Treatment

Abstract

The present invention relates to methods and compositions for preventing and / or treating pyelonephritis and / or urosepsis.

Description

[Technical field]

[0001] The present invention relates to methods and compositions for the prevention and / or treatment of pyelonephritis and / or urosepsis. [Background technology]

[0002] Urinary tract infections (UTIs) are common and can be dangerous. Clinical symptoms and severity vary depending on the site of infection and the molecular mechanism of disease. In acute pyelonephritis (APN), bacteria ascend to the renal pelvis where they cause an intense inflammatory response in the mucosa as they progress into the renal parenchyma. Symptoms include high fever, malaise, lower back pain, and poor feeding and irritability in infants. APN can lead to urosepsis.

[0003] In acute pyelonephritis, pathogen-specific TLR4 responses are activated by P-type E. coli, resulting in the release of ceramide and sequential phosphorylation of TICAM-1 (TRIF) and TICAM-2 (TRAM) adaptor molecules, CREB-1, c-FOS, and c-JUN activating IRF- and API-dependent transcription. Further involvement of MyD88, TIRAP, and NF-kB depends on the virulence repertoire of the infecting strain. Genetic studies in mouse UTI models have identified IRF3-dependent gene expression and mCXCR2-dependent neutrophil activation as determinants of bacterial clearance and tissue homeostasis. Infected Irf3 - / - or mCxcrl - / - Mice develop severe APN and tissue damage after one week, and a link to human APN susceptibility has been demonstrated through disease-associated IRF3 and CXCR1 polymorphisms in APN-prone patients. Summary of the Invention

[0004] The inventors have now identified drugs that are effective in treating or preventing pyelonephritis, and thus in preventing urosepsis. These drugs include inhibitors of IL-1 receptors, particularly IL-1β, and NlpD protein. Based on an understanding of IL-1β processing, without being bound by theory, the inventors have identified that MMP7 inhibitors and drugs that suppress the expression of MMP7 may also be useful in the present invention. It is particularly surprising that these drugs, known for their anti-inflammatory effects, can be used to treat infections.

[0005] The present invention provides a method for preventing or treating pyelonephritis and / or urosepsis, comprising administering to a patient in need thereof an effective amount of a drug selected from the group consisting of an IL-1 inhibitor, an MMP inhibitor, and an NlpD protein.

[0006] The present invention also provides an agent selected from the group consisting of an IL-1 inhibitor, an MMP inhibitor, and an NlpD protein, for treating or preventing pyelonephritis and / or urosepsis.

[0007] In certain embodiments, the method or medicament is for treating or preventing, preferably treating, pyelonephritis.In certain embodiments, the pyelonephritis is acute pyelonephritis.In certain embodiments, the pyelonephritis is chronic or long-term.In certain embodiments, the method or medicament is for treating or preventing, preferably preventing, urosepsis, particularly urosepsis caused by pyelonephritis.

[0008] In certain embodiments, the agent may be provided as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

[0009] In particular, the agent is an IL-1 inhibitor. In certain embodiments, the agent is an IL-1β inhibitor. In certain embodiments, the agent is an IL-1 receptor antagonist inhibitor. Many IL-1 inhibitors are known in the art. These include small molecules such as anthraquinones, for example, exemplified in USP 4,244,968, including diacerin, and proteins and peptides such as interleukin-1 receptor antagonists (IL-1 RA), for example, anakinra and rilonacept, or pharma-ceutically acceptable salts thereof, or prodrugs thereof, and combinations thereof. In particular, the agent is an IL-1β receptor antagonist, such as anakinra (US Pat. No. 5,075,222).

[0010] Alternatively, the agent is an MMP inhibitor, in particular an MMP7 inhibitor.A wide range of MMP inhibitors are known, for example as described in Durrant et al. Chem. Biol. Drug Des 20111; 78; 191-198, the contents of which are incorporated herein by reference.Specific examples include batimastat, periostat (doxycycline hyclate), marimastat, or salts or prodrugs thereof, in particular batimastat.It may also be an agent that reduces the expression of MMP, in particular MMP-7, such as a protein selected from ASC or NLRP-3, or an active fragment or variant thereof.

[0011] In certain embodiments, the NlpD protein is a bacterial protein, preferably a protein from a commensal bacterium or an asymptomatic carrier. In certain embodiments, the NlpD protein is from a commensal bacterium or an asymptomatic carrier in a human host. The bacterium may be asymptomatic bacteriuria (ABU). In certain embodiments, the bacterial strain is an E. coli strain, such as E. coli 83972.

[0012] In certain embodiments, the NlpD protein may consist of or consist of SEQ ID NO:1 or a variant or active fragment thereof.

[0013] [Table 1]

[0014] The particular fragment of SEQ ID NO:1 is shown in bold (SEQ ID NO:2).

[0015] In certain embodiments, the NlpD protein, or a variant or active fragment thereof, is of low molecular weight, for example, less than 3 kDa in molecular weight, in other embodiments, the protein is larger, for example, about 40 kDa.

[0016] As used herein, the term "fragment" refers to a peptide or protein that lacks one or more amino acids present in the full-length protein, but retains the function of the full-length protein.

[0017] The term "variant" refers to a protein or polypeptide having a similar biological function, but whose amino acid sequence differs from the base sequence from which it is derived in that one or more amino acids in the sequence have been replaced by other amino acids. An amino acid substitution may be considered "conservative" if an amino acid is replaced by another amino acid having similar properties. In a non-conservative substitution, an amino acid is replaced by another amino acid of a different type.

[0018] "Conservative substitution" means the replacement of an amino acid with another amino acid of the same class, the categories being defined as follows:

[0019] [Table 2]

[0020] As is well known to those of skill in the art, alterations in the primary structure of a polypeptide through conservative substitutions may not significantly alter the activity of the polypeptide, since the side chain of the amino acid inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid that is lost by substitution, even if the substitution is in a region that is important in determining the three-dimensional structure of the peptide.

[0021] Non-conservative substitutions are possible as long as they do not interfere with activity. In general, there are few non-conservative substitutions that do not change the biological activity of a polypeptide.

[0022] Determining the effect of any substitution (and indeed any amino acid deletion or insertion) is well within the ordinary skill of the art, who can easily determine whether a mutant polypeptide retains the basic properties and activity of the original polypeptide. For example, when determining whether a polypeptide variant falls within the scope of the present invention, the art will determine whether the variant retains the biological activity of the native protein, and whether the variant retains at least 60%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the activity of the native protein.

[0023] A variant of a polypeptide may comprise or consist essentially of an amino acid sequence having at least 75%, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity to a native polypeptide sequence. The level of sequence identity is suitably determined by a BLASTP computer program using the native polypeptide sequence as a base sequence. This means that the native polypeptide sequence forms the sequence against which the percentage of identity is determined. BLAST software is publicly available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi (accessed October 13, 2016).

[0024] The NlpD protein may be isolated from bacteria. When the NlpD protein is a mutant or an active fragment, it may be obtained by recombinant expression. When the protein is obtained by recombinant expression, the protein sequence may include a sequence for use in purification, such as an N-terminal or C-terminal His tag. The use of purification tags is well known in the art. In a preferred embodiment, the NlpD protein, or its mutant or active fragment, is synthetic. Usually, the NlpD protein, or its mutant or active fragment, is isolated or synthesized.

[0025] For administration to a patient, the agents are suitably administered in the form of a pharmaceutical composition, which further comprises a pharma- ceutical acceptable carrier, Such compositions are well known in the art.

[0026] Suitable pharmaceutical compositions are in solid or liquid form. They can be adapted for administration by any convenient route, such as parenteral, oral, topical, or by inhalation or insufflation. Pharmaceutically acceptable carriers include diluents or excipients that are physiologically acceptable and compatible with the active ingredient.

[0027] Parenteral compositions are prepared for, for example, subcutaneous or intravenous injection. They may be in liquid solutions or suspensions, or in solid forms suitable for dissolving or suspending in liquid prior to injection. Suitable diluents and excipients include, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizing agents, or pH buffering agents.

[0028] Oral preparations may be in solid or liquid form, and may be solutions, syrups, suspensions, tablets, pills, capsules, sustained release formulations, or powders. Oral preparations include commonly used excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0029] Topical formulations generally take the form of suppositories or nasal aerosols. For suppositories, traditional binders and additives include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed with a mixture containing the active ingredient as a base.

[0030] The amount of drug administered will vary in accordance with normal clinical practice, depending on factors such as the nature of the drug used, the size and health of the patient, and the nature of the condition being treated. Typically, doses in the range ^g-50 mg / kg, for example 2-20 mg / kg, for example 5-15 mg / kg, are expected to produce suitable effects.

[0031] Throughout the description and claims of this specification, the words "comprise" and "contain," and variations of these words, such as "comprising" and "comprises," mean "including, but not limited to," and do not exclude other elements, integers, or steps. Further, unless the context requires otherwise, the singular includes the plural: in particular, where the indefinite article is used, the specification is to be interpreted as contemplating the singular and the plural, unless the context requires otherwise.

[0032] Preferred features of each aspect of the invention are as described in relation to any of the other aspects. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples and alternatives described in the preceding paragraphs, claims and / or the following description and drawings, in particular their individual features, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any way and / or combination, except where those features are incompatible.

[0033] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings. [Brief description of the drawings]

[0034] [Figure 1] Figure 1 shows the effect of NlpD and IL-1RA in infected Irf3- / - mice, a genetic model of acute pyelonephritis and urosepsis. Figure 1A shows the experimental protocol, 1B shows bacterial and neutrophil counts in urine, 1C shows gross pathology of kidneys on the day of sacrifice, and 1D shows bacterial counts in kidneys on the day of sacrifice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Example 1 Methods and Materials Irf3 - / - Mice were infected with E. coli CFT073 (2.10 9 Mice were infected by intravesical instillation of 100 μl ...

[0036] Animals were sacrificed under anesthesia, kidneys and bladders were aseptically removed, and gross pathology was documented photographically in the case of the bladder. Tissues were fixed in 4% paraformaldehyde or frozen, sectioned, and RNA was extracted. Viable bacterial counts in homogenized tissues (Stomacher 80, Seward Medical) were determined using TSA (37°C, overnight). Urine samples were collected pre- and post-infection at regular intervals and quantitatively cultured. Neutrophils in uncentrifuged urine were counted using a hemocytometer.

[0037] [result] <Urine bacteria and neutrophil counts> Treatment increased bacterial clearance from the urinary tract and reduced urinary neutrophil infiltration, as shown in Figure 1 B. Results were evaluated using two-way analysis of variance and Sidak's multiple comparison test.

[0038] <General Kidney Pathology> Untreated Irf3 - / - Control mice developed severe kidney pathology with evidence of renal abscesses. Treatment protected mice from kidney pathology.

[0039] <Bacterial count in the kidney (Kruskal-Wallis test)> Kidney pathology in untreated mice was associated with high bacterial counts, whereas treated mice had very low or no bacterial growth in the kidneys.

[0040] <Urine bacteria and neutrophil counts> As shown in Figure 2, treatment of mice with IL-1RA or NlpD conferred long-term protection against infection and inflammation. Infected and treated mice had no bacterial growth in the urine and low urinary neutrophil counts on days 21 and 42.

Claims

1. selected from the group consisting of an IL-1 inhibitor, an MMP inhibitor, and an NlpD protein; For use in the treatment or prevention of pyelonephritis, in particular acute pyelonephritis and / or urosepsis, Drugs.

2. the medicament is for the treatment or prevention of acute pyelonephritis, The medicament is for the prevention of urosepsis caused by acute pyelonephritis, The drug according to claim 1.

3. the agent is an IL-1β inhibitor; The drug according to claim 1.

4. the IL-1 inhibitor is an interleukin-1 receptor antagonist (IL-1 RA); The drug according to claim 1.

5. The IL-1RA is anakinra or rilonacept, or a pharmaceutically acceptable salt or prodrug thereof; The drug according to claim 4.

6. the drug is an MMP inhibitor; The drug according to claim 1.

7. the MMP inhibitor is an MMP7 inhibitor; The MMP inhibitor is batimastat, periostat (doxycycline hyclate), marimastat, or a salt or prodrug thereof; The drug according to claim 6.

8. The agent is an NlpD protein or a fragment or variant thereof. The drug according to claim 1.

9. the NlpD protein is derived from a bacterium, in particular a commensal bacterium or an asymptomatic carrier; The NlpD protein comprises or consists of an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, or a variant or active fragment thereof; The drug according to claim 8.

10. 1. A method for preventing or treating pyelonephritis, particularly acute pyelonephritis and / or urosepsis, comprising: administering to a patient in need thereof an effective amount of an agent selected from the group consisting of an IL-1 inhibitor, an MMP inhibitor, and an NlpD protein; method.

11. the method is for the treatment or prevention of acute pyelonephritis, or The method is for preventing urosepsis due to acute pyelonephritis. The method of claim 10.

12. the agent is an IL-1β inhibitor; The method of claim 10.

13. the IL-1 inhibitor is an interleukin-1 receptor antagonist (IL-1 RA); The method of claim 10.

14. The IL-1RA is anakinra or rilonacept, or a pharmaceutically acceptable salt or prodrug thereof; The method of claim 13.

15. the drug is an MMP inhibitor; The method of claim 10.

16. the MMP inhibitor is an MMP7 inhibitor; The MMP inhibitor is batimastat, periostat (doxycycline hyclate), marimastat, or a salt or prodrug thereof; 16. The method of claim 15.

17. The agent is an NlpD protein or a fragment or variant thereof. The method of claim 10.

18. the NlpD protein is derived from a bacterium, in particular a commensal bacterium or an asymptomatic carrier; The NlpD protein comprises or consists of an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, or a variant or active fragment thereof; 18. The method of claim 17.

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