WO2021116420A1

WO2021116420A1 - Use of tlr7 and/or tlr8 agonists for the treatment of leptospirosis

Info

Publication number: WO2021116420A1
Application number: PCT/EP2020/085783
Authority: WO
Inventors: Axel Joseph Perianin; Catherine WERTS; Frédérique VERNEL-PAUILLAC
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Centre National De La Recherche Scientifique (Cnrs); Institut Pasteur; Université de Paris
Priority date: 2019-12-13
Filing date: 2020-12-11
Publication date: 2021-06-17

Abstract

Leptospirosis is a zoonotic disease that is widespread throughout the world and is caused by pathogenic strains of leptospires. It affects more than one million people each year with a patient mortality rate that remains high despite treatment with antibiotics. The inventors evaluated the effects of a TLR7/8 (i.e. R848) agonist in an acute infection murine model of leptospirosis. Strikingly, intraperitoneal administration of the agonist keeps alive mice infected with lethal doses of leptospires. In addition, the agonist strongly inhibits the early proliferation of leptospires as well as their late colonization of the kidneys. In vitro studies show that R848 stimulates ROS production in mouse whole blood and potentiates the production of ROS induced by powerful activators of the oxidative burst. In purified mouse leukocytes, as in human neutrophils, R848 stimulates basal ROS production and potentiates ROS production induced by PMA or leptospires. However, prolonged exposure of human neutrophils to leptospires leads to desensitization of neutrophils characterized by low production of superoxide anion secondarily stimulated by the fMLP. This adverse effect of leptospires is reversed in neutrophils treated with R848. Treatment of mice with R848 also stimulated ROS production by peritoneal neutrophils from mice infected by leptospires. In conclusion, these results show that TLR7/8 agonists can promote the production of antibacterial oxidants in the whole blood of mice and indicate that such compounds would be suitable for treating early infection, which drastically reduces renal colonization by leptospires.

Description

USE OF TLR7 AND/OR TLR8 AGONISTS FOR THE TREATMENT OF

LEPTOSPIROSIS

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular microbiology.

BACKGROUND OF THE INVENTION:

Leptospirosis is a zoonotic disease that is widespread throughout the world and is caused by pathogenic strains of leptospires (1). It affects more than one million people each year with a patient mortality rate that remains high, 5 to 20% (2), despite treatment with antibiotics. In animals, the economical impact is also considerable, the disease being responsible for reproductive problems in farm animals (ruminants or pigs) or for serious clinical signs that impair the health of pets, namely dogs, or that of animals in sport, namely horses.

Leptospirosis involves an initial phase of invasion and proliferation in the blood by bacteria (sepsis) and then their spread to various tissues such as the lungs, liver, brain and kidneys (3-5). The bacteria then disappear from the circulation and-reappear as persistent colonies in the kidneys (6) and impact renal function (7). The bacteria are excreted through the urines. Rats and mice are asymptomatic carriers and the main known reservoir of leptospires. Because of global warming, the disease is re-emerging, associated with floods and rains, favouring the contact of animals or humans with leptospires excreted in the environment. More than 400 serovars of leptospires have been described and available human or animal vaccines (inactivated bacteria) provide only serovar specific and short-term (one year) protection, conferred by neutralizing antibodies.

The persistence of pathogenic leptospires suggests possible failures of the host's antibacterial defense systems. Several examples of evasion of innate immune responses have been described. Due to modified cell wall components, Leptospira are able to escape the complement system, but also the TLR4 receptor in humans, as well as the NODI and NOD2 responses, known to be critical for the host phagocyte functions (8, 9).

Bacteria are normally eliminated by phagocytes, mainly neutrophils and macrophages via several major mechanisms (8) including reactive oxygen species (ROS) (10-12). The latter are produced massively via the NADPH oxidase (NOX2) of neutrophils, a phenomenon called an oxidative burst (12). The activation of NOX2 in neutrophils is triggered instantly when they are in contact with various agents such as bacteria, substances derived from bacteria (N-formylated peptides, LPS, RNA debris) or various pro-inflammatory substances (complement factors, cytokines, chemokines, lipids).

In some pathological situations, the production of ROS by phagocytes is deficient due to various abnormalities, including signaling pathways leading to NOX2 activation and/or NOX2 expression (13-15), which contribute to microbial infection and patient mortality. The inventors have recently shown that these two types of abnormalities can be effectively corrected ex vivo using TLR7/8 agonists in neutrophils from immunocompromised patients with advanced cirrhosis (14,15). This treatment has also been shown to be effective in vivo in a rat cirrhosis model and to reduce infection and mortality in animals (16).

SUMMARY OF THE INVENTION:

As defined by the claims, the present invention relates to a method of treating leptospirosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a TLR7 and/or TLR8 agonist.

DETAILED DESCRIPTION OF THE INVENTION:

The first object of the present invention relates to a method of treating leptospirosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a TLR7 and/or TLR8 agonist.

As used herein, “subject” refers to a mammal, preferably a human. In the present invention, the terms subject and patient may be used with the same meaning. Examples of non-human mammal include a pet such as a dog, a cat, a domesticated pig, a rabbit, a ferret, a hamster, a mouse, a rat and the like; a primate such as a chimp, a monkey, and the like; an economically important animal such as cattle, a pig, a rabbit, a horse, a sheep, a goat. In some embodiments, the subject is an adult (for example a subject above the age of 18). In some embodiments, the subject is a child (for example a subject below the age of 18). In some embodiments, the subject is a male. In some embodiments, the subject is a female.

As used herein, the term "leptospirosis" has its general meaning in the art and refers to infectious disease caused by pathogenic spirochete, which belongs to the genus Leptospira.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

In particular, the TLR 7 or TLR8 agonist is particularly suitable for the treatment of early infections and for preventing renal colonization by leptospires.

As used herein, the expressions “agonist of TLR7” and “agonist of TLR8” refer to compounds which bind to and activate TLR7 or TLR8 respectively. The terms “activation” and “stimulation” are used indifferently. "TLR7 and/or TLR8 agonist” or “TLR7/8 agonist” refers to a molecule that is an agonist of TLR7 only, TLR8 only or both TLR7 and TLR8.

TLR7 and/or TLR8 agonists are well known in the art (Waleed M Hussein , Tzu-Yu Liu , Mariusz Skwarczynski , Istvan Toth. Toll-like receptor agonists: a patent review (2011 - 2013) Expert Opinion on Therapeutic Patents Apr 2014, Vol. 24, No. 4, Pages 453-470: 453- 470). Moreover, methods for identifying agonists of TRL7 or agonists of TLR8 are well known in the art and are described for example in document W02004/075865 (3M Innovative Properties Company). Natural agonists of TLR7 and TLR8 have been identified as guanosine- and uridine- rich ssRNA (Diebold, Science 2004, Heil Science, 2004). In addition, synthetic agonists of TLR7 are described in the prior art.

For example, TLR7 agonists include, but are not limited to: imidazoquinoline-like molecules, imiquimod, resiquimod, gardiquimod, S-27609; and guanosine analogues such as loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine), 7-Thia-8-oxoguanosine and 7- deazaguanosine, UC-1V150, ANA975 (Anadys Pharmaceuticals), SM-360320 (Sumimoto), 3M-01 and 3M-03 (3M Pharmaceuticals) (see for example Gorden et al, J Immunology, 2005; Schon, Oncogene, 2008; Wu et al, PNAS 2007). TLR7 agonists include imidazoquinoline compounds; guanosine analogs; pyrimidinone compounds such as bropirimine and bropirimine analogs; and the like. Imidazoquinoline compounds that function as TLR7 ligands include, but are not limited to, imiquimod, (also known as Aldara, R-837, S- 26308), and R-848 (also known as resiquimod, S-28463; having the chemical structure: 4- amino-2-ethoxymethyl-a, a. -dimethyl- 1 H- imidazol[4,5-c]quinoline-l-ethanol). Suitable imidazoquinoline agents include imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2 bridged imidazoquinoline amines. These compounds have been described in U.S. Patents 4,689,338, 4,929,624, 5,238,944, 5,266,575, 5,268,376, 5,346,905, 5,352,784, 5,389,640, 5,395,937, 5,494,916, 5,482,936, 5,525,612,

6,039,969 and 6,110,929. Particular species of imidazoquinoline agents that are suitable for use in a subject method include R-848 (S-28463); 4-amino-2ethoxymethyl-a, a-dimethyl-lH- imidazo[4,5-c]quinoline-s-i-ethanol; and l-(2-methylpropyl)-lH-imidazo[4,5-c]quinolin-4- amine (R-837 or Imiquimod). Also suitable for use is the compound 4-amino-2- (ethoxymethyl)-a, a-dimethyl-6, 7,8,9- tetrahydro-lH-imidazo[4,5-c]quinoline-l-ethanol hydrate (see, e.g., BM-003 in Gorden et al. (2005). Guanosine analogs that function as TLR7 ligands include certain C8-substituted and N7,C8-disubstituted guanine ribonucleotides and deoxyribonucleotides, including, but not limited to, Loxoribine (7-allyl-8-oxoguanosine), 7- thia-8-oxo-guanosine (TOG), 7- deazaguanosine, and 7-deazadeoxy guanosine (Lee et ah, 2003). Bropirimine (PNU-54461), a 5-halo-6-phenyl-pyrimidinone, and bropirimine analogs are described in the literature and are also suitable for use. See, e.g., Vroegop et al. (1999). Additional examples of suitable C8- substituted guanosines include but are not limited to 8- mercaptoguanosine, 8- bromoguanosine, 8-methylguanosine, 8-oxo-7,8-dihydroguanosine, C8-arylamino-2'- deoxyguanosine, C8-propynyl-guanosine, C8- and N7-substituted guanine ribonucleosides such as 7-allyl-8-oxoguanosine (loxoribine) and 7-methyl-8-oxoguanosine, 8- aminoguanosine, 8-hydroxy-2'-deoxyguanosine, and 8-hydroxyguanosine. TLR7- selective agonists also include those shown in U.S. Patent Publication 2004/0171086. Additional suitable TLR7 agonists include, but are not limited to, 2- (ethoxymethyl)- 1 -(2- methylpropyl)- 1 H-imidazo [4,5 -c] quinolin-4-amine (U.S. Patent 5,389,640); 2-methyl-l-[2- (3-pyridin-3-ylpropoxy)ethyl]-lH-imidazo [4,5-c]quinolin-4- amine (WO 02/46193); N-(2-{2- [4-amino-2-(2-methoxyethyl)-lH-imidazo[4,5-c]quinolin-l- yljethoxy} ethyl-ethylN- methylcyclohexanecarboxamide (U.S. Patent Publication 2004/0171086); 1 -[2-

(benzyloxy)ethyl]-2-methyl- lH-imidazo[4,5-c]quinolin-4-amine (WO 02/46189); N-{8-[4- amino-2-(2-methyoxyethyl)-lH-imidazo[4,5-c]quinolin-l-yl]octyl}-N- phenylurea (U.S. Patent Publication 2004/0171086 (IRM5)); 2-butyl-l-[5- (methylsulfonyl)pentyl]-lH- imidazo[4,5-c]quinolin— 4-amine (WO 02/46192); N-{3-[4- amino-2-(2-methoxyethyl)-lH- imidazo[4,5-c]quinolin-l-yl]propyl}-4- methylbenzenesulfonamide (U.S. Patent 6,331,539); and N-[4-(4-amino-2-ethyl-lH- imidazo[4,5-c]quinolin-l- yl)butyl]cyclohexanecarboxamidecarboxamide (U.S. Patent Publication 2004/0171086 (IRM8)). Also suitable for use is the TLR7- selective agonist N-[4-(4-amino- 2-ethyl-lH- imidazo[4,5-c]quinolin-l-yl)butyl-]methanesulfon- amide.

For example, TLR8 agonists include, but are not limited to, the compounds shown in U.S. Patent Publication No. 2004/0171086 that include N-{4-[4-amino- 2-(2-methoxyethyl)- lH-imidazo[4, 5-c]quinolin-l-yl]butyl } quinolin-3 -carboxamide, N- (4-[4- amino-2-(2- methoxyethyl)-lH-imidazo[4,5-c]quinolin-l-yl]butyl}quinoxoline-2- carboxamide, and N-[4- (4-amino-2 -propyl- lH-imidazo[4,5-c]quinolin-l- yl)butyl]morpholine-4-carboxamide. Other suitable TLR8- selective agonists include, but are not limited to, 2- propylthiazolo[4,5- c]quinolin-4-amine (U.S. Patent 6,110,929); Nl-[2-(4-amino-2-butyl-lH- imidazo[4,5-c] [ 1 ,5]naphthyridin- 1 -yl)ethyl]~2-amino-4-methylpentanamide (U.S. Patent 6,194,425); Nl- [4-(4-amino-lH-imidazo[4,5-c]quinolin-l-yl)butyl]-2-phenoxy-benzamide (U.S. Patent 6,451,810); Nl-[2-(4-amino-2-butyl-lH-imidazo[4,5-c]quinolin-l-yl)ethyl]-l- propanesulfonamide (U.S. Patent 6,331,539); N-{2-[2-(4-amino-2-ethyl-lH-imidazo[4,5- c]quinolin-l-yl)ethyoxy]ethyl}-N' -phenylurea (U.S. Patent Publication 2004/0171086); l-{4- [3,5-dichlorophenyl)thio]butyl}-2-ethyl-lH-imidazo[4,5-c]quinolin-4~ amine (U.S. Patent Publication 2004/0171086); N- {2-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin- 1 - yljethyl }-N'-(3-cyanophenyl)urea (WO 00/76518 and U.S. Patent Publication No. 2004/0171086); and 4-amino-a,a-dimethyl-2-methoxyethyl- lH-imidazo[4,5-c]quinoline-l - ethanol (U.S. Patent 5,389,640). Included for use as TLR8- selective agonists are the compounds in U.S. Patent Publication No. 2004/0171086. Also suitable for use is the compound 2-propylthiazolo-4,5-c]quinolin-4-amine. Specific examples of TLR7/8 agonists of the present invention include:

Thiazoloquinoline derivative CL097 R848 TLR8 agonist TLR7/8 agonist TLR7/8 agonist

In some embodiments, the TLR7/8 agonist of the present invention is R848. By "R848" is meant an imidazoquinoline compound with the structure above described.

In some embodiments, the TLR7/8 agonist of the present invention is incorporated in a particle appropriate for ingestion by neutrophils. Accordingly, the dimensions of the particle (e.g. diameter) are selected to promote phagocytosis of the particles by neutrophils. In some embodiments, the present invention provides a population of particles wherein the TLR7/8 agonist of the present invention is incorporated and, wherein at least 50% of the particles have a size appropriate for ingestion by neutrophils. In some embodiments, the desired size ranges within ±10%, ±20%, ±30%, ±40%, or ±50% of a given value. The value may be, e.g. 20 nm, 100 nm, 500 nm, 1, 5, 10, 20, 50 microns, etc. The particles in any of these embodiments can result from any composition. Typically, the particles comprise denatured proteins (e.g. human serum albumin (Benacerraf et al., 1957 Brit. J. Exp. Path, 38:35)), insoluble materials (e.g. carbon black, silica, silicon dioxide, polystyrene, latex), metal oxides (e.g. titanium oxides, iron oxides), and India ink (i.e., suspension of colloidal carbon particles) (described in Rei chard and Filkins, 1984, The Reticuloendothelial System; A Comprehensive Treatise, pp. 73-101 (Plenum Press), and references therein), hydrogels, (for example as described in US Patent Publication No. 20050191361 ), sepharose or agarose beads or microparticles. In some embodiments, the particles are formed from materials that are biodegradable and non-toxic (e.g. a poly(a-hydroxy acid) such as poly(lactide-co-glycolide), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.). The particles of the present invention may comprise red blood cells (RBCs) that have been purged of their cytoplasm, known as 'Ghost' RBCs, bacteria (as bacteria are cleared by the RES; see, e.g. Benacerraf and Miescher, 1960, Ann NY Acad Sci, 88:184-195), cell fragments, liposomes, bacteriophages, bacteriophage fragments, and viral capsids devoid of the viral nucleic acids (e.g. hepatitis B virus surface antigen particles), etc.

As used herein, the term "effective amount" refers to a quantity sufficient to achieve a therapeutic effect (e.g. treating the bacterial infection). In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, an effective amount of TLR7/8 agonist for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Typically, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In some embodiments, a single dosage of the agonist ranges from 0.1-10,000 micrograms per kg body weight. In some embodiments, aromatic- cationic peptide concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.

Typically, the TLR7/8 agonist of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Typically, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the TLR7 and/or TLR8 agonist of the present invention in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In some embodiments, the TLR7/8 agonist of the present invention is administered concomitantly with an antibiotic. In some embodiments, the antibiotic is selected from the group consisting of aminoglycosides, beta lactams, quinolones or fluoroquinolones, macrolides, sulfonamides, sulfamethaxozoles, tetracyclines, streptogramins, oxazolidinones (such as linezolid), rifamycins, glycopeptides, polymixins, lipo-peptide antibiotics. Typically beta lactames include 2-(3-alanyl)clavam, 2-hydroxymethylclavam, 7-methoxycephalosporin, epi-thienamycin, acetyl-thienamycin, amoxicillin, apalcillin, aspoxicillin, azidocillin, azlocillin, aztreonam, bacampicillin, blapenem, carbenicillin, carfecillin, carindacillin, carpetimycin A and B, cefacetril, cefaclor, cefadroxil, cefalexin, cefaloglycin, cefaloridine, cefalotin, cefamandole, cefapirin, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefmenoxime, cefmetazole, cefminox, cefmolexin, cefodizime, cefonicid, cefoperazone, ceforamide, cefoselis, cefotaxime, cefotetan, cefotiam, cefoxitin, cefozopran, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefradine, cefroxadine, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cephalosporin C, cephamycin A, cephamycin C, cephalothin, chitinovorin A, chitinovorin B, chitinovorin C, ciclacillin, clometocillin, cloxacillin, cycloserine, deoxy pluracidomycin B and C, dicloxacillin, dihydro pluracidomycin C, epicillin, epithienamycin D, E, and F, ertapenem, faropenem, flomoxef, flucloxacillin, hetacillin, imipenem, lenampicillin, loracarbef, mecillinam, meropenem, metampicillin, meticillin (also referred to as methicillin), mezlocillin, moxalactam, nafcillin, northienamycin, oxacillin, panipenem, penamecillin, penicillin G, N, and V, phenethicillin, piperacillin, povampicillin, pivcefalexin, povmecillinam, pivmecillinam, pluracidomycin B, C, and D, propicillin, sarmoxicillin, sulbactam, sultamicillin, talampicillin, temocillin, terconazole, thienamycin, andticarcillin. Typically, quinolones include nalidixic acid, cinoxacin, oxolinic acid, flumequine, pipemidic acid, rosoxacin, norfloxacin, lomefloxacin, ofloxacin, enrofloxacin, ciprofloxacin, enoxacin, amifloxacin, fleroxacin, gatifloxacin, gemifloxacin, clinafloxacin, sitafloxacin, pefloxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, grepafloxacin, levofloxacin, moxifloxacin, and trovafloxacin.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: R848 provides protection for survival in mice infected with leptospires.

Mice were treated each 2 days without (control) or with R848 (0,25mg/kg) injected intraperitoneally. One day after the first R848 treatment, mice were infected with a single lethal dose of bioluminescent Leptospira interrogans serovar Manilae (Lepto) (2xl0⁷ bacteria/per mouse. Two other groups of mice were not infected but received PBS or R848 only. Data show the percentage of surviving mice among the 2 infected groups (n=5 per group).

Figure 2: R848 provides protection for renal infection in mice infected with leptospires. Mice were treated each 2 days without (control) or with R848 (0,25mg/kg) injected intraperitoneally. One day after the first R848 treatment, mice were infected with a single non-lethal dose of bioluminescent Leptospira interrogans serovar Manilae (Lepto). The figure shows the intensity of animal infection visualized and quantified using a bioluminescence imager for live animals (n=5, representative of 2 experiments). Figure 3: R848 stimulates a weak production of Reactive Oxygen Species (ROS) in mouse whole blood in vitro and promotes ROS production induced by Phorbol Myristate Acetate (PMA). ROS production was measured by a chemiluminescence (CL) assay adapted for whole blood (5m1) of normal mice. Blood cells were first treated without (control) or with various R848 concentrations for 10 min (Panel A), then with 50 nM PMA (Panel B). Results represent the CL response expressed as a percentage of control values (i.e 2400±467 cpm for Panel A and 33000± 7520 cpm for Panel B, (n = 5-7, * P<0,05).

Figure 4: R848 improves suboptimal but not optimal ROS production induced by the formylpeptide fMLP in whole mouse blood. ROS production was measured by a chemiluminescence (CL) assay with 5m1 whole blood of mice. Blood was treated first without (control) or with R848 (1 pg/ml) for 15 min, then with ImM fMLP. Results represent the peak of CL response expressed in cpm (n=6-10, *P<0,05). Two groups of mice were distinguished; those providing low (Panel A) and high (Panel B) ROS production.

Figure 5: R848 stimulates a weak production of ROS by mouse leucocytes in vitro and promotes ROS production induced by PMA or Leptospira. ROS production was measured by a chemiluminescence (CL) assay. Leucocytes (6-9x10⁵ phagocytes/tube) were first treated without (control) or with various R848 concentrations for 10 min (Panel A), then with 50 nM PMA (Panel B) or Leptospira interrogans serovar Copenhageni strain Fiocruz Ll-130 (2xl0⁷ heat-inactivated bacteria) (Panel C, n=4, R848= 1 pg/ml/l O in) Results represent the mean maximal CL response expressed as % of basal or control values (Basal values for Panel A: 5571±1896 cpm (n=10) and for Panel B: 14550±4252 (n=4), * P<0,05).

Figure 6: Comparison of ROS production by human neutrophils exposed to Leptospira and the bacterial peptide fMLP. Human polymorphonuclear leucocytes (PMN, 0,5xl0⁶ cells/0, 5ml) were treated for 45 min with various amounts of Leptospira interrogans serovar Copenhageni strain Fiocruz Ll-130 ranging from 5 to 40x10⁶ heat-inactivated bacteria (Panel A, B), or with ImM fMLP alone (Panel C, a representative response). Data represent the chemiluminescence response expressed in cpm. The early peak of CL response induced by Leptospira (Panel A) was expressed as a function of Leptospira amounts (Panel B, n=4 P<0,05).

Figure 7: Treatment of human neutrophils in the presence of Leptospira inhibited ROS production secondarily induced by fMLP. Human polymorphonuclear leucocytes (PMN, 0,5xl0⁶ cells/0, 5ml) were treated for 30 min in the absence (control) or presence of various amounts of Leptospira Fiocruz strain Ll-130 ranging from 5 to 40xl0⁶ heat- inactivated bacteria, then stimulated with 1 mM fMLP. Data represent the chemiluminescence response expressed as percentage of control values (100%= 268xl0⁶ cpm, n=3).

Figure 8: R848 restores fMLP-induced superoxide production by NOX2 in human neutrophils pretreated with Leptospira. Neutrophils (PMN, lxlO⁶ cells/lml) were treated in the absence (Control) or presence of Leptospira strain Verdun (40xl0⁶ heat- inactivated bacteria) for 30min, then with R848 (lpg/ml) for 15 min before stimulation with fMLP (ImM). Data represent the total amount of superoxide quantified with the cytochrome c reduction assay. Data are expressed in nmole superoxide /10⁶ PMN (n=4, *:P<0.05).

Figure 9: In vivo potentiating effect of R848 on ROS production by peritoneal neutrophils from Leptospira- infected mice. C57BL6/J mice (n=2) were intraperitoneally injected with PBS or R848 (0.1 mg/kg), 24 hours before infection with a single non-lethal dose of live leptospires in PBS (2xl0⁷ L. interrogans serovar Manilae (Lepto). 4h after infection, mice were sacrificed and the peritoneal exudates were collected. Cells were washed and incubated under standard conditions in HBSS with the ROS marker 2’7- dichlorofluorescein-diacetate (FLDC-FDA) (10 mM) for 30 min at room temperature, washed and subsequently labelled for 20 min on ice with the anti-neutrophil antibodies CD1 Ib-Vb450 and Ly6G-PE Cy7. Cells were then incubated for 5 min on ice with efluor 780, a viability marker, and fixed with 4% PFA. Fluorescence emitted by the 2’7-dichlorofluorescein (DCF) was indicator for ROS production by neutrophils and measured by FACS (Cytoflex Beckman Coulter) and analysed with FlowJo software. Analysis was done on viable singlet cells. The data represent the mean fluorescence intensity (MFI) in the neutrophil (LY6-G⁺) population of n=2 mice labelled without (wo DCF) or with (w) the DCF probe. Similar results were obtained in a second experiment with 2 mice per group.

EXAMPLE:

In this study, we evaluated the effects of R848 (Resiquimod) in an acute infection murine model of leptospirosis. Strikingly, intrap eritoneal administration ofR848 (0.25mg/kg) keeps alive mice infected with lethal doses of leptospires (strain Manilae) alive (Figure 1). In addition, R848 strongly inhibits the early proliferation of leptospires as well as their late colonization of the kidneys (Figure 2).

In vitro studies show that R848 (0 25-4pg/ml) stimulates ROS production in mouse whole blood (Figure 3A) and potentiates the production of ROS induced by potent activators of the oxidative burst such as phorbol myri state acetate (PM A) (Figure 3B) and the bacterial peptide, formyl-Met-Leu-Phe (fMLP) (Figure 4A-B). In purified mouse leukocytes, as in human neutrophils, R848 stimulates basal ROS production (Figure 5A) and potentiates ROS production induced by PMA (Figure 5B) or leptospires (Figures 5C, 6A-B). However, prolonged exposure of human neutrophils to leptospires leads to desensitization of neutrophils characterized by low production of superoxide anion secondarily stimulated by the fMLP (Figure 6C, Figure 7). This adverse effect of leptospires is reversed in neutrophils treated with R848 (Figures 8). Pretreatment of mice with R848 (0,lmg/kg) one day before infection with Leptospira also potentiated the ROS production by peritoneal neutrophils collected 4h post-infection (Figure 9).

In conclusion, these results show that Resiquimod promotes the production of antibacterial oxidants in the whole blood of mice ex vivo and in vivo in peritoneal neutrophils from infected mice, which indicates that TLR7/8 activation is an effective method to treat early infection, which drastically reduces renal colonization by leptospires.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of treating leptospirosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a TLR7 and/or TLR8 agonist.

2. The method of claim 1 wherein the TLR7 and/or TLR8 agonist is selected from imidazoquinoline compounds.

3. The method of claim 1 wherein the TLR7 and/or TLR8 agonist is R848

4. The method of claim 1 wherein the TLR7/8 agonist is incorporated in a particle appropriate for ingestion by neutrophils.

5. The method of claim 1 wherein the TLR7/8 agonist is administered concomitantly with an antibiotic.