Use of metalloproteinase inhibitors against bacterial infections

Abstract

A composition comprising as components a polypeptide IMPI (including wild type) and/or a polypeptide IMPI-fusion and at least one antibiotic compound, in particular an aminoglycoside antibiotic, and/or at least one bactericidal compound, wherein the polypeptides, the at least one antibiotic and the at least one bactericidal compound is present in the composition in concentrations which exhibit in combination a synergistic effect against resistant bacteria.

Claims

1. A composition comprising: a polypeptide comprising at least 80% sequence identity to SEQ ID NO. 2, wherein the polypeptide is selected from the group consisting of IMPI, an IMPI-fusion, and both an IMPI and an IMPI-fusion; and at least one antibiotic compound, wherein the at least one antibiotic compound comprises an aminoglycoside antibiotic, further wherein the polypeptide and the at least one antibiotic compound are in concentrations which exhibit in combination a synergistic effect against resistant bacteria.

2. The composition of claim 1, wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 10, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and IMPI-fusions, having amino acid sequences selected from the group consisting of SEQ ID NOs: 6, 8, 12, 86, 88, 90, and 92.

3. A composition of claim 1, wherein the IMPI polypeptide has at least 90% sequence identity to SEQ ID No: 2, representing the wild-type of the protein insect metalloproteinase inhibitor IMPI.

4. A method of treating or preventing a bacterial infection in a subject, the method comprising administering the composition of claim 1 to a subject suffering from or at risk of suffering from a bacterial infection.

5. The method of claim 4, wherein the at least one antibiotic compound: (i) is administered in doses lower than inhibitory upon solitary application, or in higher doses up to maximally tolerable doses, and (ii) is administered essentially simultaneous with IMPI or separately in an individual dosing scheme, frequency, and treatment duration.

6. A pharmaceutical composition comprising the composition of claim 1, and a carrier suitable for injection, inhalation or topical application.

7. A method of sterilizing a device comprising coating the device with the composition of claim 1.

8. The method of claim 7, wherein the device is an implant.

9. The composition of claim 1, wherein the polypeptide is SEQ ID NO. 2.

10. The composition of claim 1, wherein the IMPI concentration is at 20 M.

11. The composition of claim 1, wherein the IMPI concentration is at 45 M.

12. The composition of claim 1, wherein the polypeptide is the IMPI-fusion.

13. The composition of claim 1, further comprising a bactericidal compound.

Description

BRIEF DESCRIPTION THE DRAWINGS

(1) FIG. 1: Comparison of growth curves of P. aeruginosa cultures (DSM No. 50071) in the presence (long dashed line) and absence (short dashed line) of the M4-metalloprotease inhibitor IMPI (45 M; NB-Medium No. 5) in presence of (a) 1.000, (b) 500, (c) 250 and (d) 125 ng/ml Gentamycin.

(2) FIG. 2: Growth curves of P. aeruginosa cultures (DSM No. 50071) in presence of different Gentamycin concentrations (8,000; 4,000; 2,000; 1,000; 500; 250; 125; 62.5 ng/ml; NB-Medium) (a) without and (b) in presence of M4-metalloprotease inhibitor IMPI (45 M; NB-Medium No. 5)

(3) FIG. 3: Growth curves of P. aeruginosa cultures (DSM No. 50071) in presence of 45 (black, diamond symbols) and 20 (dark grey, triangles) M IMPI and absence (light grey, horizontal bars) of IMPI (NB-Medium No. 5), in presence of different gentamycin concentrations (8000; 4000; 2000; 1000; 500; 250; 125; 62.5 ng/ml) (a) after 12 h (b) after 24 h (c) after 36 h and (d) after 48 h.

(4) FIG. 4: The formation of freely suspended biofilm in flocs is a successful strategy used by Pseudomonas aeruginosa to overcome stressfull envionmental conditions (e.g. addition of Gentamicin Gm to buffer; Panel A (62.5 ng/ml) and B (125 ng/ml)). When incubated with Insect Metalloprotease Inhibitor (IMPI), biofilm formation is prohibited (Panel C and D at 62.5 and 125 ng/ml Gm doses, respectively).

(5) FIG. 5: Comparison of growth curves of P. aeruginosa cultures (VB7623, Cllinical isolate from tracheal secrete) in presence (long dashed line) and absence (short dashed line) of the M4-metalloprotease inhibitor IMPI (25 M; NB-Medium No. 5) in presence of (a) 1,000 (b) 500, and (c) 250 ng/ml Gentamycin

(6) FIG. 6: At low concentrations of gentamycin (dashed lines) the growth rate of P. aeruginosa cultures VB7623 seem to enhance growth after 26 h in NB-medium, a growth which could be part of general stress response.

(7) FIG. 7: At very low concentrations gentamycin has no effect on the growth rate of P. aeruginosa cultures VB7623. Furthermore no stress response can be observed.

(8) FIG. 8: Growth curves of Pseudomonas aeruginosa cultures (Clinical Isolate VB7623 from tracheal secrete, assessed as 4MRGN strain [EUCAST]) in presence (dark grey, triangles) and absence (light grey, horizontal bars) of the M4-metalloprotease inhibitor IMPI (25 M, NB-Medium No. 5) in presence of different gentamycin concentrations (8000; 4000; 2000; 1000; 500, 250, 125, 62.5 ng/ml) (a) after 12 h (b) after 24 h (c) after 36 h and (d) after 48 h.

(9) FIG. 9: Comparison of the development of pyoveridine (dark grey background in circular light grey structure, i.e. the plate wells) during growth of P. aeruginosa (Clinical Isolate VB7623 from tracheal secrete, assessed as 4MRGN strain [EUCAST]) in presence (lower rectangle) and absence (upper rectangle) of the M4-metalloprotease inhibitor IMPI (73 M; NB-Medium No. 5), and different Gentamycin concentrations (32; 8; 4; 2; 1; 0.5; 0.25; 0.125 g/ml) in a 96-well plate after 48 h. Pyoveridine is not expressed in the concurrent presence of IMPI and Gentamycin, not even at gentamycin concentrations as low as 0.125 g/

(10) FIG. 10: Comparison of growth curves of P. aeruginosa P. aeruginosa cultures (VB7623, Clinical isolate from tracheal secrete) in the presence (long dashed line) and absence (short dashed line) of the M4-metalloprotease inhibitor IMPI (73 M; NB-Medium No. 5) in presence of 125 ng/ml gentamycin.

(11) FIG. 11: Comparison of growth curves of P. aeruginosa cultures (VB7623, Clinical isolate from tracheal secrete) in presence (dark thick line) and absence (short dashed line) of the M4-metalloprotease inhibitor IMPI (73 M; NB-Medium No. 5) in presence of different gentamycin-concentrations (500; 250; 125 ng/ml GM), positive control in grey.

(12) FIG. 12: Comparison of growth curves of P. aeruginosa cultures (clinical isolate VB7623 from tracheal secrete, assessed as 4MRGN strain [EUCAST]) in the presence (dark grey) and absence (light grey) of the M4-metalloprotease inhibitor IMPI (73 M; NB-Medium No. 5). in the presence of different Gentamycin concentrations (32; 8; 4; 2; 1 g/ml; 500; 250; 125; 62.5 ng/ml) after (a) 12 h, (b) 24 h, (c) 36 h, (d) 48 h.

(13) FIG. 13: Antibiogram of the Pseudomonas aeruginosa strain VB7623 which was isolated from the tracheal secrete of a patient. The strain was assessed as multiresistant Gram-negative strain (4MRGN [EUCAST]).

(14) FIG. 14: Comparison of growth curves of P. aeruginosa cultures (VB7623, clinical isolate from tracheal secrete) in the presence (black line) and absence (grey line) of the M4-metalloprotease inhibitor IMPI without the use of gentamicin or any other antibiotics. IMPI concentration is (a) 25 M; NB-Medium No. 5 and (b) 60 M; NB-Medium No. 5.

(15) FIG. 15: Comparison of the development of planctonic biofilm and pyoveridine (grey colour in the wells) during growth of P. aeruginosa (clinical isolate VB7623 from tracheal secrete, assessed as 4MRGN strain [EUCAST]) in the presence (black outlined rectangle) and absence (double outlined rectangle) of the M4-metalloprotease inhibitor IMPI (60 M; NB-Medium No. 5) in a 96-well plate after 48 h. Pyoveridine (grey colour) is secreted less in presence of IMPI, in which case also no plantonic biofilm can be observed.

(16) FIG. 16: Excitation and emission scan of the fluorophores (a) pyochelin and (b) pyoveridin. Both molecules are essential for virulence and are expressed upon quorum sensing. A difference spectrum was taken by centrifuging the cultivation medium and measuring the supernatant against medium.

DETAILED DESCRIPTION OF THE INVENTION

(17) The inventors discovered that applying IMPI, having for example the amino acid sequences of SEQ ID NOs: 10, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and IMPI-fusions, having for example the amino acid sequences of SEQ ID NOs: 6, 8, 12, 86, 88, 90, 92, reduce and stop growth of resistant bacteria at any stage of the infection, especially when applied in combination with at least one further bactericidal compound, surprisingly when the composition of the invention was applied at early stages of the infection. This observation is surprising because IMPI interfere with M4 protease activity, for example with thermolysin, pseudolysin, aureolysin, vibriolysin, bacillolysin and npr599, which are shed only at high bacterial concentrations, e.g. during or after biofilm formation as is the case for pseudomonas aeruginosa. Therefore one has to expect that M4 protease inhibitors only interfere with bacteria after biofilm growth, as several publications on non resistant bacteria suggest.

(18) The inventors discovered, however, that IMPI and IMPI fusions or its combination with bactericidal compounds delays, stops or even reverses growth of resistant bacteria in a solution containing just non adherent bacteria. In this experiment, care was taken by visual inspection that biofilm formation and hence protease shedding had not yet started. More precisely it was observed that a delay of bacterial growth starts much earlier during the so called late log (late logarithmic) phase, when bacterial growth kinetics still follows a logarithmic function of time.

(19) The inventors also discovered that IMPI and IMPI fusions or its combination with at least one bactericidal compound effectively prohibits biofilm formation, even for resistant bacterial strains.

(20) Furthermore the inventors discovered that combining IMPI or IMPI-fusions and antibiotics always inhibits growth of resistant bacteria synergistically, regardless of the antibiotics concentration and the stage of the infections. Even at sub inhibitory antibiotic doses IMPI or IMPI-fusions interfere with bacterial growth.

(21) The inventors observed, for example, that the synthesis of the P. aeruginosa siderophore pyoverdine, known to be involved in the synthesis of virulence factors, is inhibited synergistically by IMPI and the antibiotic gentamycin.

(22) Thus, subject matter of the invention are compositions containing IMPI or IMPI-fusions and at least one further bactericidal compound, and the use of any IMPI against partially or completely resistant or even multiresistant strains of bacteria, such as multiresistant Staphylococcus aureus (MRSA) alone or in combination with antibiotics or other bactericidal compounds to treat patients or protect devices, especially implants. This use of IMPI or IMPI-fusions is particularly advantageous since it is bactericidal even without applying antibiotics in parallel, or even with very low doses of antibiotics. IMPI or IMPI-fusions may be used to affect bacteria being planktonic, isolated sessile or forming biofilms, at any stage of an infection including early stages. Thus, bacteria resistant against antibiotics will still be affected by IMPI. Moreover, IMPI can act synergistically with antibiotics in areas of the patient's body where antibiotics concentrations are low due to, e.g., rapid dilution or low drug influx related to low diffusion rate or mechanisms inducing active outflow of the drug. So by use of IMPI and IMPI fusions and combinations with bactericidal compounds according to the invention these areas cannot become areas where, due to sub inhibitory concentrations of the antibiotic, bacteria could respond to the challenge by developing resistance.

(23) Further contemplated is the use of IMPI or IMPI-fusions with the IMPI element exhibiting additional modifications, such as chemical modifications in the side chain or at the N and/or C terminal for improving biological or chemical properties such as bioavailability, stability, and effectivity. The modification may also provide for a detectable label, for example a chemiluminescent structural element, one or more radioactive isotopes in one or more side chains of an amino acid in the polypeptide, an enzyme which is able to generate a colour reaction and the like. A cystein, for example, may be added for linking a water soluble polymer such as polyethylene glycol, or other amino acids like lysine, cysteine, histidine, arginine, asparaginic acid, glutamic acid, serine, threonine, or tyrosin could also be used for coupling polymers to the peptide. Another example is the insertion of tripeptide sequences NXT or NXS or fragments thereof with X designating any amino acid except P, which may be recognized by a cellular enzyme adding glycosylation elements. Suitable, clinically acceptable, water soluble polymers include polyethylenglycol (PEG) and polysialic acid (PSA).

(24) IMPI-fusions according to the invention comprise IMPI and at least one polypeptide having a physiological function, in particular IMPIR, an antibody or antibody fragment, scaffolds such as lipocalin, ankyrin, fibronectin, transferrin, tetranectin, adnectin, albumin, uteroglobin, or protein A, functional peptides such as transferrin, peptides useful for diagnostic applications, such as green fluorescent protein (GFP), or peptide tags enabling immobilization on technical surfaces, such as hexahistidine, or glutathione-S-transferase (GST).

(25) There are three super families (cytosolic, mitochondrial, and MAPEG) of GSTs: while classes from the cytosolic super family of GSTs possess more than 40% sequence homology, those from other classes may have less than 25%. Cytosolic GSTs are divided into 13 classes based upon their structure: alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi, and omega. Mitochondrial GSTs are in class kappa. The MAPEG super family of microsomal GSTs consists of subgroups designated I-IV, between which amino acid sequences share less than 20% identity. Human cytosolic GSTs belong to the alpha, zeta, theta, mu, pi, sigma, and omega classes, while six isozymes belonging to classes I, II, and IV of the MAPEG super family are known to exist:

(26) TABLE-US-00002 GST Class Homo sapiens GST Class Members Alpha GSTA1, GSTA2, GSTA3, GSTA4, GSTA5 Kappa GSTK1 Mu GSTM1, GSTM1L (RNAi), GSTM2, GSTM3, GSTM4, GSTM5 Omega GSTO1, GSTO2 Pi GSTP1 Theta GSTT1, GSTT2, GSTT4 Zeta GSTZ1 (aka GSTZ1 MAAI-Maleylacetoacetate isomerase) Microsomal MGST1, MGST2, MGST3

(27) An IMPI fusion may also comprise a linker of 1-100 amino acids between IMPI and the polypeptide.

(28) Another subject matter of the invention are nucleic acids, especially single stranded RNA, coding for IMPI or IMPI-fusion, which are administered into a patient and taken up by cells into their cytoplasm, where the cellular protein expression machinery expresses the IMPI or IMPI-fusion from the nucleic template. Preferred are nucleic acids coding for an IMPI-fusion, wherein the fused element comprises a signal peptide inducing secretion of the assembled and posttranslationally modified IMPI-fusion protein. Once secreted, the IMPI-fusion protein acts in a manner similar to an IMPI or IMPI-fusion protein directly applied to the patient.

(29) Nucleic acids according to the invention may be modified to resist degradation and improve delivery. Useful modifications include LNA (Locked nucleic acids) or PNA (peptide nucleic acids), and phosphodiester or phosphorothioate modified backbones. Specific formulations for nucleic acid administration in a pharmaceutical composition include liposomes.

(30) The use of the polypeptide or fusion polypeptide comprising IMPI according to the invention includes treating patients, such as humans or animals infected by microorganisms capable of secreting bacterial toxins of the M4 or Metzincin family of metalloproteinases, in particular thermolysine, aureolysin, bacillolysin, pseudolysin, vibriolysin, Msp peptidase, Mpl Peptidase, or anthrax npr599.

(31) In another aspect of the invention, the simultaneous use of antibiotics or other bactericidal compounds, and IMPI or IMPI-fusions is provided at any time of infection, including early stages. Simultaneous application may comprise dosing schemes with a delay between application of antibiotics and M4 protease inhibitors, a different application frequency or different and individually evolving dosings.

(32) These drug application schemes may prove beneficial for the patient or facilitate the application.

(33) Bactericidal compounds amenable for use according to the invention include all antibiotics, such as listed in http://en.wikipedia.org/wiki/List_of_antibiotics, for example. They further include antibodies like the anti-Pseudomonas-PcrV antibody Fab fragment (KB001, Kalos Therapeutics, Inc.), and a fully human IgG1 antibody highly specific for S. aureus Exotoxin (KBSA 301, Kenta Biotech Ltd.)

(34) Subject matter of the invention are also the use of IMPI or IMPI fusions or a nucleic acid comprising a section coding for IMPI or IMPI-fusions in a suitable pharmaceutical composition and the use thereof to treat bacterial infections, especially in combination with antibiotics in a single pharmaceutical composition so that they are always applied simultaneously to the patient.

(35) Another embodiment of the invention comprises the use of antibiotics or other bactericidal compounds in one pharmaceutical composition and of IMPI or IMPI-fusions or a nucleic acid comprising a section coding for IMPI or IMPI or IMPI-fusions in a separate one. The separate entities can be advantageous for treating patients since the doses relative to each other can be varied independently over time, as well as the individual frequency of administration. A delayed application of the two compositions may be beneficial to prohibit side effects to occur or to limit their strength. Separate entities exhibit the further advantage that different formulations can be chosen, which may even be required for particular molecule combinations.

(36) Subject matter of the invention are also particular dosing schemes, such as combining maximum doses for both, IMPI and bactericidal compounds. Another dosing scheme may include reduction of the applied dose of the bactericidal compound for some time, even down to sub inhibitory doses, where the combination of IMPI and the bactericidal compound is still active. The benefit of such temporary dose regimen would be to encounter development of resistance against the compounds.

(37) IMPI or IMPI-fusions may be combined with ingredients to form a pharmaceutical composition. The pharmaceutical composition may include water and salts at physiological concentrations, solubilizing or dispersing agents, or anti-oxidant, or particles forming micelles, such as liposomes. This pharmaceutical composition may be filled in a glass or plastic vials, or in a syringe. The pharmaceutical composition may also contain additives supporting drying or freeze-drying of the pharmaceutical composition, for example cyclodextrins or saccharides, in particular disaccharides.

(38) IMPI or IMPI-fusions or nucleic acids encoding for IMPI or IMPI-fusions and combinations with bactericidal compounds may be administered parenterally, orally, or topically using suitable pharmaceutical compositions, or attached to a patch or wound debridement from where the medication elutes into a wound of the patient.

(39) IMPI or IMPI-fusions or nucleic acids encoding for IMPI or IMPI-fusions and combinations with bactericidal compounds may be administered in biodegradable containers suitable for implantation into patients, or a reservoir attached or included in a device may contain IMPI or IMPI-fusions actively or passively deployed so that the device is situated in an area with known high load of target bacteria.

EXAMPLES

(40) 1. Measurements of Extracellular DNA in Microtitre Tray Cultures

(41) A method for tracking eDNA (extracellular DNA) was derived and compiled partly from similar procedures and conditions found in the literature and modified by the inventors.

(42) The reference strain P. aeruginosa (DSM No. 50071; MIC 8 mg/liter), partially resistant to Gentamycin, was studied in parallel with a clinical isolate of P. aeruginosa PAO1 (DSM No. 19880) to evaluate eDNA accumulation over time in the presence and absence of IMPI. First the strains were cultured in NB medium (Nutrient Broth No. 4) overnight at 37 C. and grown to stationary phase. From these bacteria, 5 l were used to inoculate 96-well black flat bottom plates (Greiner) containing NB medium (200 l). The NB medium contained either double concentrated NB medium (2; 100 l) diluted with 100 l TBS-buffer (Negative Control) or medium diluted with IMPI, which was previously solved in TBS (100 l; Positive Control). The final concentration of IMPI per well was 35 M for experiments with P. aeruginosa DSM 50071 and DSM 19880 respectively. To stain extracellular DNA and membrane-compromised (dead) bacteria in aggregates of P. aeruginosa, 1 L of 1 mm stock solution of BOBO-3 stain (Life Technologies) was added to 5 mL cultures at the start of growth experiments (incubation in the dark). BOBO-3 is a membrane-impermeable fluorescent dye (Aex570, em602) that binds to DNA and therefore specifically stains extracellular DNA which Images were taken over 48 h by a high-definition area scan (9999 points) on a well of a 96-well microplate using a synergy H4 plate reader (Biotek).

(43) 2. MIC Determination and OD-Measurement

(44) Gentamycin MIC (minimal inhibitory concentration) values were determined using a standard two fold microtiter broth dilution protocol with Nutrient Broth as medium. Midexponential phase cultures of P. aeruginosa reference strain DSM No.: 50071 and two antibiotic-resistant clinical isolates (P. aeruginosa VB7623 and VB7444), which were isolated from the tracheal secrete of a patient, were tested. Antimicrobial susceptibility testing of the clinical isolates was performed by the University Clinic Tubingen. Typically gentamicin concentrations between 8 g/ml and 0.0625 g/ml were chosen to estimated bacterial growth and determine the MIC values. Further, the OD (optical density) at 600 nm in the presence and absence of the insect metalloprotease inhibitor IMPI was investigated. For assessing influences of IMPI on bacterial growth final concentrations of IMPI between 20 and 75 M were used. Bacterial growth was monitored at 37 C. over 48 h. All MIC values were done as triplicates.

(45) The subsequent table lists the sequences printed in the ensuing sequence protocol. The leading number denotes the SEQ ID NO for the nucleotide sequence, the subsequent even number missing number would denote the SEQ ID NO of the respective peptide sequence.

(46) 1 IMPIalpha (wild type or wtIMPIalpha)

(47) 3 IMPIbeta (wild type or wt IMPIbeta)

(48) 5 GST/IMPIalpha

(49) 7 GST/IMPIbeta

(50) 9: IMPIalpha Pos37 NnG Pos38 InL Pos39 RnA

(51) 11: GST/IMPIalpha Pos37 NnG Pos38 InL Pos39 RnA

(52) 13: IMPIalpha Pos52 RnK

(53) 15: GST/IMPIalpha Pos52 RnK

(54) 17: IMPI Pos35 InL

(55) 19: IMPI Pos35 InM

(56) 21: IMPI Pos35 InF

(57) 23: IMPI Pos35 InC

(58) 25: IMPI Pos35 InN

(59) 27: IMPI Pos35 InQ

(60) 29: IMPI Pos35 InH

(61) 31: IMPI Pos35 InK

(62) 33: IMPI Pos35 InR

(63) 35: IMPI Pos36 InV

(64) 37: IMPI Pos36 InM

(65) 39: IMPI Pos36 InF

(66) 41: IMPI Pos36 InW

(67) 43: IMPI Pos36 InY

(68) 45: IMPI Pos36 InS

(69) 47: IMPI Pos36 InT

(70) 49: IMPI Pos36 InN

(71) 51: IMPI Pos36 InQ

(72) 53: IMPI Pos36 InH

(73) 55: IMPI Pos36 InR

(74) 57: IMPI Pos36 InK

(75) 59: IMPI Pos39 InV

(76) 61: IMPI Pos39 InK

(77) 63: IMPI Pos35 InW

(78) 65: IMPI Pos35 InY

(79) 67: IMPI Pos39 RnA

(80) 69: IMPI Pos35 InC

(81) 71: IMPI Pos35 InK

(82) 73: IMPI Pos35 InR

(83) 75: IMPI Pos35 InL

(84) 77: IMPI Pos35 InM

(85) 79: IMPI Pos35 InF

(86) 81: IMPI Pos35 InQ

(87) 83: IMPI Pos35 InH

(88) 85: GST/IMPI Pos35 InN

(89) 87: GST/IMPI Pos36 InS

(90) 89: GST/IMPI Pos39 InK

(91) 91: GST/IMPI Pos35 InC

(92) 93: IMPI signal peptide,

(93) 95: IMPI like (Solenopsis, Peptide only, no nucleotide sequence provided)

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