Methods for treatment of chronic inflammatory diseases including atherosclerosis and periodontitis caused by intracellular bacteria

Abstract

Methods for treating or preventing a chronic inflammatory diseases in an individual which includes administering a therapeutically effective amount of an inhibitor to the individual, wherein the inhibitor reduces or prevents intracellular pathogen formation or activity of a drug-resistant biofilm-associated pathogen, thereby treating or preventing the chronic inflammatory disease resulting from the intracellular pathogen or drug-resistant biofilm-associated pathogen. Examples include the chronic inflammatory disease atherosclerosis and the intracellular pathogen Porphyromonas gingivalis internalized in primary human coronary endothelial cells. Methods used to screen for inhibitor(s) of the intracellular pathogen formation or activity or formation or activity of a drug-resistant biofilm-associated pathogen are also included.

Description

Methods for treatment of chronic inflammatory diseases such as atherosclerosis and periodontitis caused by intracellular bacteria and multidrug-resistant pathogensCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Provisional Application Serial No. 63 / 689,662, filed August 31, 2024, which is incorporated herein by reference for any purpose.BACKGROU ND OF THE I NVENTION

[0002] Atherosclerosis (AS), a chronic inflammatory condition underlying cardiovascular disease (CVD), is the number one cause of morbidity and mortality in adults. Despite meaningful progress in the identification of risk factors and the development of clinical tools, such as statins, deaths from CVD continue to increase worldwide, due in part to the moderate efficiency of the available drugs. For example, after 104 weeks of maximal dose therapy, the primary efficacy end point, percent atheroma volume decreased by only 0.99% (95% Cl, -1.19 to -0.63) with atorvastatin and by 1.22% (95% Cl, -1.52 to -0.90) with rosuvastatin (P=0.17) (Nicholls et al., 2011).

[0003] Myocardial infarction and stroke continue to occur in as many as two thirds of all patients (Libby, 2005). CVD cost in the US alone is $468B (in 2008, American Heart Association), more than any other condition. Further, CVD was estimated to cost the EU169B euros a year, making it the 'largest cause of sickness and morbidity’ in the EU as well (2007, http: / / www.herc.ox.ac.uk / research / cvd).

[0004] Genome-wide association studies have so far identified 36 genetic variants with increased risk for CVD (Roberts and Stewart, 2012), which immensely complicates targeting the genetics of the predisposition to AS. In addition, many cardiovascular events have not been explained by genetics or other risk factors.

[0005] Current science has outlined a list of risk factors that are associated with atherosclerosis. Common risk factors include obesity, cholesterol levels, and blood pressure. However, these current risk factors for CVD do not account for a majority of atherosclerosis (AS) cases. So, how do we address these cases if reducing the commonly-known risk factors will not account for most AS cases?

[0006] In AS, current treatment is confined to alleviating the symptoms or targeting such risk factors. Most innovation in CVD is focused on reducing LDL cholesterol using lipids. For this reason, drug companies crowd the lipids field. However, as noted, these solutions address onlypart of the issue. Current treatments addressing cholesterol decreased mortality in less than 1 out of 10 clinical trials.

[0007] The main problem is that current treatment modalities targeting hypertension, hyperlipidemia and controlling hemostasis do not directly address the inflammatory origin of atherosclerosis, even though multiple epidemiological studies have consistently suggested an infectious bacterial component.SUMMARY OF THE INVENTION

[0008] The present invention is directed to methods for treating or preventing a chronic inflammatory diseases in an individual which includes administering a therapeutically effective amount of an inhibitor to the individual, wherein the inhibitor reduces or prevents intracellular pathogen formation or activity or reduces or inhibits a drug-resistant biofilm-associated pathogen, thereby treating or preventing the chronic inflammatory disease resulting from the intracellular pathogen.

[0009] In some embodiments, chronic inflammatory disease tissue includes atherosclerosis, infectious endocarditis (IE), reactive or rheumatoid arthritis (RA), a neurological disease selected from the group of Alzheimer's disease and multiple sclerosis, an ocular disease selected from the group of a retinal disorder, keratitis, glaucoma, and a urinary tract infections (UTI).

[0010] In some embodiments, the intracellular pathogen is Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythus, Treponema denticola, Eikenella corrodens, Prevotella intermedia, Prevotella nigrescens, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Pseudomonas, Legionella, Chlamydophila, Coxiella, Listeria, Enterobacter, Staphylococcus, Streptococcus, Enterobacter, Propionibacterium or Porphyromonas genus species.

[0011] In one preferred embodiment, the intracellular pathogen is Porphyromonas gingivalis which is internalized in primary human coronary endothelial cells.

[0012] In some embodiments, the inhibitor is oracillin, 4-acetamido-N-(5-nitro-l,3-thiazol-2-yl) benzamide or 2-(4-ethoxyphenoxy)-N-[l-(5-ethyl-4-methyl-6-oxo-l,6-dihydropyrimidin-2-yl)-3- methyl-lH-pyrazol-5-yl] acetamide.

[0013] In some embodiments, the inhibitor further includes a pharmaceutically acceptable carrier.

[0014] In certain embodiments, the pathogen associated with a biofilm is Porphyromonas gingivalis.

[0015] In a separate aspect of the invention, screening methods are included which include the steps of (i) obtaining a library of candidate drugs and / or drug analogues, (ii) obtaining a sample from an individual having a chronic inflammatory disease such as one of the diseases stated above and wherein the sample further includes an intracellular pathogen or a drug-resistant biofilm-associated pathogen, (iii) screening for drug candidate(s) which inhibit activity of the intracellular pathogen formation or activity or drug-resistant biofilm-associated pathogen and (iv) selecting the drug candidate(s) which show inhibitory activity against the intracellular pathogen formation or activity of the drug-resistant biofilm-associated pathogen. In some embodiments, the intracellular pathogen or drug-resistant biofilm-associated pathogen is Porphyromonas gingivalis and / or is internalized in primary human coronary endothelial cells.BRI EF DESCRI PTION OF DRAWI NGS

[0001] Fig. 1: shows inhibition of intracellular bacteria internalized into primary human coronary artery endothelial cells upon treatment with compound 0342-0049 ("TAI", Penicillin V).

[0002] Fig. 2: shows inhibition of intracellular bacteria internalized into primary human coronary artery endothelial cells upon treatment with compound 0342-0049 ("TAI", Penicillin V).

[0003] Fig. 3: shows inhibition of intracellular bacteria internalized into primary human coronary artery endothelial cells upon treatment with compound Y031-9897 ("TA2").

[0004] Fig. 4 shows inhibition of intracellular bacteria internalized into primary human coronary artery endothelial cells upon treatment with compound Y031-9897 ("TA2).

[0005] Fig. 5 shows an animal study design according to certain embodiments of the current invention.

[0006] Fig. 6 shows animal study results with respect to atherosclerotic plaque area for animals on a high-fat diet (HFD). Both TAI and Ta2 reduced aortic plaque areas in ApoE - / - mice infected with Porphyromonas gingivalis, suggesting their potential efficacy in reducing atherosclerosis.

[0007] Fig. 7 shows animal study results with respect to IL-lbeta concentration in serum with animals on a high-fat diet (HFD). Both TAI and TA2 reduced IL-lbeta expression in ApoE - / - mice infected with Porphyromonas gingivalis, suggesting their potential efficacy in reducing atherosclerosis.DETAI LED DESCRIPTION OF THE I NVENTION

[0008] Atherosclerosis (AS): An abundance of evidence supporting infections as a potential cause of AS has been published (Ross, 1999), (Libby et al., 2002), (Epstein et al., 2009). First, the accumulating epidemiological, seroepidemiological, and animal model data support thehypothesis that infections contribute to AS (Harskamp and van Ginkel, 2008) and the Northern Manhattan Study (NOMAS), a prospective cohort study of stroke incidence and prognosis, indicated that "infectious burden" is associated with risk of stroke and carotid plaque thickness (Elkind, 2010). Second, AS has many of the characteristics of a chronic inflammatory disease. Clinical studies have indicated that infections with multiple pathogens result in chronic persistent inflammation (Epstein et al., 2009). Third, internalization of many types of bacteria allows them to persist in a dormant, uncultivable non-replicating state, sheltered from humoral and cellular immune responses, the most studied case being Mycobacterium tuberculosis (Tufa riello et al., 2003). Fourth, DNA data suggest that various pathogens are associated with atheromas (Kozarov et al., 2006), (Ott et al., 2006), yet clinical strains could not be cultivated to provide any key mechanistic link suggesting causality (Fiehn et al., 2005). However, only cultivation can enable the necessary experiments for establishing causality, and lead to opening of a new field in vascular medicine.

[0009] We demonstrated at several levels that microbial infection of arterial plaques may be the missing link, a major contributing factor to acute ischemic events. Using immunology, cellular microbiology, genomics and fluorescence microscopy, we were the first in the medical literature to demonstrate vascular cell transmission of bacterial pathogens (Li et aL, 2008), and the first to show association of uncultivable invasive bacteria with atheromatous tissue (Kozarov, 2005). Typical periodontal pathogen such as Porphyromonas gingivalis can stimulate cytokine production and survive in monocytes, thus spreading in the circulatory system (Suwatanapongched et al., 2010), (Nicu et al., 2009).

[0010] Macrophage cell culture has been used to isolate Francisella tularensis from foodstuffs (Day et al., 2009; Day and Whiting, 2009). Similarly, Tropheryma whippelii has been isolated from Whipple disease patients (Schoedon et al., 1997), and Chlamydophila pneumoniae has been isolated from atheromatous tissue (Jackson et al., 1997). Based on that, we were the first to develop a technology to isolate and identify previously uncultivable intracellular bacterial pathogens, targets for therapy, from such tissue from patients (Rafferty et al., 2011a), (Rafferty et al., 2011b), confirming earlier data using genomic analysis approaches (Kozarov et al., 2006).

[0011] Arthritides. Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease of unclear etiology {Chen, 2019 #7013}. Septic arthritis is an infectious complication known to be overrepresented in RA {Ostensson, 1991 #7015}. Septic arthritis is an important medical emergency, with high morbidity and mortality, increasing frequency of the disorder and evolvingantibiotic resistance. Management is complicated due to newly discovered and antibioticresistant causative microorganisms {Mathews, 2010 #7000}, {Garcia-Arias, 2011 #7003}.

[0012] The hematogenous route of infection dissemination (bacteremia) from the cardiovascular system, skin and soft tissue, oral cavity, urogenital and gastrointestinal tracts, present common origins of periprosthetic joint infections {Rakow, 2019 #7004}. Their treatment is complicated due to multi-drug resistance {Siljander, 2018 #7006}.

[0013] Bacteria that produce symptoms in multiple joints during bacteremia may also induce inflammation in the neighboring tendon sheaths. Any patient who presents with an acute articular arthritis is suspected of having a bacterial process {Smith, 1990 #6992}. Because synovial fluid findings (leukocyte counts and glucose) may not be predictive of infection, bacteriologic analysis is necessary in the evaluation {Smith, 2006 #6991}, {Smith, 1995 #6990}. Related, bacterial persistence in reactive arthritis has been described. The most common pathogen causing septic arthritis is Staphylococcus aureus {Ross, 2003 #6995}, {Horowitz, 2011 #7002}, {Belkhir, 2014 #6994}. Streptococcal species and polymicrobial infection are the second most common cause of native joint septic arthritis {Lotz, 2019 #6993}, followed by Neisseria meningitides {Rosen, 1985 #6999}, {Rodn'guez, 2012 #6996}, {Siow, 2018 #6997}, including from uncultivable species {Peralta, 2012 #6998}. Of note, extended culture duration in acute hip and knee periprosthetic joint infections did not change the rate of culture-negative samples {Klement, 2019 #7007}, indicating the presence of uncultivable species that are target of this application. Staphylococcus epidermidis and Enterococcus were also identified in periprosthetic joint infections {Rosteius, 2018 #7005}. A significant percent of patients with osteoarthritis, and no signs of infection, have bacterial biofilms present in the knee {Palmer, 2016 #7001}.

[0014] In addition, with rising numbers of anti-tumor necrosis factor a (TNF-a) treatments for rheumatoid arthritis (RA), associated severe infections are encountered {Kroesen, 2003 #7008}, {Inane, 2006 #7011}, {Chen, 2020 #7014}, often skin and soft tissue infections {Dixon, 2006 #7009}, ocular adverse events {Nicolela Susanna, 2020 #7012} and increased risk of malignancies {Bongartz, 2006 #7010} and thus this treatment method targeting intracellular bacteria can be applied to arthritides as well (Toivanen, 2001), (Toivanen and Toivanen, 2004) (Astrauskiene and Bernotiene, 2007).

[0015] Our studies have found inflammatory bacterial pathogen, Porphyromonas gingivalis (P. gingivalis) in atherosclerotic plaques from vascular surgery patients. This pathogen causesinflammation and plaque buildup in animal models as well. The presence of this infectious pathogen in cardiovascular tissue is linked to inflammation and plaque buildup.

[0016] Accordingly and in some embodiments, the current invention is directed to high- throughput screening and identification drug and small molecule libraries targeting key pathogen such as P. gingivalis to identify and confirm drug leads.

[0017] In separate embodiments, the current invention is directed to methods of treating and / or preventing diseases such as AR by administering a compound which inhibits or eliminates key pathogen(s), including but not limited to, the microbe P. gingivalis and thereby prevent and / or treat AR diseases.

[0018] In separate embodiments, the key pathogen, including but not limited to P. gingivalis is in a biofilm. In further embodiments, the biofilm is periodontitis.

[0019] In some embodiments, the compounds which inhibit or eliminate key pathogen(s) are part of pharmaceutical compositions. These compositions comprise a pharmaceutically acceptable carrier and an amount of one or more of the inhibitors effective to treat or prevent one of the chronic inflammatory diseases described herein in an individual.

[0020] Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically in oral solid formulations, parenteral, intravenous, ophthalmic, suppository, aerosol, topical or other similar formulations.Such pharmaceutical compositions may contain pharmaceutically-acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer an inhibitor according to the methods of the invention.

[0021] Neurological Diseases: Similarly, bacterial infection has been implicated in debilitating brain disorders. Much experimental and epidemiological data support the involvement of bacterial pathogens in the development of Alzheimer's dementia (Kagan, 2011), (Fulop et al., 2018), (Fulop et al., 2018), (Moir et al., 2018), (Osorio et al., 2019), (Panza et al., 2019), (Fulop et al., 2020), (Kim et al., 2020). Specifically, extracellular proteinases from the periodontal pathogen P. gingivalis have been a target of technology development (US Patent 9,758,473 of Cortexyme, Inc). However, our target as described herein are not bacterial proteins, but the intracellular bacteria, internalized in host cells.

[0022] Preterm delivery / low birth weight (PTD / LBW): In adverse pregnancy outcomes, a predictive value of markers of inflammation such as C-reactive protein has been shown for intrauterine infection and preterm delivery (Greksova et al., 2009), (Herbst and Nilsson, 2006).

[0023] The placental membrane microbiome (Solt, 2015), (Dunlop et al., 2015) was shown to be altered among subjects with spontaneous preterm birth (Prince et al., 2014a; Prince et al., 2014b; Prince et al., 2016). Moreover, a specific association between maternal periodontitis and low birth weight has been reported (Foratori-Junior et al., 2020). Importantly, the periodontal pathogen P. gingivalis has been detected in placenta from preterm delivery patient (Katz et al., 2009) and associated with adverse pregnancy outcomes (Vanterpool et al., 2016). P. gingivalis infection during the third trimester caused low birth weight and inflammation in the placenta and umbilical cord (Udagawa et al., 2018).

[0024] Ocular pathologies. Infections at this immunologically privileged site have also been communicated, such as in retina (Arjunan et al., 2020). In cases of bacterial keratitis that are contact lens associated, Pseudomonas aeruginosa has been the most common causative organism (Tang et al., 2013). Of note, periodontal pathogens such as Aggregatibacter actinomycetemcomitans (Hong et al., 2016) and uncultivable P. gingivalis have been detected in keratitis (Rudolph et aL, 2004). Thus, intracellular periodontal and other bacteria from the ocular microbiome are another significant target for treatment using our method (Ozkan et al.,2017), (Kugadas et aL, 2016; Kugadas and Gadjeva, 2016), (Lu and Liu, 2016), (Rowan and Taylor,2018), (Bairn et al., 2019; Doulberis et aL, 2019), (Ozkan and Willcox, 2019).

[0025] Cancer. Bacteria were first detected in human tumors more than 100 years ago and information has been accumulating since then (Rajagopala et aL, 2017), (Picardo et aL, 2019), (Peters et aL, 2019), (McAllister et aL, 2019), (Elinav et aL, 2019), (Helmink et aL, 2019) but only recently it was reported that that each tumor type has a distinct microbiome composition (Nejman et aL, 2020). Most importantly, intratumor bacteria were mostly intracellular and present in both cancer and immune cells and include periodontal bacteria such as Fusobacterium nucleatum, opening an opportunity for tumor type-specific treatment targeting again intracellular bacteria, including periodontal.

[0026] Urinary tract infections (UTI). The intracellular bacteria-mediated antibiotic tolerance where bacteria act as "Trojan horses" play a critical and underappreciated role in the disease burden of bacterial infections (Lewis et aL, 2016), (Liu et aL, 2020), (Vestby et al., 2020). Uropathogenic Escherichia coli can survive within the epithelial bladder cells for months afterinitially infecting (Anderson et al., 2004a; Anderson et al., 2003). To accomplish this feat, uropathogenic E. coli forms intracellular bacterial communities, with many biofilm-like properties, within the bladder epithelium. These bacteria are able to subvert host defenses and form a persistent reservoir in the bladder (Anderson et al., 2004b), (Rosen et al., 2007), (Garofalo et al., 2007), (Anderson et al., 2010), (Goller and Seed, 2010), (Horvath et al., 2011), (Bokil et al., 2011), (Robino et al., 2013). Urine specimens obtained from human female volunteers with or without an active urinary tract infection were found to contain significant number of uncultivable bacteria (Anderson et al., 2004c).

[0027] We were also the first to isolate and identify previously uncultivable bacterial pathogens, mostly periodontal species, i.e., therapeutic targets, from diseased vascular tissue from patients (Rafferty et al., 2011a; Rafferty et al., 2011b), confirming earlier data where we used genomic analysis approaches (Kozarov et al., 2006).

[0028] This discovery opens the opportunity to discover the small molecule / drug sensitivity of the identified intracellular pathogens, many with periodontal origin. Intracellular location shields bacteria from most antimicrobials, leading to persistent inflammation, presented as a variety of chronic debilitating diseases including but not limited to vascular pathologies [atherosclerosis, infectious endocarditis (IE)], reactive or rheumatoid arthritis (RA), brain disorders (Alzheimer's, multiple sclerosis), ocular disorders (retinal disorders, keratitis, glaucoma) and preterm delivery / low birth weight (PTD). This made possible the identification of new antimicrobial drugs (with a low inhibitory concentration, IC50), specific for intracellular bacteria such as periodontal pathogens.

[0029] The intracellular bacteria-mediated antibiotic tolerance where bacteria act as "Trojan horses" play a critical and underappreciated role in the disease burden of bacterial infections (Liu et al., 2020). Antibiotic therapy of intracellular bacteria has sometimes been successful for respiratory species (Legionella pneumophila, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Chlamydia psittaci and Coxiella burnetii) (Lamoth and Greub, 2010), Tropheryma whipplei (Moos and Schneider, 2011), Listeria monocytogenes (Carryn et al., 2003). Few examples are also found for other bacteria from Mycobacterium, Helicobacter, Bordetella, Staphylococcus (Yang et al., 2017), (Ghosh et al., 2017), (Kamaruzzaman et al., 2017), (Cai et al., 2018), Salmonella (Ellis et al., 2019; Tuli and Sharma, 2019), Streptococcus, Anaplasma, Bacillus, Pseudomonas, Escherichia, Neisseria (Bravo-Santano et al., 2019) and others (US15 / 380,648) but not for periodontal species (Eick and Pfister, 2004) or in primary human epithelial and smoothmuscle cells or synovial cells (for atherosclerosis and arthritis drugs, resp.), main targets as will be described herein in the current application.

[0030] Determination of the most efficient inhibitors of intracellular bacteria has been demonstrated in in vitro cell-based systems using high-throughput screening (HTS) of drug, chemical and peptide libraries. The cell lines used were murine bone marrow-derived macrophages (Lieberman and Higgins, 2009), Vero cells (African green monkey), MDCK cells (canine), HEK 293T (human embryonic kidney cells), J774.1 mouse macrophage cell line [(Madrid et al., 2013) and US Application US15 / 380,648], HeLa human cervical cancer cells, human pulmonary adenocarcinoma epithelial 549 cells, RAW264.7 mouse leukemia cells (Wu et al., 2017), RAW264.7 murine leukemia cells (Ellis et al., 2019) and KB immortalized epithelial cells (Eick and Pfister, 2004). Drugs that are not antibiotics can be effective in inhibiting intracellular bacterial growth in leukemic monocytes THP-1 cells (Czyz et al., 2014).

[0031] Accordingly, the current application as described here in relates in some embodiments to screening methods for inhibitors of intracellular bacterial infection using disease-specific cell lines that have not been reported above. In some embodiments, the disease-specific cell lines are primary human endothelial and smooth muscle cells for atherosclerosis drugs, or human synovial fibroblasts (SW-982) for anti-arthritis drugs.

[0032] As described here, the target intracellular pathogens we focus on have not been covered. In some embodiments, the target intracellular pathogens include, but are not limited to, periodontal bacteria such as Porphyromonas gingivalis.

[0033] Even while uncultivable as they often are in intracellular location, bacteria continue to produce proteins at a surprisingly constant rate (Balaban et al., 2013), (Gefen et al., 2014). This constant activity stationary phase is a rare opportunity to identify small molecules - inhibitors of intracellular bacteria. Identification of specific for the identified pathogens immune response boosters / modulators enhancing bacterial killing using in vitro screening system can also be employed (Martins, 2011).

[0034] A global approach that targets the microbiome in inflamed tissues such as arterial, joint, brain or ocular tissue is lacking, in contrast to the recognition of the oral, gut and skin microbiome (Sansonetti, 2008). Here we intend to extend for the first time the microbiome concept for treatment of atherosclerosis, and of other chronic inflammations, including but not limited to arthritides, Alzheimer's, periodontitis and others listed herein. Such knowledge willprovide manageable targets of intervention as the basis for an entirely new approach to the prevention and treatment of chronic inflammations.

[0035] As an intermediate stage before clinical trials, formulation and preclinical studies using animal model of disease are being employed, as described (Tonomura et al., 2008).

[0036] Another major obstacle for early antibiotic clinical trials of patients with chronic vascular or other inflammations has been the lack of accurate and rapid diagnosis, exacerbated by the difficulty of monitoring the effects of the therapy. A discovery of biomarkers for vascular infection will have a crucial impact on the diagnosis, risk stratification and management of individuals with chronic inflammatory disease such as atherosclerosis. Once identified, infection agents can be targeted with individualized treatment regimen. A similar approach will be applied to the other conditions listed herein.

[0037] Human transcriptome contains circulating markers of systemic inflammation. Most importantly, pathogen-specific host transcriptome responses have already been reported, the presence of specific microbial "signatures" in human phagocytes has been shown in 2002 at Whitehead Institute (Huang et al., 2001; Nau et al., 2002), and also at Stanford, Baylor, Weill Cornell and Los Alamos (Boldrick et al., 2002; Chaussabel et al., 2005; Schnappinger et al., 2006; Zeytun et aL, 2007). Most importantly, it has been shown that during surveillance, peripheral blood monocytic cells (PBMCs) respond to a broad range of pathogens with a robust, shared pattern of gene expression (Nau et al., 2002), turning this approach to a promising field for biomarker and diagnostics developments. The transcriptional signature for different biologically significant pathogens bears stark differences (Zhang et al., 2010). Furthermore, it has been demonstrated that the transcriptome contains markers of AS (Kang et al., 2006; Patino et al., 2005), (Patino et al., 2006), (Llorente-Cortes et aL, 2014), (de Gonzalo-Calvo et al., 2014), (Gratchev et al., 2013), (Orekhov et al., 2015)

[0038] However no species-specific molecular signature investigation has been reported and no application in minimally invasive testing is under development.

[0039] The area is ripened to bear fruit since differentially expressed "signatures" have been also demonstrated in other areas, such as in peripheral blood of H7N9 flu patients (Mei et al., 2014). Related, lymphoid gene expression signature was found to correlate with a person's likelihood of experiencing adverse events after receiving a seasonal flu vaccine (Sobolev et al., 2016). Yet there has been little advancement in this critical area since then (Sim et al., 2008), apparently due to the lack of data on association of infection agents with atheromas. However,when we added to this solid experimental base our discovery of live pathogens in arterial atherosclerotic lesions, coupled with latest advancements in high-throughput (HT) omic technologies (RNASeq, high-density arrays), propelled our ground-breaking idea of an expedited approach to CVD diagnosis to a realistic plan of action.

[0040] Unique feature of the approach is the ability to detect a subclinical infection, such as in chronic inflammations. In a study of typhoid fever patients from Vietnam, Salmonella enterica serovar Typhi induced a distinct reproducible signature in the peripheral blood that changed during treatment and convalescence, returning in the majority of cases to the "normal" profile as measured in healthy uninfected controls (Thompson et al., 2009). Strikingly, a distinct signature of convalescence appeared at day 9 after infection and remained unchanged one month after the acute infection. The signature persisted in some cases as long as nine months despite a complete clinical recovery, suggesting a carrier state with a subclinical infection.

[0041] In another example of RNA biomarkers associated with infection, a whole-blood 393- transcript signature for active tuberculosis (TB) infection was identified (Berry et al., 2010). It correlated with the radiological extent of disease and reverted to that of healthy controls after treatment and included a specific 86-transcript signature discriminating active TB from other inflammatory and infectious diseases. Notably, similarly to the S. enterica study, there was a subgroup of latent patients having a transcriptional signature similar to that of active TB; the proportion of latent patients having this signature was equal to the expected frequency of patients at risk of progression to active disease, potentially identifying latent patients with subclinical disease or higher burden latent infection (Berry et al., 2010).

[0042] Circulating DNA markers of apoptosis and neutrophil extracellular trap formation have recently also been independently associated with coronary artery disease, prothrombotic state, and adverse cardiac events (Borissoff et al., 2013). However, there is an overall paucity of studies focusing on nucleic acid markers of infection in atherosclerosis, brain disorders, arthritides, ocular, PTD and other chronic infections.

[0043] Accordingly, an important feature of the current invention is to identify and then validate by quantitative RT-PCR analysis the monocyte transcriptional response to infection with the clinically important bacterial species we cultivated from atheromatous tissues. Using standard RNA Seq transcriptomics and multivariate analysis, these pathogen-specific transcriptome signatures ( PSTS), host biomarkers of disease can be identified and then validated by quantitative RT-PCR analysis, including with patient specimens. Next, signature probes can bedesigned and Agilent spotted e-array service can be used to create, test, optimize and validate in in vitro analyses a prototype theranostic microdevice. Using this array with printed PSTS (and any other, such as 16S rRNA and multiple conserved specific for wide range of pathogens probes), infection agents residing in chronic subclinical infection sites including but not limited to atherosclerosis can be identified quickly using a blood sample. For CNS (Alzheimer's, multiple sclerosis) or joint inflammations (arthritides) including infectious (Reiter's) arthritis; this could be CSF or synovial fluid, respectively. The device will be readied for the market using a pilot trial and then a larger scale case-control validation with ASVD patient RNA specimens from a cohort of healthy and diseased clinically annotated individuals (a collection of 2,000 RNAs is now available).

[0044] One of the challenges in clinical infectious diseases is the problem of chronic infections, which can require long durations of antibiotic treatment and often recur. In addition, they are often unperturbed by host immune responses such as phagocytosis, despite a sustained presence of host inflammation. These infections include chronic and recurrent otitis media, chronic wounds and vascular infections, infected joints, dental plaque, ocular infections, implant-, catheter- or prosthesis-associated biofilm infections, and urinary tract infections, among others. An emerging explanation for the refractoriness of some infections to treatment is the existence - often in biofilm - of subpopulations of drug tolerant cells (Vestby et al., 2020), (Anderson and O'Toole, 2008), (Kester and Fortune, 2014). Typically discussed as "persisters", the biofilm bacteria are 100 to 1,000 times more tolerant to antimicrobials than corresponding cultivable cells (Lewis et al., 2016; Lewis, 2000, 2007, 2008, 2012, 2016), (Hoiby et al., 2010; Hoiby et aL, 2011; Olsen, 2015). Antibiotic tolerance, the capacity of genetically susceptible bacteria to survive multiple drug treatment, plays a critical and underappreciated role in the disease burden of bacterial infections (Meylan et al., 2018). Biofilms are recalcitrant to antibiotic treatment due to multiple tolerance mechanisms (phenotypic resistance) of biofilm bacteria, causing persistence of biofilm infections in spite of antibiotic exposure. Thus, the phenotypic drug resistance (tolerance) predisposes to genetic drug resistance development (Ciofu et al., 2017). Non-growing uncultivable bacteria that survive both antibiotics and host immune responses persist at the infected site, thereby most likely contributing to the relapse of many infections (Gollan et aL, 2019). These uncultivable persisting bacteria survive exposure to (aka tolerate) high concentrations of antibiotic, compromise successful antibiotic therapy of bacterial infections and are thought to contribute to the development of antibiotic resistance. In addition,drug-tolerant persisters have also been identified as a factor underlying failure of chemotherapy in tumor cell populations (Michiels et al., 2016). Thus, new approaches are urgently needed (Wilkins et al., 2014).

[0045] Our latest data using clinical periodontal specimens of complex disease biofilm demonstrate i) the presence in biofilm of uncultivable bacterial pathogens, including anaerobes, ii) the resistance of these uncultivable bacteria to a cocktail of six antibiotics that kills all cultivable bacterial species, and iii) our ability to cultivate ("resuscitate") these uncultivable multiple drug resistant (MDR) species, allowing for the identification of the pathogens underlying drug resistant infections and the study of their virulence and drug resistance determinants in order to inform a treatment strategy. A number of compounds may counteract the MDR bacteria by targeting indispensable cellular processes or by activating resuscitation (cultivation) (Sanz and van Winkelhoff, 2011).

[0046] EXAMPLES:

[0047] Example: 1

[0048] Porphyromonas qinqivalis (Pg) Infection and High fat diet (HFD) induced atherosclerosis in ApoE- / - mouse (Study NO: MAGN-20240125)

[0049] Study Protocol: Porphyromonqs gingivqlis (Pg) Infection and High fat diet (HFD) induced atherosclerosis in ApoE- / - mouse

[0050] This study was conducted at WuXi AppTec Co., Ltd., (hereafter WuXi) and in accordance with the WuXi IACUC standard animal procedures along with the IACUC guidelines that are in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare (OLAW). This activity was conducted as a non-GLP activity. The study was conducted in accordance with this protocol and the associated study specific procedures, with applicable WuXi Standard Operating Procedures (SOPs) and generally recognized good laboratory practices. 1.4 Proposed in-life Study Schedule

[0051] The In-life study had a duration of 15 weeks after animals were ready with a start date ofApril 11 2024 and an ed date of September 23 2024

[0052] Test Article and Vehicle Information

[0053] The test articles was supplied by the Sponsor. Test article characterization (purity, identify, composition, and strength) was documented by the sponsor. Test article description and handling procedures, as well as other pertinent information was documented as part of the study data.

[0054] Compounds Information: The Products TAI and TA2 were provided by Magnacor LLC as the supplier. The vehicle was saline.

[0055] TA preparation: Dissolved TAI, TA2 in saline to prepare the 10 mg / ml stock solution. Thus, for each 100 ul injection with 1 mg TAI or 2, Adjust TA solution to PH8.

[0056] Study design:

[0057] Instruments: Centrifuge (model 5424R, supplied by Eppendorf); Electronic scales (model SOP, supplied by Sartorius Scientific Instruments (Beijing) Co., Ltd, Electronic scales, YH-2000, supplied by Changzhou Tianzhiping instrument and equipment Co., Ltd., Thermostatic magnetic stirrer (85-2, supplied by Shanghai Sile Instrument Co., LTD. Rockers (TS-1, supplied by Haimen Qilin Medical Instrument Factory.

[0058] Reagents: 0.9% Sodium Chloride Injection, Batch No. 20220228, supplied by Anhui Fengyuan Pharmaceutical Co., Ltd. HFD(D12079B), Bach No: 230515071, supplied by Research Diets.

[0059] Animals: ApoE- / - mice, level -SPF, age (ordered) -3 weeks old; sex -male; source - Vital River Laboratory Animal Technology Co., Ltd., Beijing China; Method of Identification -Ear marker; Animal Number: 40

[0060] The Male ApoE- / - mice obtained from Vital River Laboratory Animal Technology Co., Ltd, after arriving at Wuxi, the animal room environment was controlled (target conditions: temperature 20 to 24°C, relative humidity 40 to 70%). Temperature and relative humidity was monitored daily. An electronic time-controlled lighting system was used to provide a 12 hour I ight / 12 hour dark cycle. Animal were housed in plastic cage which is in accordance with the National Research Council "Guide for the Care and Use of Laboratory Animals". Enrichment toys were provided. All mice were single-housed throughout the study. After arrival, the animals were acclimated to environment for 1 week.

[0061] Animals were grouped into four groups and treated as follows:

[0062] Group 1: Treatment: HFD + Vehicle, Number of animals: 10; Dose (mg / kg): Vehicle (Saline, lOOuL / mouse); dose route (IP), Schedule: 23 weeks;

[0063] Group 2: Treatment: HFD + Pg (i.v.); Number of animals: 10; Dose (mg / kg): Vehicle (Saline 100pL / mouse)); dose route (IP), Schedule: 23 weeks;

[0064] Group 3: Treatment: HFD+TA1: CDI-MAGN001; Number of animals: 10; Dose (mg / kg): 1 mg / shot, each 6 hours (1 mg / shot, each 6 hours).; dose route (IP), Schedule: 23 weeks;

[0065] Group 4: Treatment: HFD+TA2: CDI-MAGN002; Number of animals: 10; Dose (mg / kg): 1 mg / shot, each 6 hours (1 mg / shot, each 6 hours).; dose route (IP), Schedule: 23 weeks;

[0066] After acclimation, 40 male ApoE- / - weaned at 4 weeks of age are assigned to a high-fat diet (HFD) (Western diet, D12079B) (n=40). At 14 weeks of age, the mice are randomly divided into 4 groups (n=10 / group) as follows:

[0067] Group 1: 10 wks HFD only, then 10 wks vehicle once a week, then 2 weeks of 50 100 ul i.p. injections of control solution saline, then sacrifice. Total of 23 weeks.

[0068] Group 2: 10 wks HFD only, then 10 wks Pg + vehicle in tail vein once a week, then 2 weeks of 100 ul i.p. injections of control solution saline, then sacrifice. Total of 23 weeks.

[0069] Group 3: 10 wks HFD only, then 10 wks Pg + vehicle in tail vein, then 2 weeks TAI Tx i.p. (without Pg), then sacrifice. Total of 23 weeks.

[0070] Group 4: 10 wks HFD only, then 10 wks Pg + vehicle in tail vein, then 2 weeks TA2 Tx i.p. (without Pg), then sacrifice. Total of 23 weeks.

[0071] At week 11. Iv. inoculation is with live Pg (FDC381 50 uL / mouse) or vehicle containing diluted medium (50 uL / mouse). The inoculation is performed intravenously once per week for 10 consecutive weeks via intravenous administration in the tail vein.

[0072] Intraperitoneal treatment with TAI and TA2 starts to be administered in G3 and G4 mice at the week 21. (Pg is no longer used). Both G3 and G4 are treated for 2 weeks. Four 100 ul shots per day (1 mg TAI or TA2 / shot, each 6 hours) are administered. Both TAI and TA2 are dissolved in saline and inject peritoneally.

[0073] All 4 groups are on HFD, ALL the time (no standard chow).

[0074] After mice are sacrificed, the post-life quantitative studies are performed.

[0075] Body weight of mice are monitored at every week.

[0076] Post-Life Tests:

[0077] Exploratory Serum Biomarkers:

[0078] Blood was be collected and stand for 2 hours; serum is separated by centrifuge at 7000 rpm 4°C for 10 min. Then quickly froze in dry ice, stored in -80°C till transfer to the vitro team for determination. Serum biochemistry of Serum I Lip and serum amyloid A are determined by using ELISA Kit.

[0079] Atherosclerotic plaques-Oil Red O staining in whole aortic roots:

[0080] First, aortic collection. The mice were euthanasia by carbon dioxide, Open the chest and abdominal cavity. Discarded liver, gastrointestinal tract and other organs, expose the aorta from the heart to the iliac artery. Carefully isolate the mouse aorta and remove fat from aorta surface. Open the aorta from the arch of the aorta to the iliac artery, the branches of the aortic arch (innominate artery, left common carotid artery, left subclavian artery) were cut successively.

[0081] Second, Oil-red O staining. The inner surface of the aorta was upward, fasten to the black dissecting tray with capillary needle, the aortas were fixed for 30 min in 4% paraformaldehyde.

[0082] Removed 4% paraformaldehyde, then, add 60% isopropanol and shake for 10 min. Removed 60% isopropanol, add Oil-red O stock solution (Oil-red O: ddH2O=6:4) and shake for 30-60 min. Eluted with 60% isopropanol for 3 times, 10 min / time. Eluted with ddH2O for 3 times, 10 min / time.

[0083] Third, Aorta photograph analysis. Finally, Images were captured by use of a canon camera and analyzed by a single technician who was blinded to the study protocol and used Image J analysis software.

[0084] Data Processing and Analysis:

[0085] Manual data was transferred into an excel spreadsheet. All values were expressed as mean ± SEM. The significances of the differences among groups and within groups was evaluated by T test or one-way ANOVA followed by Dunnett's test using Graph Pad statistic software. A p value of less than 0.05 is considered statistically significant.

[0086] Study protocol and Final report; Excel summary of data

[0087] Example 2:

[0088] Minimum Inhibitory Concentration Screen

[0089] Part A: MIC test Primary compound screening

[0090] 1. Strain:

[0091] Strain name FDC381

[0092] Porphyromonas gingivalis

[0093] 2. Procedures

[0094] 2.1 Bacterial stock solution preparation

[0095] 2.1.1 Grew P. gingivalis glycerol stock in supplemented TSB (Tryptic soy broth + 5mg / ml yeast

[0096] extract +0.5mg / ml L-cys+5pg / ml hemin+2pg / ml vitaminKl) 48h anaerobically. This was the

[0097] primary culture.

[0098] 2.1.2 Sub-cultured the primary culture into new supplemented TSB and incubated 6h

[0099] anaerobically. Collected bacteria pellet by centrifuging and re-suspended in supplemented TSB with 20% glycerol to have bacterial concentration ~l*10 9 CFU / ml.

[0100] 2.1.3 Stored them -80°C as the bacterial stock solution for use.

[0101] 2.1.4 The actual bacteria concentration was determined by CFU counting assay.

[0102] 2.2 Compound testing plate preparation

[0103] 2.2.1 The stock solution of reference metronidazole was manually diluted to 0.2, 0.1,0.05, 0.025, 0.0125, 0.006, 0.003, 0.0016 mg / mL in 100% DMSO in a 96-well V plate followed by transferring 40 pl to a source plate.

[0104] 2.1.2 Transferred 0.5 ul of metronidazole (0.2, 0.1, 0.05, 0.025, 00125, 0.006, 0.003,0.0016 mg / mL) and 10 mM compounds per well from the source plates to the 384-well testing plate by ECH0655. Each concentration was done in duplicate. Stored them at -20°C for use.

[0105] 2.1.3 On the day of the testing, added 25pl testing medium (Tryptic soy broth +5mg / ml yeast extract+0.5mg / ml L-cys+5pg / ml hemin+2ug / ml vitaminKl) to the 384-well testing plates prepared in the step 2.1.2 and pre-reduced in the anaerobic chamber for at least 2 h.

[0106] 2.3 Addition of bacteria

[0107] 2.3.1 Thawed the bacterial stock solution prepared in the step 2.1 and diluted strain stock solution with the pre-reduced testing medium to a concentration of ~2xl06 CFU / mL in anaerobic chamber.

[0108] This was inoculum.

[0109] 2.3.2 Added 25 pl inoculum into each 384-well testing plate to get a final concentration of ~lxl0 6 CFU / mL. Growth control wells only containl% DMSO and bacteria. Blank wells only contain testing medium.

[0110] 2.4 Incubation

[0111] 2.4.1 Incubated all the testing plates at 37°C, anaerobically for 72h.

[0112] 2.5 Endpoint

[0113] 2.5.1 Measured the testing plates at OD600nm by microplate reader and calculated the inhibition rate (%), compared to the growth control. Observed the MIC value of positive control metronidazole by naked eyes.

[0114] Part B: MIC test Secondary compound screening

[0115] 1. Strains

[0116] ATCC Strain name FDC381

[0117] P. gingivalis

[0118] 2. Procedures

[0119] 2.1 Bacterial stock solution preparation

[0120] 2.1.1 Grew P. gingivalis glycerol stock in supplemented TSB (Tryptic soy broth + 5mg / ml yeast extract +0.5mg / ml L-cys+5pg / ml hemin+2pg / ml vitaminKl) 48h anaerobically. This was the primary culture.

[0121] 2.1.2 Sub-cultured the primary culture into new supplemented TSB and incubated 6h

[0122] anaerobically. Collected bacteria pellet by centrifuging and re-suspended in supplemented TSB with 20% glycerol to have bacterial concentration ~l*10 9 CFU / ml.

[0123] 2.1.3 Stored them -80°C as the bacterial stock solution for use.

[0124] 2.1.4 The actual bacteria concentration was determined by CFU counting assay.

[0125] 2.2 Compounds and metronidazole preparation

[0126] 2.2.1 The compounds were transferred from the source plate to the 385-well testing plate according to the layout below by ECHO655. The volume of compounds per well was 0.5 pl.

[0127] 2.2.2 The stock solution of reference metronidazole was manually diluted to 0.2, 0.1,0.05, 0.025, 0.0125, 0.006, 0.003, 0.0016 mg / mL in 100% DMSO in a 96-well V plate followed by transferring 40 pl to a source plate. Transferred 0.5 pl of metronidazole (0.2, 0.1, 0.05, 0.025, 0.0125, 0.006, 0.003, 0.0016 mg / mL) per well from the source plates to the 384-well testing plate by ECHO655. 2.2.3. Each concentration of compounds and metronidazole was done in a single test. Stored all the 384-well testing plate prepared above at -20°C for use.

[0128] 2.2.4 On the day of the testing, added 25pl testing medium (Tryptic soy broth +5mg / ml yeast extract +0.5mg / ml L-cys+5pg / ml hemin+2ug / ml vitaminKl) to the 384-well testing plates prepared in the step 2.2.3 and pre-reduced in the anaerobic chamber for at least 2 h.

[0129] 1234567891011.0012.00131415161718192021222324

[0130] 1A

[0131] 2Bcompoundsl00502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0132] 3C100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0133] 4D100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0134] 5E100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0135] 6F100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0136] 7G100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0137] 8H100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0138] 91100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0139] 10J100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0140] 11K100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0141] 12L100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0142] 13M100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0143] 14N100502512.56.253.131.560.780.390.200.10100502512.56.253.131.560.780.390.20.1

[0144] 150210.50.250.130.060.030.020.010.000.00GCGCGCGCGCGCBIankBlankBlankBlankBlan k

[0145] 16P

[0146] O2-O12Metronidazole(pg / ml)

[0147] O13-O18GC: Growth Control

[0148] O19-O23BK: Blank Testing compounds (100-0.1 pM)

[0149] 2.3 Addition of bacteria

[0150] 2.3.1 Thawed the bacterial stock solution prepared in the step 2.1 and diluted strain stock solution with the pre-reduced testing medium to a concentration of ~2xl06 CFU / mL in anaerobic chamber.

[0151] This was inoculum.

[0152] 2.3.2 Added 25 pl inoculum into each 384-well testing plate to get a final concentration of ~lxl0 6 CFU / mL. Growth control wells only containl% DMSO and bacteria. Blank wells only contain testing medium.

[0153] 2.4 Incubation

[0154] 2.4.1 Incubated all the testing plates at 37°C, anaerobically for 72h.

[0155] 2.5 Endpoint

[0156] 2.5.1 The MIC was read and recorded as the lowest concentration of each agent that completely inhibits visible growth of the microorganism after incubation. A magnifying mirror device was used for ease of scoring the presence or absence of growth in the wells.

[0157] Example 3:

[0158] WuXi P. qinqivalis Biofilm Assay:

[0159] Prepare the inoculum:

[0160] P. gingivalis FDC381 glycerol stock is inoculated into supplemented TSB (Tryptic soy broth +5 mg / ml yeast extract+0.5 mg / ml L-cys+5 pg / ml hemin+2 pg / ml vitaminKl) and grow anaerobically for ~48 h until reaching the stationary phase. Collect bacteria pellets from the culture by centrifuging and re-suspend the pellets in pre-reduced testing medium dTSB (supplemented TSB / PBS ratio, 1:2). Adjust the bacterial density to 0.7~l*108 CFU / ml. This is the inoculum.

[0161] Pre-coat the microplate with artificial saliva:

[0162] The 96-well polystyrene microplates are pre-coated with artificial saliva, 45~50 pl per well at room temperature for 1 h followed by discarding the artificial saliva. The coated microplates were air-dried for use.

[0163] Prepare the compound dilutions

[0164] Serial 2-fold compound dilutions are prepared in DMSO, and 1.5 pl of each dilution is added into the well of pre-coated 96-well plate (section 2). For each compound, 9 doses in triplicates are tested. 148.5 pl inoculum (section 1) is then added to the compound plate. It is incubated anaerobically at 37 °Cfor 72 h. Moxifloxacin is used as a positive control.

[0165] Biofilm measurement

[0166] After incubation, the culture is discarded, and the plate is washed three times with water. Then, 200 pl 0.1% crystal violet (CV) is added into the 96-well plate. Stain the biofilm for 10 min at room temperature, followed by washing with water for 3 times. Then, 200 pl 30% acetic acid is added to elute the CV (biofilm) by incubating at room temperature for 10 min. OD550 is measured to quantify the biofilm biomass.

[0167] Artificial Saliva:

[0168] 1 L contained 0.7 g sodium chloride, 0.26 g disodium phosphate, 0.33 g potassium thiocyanate, 1.2 g potassium dihydrogen phosphate, 1.5 g sodium hydrogen carbonate and 1.2 g potassium chloride: supplemented with 4 g porcine mucin and 50 g albumin. Sterilize before use.

[0169] Table 1: Study Timeline:

[0170] Results:

[0171] MIC screen of 14,600 compounds yielded 115 compounds inhibiting planktonic P. gingivitis. A cytotoxicity screen of these 115 compounds yielded 85 compounds non-toxic to primary human coronary artery endothelial cells. A screen for inhibitory activity of 25 (out of those 85) against intracellular P. gingivitis at 2-hour treatment yielded 2 drug candidates (TAI, TA2).

[0172] Another screen of 6 compounds at 6 hour treatment yielded 3 more drug candidates (TA3, TA4, Ta5). This demonstrated potentially much higher activity of the selected 85 compounds at 6 hours compared to 2 hours. 4,600 compound libraries including anti-infectives and FDA-approved drugs.

[0173] Results showed inhibition of intracellular bacteria internalized into primary human coronary artery endothelial cells upon treatment with TAI and TA2. Figure 2 for example shows inhibition of intracellular bacteria internalized into primary human coronary artery endothelial cells upon treatment with compound 0342-0049 ("TAI", Penicillin V). Figure 3 shows inhibition of intracellular bacteria internalized into primary human coronary artery endothelial cells upon treatment with compound Y031-9897 ("TA2"). Figure 4 shows inhibition of intracellular bacteria internalized into primary human coronary artery endothelial cells upon treatment with compound Y031-9897 ("TA2).

[0174] In addition, there was atherosclerotic plaque reduction in Porphyromonas gingivalis- exacerbated atherosclerosis in ApoE - / - murine model of atherosclerosis.

[0175] Both TAI and Ta2 reduced aortic plaque areas as well as IL-lbeta expression in ApoE - / - mice infected with Porphyromonas gingivalis, suggesting their potential efficacy in reducing atherosclerosis.

[0176] Table 2 below lists several molecules with particularly effective MIC with respect to our CVD model

[0177] Table 2:

[0178] Tab e 3 below 1st several promising drug candidates with respect to inhibition of P. gingiva I is in biofilm.

[0179] Table 3

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Claims

Claims1. A method for treating or preventing a chronic inflammatory disease in an individual, the method comprising administering a therapeutically effective amount of an inhibitor to the individual, wherein the inhibitor reduces or prevents an intracellular pathogen or its activity, or reduces or inhibits a drug-resistant biofilm-associated pathogen, thereby treating or preventing the chronic inflammatory disease resulting from the intracellular or biofilm-associated pathogen.

2. The method of claim 1, wherein the chronic inflammatory disease is atherosclerosis, infectious endocarditis (IE), reactive or rheumatoid arthritis (RA), a neurological disease selected from the group of Alzheimer's disease and multiple sclerosis, an ocular disease selected from the group of a retinal disorder, keratitis, glaucoma, preterm delivery / low birth weight (PDLBW), periodontitis, a urinary tract infection (UTI), or a biofilm-associated infection such as chronic wound infection selected from the group of a diabetic foot ulcer, a pressure ulcer (bedsore), a venous leg ulcer, and a battlefield wound, a chronic respiratory infection selected form the group of a cystic fibrosis-associated lung infection, a chronic obstructive pulmonary disease (COPD)-related infection, sinusitis, a UTI, an infection in patients with catheters, a heart valve infection associated with biofilms on native or prosthetic heart valves, osteomyelitis (bone infection), a prosthetic joint infection selected from the group of knee and hip implant infections, pacemaker and defibrillator infections, infections of intrauterine devices (IUDs), and a chronic middle ear infection (otitis media).

3. The method of claim 1, wherein the intracellular pathogen is selected from the group of Porphyromonas gingival is, Aggregatibacter actinomycetemcomitans, Tannerella forsythus, Treponema denticola, Eikenella corrodens, Prevotella intermedia, Prevotella nigrescens, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Pseudomonas, Legionella, Chlamydophila, Coxiella, Listeria, Enterobacter, Staphylococcus, Streptococcus, Enterobacter, Propionibacterium and Porphyromonas genus species.

4. The method of claim 1, wherein the intracellular pathogen or drug-resistant biofilm-associated pathogen is Porphyromonas gingivalis.

5. The method of claim 4, wherein the intracellular pathogen is internalized in primary human coronary endothelial cells.

6. The method of claim 1, wherein the inhibitor is 3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4- thia-l-azabicyclo[3.2.0]heptane-2-carboxylate.

7. The method of claim 1, wherein the inhibitor is 4-acetamido-N-(5-nitro-l,3-thiazol-2-yl) benzamide.

8. The method of Claim 1, where the inhibitor of the intracellular pathogen is l-cyclopropyl-6- fluoro-8-methoxy-7-[3-methyl-4-(methylcarbamothioyl)piperazin-l-yl]-4-oxo-l,4- dihydroquinoline-3-carboxylic acid.

9. The method of claim 1, wherein the inhibitor of the intracellular pathogen is 3-{[(5-nitrofuran-2- yl)methylidene]amino}-l,3-oxazolidin-2-one.

10. The method of claim 1, wherein the inhibitor of the intracellular pathogen is 2-(4- ethoxyphenoxy)-N-[l-(5-ethyl-4-methyl-6-oxo-l,6-dihydropyrimidin-2-yl)-3-methyl-lH-pyrazol- 5-yl]acetamide.

11. The method of claim 1, wherein the inhibitor of the pathogen associated with a drug-resistant biofilm is l-cyclopropyl-6-fluoro-8-methoxy-7-(3-methylpiperazin-l-yl)-4-oxo-l,4- dihydroquinoline-3-carboxylic acid.

12. The method of claim 1, wherein the inhibitor of the pathogen associated with a drug-resistant biofilm is 7-(4-acetyl-3-methylpiperazin-l-yl)-l-cyclopropyl-6-fluoro-8-methoxy-4-oxo-l,4- dihydroquinoline-3-carboxylic acid.

13. The method of claim 1, wherein the inhibitor of the pathogen associated with a drug-resistant biofilm is 3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-l-azabicyclo[3.2.0]heptane-2- carboxylate.

14. The method of claim 1, wherein the inhibitor of the pathogen associated with a drug-resistant biofilm is 7-{[(2Z)-2-(2-amino-l,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino}-3-{[(6-hydroxy-2- methyl-5-oxo-2,5-dihydro-l,2,4-triazin-3-yl)sulfanyl]methyl}-8-oxo-5-thia-l-azabicyclo[4.2.0]oct- 2-ene-2-carboxylate.

15. The method of claim 1, wherein the inhibitor of the pathogen associated with a drug-resistant biofilm is 6-{[3-(2,6-dichlorophenyl)-5-methyl-l,2-oxazole-4-carbonyl]amino}-3,3-dimethyl-7- oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate.

16. The method of claim 1, wherein the inhibitor of the pathogen associated with a drug-resistant biofilm is l-(3',5'-dimethyl[l,l'-biphenyl]-4-yl)-N-{2-[(propan-2-yl)oxy]ethyl}-lH-imidazole-5- carboxamide.

17. The method of claim 1, wherein the individual is a human.

18. The method of claim 1, wherein the drug-resistant biofilm-associated pathogen is an intracellular pathogen.

19. The method of claim 18, wherein the intracellular drug-resistant biofilm-associated pathogen is Porphyromonas gingivalis.

20. A method of screening for an inhibitor which reduces or prevents intracellular pathogen formation or activity or reduces or inhibits a drug-resistant biofilm-associated pathogen, which includes the steps of (i) obtaining a library of candidate drugs and / or drug analogues, (ii) obtaining a sample from an individual having a chronic inflammatory disease and wherein the sample further includes an intracellular pathogen or a drug-resistant biofilm-associated pathogen, (iii) screening for drug candidate(s) which inhibit activity of the intracellular pathogen formation or activity or drug-resistant biofilm-associated pathogen and (iv) selecting the drug candidate(s) which show inhibitory activity against the intracellular pathogen formation or activity of the drug-resistant biofilm-associated pathogen.

21. The method of claim 20, wherein the chronic inflammatory disease tissue includes atherosclerosis, infectious endocarditis (IE), reactive or rheumatoid arthritis (RA), a neurological disease selected from the group of Alzheimer's disease and multiple sclerosis, an ocular disease selected from the group of a retinal disorder, keratitis, and glaucoma, preterm delivery / low birth weight (PDLBW), and urinary tract infections (UTI).

22. The method of claim 21, wherein the intracellular pathogen includes Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythus, Treponema denticola, Eikenella corrodens, Prevotella intermedia, Prevotella nigrescens, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Pseudomonas, Legionella, Chlamydophila, Coxiella, Listeria, Enterobacter, Staphylococcus, Streptococcus, Enterobacter, Propionibacterium and Porphyromonas genus species.

23. The method of claim 21, wherein the intracellular pathogen is internalized in primary human coronary endothelial cells Porphyromonas gingivalis.

24. The method of claim 21, wherein the pathogen associated with a drug-resistant biofilm is Porphyromonas gingivalis.

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