Therapeutic agent

Abstract

The present invention relates to methods and compositions for the prevention and / or treatment of pyelonephritis and / or urinary sepsis.

Description

Technical Field

[0001] The present invention relates to methods and compositions for preventing and / or treating pyelonephritis and / or urosepsis. Background Art

[0002] Urinary tract infections (UTIs) are common and can be dangerous. Clinical presentation and severity depend on the site of infection and the molecular basis of the disease. In acute pyelonephritis (APN), bacteria enter the renal pelvis where they induce an intense mucosal inflammatory response that progresses into the renal parenchyma. Symptoms in infants include high fever, malaise, flank pain, as well as poor feeding and irritability. APN can lead to urosepsis.

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

[0004] The present inventors have now unexpectedly discovered agents that can be used to treat or prevent pyelonephritis (including APN) and thus prevent the urosepsis caused thereby. These agents include inhibitors of the IL-1 receptor, in particular inhibitors of IL-1β and NlpD protein. Without being bound by theory, based on the understanding of IL-1β processing, the inventors have also discovered that MMP7 inhibitors and agents that modulate MMP7 expression can also be used in the present invention. It is particularly surprising that these agents known for their anti-inflammatory activity can be used to treat infection.

[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 an agent selected from the group consisting of an IL-1 inhibitor, a MMP inhibitor and an NlpD protein.

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

[0007] In certain embodiments, the method or medicament is used to treat or prevent pyelonephritis, preferably to treat pyelonephritis. In certain embodiments, pyelonephritis is acute pyelonephritis. In certain embodiments, pyelonephritis is chronic or long-term. In certain embodiments, the method or medicament is used to treat or prevent, preferably prevent, urosepsis, particularly urosepsis caused by pyelonephritis.

[0008] In certain embodiments, the agent may be provided in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0009] Specifically, 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. IL-1 inhibitors include, for example, small molecules such as anthraquinones, such as described in USP 4,244,968, including diacerein; and proteins and peptides such as interleukin-1 receptor antagonists (IL-1RA), such as anakinra and rilonacept; or a pharmaceutically acceptable salt thereof, or a prodrug thereof, and combinations of these. Specifically, the agent is an IL-1β receptor antagonist, such as anakinra (U.S. Pat. No. 5,075,222).

[0010] Alternatively, the agent is an MMP inhibitor, particularly an MMP7 inhibitor. As described in Durrant et al. Chem. Biol. Drug Des 20111; 78; 191-198 (the entire contents of which are incorporated herein by reference), a variety of MMP inhibitors are known. Specific examples include batimastat, doxycycline (doxycycline hydrochloride), marimastat, or a salt or prodrug thereof, but particularly batimastat. The agent can also be an agent that reduces the expression of MMP, particularly MMP-7, such as a protein selected from ASC or NLRP-3 or an active fragment or variant thereof.

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

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

[0013] MSAGSPKFTV RRIAALSLVS LWLAGCSDTS NPPAPVSSVN GNAPANTNSG MLITPPPKMGTTSTAQQPQIQPVQQPQIQA TQQPQIQPMQ PVAQQPVQME NGRIVYNRQY GNIPKGSYSG STYTVKKGDTLFYIAWITGN DFRDLAQRNNIQAPYALNVG QTLQVGNASG TPITGGNAIT QADAAEQGVVIKPAQNSTVAVASQPTITYS ESSGEQSANK MLPNNKPTAT TVTAPVTVPT ASTTEPIVSSTSTSTPISTWRWPTEGKVIE TFGASEGGNK GIDIAGSKGQ AIIATADGRVVYAGNALRGY GNLIIIKHND DYLSAYAHNDTMLVREQQEV KAGQKIATMGSTGTSSTRLH FEIRYKGKSV NPLRYLPQR(SEQ ID NO:1)

[0014] One specific fragment of SEQ ID NO: 1 is indicated in bold (ie, SEQ ID NO: 2).

[0015] In certain embodiments, the NlpD protein or variant or active fragment thereof has a low molecular weight, such as a molecular weight of less than 3 kDa. In other embodiments, the protein may be larger, such as about 40 kDa.

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

[0017] The expression "variant" refers to a protein or polypeptide having a similar biological function but with an amino acid sequence that differs from the base sequence from which it is derived, in that one or more amino acids in the sequence are replaced by other amino acids. An amino acid substitution may be considered "conservative" when an amino acid is replaced by a different amino acid with roughly similar properties. A non-conservative substitution is one in which an amino acid is replaced by an amino acid of a different type.

[0018] A "conservative substitution" is one in which an amino acid is replaced by another amino acid of the same class, where the classes are defined as follows:

[0019]

[0020] It is well known to those skilled in the art that altering the primary structure of a polypeptide by conservative substitutions may not significantly alter the activity of the polypeptide, because the amino acid side chains in the inserted sequence may be able to form similar bonds and contacts as the amino acid side chains being replaced, even if the substitutions are located in key regions for determining the conformation of the peptide.

[0021] Non-conservative substitutions are also possible as long as they do not destroy the activity. In broad terms, a small number of non-conservative substitutions can be made without changing the biological activity of the polypeptide.

[0022] It is well within the routine capabilities of those skilled in the art to determine the effect of any substitution (and indeed any amino acid deletion or insertion), who can readily determine whether a variant polypeptide retains the essential properties and activity of the base polypeptide. For example, in determining whether a variant of a polypeptide falls within the scope of the present invention, one skilled in the art will determine whether the variant retains the biological activity of the native protein, and whether the variant has at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably 90%, 95%, 96%, 97%, 98%, 99% or 100% of the activity of the native protein.

[0023] The variant of polypeptide can comprise or consist essentially of: have at least 70% homology, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98% or 99% homology to the amino acid sequence of natural polypeptide sequence. The level of sequence homology can be suitably determined using the natural polypeptide sequence as the basic sequence using the BLASTP computer program. This means that the natural polypeptide sequence forms a sequence that determines the percentage homology. BLAST software is publicly available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi (opened on October 13, 2016).

[0024] The NlpD protein can be isolated from bacteria. When the NlpD protein is a variant or an active fragment, it can be obtained by recombinant expression. When the protein is obtained by recombinant expression, the protein sequence may contain a sequence for 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 variant or active fragment is synthesized. Typically, the NlpD protein or its variant or active fragment is isolated or synthesized.

[0025] For administration to a patient, the agent is suitably administered in the form of a pharmaceutical composition which also comprises a pharmaceutically acceptable carrier. Such compositions are known in the art.

[0026] Suitable pharmaceutical compositions may be in solid or liquid form. The pharmaceutical composition may be suitable for administration by any convenient route, such as parenteral, oral or topical administration, or administration by inhalation or insufflation. A pharmaceutically acceptable carrier may include a diluent or excipient that is physiologically tolerable and compatible with the active ingredient.

[0027] Parenteral compositions are prepared for injection, such as subcutaneous injection or intravenous injection. Parenteral compositions can be liquid solutions or suspensions, or can be solid forms suitable for dissolving or suspending in liquids before injection. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, etc. and combinations thereof. In addition, if desired, the compositions may include a small amount of auxiliary substances, such as wetting agents or emulsifiers, stabilizers, or pH buffers, etc.

[0028] Oral preparations will be in solid or liquid form and can be solutions, syrups, suspensions, tablets, pills, capsules, sustained release preparations or powders. Oral preparations include commonly used excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0029] Topical formulations are usually in the form of suppositories or intranasal aerosols. For suppositories, traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from a mixture containing the active ingredient.

[0030] According to normal clinical practice, the amount of the administered agent will vary depending on factors such as the nature of the agent used, the size and health of the patient, the nature of the condition being treated, etc. Generally, it is expected that a dosage within the range of 2 mg-50 mg / Kg, such as 2 mg / Kg to 20 mg / Kg, such as 5 mg / Kg to 15 mg / Kg, will produce a suitable effect.

[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 components, integers or steps. In addition, unless the context requires otherwise, the singular encompasses the plural, and in particular, when the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity unless the context requires otherwise.

[0032] Preferred features of various aspects of the invention may be described in combination with any other aspect. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples and alternatives presented in the preceding paragraphs, claims and / or the following description and drawings, in particular their single features, may be used independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined and / or combined in any way, unless these features are incompatible. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] One or more embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0034] Figure 1 NlpD and IL-1RA are shown to be involved in the Irf3 - / - Genetic models of acute pyelonephritis and urosepsis in mice.

[0035] Figure 1 A shows the experimental scheme, Figure 1 B shows the bacterial and neutrophil counts in urine, Figure 1 C shows the gross pathology of kidneys on the day of sacrifice. Figure 1 D shows the number of kidney bacteria on the day of sacrifice. DETAILED DESCRIPTION

[0036] Example 1

[0037] Methods and Materials

[0038] Irf3 - / - Mice were infected with E. coli CFT073 (50 μl, 2.10 9 cfu / ml). Intraperitoneal treatment was administered daily starting 6 hours after infection for 7 days. Mice were treated with recombinant NlpD protein (105 μg, 100 μl) or IL-1RA (Anakinra, 1 mg / kg, 100 μl). Control mice received PBS. Urine was collected on days 1, 3, 5, and 7 from one group of animals (sacrificed on day 7), while urine was collected on days 1, 3, 5, 7, 21, and 42 from the other groups of animals for bacterial and neutrophil counts.

[0039] Animals were sacrificed under anesthesia; kidneys and bladders were removed aseptically, and for the bladder, macroscopic pathology was recorded by photography. Tissues were fixed with 4% paraformaldehyde or frozen for sectioning and RNA extraction. The number of viable cells in homogenized tissues (Stomacher 80, Seward Medical) was determined based on TSA (37°C, overnight). Urine samples were collected regularly before and after infection and quantitatively cultured. Neutrophils in uncentrifuged urine were counted using a hemocytometer.

[0040] result

[0041] Urine bacterial and neutrophil counts

[0042] like Figure 1 Treatment increased bacterial clearance from the urethra and decreased urinary neutrophil infiltration, as shown in B. Results were evaluated using two-way ANOVA and Sidak's multiple comparison test.

[0043] Renal gross pathology

[0044] Unprocessed Irf3 - / - Control mice developed severe renal lesions with renal abscesses. Treatment protected mice from renal lesions.

[0045] Kidney bacterial count (Kruskal-Wallis test)

[0046] Kidney lesions in untreated mice were associated with high bacterial counts. Kidneys of treated mice showed little or no bacterial growth.

[0047] Urine bacterial and neutrophil counts

[0048] like Figure 2 As shown, for mice treated with IL-1RA or NlpD, treatment provided long-term protection against infection and inflammation. Infected and treated mice remained disease-free at days 21 and 42, with no bacterial growth in the urine and low numbers of urinary neutrophils.

Claims

1. A medicament selected from the group consisting of an IL-1 inhibitor, a MMP inhibitor and an NlpD protein, for treating or preventing pyelonephritis, particularly acute pyelonephritis and / or urosepsis.

2. A method for preventing or treating pyelonephritis, especially 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, a MMP inhibitor and an NlpD protein.

3. The agent according to claim 1 or the method according to claim 2, wherein: The medicament or the method is used for treating or preventing acute pyelonephritis.

4. The agent according to claim 1 or the method according to claim 2, wherein: The medicament or the method is used for preventing urosepsis caused by acute pyelonephritis.

5. The agent according to claim 1, 3 or 4, or the method according to claim 2, 3 or 4, wherein: The agent is an IL-1β inhibitor.

6. The agent according to claim 1, 3, 4 or 5, or the method according to claim 2, 3, 4 or 5, wherein The IL-1 inhibitor is an interleukin-1 receptor antagonist (IL-1RA).

7. The agent or method according to claim 6, wherein: IL-1RA is anakinra or rilonacept, or a pharmaceutically acceptable salt or prodrug thereof.

8. The agent according to claim 1, 3 or 4, or the method according to claim 2, 3 or 4, wherein The agent is a MMP inhibitor.

9. The agent or method according to claim 8, wherein: The MMP inhibitor is an MMP7 inhibitor.

10. The agent or method according to claim 8, wherein: The MMP inhibitor is batimastat, doxycycline hydrochloride, marimastat, or a salt or prodrug thereof.

11. The agent according to claim 1, 3 or 4, or the method according to claim 2, 3 or 4, wherein The agent is NlpD protein or a fragment or variant thereof.

12. The agent or method according to claim 11, wherein: The NlpD protein is derived from bacteria, in particular from commensal bacteria or asymptomatic carriers.

13. The agent or method according to claim 11, wherein: The NlpD protein comprises an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, or a variant or active fragment thereof, 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.

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