pH meter (Mettler Toledo AG, Seven Compact). The pH meter was first calibrated using two buffer solutions (pH 4.0 and 7.0; see Example 10A, supra). The electrode was then inserted into the gel and remained there until the measurement was completed. Each gel was measured in triplicate.

[0323] The pH value of the two hydrogels remained stable over the course of 4 weeks (Figure 15). The poloxamer gel with pentamidine isethionate exhibited pH values around 7.6 while the HEC gel exhibited values around 7.1 - 7.2 (Figure 15). These values represent approximately neutral pH which is suitable for example for application on human rectal mucosa (Kong F.Y.S. et al. (2021), Sexual Health 18(2): 140-146) or murine vaginal tract (Mejia M.E. et al. (2023), NPJ Biofilms Microbes 9(1):87) as model. For application to the human vaginal tract, which has acidic pH between 4.2 and 5 (Ravel et al. (2011), PNAS 108 (supplement_l) 4680-4687) the pH may, for example, be adjusted to 4.5-6 by slowly adding diluted citric acid before the total volume is achieved with water for injection.

[0324] Pentamidine (active ingredient) stability

[0325] To determine the active ingredient content and stability of pentamidine isethionate (Pent) in the test formulations over time, an HPLC-based assay was developed. In a first step, a calibration curve was measured to calibrate the method and to identify the limit of detection (LOD) and the limit of quantitation (LOQ). For this purpose, 1 mg / ml pentamidine isethionate was dissolved in methanol and in water and a dilution series was prepared. The measurement was carried out using a Shimadzu Nexera XR HPLC (Shimadzu, Kyoto, Japan) equipped with a Macherery-Nagel RP-18e column (EC 150 / 4.6 Nucleodur C18 Gravity-SB, 5 pm, SN N19091285, LOT 38779011) at a flow rate of 1 ml / min. The solvents used were acetonitrile (ACN) + 0.08 % formic acid and water + 0.1 % formic acid. For the measurement, 10 pl of the pentamidine isethionate solutions were injected and detected with a PDA detector at a wavelength of 265 nm or an XRF detector at an excitation / emission maximum of 270 / 345 nm (data not shown). The calibration curve measurements resulted in an LOD of 0.1043 pg / ml and an LOQ of 0.3130 pg / ml, whereby the measurements in methanol and in water were comparable and were therefore combined for the calibration curve.

[0326] To determine the concentration in the gel formulations, 5 mg of the previously described POX and HEC gels with 1 % (w / w) pentamidine isethionate were dissolved in 1 ml methanol. These solutions were again diluted 1:5 in methanol and 10 pl of the final dilutions were measured in triplicates using the method described above. The gels were measured immediately after preparation (week 0), as well as after one (week 1) and two weeks (week 2, HEC only). No significant deviations in the pentamidine isethionate concentration were observed over the measured period, which indicates good chemical stability of the active ingredient pentamidine isethionate in the gels (Figure 16).

[0327] The above characterization of the exemplary gel formulations showed adequate physical properties like viscosity and stability as well as a stable pH range. Moreover, the active ingredient pentamidine isethionate content remained stable if dissolved in the formulation which indicates good shelf-life and feasibility for a drug sold, for example, in the pharmacy and stored at home by the patient for easy self-application, such as to the anogenital region. Moreover, pH value of the final formulations is stable over time and is already suitable for the rectal region. Adaptations for other tissues and / or organs with different pH milieu can be easily achieved. Example 11 - Pentamidine activity after storage in gel formulation

[0328] The previously described exemplary gel formulations based on Poloxamer (POX) or Hydroxyethyl cellulose (HEC) (see Example 10) with or without 1 % (w / w) pentamidine isethionate were stored at room temperature (RT) or at 37°C for 48 hours. After this time period, the gels were diluted in 0.9% NaCI solution to obtain a final concentration of lOpM pentamidine isethionate in the cell cultures. For this experiment, 10,000 McCoy cells were seeded in 100 pl DMEM + 10 % FCS in 96-well plates and treated with the respective diluted pentamidine gel formulations or 0.9% NaCI solution containing lOpM pentamidine isethionate (Pent) as controls. McCoy cells are fibroblast cells originally derived from human synovial fluid and are commonly used for cultivation of Chlamydia trachomatis.

[0329] 4 hours after treatment, McCoy cells were infected with 100 pl medium containing Chlamydia trachomatis L2-GFP at a MOI of 2.5. Subsequently, the cells were cultured for 24 hours and inclusions per field of view (FOV) (Olympus 1X53, LUCPIanFL N, 40x) were counted. From each condition, 5 FOV were counted (Figure 17).

[0330] The data shows that pentamidine remains stable and active in the formulations, independently of storage at room temperature or 37°C, for at least for 48 hours. This confirms that pentamidine is stable and maintains its protective effect for at least about 48 hours at 37 °C (i.e. at body temperature). The conditions tested resemble storage conditions as well as conditions representative of administration, particularly administration into body cavities such as the anogenital tract, mouth, or eyes. Accordingly, pentamidine (or an analogue thereof) may be advantageously employed in the prevention (i.e. prophylaxis) and / or treatment of infection with Chlamydia spp. and Neisseria spp., as described herein above.

Claims

Claims1. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of thesefor use in treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp..

2. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claim 1, wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct or Neisseria gonorrhoeae {Ng}.

3. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claims 1 or 2, wherein said analogue of pentamidine is selected from the group consisting of 4,4'-(((phenylazanediyl)bis(ethane-2,l-diyl))bis(oxy))dibenzimidamide, 3,3'-(heptane-l,7-diyl)dibenzimidamide,3-(7-(3-(pyrrolidin-3-yl)phenyl)heptyl)benzimidamide, 4,4'-(2,2'-((lR,3S)-cyclohexane-l,3-diyl)bis(ethane-2,l-diyl))dibenzimidamide, 4,4'-(2,2'-((lR,3R)-cyclohexane-l,3-diyl)bis(ethane-2,l-diyl))dibenzimidamide,4-(2-((lS,3R)-3-(4-(pyrrolidin-3-yl)phenethyl)cyclohexyl)ethyl)benzimidamide, 3,3'-(((lR,3S)-cyclohexane-l,3-diyl)bis(ethane-2,l-diyl))dibenzimidamide, 7,7'-(heptane-l,7-diyl)bis(isoquinolin-l-amine), and 4-(7-(4-(pyrrolidine-3-yl)phenyl)heptyl)benzimidamide.

4. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claims 1 to 3, wherein said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine inhibits growth of the respective Gram-negative bacteria.

5. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claims 1 to 4, wherein said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine inhibits formation of Ct elementary bodies.

6. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claims 1 to 5, wherein said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine inhibits formation of Ct inclusions.

7. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of any one of claims 1 to 6, wherein pentamidine (or an analogue thereof) is to be administered once or multiple times.

8. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claims 1 to 7, wherein said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is to be administered topically.

9. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claim 8, wherein the topical administration is intravaginal, intrarectal, ocular, pharyngal, or onto a primary sex organ.

10. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claim 9, wherein the intravaginal administration is by ointment, gel, cream, foam, lubricant, suppository, clyster, pessary, diaphragm, vaginal ring, or irrigation.

11. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claim 9, wherein the intrarectal administration is by ointment, gel, cream, lubricant, suppository, clyster, or irrigation.

12. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claim 9, wherein the ocular administration is by eye ointment, eye gel, or eye drops.

13. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use ofclaim 9, wherein the pharyngal administration is by lozenge, troche, chewing gum, spray, mouthwash, gargle, or powder for resuspension in liquid.

14. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claim 9, wherein the administration onto a primary sex organ is by ointment, cream, or lubricant.

15. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claims 10, 11 or 14, wherein said ointment, cream, or lubricant is (pre-)administered to a mechanical contraceptive.

16. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of any one of claims 1 to 15, wherein said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine inhibits growth of the respective Gram-negative bacteria after a single administration.17.. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claims 1 to 16, wherein said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is to be administered at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours before a possible contact or infection with the respective Gram-negative bacteria.

18. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claims 1 to 17, wherein said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is to be administered at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours after a possible contact or infection with the respective Gram-negative bacteria.

19. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of any one of claims 1 to 18, wherein the minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine against the respective Gram-negative bacteria is at least between about 1 and about 50 pM,20. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of any one of claims 1 to 19, wherein the minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine against the respective Gram-negative bacteria is at least between about 1 and about 25 pM,21. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of any one of claims 1 to 20, wherein the minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine against the respective Gram-negative bacteria is at least between about 1 and about 10 pM,22. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of any one of claims 1 to 21, wherein the minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine against the respective Gram-negative bacteria is at least between about 1 and about 5 pM.

23. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of any one of claims 1 to 22, wherein said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is to be used as a pre- and / or post-exposure prophylaxis (PrEP and / or PEP) treatment.

24. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claims 23, wherein said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is to be used in combination with one or more further PrEP and / or PEP treatment(s) (e.g., doxycycline).

25. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of claim 24, wherein the one or more further PrEP and / or PEP treatment(s) is / are selected from doxycycline PrEP, doxycycline PEP, HIV-PrEP, and HIV-PEP.

26. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of any one of claims of 1 to 25, wherein the treatment and / or prevention of a STI is in a HIV-positive patient or a subject having AIDS.

27. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use claim 26, wherein the HIV-positive patient is receiving doxycycline PrEP and / or PEP.

28. Pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these for use of any one of claims of 1 to 27, wherein said pentamidine (or an analogue thereof) is formulated in a pharmaceutically acceptable carrier.

29. An agent for use in treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the agent comprises at least pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these.

30. The agent for use of claim 29, wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct) or Neisseria gonorrhoeae {Ng).

31. A composition for use in treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the composition comprises at least pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these.

32. The composition for use of claim 31, wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct) or Neisseria gonorrhoeae {Ng).

33. A pharmaceutical composition for use in treatment and / or prevention of a sexually transmitted infection (STI), wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., wherein the pharmaceutical composition comprises at least pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these.

34. The pharmaceutical composition for use of claim 33, wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct) or Neisseria gonorrhoeae Ng).

35. The agent for the use of any one of claims 29 or 30, the composition for use of any one of claims 31 or 32, or the pharmaceutical composition for the use of any one of claims 33 or 34, wherein said agent, said composition, or said pharmaceutical composition is administered once or multiple times.

36. The agent for the use of any one of claims 29, 30, or 35, the composition for use of any one of claims 31, 32, or 35, or the pharmaceutical composition for the use of any one of claims 33 to 35, wherein said agent, said composition, or said pharmaceutical composition is to be administered topically.

37. The agent for the use of claim 36, the composition for use of claim 36, or the pharmaceutical composition for the use of claim 36, wherein the topical administration is intravaginal, intrarectal, ocular, or pharyngal.

38. The agent for the use of any one of claims29, 30, or 35 to 37, the composition for use of any one of claims 31, 32, or 35 to 37, or the pharmaceutical composition for the use of any one of claims 33 to 37, wherein said agent, said composition, or said pharmaceutical composition is an ointment, gel, cream, lotion, foam, lubricant, clyster, suppository, liquid, irrigation, eye lotion, eye gel, eye drops, lozenge, troche, chewing gum, spray, mouthwash, gargle, or a powder for resuspension in liquid, or is applied onto a pessary, vaginal ring, or a mechanical contraceptive.

39. The agent for the use of any one of claims 29, 30, or 35 to 38, the composition for use of any one of claims 31, 32, or 35 to 36, or the pharmaceutical composition for the use of any one of claims33 to 38, wherein said agent, said composition, or said pharmaceutical composition is to be administered at least 0 hours, at least 6 hours, at least 24 hours, or at least 48 hours before a possible contact or infection with the respective Gram-negative bacteria.

40. The agent for the use of any one of claims 29, 30, or 35 to 39, the composition for use of any one of claims 31, 32, or 35 to 39, or the pharmaceutical composition for the use of any one of claims 33 to 39, wherein said agent, said composition, or said pharmaceutical composition is to be administered at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours after a possible contact or infection with the respective Gram-negative bacteria.

41. The agent for the use of any one of claims 29, 30, or 35 to 40, the composition for use of any one of claims 31, 32, or 35 to 40, or the pharmaceutical composition for the use of any one of claims 33 to 40, wherein the concentration of said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is at least between about 1 and about 50 pM,42. The agent for the use of any one of claims 29, 30, or 35 to 41, the composition for use of any one of claims 31, 32, or 35 to 41, or the pharmaceutical composition for the use of any one of claims 33 to 41, wherein the concentration of said pentamidine (or an analogue thereof) is at least between about 1 and about 25 pM,43. The agent for the use of any one of claims 29, 30, or 35 to 42, the composition for use of any one of claims 31, 32, or 35 to 42, or the pharmaceutical composition for the use of any one of claims 33 to 42, wherein the concentration of said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is at least between about 1 and about 10 pM,44. The agent for the use of any one of claims 29, 30, or 35 to 43, the composition for use of any one of claims 31, 32, or 35 to 43, or the pharmaceutical composition for the use of any one of claims 33 to 43, wherein the concentration of said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is at least between about 1 and about 5 pM.

45. The agent for the use of any one of claims 29, 30, or 35 to 44, the composition for use of any one of claims 31, 32, or 35 to 44, or the pharmaceutical composition for the use of any one of claims 33 to 44, wherein the concentration of the pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine within said agent, composition or pharmaceutical composition is between about 10 % and about 0.01 %.

46. The agent for the use of any one of claims 29, 30, or 35 to 45, the composition for use of any one of claims 31, 32, or 35 to 45, or the pharmaceutical composition for the use of any one of claims 33 to 45, wherein the concentration of the pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine within said agent, composition or pharmaceutical composition is between about 5 % and about 0.1 %,47. The agent for the use of any one of claims 29, 30, or 35 to 46, the composition for use of any one of claims 31, 32, or 35 to 46, or the pharmaceutical composition for the use of any one of claims 33 to 46, wherein the concentration of the pentamidine, said analogue of pentamidine, or saidpharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine within said agent, composition or pharmaceutical composition is between about 1.3 % and about 0.32 %.

48. The agent for the use of any one of claims 29, 30, or 35 to 47, the composition for use of any one of claims 31, 32, or 33 to 47, or the pharmaceutical composition for the use of any one of claims 33 to 47, wherein the concentration of the pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine within said agent, composition or pharmaceutical composition is between about 1.3 % (w / w) and about 0.32 % (w / w).

49. The agent for the use of any one of claims 29, 30, or 35 to 48, the composition for use of any one of claims 31, 32, or 35 to 48, or the pharmaceutical composition for the use of any one of claims 33 to 48, wherein the agent, composition, or pharmaceutical composition further comprises poloxamer 407 (POX), propylene glycol, and water.

50. The agent for the use of claim 49, the composition for use of claim 49, or the pharmaceutical composition for the use of claim 49, wherein concentration of the pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is 1 % (w / w), wherein concentration of the poloxamer 407 (POX) is 15 % (w / w), wherein concentration of the propylene glycol is 20 % (w / w), and wherein the concentration of the water is 64 % (w / w).

51. The agent for the use of any one of claims 29, 30, or 35 to 48, the composition for use of any one of claims 31, 32, or 35 to 48, or the pharmaceutical composition for the use of any one of claims 33 to 48, wherein the agent, composition, or pharmaceutical composition further comprises hydroxyethyl cellulose 250G (HEC), propylene glycol, and water.

52. The agent for the use of claim 51, the composition for use of claim 51, or the pharmaceutical composition for the use of claim 51, wherein concentration of the pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is 1 % (w / w), wherein concentration of the hydroxyethyl cellulose 250G (HEC) is 3 % (w / w), wherein concentration of the propylene glycol is 20 % (w / w), and wherein the concentration of the water is 76 % (w / w).

53. The agent for the use of any one of claims 29, 30, or 35 to 52, the composition for use of any one of claims 31, 32, or 35 to 52, or the pharmaceutical composition for the use of any one of claims 33 to 52, wherein said agent, said composition, or said pharmaceutical composition is to be used as a pre- and / or post-exposure prophylaxis (PrEP and / or PEP) treatment.

54. The agent for the use of claim 53, the composition for the use of claim 53, or the pharmaceutical composition for the use of claim 53, wherein said agent, said composition, or said pharmaceutical composition is to be used in combination with one or more further PrEP and / or PEP treatment(s) (e.g., doxycycline).

55. The agent for the use of claim 54, the composition for the use of claim 54, or the pharmaceutical composition for the use of claim 54, wherein the one or more further PrEP and / or PEP treatment(s) is / are selected from the group consisting of doxycycline PrEP, doxycycline PEP, HIV-PrEP, and HIV- PEP.

56. The agent for the use of any one of claims 29, 30, or 35 to 55, the composition for use of any one of claims 31, 32, or 35 to 55, or the pharmaceutical composition for the use of any one of claims 33 to 55, wherein the treatment and / or prevention of a STI is in a HIV-positive patient or a subject having AIDS.

57. The agent for the use of claim 56, the composition for the use of claim 56, or the pharmaceutical composition for the use of claim 56, wherein the HIV-positive patient is receiving doxycycline PrEP and / or PEP.

58. A method of treatment and / or prevention of a sexually transmitted infection (STI) in a subject in need thereof, wherein the STI is caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp., the method comprising administering to the subject an effective amount of pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these.

59. The method of claims 58, wherein said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp. is Chlamydia trachomatis Ct) or Neisseria gonorrhoeae Ng .

60. The method of any one of claims 58 or 59, wherein the minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) of said pentamidine, said analogue of pentamidine, or said pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine is at least between about 1 and about 50 pM.

61. The method of any one of claims 58 to 60, wherein the administration is to the vagina, rectum, eye, pharynx, or onto a primary sex organ.

62. The method of any one of claims 58 to 61, wherein the administration is performed as pre- and / or post-exposure prophylaxis (PrEP and / or PEP).

63. The method of any one of claims 58 to 62, wherein the administration is performed at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours before a possible contact or infection with the respective Gram-negative bacteria.

64. The method of any one of claims 58 to 63, wherein the administration is performed at least about 0 hours, at least about 6 hours, at least about 24 hours, or at least about 48 hours after a possible contact or infection with the respective Gram-negative bacteria.

65. The method of any one of claims 58 to 64, wherein said effective amount of pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these is administered once or multiple times.

66. The method of any one of claims 58 to 65, wherein said effective amount of pentamidine, an analogue of pentamidine, or a pharmaceutically acceptable salt of said pentamidine or of said analogue of pentamidine, or a combination of any one of these is administered topically.

67. The method of claim 66, wherein the topical administration is intravaginal, intrarectal, ocular, pharyngal, or onto a primary sex organ.

68. The method of claim 67, wherein the intravaginal administration is by ointment, gel, cream, foam, lubricant, clyster, suppository, or irrigation.

69. The method of claim 68, wherein the intravaginal administration is via a pessary, diaphragm, or vaginal ring.

70. The method of claim 67, wherein the intrarectal administration is by ointment, gel, cream, lubricant, suppository, clyster, or irrigation.

71. The method of claim 67, wherein the ocular administration is by eye ointment, eye gel, or eye drops.

72. The method of claim 67, wherein the pharyngal administration is by lozenge, troche, chewing gum, spray, mouthwash, gargle, or powder for resuspension in liquid.

73. The method of claim 67, wherein the administration onto a primary sex organ is by ointment, cream, or lubricant.

74. The method of any one of claims 68, 70, or 73, wherein said ointment, cream, or lubricant is (pre-)administered to a mechanical contraceptive.

75. The method of any one of claims 58 to 74, wherein the subject is at high risk of an STI caused by Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp..

76. The method of any one of claims 58 to 75, wherein the subject is engaging in high-risk sex practices, such as unprotected sex with changing and / or multiple partners or unprotected sex with an unknown person.

77. The method of any one of claims 58 to 76, wherein the subject has an incompatibility to an existing treatment option for an STI caused by said Gram-negative bacteria selected from the group of Chlamydia spp. and Neisseria spp..

78. The method of any one of claims 57 to 77, wherein the subject is receiving one or more further pre- and / or post-exposure prophylaxis (PrEP and / or PEP) treatment(s) (e.g., doxycycline).

79. The method of claim 78, wherein the one or more further PrEP and / or PEP treatment is selected from doxycycline PrEP, doxycycline PEP, HIV-PrEP, and HIV-PEP.

80. The method of any one of claims 58 to 79, wherein the subject has a reduced / weakened immune system.

81. The method of any one of claims 58 to 80, wherein the subject is HIV-positive or has AIDS.

82. The method of claim 81, wherein the subject is receiving a doxycycline pre- and / or post-exposure prophylaxis (PrEP and / or PEP) treatment.

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