US 20090304673A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0304673 A1 Buchberger et al. (43) Pub. Date: Dec. 10, 2009

(54) METHOD OF TREATMENT OF Publication Classification NFLAMMLATORY DISEASES (51) Int. Cl. (76) Inventors: Bernd Buchberger, Zeitlarn (DE); A638/48 (2006.01) Holger Grallert, Regensburg (DE); A638/43 (2006.01) Ingrid Wanninger, Regensburg CI2N 9/00 (2006.01) (DE) (52) U.S. Cl...... 424/94.67; 424/94.1; 435/183 Correspondence Address: FULBRIGHT & UAWORSK L.L.P. 600 CONGRESSAVE., SUITE 2400 (57) ABSTRACT AUSTIN, TX 78701 (US) (21) Appl. No.: 12/482,174 The present invention relates to a method of treatment of an inflammatory disease, in particular sepsis, in particular sepsis (22) Filed: Jun. 10, 2009 caused by gram-positive bacteria, wherein the method com Related U.S. ApplicationO O Data ERNrises the ANAstep of administeringof El to aWild subject in need enzymes. thereof a (60) Provisional application No. 61/060,314, filed on Jun. The present invention relates further to pharmaceutical com 10, 2008. positions comprising bacterial cell wall degrading enzymes. Patent Application Publication Dec. 10, 2009 Sheet 1 of 7 US 2009/0304673 A1

1e--6 ...is 60min 360min e5

1e--3

1e-2

1e-1 control PRF-100

F.G. 1

cfulg heart 360min

6e-A -

3e-4 -

2e+4 -

1.e4 -

control PRF-100

FIG. 2 Patent Application Publication Dec. 10, 2009 Sheet 2 of 7 US 2009/0304673 A1

TNFa. Values after S. aureus infection +l- PRF-100 treatment

500 450 400 350

300 Control 250 200 150 100 50

5 min 60 min 360 min

FIG. 3A

L6 values after S. aureus infection - PRF-100 treatment

Control

FIG. 3B Patent Application Publication Dec. 10, 2009 Sheet 3 of 7 US 2009/0304673 A1

1,00E+08

1,00E+07

1,00E+06

1,00E+05

1,00E+04

1,00E+03

1 OOE+02

1,00E+01

1,00E+00 injected cfu/ml animals without PRF. 100 Lysostaphin treatment

FG, 4 Patent Application Publication Dec. 10, 2009 Sheet 4 of 7 US 2009/0304673 A1

Figure 5A

A 1800,00 1600,00 ir 1400,00 1200,00 rho 1000,00 800,00 ir

Figure 5B

10000,00 9000,00 8000,00 rhir 700000 rho 6000,00 5000,00 4000,00 r

3000,00 ir 2000,00 ir 1000.00 or 0.00 ir------Patent Application Publication Dec. 10, 2009 Sheet 5 of 7 US 2009/0304673 A1

Figure 5C

4000,00 3500,00 r 3000,00 irr 2500,00 irr

Patent Application Publication Dec. 10, 2009 Sheet 6 of 7 US 2009/0304673 A1

A 6 h 300 splenocytes culture A 250 3: E. Culture B

200

150

100 50

2 3 4. 5 6 LPS neg.

350 splenocytes Culture A 300 : Culture B

250

200

150

100

50

2 3 4 5 6 LPS neg. group

FIG. 6A Patent Application Publication Dec. 10, 2009 Sheet 7 of 7 US 2009/0304673 A1

B 6 h 140 splenocytes culture A

120 Culture B 100 E G 80

9t O 60

40

20

1 2 3 4. 5 6 LPS neg. group

24h 1000 splenocytes culture A is culture B 800 3 600 up 400

200

FIG. 6B US 2009/0304673 A1 Dec. 10, 2009

METHOD OF TREATMENT OF promised patients, including individuals infected with HIV. NFLAMMATORY DISEASES cancer and transplant patients receiving immunosuppressive therapy. One of the key risk factors is the presence of foreign CROSS-REFERENCE TO RELATED objects in patients undergoing Surgery, for example intrave APPLICATION nous lines and urinary catheters. 0008. In the US, severe sepsis has been estimated to affect 0001. This application claims benefit of U.S. Provisional over 750,000 individuals per year, equating to 300 cases per Application Ser. No. 61/060,314, filed Jun. 10, 2008, the 100,000 population. Importantly, surveillance in the US has entire contents of which are incorporated by reference. shown that the incidence of severe sepsis has doubled in the past 20 years and is expected to increase by 1.5% per annum, BACKGROUND Such that the total number of cases might rise to one million 0002 A. Field of the Invention by 2010 (www.emedicine.com, 2003). Although European 0003. The present invention relates to a method of treat studies have estimated the sepsis incidence at 51-115 cases ment of an inflammatory disease, in particular sepsis, in par per 100,000 population, experts agree that there is no reason ticular sepsis caused by gram-positive bacteria, wherein the for the incidence in these two geographical areas to differ. method comprises the step of administering to a Subject in 0009. The rise in sepsis is following advances in medical need thereof a sufficient amount of bacterial cell wall degrad practice, which have led to the extension of the normal ing enzymes. The present invention relates further to phar lifespan, the increase in the number of invasive Surgical pro maceutical compositions comprising bacterial cell wall cedures, and the size of the patient pool with weakened degrading enzymes. immune systems: 0004 B. Background of the Invention 0.010 the aging of the population has led to an increase 0005 Sepsis is a complex, multifactoral and rapidly pro in the number of people with a weaker immune system gressing disease characterized by an excessive inflammatory or Suffering from chronic conditions that increase the response to infection that leads to organ failure and death. likelihood of developing sepsis, such as diabetes, cancer, Severe forms of sepsis such as septic shock result in up to 80% blood diseases, and liver, kidney and lung infections; mortality. In the US, sepsis is the 10th leading cause of death, 0.011 the number of immunosuppressed patients is killing over 200,000 people annually. increasing, including cancer, HIV-infected, and trans 0006 Sepsis is often confused with blood poisoning, a plant patients; condition also associated with the presence of bacteria in a 0012 the overuse of antibiotics for infections not patient's blood. Sepsis is a complex illness consisting in the caused by bacteria have led to high levels of resistant overreaction of the body to an infection. Central to the patho bacteria, rendering the treatment of sepsis very difficult. genesis of sepsis is the host immune response, which triggers 0013 For many years the terms sepsis, septicemia, bacte a systemic chain reaction that leads to the activation of the remia, infection, septic shock and toxic shock were used inflammatory response and the coagulation cascade, ulti interchangeably. However, with time it became clear that the mately leading to multiple organ damage, organ failure, and reason for the ultimate multiple-organ failures was systemic eventually death. The high mortality associated with sepsis is inflammation, rather than the original infection itself. In order partly a consequence of its rapid progression of the disease, to eliminate the prevailing climate of confusion and ambigu with patients often progressing from sepsis to severe sepsis, ity, in 1992 the American College of Chest Physicians and the septic shock and organ damage in a matter of hours. Despite Society of Critical Care Medicine organized a consensus significant progress in the management of sepsis, mortality conference aimed at establishing a set of definitions that remains unacceptably high, estimated at 20-50% for severe could be applied to patients with sepsis and its sequelae sepsis and 45-80% for septic shock (EMEA, 2005). In the US, (ACCP-SCCM Consensus Conference, 1992). According to sepsis causes more than 200,000 deaths per year, more than these definitions, sepsis is a progressive disease that consists Some common forms of malignancies including lung, colon of various stages, which can be clearly differentiated based on and breast cancer (www.survivingsepsis.org). specific physiological occurrences. 0007 Up to 70% of sepsis cases are caused by bacterial 0014. The consensus meeting established the following infection, with the remainder of cases due to fungi, viruses definitions for the different stages of the disease: and parasites, although polymicrobial infections can also be 0.015 systemic inflammatory response syndrome present. Recent epidemiological studies Suggest that there (SIRS) is a widespread inflammatory response to any of might be a similar contribution of gram-negative and gram a wide variety of severe clinical insults. Visible symp positive infections, with the recent increase in gram-positive toms resulting from these conditions are: fever, chills, infections likely due beaconsequence of the increasing emer severe shaking, tachycardia, confusion, disorientation gence of drug-resistant bacterial Strains, for example methi and agitation, rash on the skin and pain in the joints; cillin-resistant Staphylococcus aureus (MRSA). Almost half 0016 sepsis is defined as the systemic inflammatory of all infections leading to sepsis originate in the lung, fol response to infection. Sepsis patients thus represent a lowed by infections of the bloodstream, the urinary tract and subset of SIRS patients: the abdomen. The body's overreaction to the infection or 0017 severe sepsis is associated with organ dysfunc toxins released by the pathogen then leads to a widespread tion, hypoperfusion or hypotension. Clinical manifesta inflammation causing extensive collateral damage to the tions of hypoperfusion may include lactic acidosis, olig host’s microcirculation, increased blood clotting, and uria or an acute alteration in mental status. Sepsis can impaired fibrinolysis, culminating in the formation of Small turn into severe sepsis within a few hours; blood clots that eventually damage vital organs. Individuals 0.018 septic shock, a subset of severe sepsis, is defined with a weakened immune system are at an increased risk of as sepsis-induced hypotension despite adequate fluid sepsis, in particular the elderly, children, and immunocom resuscitation, combined with perfusion abnormalities US 2009/0304673 A1 Dec. 10, 2009

that may include, but are not limited to, lactic acidosis, pathogens and their Susceptibility profiles are deter oliguira or an acute alteration in mental status (Patients mined, and by employing the shortest course of therapy receivinginotropic or vasopressor agents may no longer clinically acceptable. Antibiotics need to be adminis be hypotensive by the time they manifest hypoperfusion tered intravenously (IV) at doses high enough to achieve abnormalities or organ dysfunction; yet they would still bactericidal serum levels. be considered to have septic shock. These patients gen 0026. However, sepsis is as much a result of an imbalance erally present three cardiovascular upsets: vasodilation, in the coagulation and fibrinolysis cascades as an overwhelm reduced stroke Volume, and microcirculatory failure. ing inflammatory response. Thus, there is a need for new Importantly, over half of all patients with septic shock therapeutic substances for the treatment of inflammatory dis have bacteremia. Although the exact sequence of the eases like sepsis. events that lead to septic shock is not fully understood, two key factors associated with septic shock are the SUMMARY OF THE INVENTION predominance of the pro-inflammatory over the anti inflammatory response, and coagulation over fibrinoly 0027. The present invention relates to a method of treat sis); ment of an inflammatory disease, in particular sepsis, in par 0019 multiple organ dysfunction syndrome (MODS) is ticular sepsis caused by gram-positive bacteria, wherein the characterized by the presence of altered organ function method comprises the step of administering to a Subject in in an acutely-ill patient such that homeostasis cannot be need thereof a sufficient amount of bacterial cell wall degrad maintained without intervention; ing enzymes. The present invention relates further to phar 0020 multi-organ failure: the activation of the coagula maceutical compositions comprising bacterial cell wall tion cascade appears to be an essential component in the degrading enzymes. development of multi-organ failure. Organs most com 0028. One aspect of the invention concerns a method of monly affected by sepsis are the brain, heart, lungs, treating an inflammatory disease in a Subject (e.g., humans, kidneys and liver. mammals, animals, etc.) comprising administering an effi 0021 Sepsis most often starts with a localized infection cient or effective amount of bacterial cell wall degrading caused by bacteria, fungi, viruses or parasites introduced by enzymes to said Subject. The bacterial cell wall degrading insults such as trauma, burn wounds, ischemia-reperfusion enzyme can be an endolysin, autolysin, lysostaphin, mytanol and major Surgery including cardiopulmonary bypass and ysin, lytic enzyme similar in function and/or structure to abdominal Surgery. 40% of infections were due to gramposi lysostaphin or murolytic enzyme. The bacterial cell wall tive infections, while 38% were due to gram-negative infec degrading enzyme can be administered in combination with tions (Vincent et al., 2006). conventional antibacterial agents (e.g., antibiotics). The bac 0022. The management of sepsis currently relies on: Con terial cell wall degrading enzyme can be administered in trol of the infection with antibiotic treatment and source con combination with conventional antibacterial agents (e.g., trol in combination with hemodynamic stabilization by fluid antibiotics and other endolysins, mytanolysins, autolysins, administration, administering vasopressor agents and inotro lysostaphins, lytic enzymes similar to lysostaphin or muro pic agents further in combination with modulation of the lytic enzymes). sepsis response by administering recombinant protein C and 0029. Another aspect of the invention concerns a pharma steroids. ceutical composition comprising a bacterial cell wall degrad 0023. Early and appropriate administration of anti-infec ing enzyme. The bacterial cell wall degrading enzyme can be tives, predominantly intravenous (IV) antibiotics, remains the an endolysin, autolysin, lysostaphin, mytanolysin, lytic cornerstone of sepsis therapy. In addition, it is often necessary enzyme similar in structure and/or function to lysostaphin or to eliminate the focus of the infection by Surgical excision/ murolytic enzyme. The pharmaceutical composition can fur removal. This affects predominantly patients with a perfo ther include a conventional antibacterial agent (e.g., an anti rated Viscus, ruptured esophagus or diaphragm, severe burns biotic agent). The pharmaceutical composition can include or abscesses, and gangrene. The choice of the antibiotic to be different bacterial cell wall degrading enzymes. The pharma used varies from patient to patient, being selected based on ceutical composition can include conventional antibacterial the microorganisms most likely to have caused infection at agents, such as antibiotics and other endolysins, autolysins, the Suspected site, the potential risk of antibiotic resistance, lysostaphins, mytanolysins, lytic enzymes similar to lysos and known patterns of microorganism presence in the specific taphin or murolytic enzymes. The pharmaceutical composi community and the hospital. tion can include a pharmaceutical acceptable a carrier. 0024. Empirical therapy is initiated with a broad-spectrum 0030 The pharmaceutical compositions and methods for antibiotic that covers gramnegative, gram-positive and their use can “comprise.” “consist essentially of” or “consist anaerobic organisms, until the results of the blood test or of any of the ingredients disclosed throughout the specifica culture become available. Following the identification of the tion. pathogen, therapy is Switched to a narrow-spectrum agent, a 0031. It is contemplated that any embodiment discussed in process known as de-escalation: this specification can be implemented with respect to any 0025 Antibacterial de-escalation is an approach that method or composition of the invention, and vice versa. Fur attempts to balance the need to provide appropriate ini thermore, pharmaceutical compositions of the invention can tial antibacterial treatment, while limiting the emer be used to achieve methods of the invention. gence of antibacterial resistance. The goal is that the 0032. The term “about” or “approximately” are defined as initial antibacterial regimen will cover the most likely being close to as understood by one of ordinary skill in the art, bacterial pathogens while minimizing the chance of and in one non-limiting embodiment the terms are defined to resistance. The risk for the latter is reduced by narrowing be within 10%, preferably within 5%, more preferably within the scope of the antibacterial regimen as soon as the 1%, and most preferably within 0.5%. US 2009/0304673 A1 Dec. 10, 2009

0033. The term “substantially' and its variations are storage buffer. After 6 hours the hearts of the animals were defined as being largely but not necessarily wholly what is prepared, homogenised and dilution series of the homogeni specified as understood by one of ordinary skill in the art, and sate were plated onto LB agar plates. After over night culti in one non-limiting embodiment Substantially refers to ranges vation at 30°C., cell forming units were determined. within 10%, within 5%, within 1%, or within 0.5%. 0034. The terms “inhibiting or “reducing” or “prevent 0043 FIG. 3A A schematic representation of the time ing’ or "avoiding or any variation of these terms, when used dependence of TNF-C. levels post S. aureus infection with or in the claims and/or the specification includes any measurable without treatment of an endolysin as depicted in SEQ ID decrease or complete inhibition to achieve a desired result. NO:1. The mean values and standard deviation of the TNF-C. 0035. The term “effective, as that term is used in the concentration in sera of rats 5, 60 and 360 minutes after specification and/or claims, means adequate to accomplish a infection with 6.5x10 S. aureus cells are blotted (lightgrey). desired, expected, or intended result. The TNF-C. concentrations after infection and subsequent 0036. The use of the word “a” or “an when used in con treatment with the endolysin as depicted in SEQID NO:1 are junction with the term “comprising in the claims and/or the blotted in dark grey. specification may mean “one.” but it is also consistent with 0044 FIG. 3B A schematic representation of the time the meaning of"one or more.” “at least one and “one or more dependence of IL-6 levels after post S. aureus infection with than one.” or without treatment of an endolysin as depicted in SEQ ID 0037. The use of the term “or' in the claims is used to NO:1. The mean values and standard deviation of the IL-6 mean “and/or unless explicitly indicated to refer to alterna concentration in sera of rats 5, 60 and 360 minutes after tives only or the alternatives are mutually exclusive, although infection with 6.5x10 S. aureus cells are blotted (lightgrey). the disclosure supports a definition that refers to only alter The IL-6 concentrations after infection and Subsequent treat natives and “and/or.” ment with the endolysin as depicted in SEQ ID NO:1 are 0038. As used in this specification and claim(s), the words “comprising (and any form of comprising, Such as "com blotted in dark grey. prise' and "comprises”), “having (and any form of having, 0045 FIG. 4.—A schematic representation of S. aureus such as “have and “has'), “including (and any form of distribution in blood of animals with or without pre-admin including, such as “includes” and “include’) or “containing istration of endolysin PRF-100 as depicted in SEQID NO:1 (and any form of containing, Such as “contains' and “con and Lysostaphin. Animals were pretreated with endolysin tain’) are inclusive or open-ended and do not exclude addi (PRF-100: 16 mg/kg) or Lysostaphin (8 mg/kg) 15 minutes tional, unrecited elements or method steps. prior to infection with 1.0x107 S. aureus cells. Blood of ani 0039. Other objects, features and advantages of the mals were taken 10 minutes after infection with S. aureus present invention will become apparent from the following cells and dilution series of blood were plated onto LB agar detailed description. It should be understood, however, that plates. After overnight growth at 30°C., the number of colo the detailed description and the specific examples, while indi nies (cfu) were determined. cating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifica 0046 FIGS. 5A-D. A schematic representation of the tions within the spirit and scope of the invention will become time dependence of cytokine levels in animals after injection apparent to those skilled in the art from this detailed descrip of S. aureus cells with or without pre-treatment of endolysin tion. PRF-100 as depicted in SEQID NO:1 or Lysostaphin. Ani mals were infected with 6.5x107S. aureus cells or 6.5x107 S. BRIEF DESCRIPTION OF THE DRAWINGS aureus cells preincubated for 3 hours at 37°C. in the presence of endolysin (PRF-100: 150 lug/ml) or Lysostaphin (150 0040. The following drawings form part of the present ug/ml). Blood of animals were taken 1 hour or 6 hours after specification and are included to further demonstrate certain injection of S. aureus cells or lysine treated cells and serum aspects of the present invention. The invention may be better was prepared. The mean values and Standard deviation (n-3) understood by reference to one or more of these drawings in combination with the detailed description of specific embodi of A, TNF-C. concentrations: B, IL-6 concentrations: C, IFN-y ments presented herein. concentrations; D, IL-1B concentrations in Sera of rats are 0041 FIG. 1—A schematic representation of the time blotted. Control: not treated animals; SA: S. aureus treated dependence of S. aureus distribution in blood with or without animals; CLPRF 100: Cell lysat of PRF 100 treated S. aureus treatment of an endolysin as depicted in SEQID NO:1. Ani cells: CL Lys: Cell lysat of Lysostaphin treated S. aureus mals were-infected with 6.5x107 S. aureus cells (black cells. bar-cell number of inocculum). One minute later, they were 0047 FIGS. 6A-B Stimulatory properties of S. aureus treated either with the endolysin as depicted in SEQID NO:1 cell wall preparations with or without pre-treatment of cell or storage buffer (control). Blood of the animals were taken 5, wall degrading enzymes in the murine splenocyte model sys 60 and 360 minutes after infection and dilution series of the tem. Splenic cells of mice were incubated for 6 hor 24 h with blood samples were plated onto LB agar plate. After over 12.5ug untreated cell wall preparations of S. aureus (group 6) night growth at 30° C., the number of colonies (cfu) were or cell walls, preteated either with 5ug/ml PRF 100 (group 1), determined. 2.5 ug/ml Lysostaphin (group 2), 10 ug/ml Mutanolysin 0042 FIG. 2. A schematic representation of the appear (group 3) or combinations of 5 g/ml PRF 100 and 10 g/ml ance of S. aureus within heart tissue 6 hours post S. aureus Mutanolysin (group 4) or 2.5 g/ml Lysostaphin and 10 ug/ml infection with or without treatment of an endolysin as Mutanolysin (group 5). LPS of E. coli O111: B4 served as depicted in SEQID NO:1. Rats were intravenously infected positive control for activation of cytokine response of primary with 6.5x107 S. aureus cells per animal and treated 1 minute murine splenocytes. Activation of the innate immune system later either with the endolysin as depicted in SEQID NO:1 or was analysed by determining the Secretion of proinflamma US 2009/0304673 A1 Dec. 10, 2009 tory cytokines TNF-a (A) and IL-6 (B). Experiments were binding partners. It is identified via a praline-rich consensus performed in duplicate (culture A and culture B). motif. The SH3 domain is usually located within the CBD. 0056. The term “shuffling as used herein refers to the DETAILED DESCRIPTION OF THE INVENTION combination of different fragments of polypeptides from dif ferent enzymes into new polypeptide constructs. In this con A. Definitions text, the enzymes are preferentially endolysins, and the frag ments are preferentially modules. Usually, the fragments are 0048. The term “bacterial cell wall degrading enzyme’ as combined by molecular biological methods on nucleic acid used herein refers to an enzyme which is suitable to hydrolyse level. Small linker sequences may be introduced between the bacterial cell walls. Said enzyme may be an endolysin, autol fragments for structural or cloning reasons. ysin, lysostaphin, lytic enzyme similar to lysostaphin or a muralytic enzyme. B. Endolysin 0049. The term “endolysin' as used herein refers to an 0057 Endolysins are enzymes, used by bacteriophages to enzyme that comprises at least one of the following activities facilitate the release of newly assembled bacteriophages at of which the “enzymatically active domains' (EADs) of the the end of the lytic life cycle. Endolysin enzyme activities endolysins are constituted: endopeptidase, N-acetyl may be divided into five classes: (1) N-acetylmuramidases nuramoyl-L-alanine-amidase (amidase), N-acetyl-murami (lysozymes), (2) endo-B-N-acetylglucosaminidases, and (3) dase or N-acetyl-glucosaminidase (lysozyme). Either, the lytic transglycosylases, which all cleave the Sugar moiety of enzyme is phage encoded or it is derived from related peptidoglycan, (4) endopeptidases, which cleave the peptide enzymes coded by bacteria, the so-called “autolysins”. In moiety, and (5) N-acetylmuramoyl-L-alanine amidases, addition, the endolysins usually contain also regions which which cut the amide bond between Sugar backbone and pep are enzymatically inactive, and bind to the cell wall of the host tide linkers. Endolysins show a modular organization exhib bacteria, the so-called CBDs (cell wall binding domains). iting a combination of different polypeptide domains show 0050. The term “domain” as used herein refers to a subunit ing at least one enzymatic activity and a cell binding activity, of an endolysin which is ascribed a specific function and can the so-called EADS (enzymatically active domains) and also coincide with structural domains. The term domain is CBDs (cell binding domains), respectively. Mostly, EADs are preferentially used to describe the antagonism between EAD located at the N-terminal part of the endolysins, and CBDs at which can be composed of more than one module and CBD the C-terminal parts, but there are also exceptions of this rule domains. of thumb. It is also shown that modules can be exchanged 0051. The term “CBD as used herein refers to the cell between different cell wall lytic enzymes producing new wall binding domain of an endolysin, which is often found at functional enzymes, which sometimes exhibit even new func the C-terminus of the protein. CBD domains have no enzy tional properties (Diaz et al., 1990, Proc. Natl. Acad. Sci. matic activity in terms of hydrolyzing the cell wall, but often USA, 87, 8125-8129: Croux et al., 1993, Molecular Micro mediate binding of the endolysin to the bacterial cell wall. biology, 9, 1019-1025; Donovan et al., 2006, Appl. & Envi CBD may contain an SH3-domain. ronm. Microbiol., 72,2988–2996). 0052. The term “EAD as used herein refers to the enzy matically active domain of an endolysin which is responsible C. Autolysin for hydrolysis of the bacterial peptidoglycan. It contains at 0.058 Autolysins are a group of enzymes that exist in all least one of the enzymatic activities of an endolysin. The bacteria containing peptidoglycan. The peptidoglycan matrix EAD can also be composed of more than one enzymatically is very rigid, so these enzymes break down the peptidoglycan active module. matrix in Small sections so that growth and division of cells 0053 A “CHAP domain (cysteine, histidine-dependent can occur. Autolysins do have similar or same enzymatic amidohydrolases/peptidases) is a region between 110 and activities like endolysins. Autolysins are naturally produced 140 amino acids that is found in proteins from bacteria, bac by peptidoglycan containing bacteria, but excessive amounts teriophages, archaea and eukaryotes of the Trypanosomidae will degrade the peptidoglycan matrix and cause the cell to family. The proteins may function mainly in peptidoglycan burst due to osmotic pressure. Grampositive bacteria regulate hydrolysis. The CHAP domain is commonly associated with autolysins with teichoic acid molecules attached to the tet bacterial type SH3 domains and with several families of ami rapeptide of the peptidoglycan matrix. dase domains. CHAP domain containing proteins may utilize a catalytic cysteine residue in a nucleophilic-attack mecha D. Lysostaphin nism. The CHAP domain contains two invariant amino acid 0059 Lysostaphin is an endopeptidase encoded by Sta residues, a cysteine and a histidine. These residues form part phylococcus simulans. to attack Staphylococcus aureus. of the putative active site of CHAP domain containing pro teins. E. Muralytic Enzymes 0054 The term “ami’ as used herein describes an enzy 0060 Muralytic enzymes are defined as enzymes cleaving matically defined module which exhibits amidase activity, i.e. the murein or petidogylcan. it hydrolyzes the amide bond between N-acetylmuramine in the peptidoglycan backbone and the adjacent amino acid F. Mutanolysin which is usually L-ala in the peptide linker. The amidase are 0061 Mutanolysin is a N-acetylmuramidase encoded by often metal ion dependent for activity. Streptomyces globisporus. 0055. The term “SH3 domain which is sometimes also called Src homology 3 domain as used herein describes a G. Sepsis Small non-catalytic protein domain of about 60 amino acids 0062 Bacterial products commonly leading to septic which is characteristic for proteins which interact with other shock are lipopolysaccharide (LPS) or endotoxin, pepti US 2009/0304673 A1 Dec. 10, 2009

doglycan, bacterial lipoproteins, mycobacterium lipoproteins been biochemically isolated or which have been recombi and lipoarabinomannan, flagellin and heat shock proteins nantly generated. Said enzymes further may have been Syn (SOM 208 Medical Microbiology Syllabus). Once the infec thetically engineered, in particular by amino acid modifica tious agent or toxin activates the immune system, inflamma tions like substitutions, deletions or additions. Said bacterial tion characterized by the usual symptoms (pain, Swelling, cell wall degrading enzymes combine the activities of binding redness and heat) follows. to the bacterial cells, lysis of bacterial cells and neutralizing 0063. The cell wall of gram-positive bacteria contains of cell wall degradation products. lipoteichoic acid (LTA) and peptidoglycan (PepG), which can 0067. In a preferred embodiment the endolysin is com activate leukocytes, stimulate the generation of proinflamma posed of a CBD domain of one endolysin and an EAD of tory cytokines, and hence, cause a moderate systemic inflam another endolysin. In a particularly preferred embodiment matory response syndrome. The endogenous vasodilator said recombinant endolysin comprises an endolysincell bind autacoid nitric oxide (NO) is generated by three different ingdomain of the SH3 type. Preferably, the CBD domains are isoforms of NO synthase (NOS), two of which are expressed selected from the endolysin CBD domains of ply USA, or constitutively (eNOS in endothelium, nNOS in brain), the ply pitti20. As CBD of ply USA in particular the sequence third (iNOS) induced by endotoxin (LPS) or cytokines. There as denoted in SEQ ID NO:2 is preferred. As CBD of ply is now substantial evidence that an enhanced formation of NO pitti20 in particular the sequence as denoted in SEQID NO:3 by iNOS contributes to the circulatory failure (hypotension, is preferred. and vascular hyporeactivity to vasoconstrictors) and possibly the organ injury associated with endotoxemia. LTA is a mac 0068. In a particularly preferred embodiment said recom roamphiphile, equivalent to LPS in gramnegative bacteria, binant endolysin comprises an enzymatically active domain containing a substituted poly-glycero-phosphate backbone which derives from a different endolysin than the CBD. Illus attached to a glycolipid. The glycolipid content in LTA trative examples for Such recombinant endolysin variants and resembles the bacterial membrane composition, which usu consists of the EAD from ply pitt26 and the CBD from ally varies in a genus-specific manner. LTA from Staphyllo ply USA. coccus aureus can cause a moderate induction of iNOS which 0069. In a further preferred embodiment the endolysin (in murine macrophages) requires the activation of tyrosine may comprise one or more of the following modifications: kinases and NFk B. Although LTA from S. aureus can cause amino acid substitutions, deletions or additions. Particularly moderate hypotension in the rat, LTA (unlike S. aureus itself) preferred is the polypeptide according to SEQID NO:1. Said does not cause multiple organ failure or death in this species. polypeptide is derived from ply pitti26, i.e. the CBD of ply However, we have discovered recently that LTA and PepG act pitti 26 is replaced with the CDB of plyUSA as depicted in in synergy to release TNFalpha and IFNgamma, to induce SEQID NO:2 and said polypeptide having the following five iNOS, and to cause shock and multiple organ failure in anes singleamino acid substitutions L55H, L56T, E163O, R167A thetized rats. and Y20OH. 0064. In addition, it is not known which of the structural 0070. In particular said bacterial cell wall degrading components of PepG (or LTA) is essential for the observed enzymes may be administered in case the inflammatory dis synergism. PepG is a large polymer that provides stress resis ease, in particular a sepsis is caused by gram positive bacteria tance and shape-determining properties to bacterial cell e.g. Staphylococcus, Streptococcus or Enterococcus. walls. This polymer contains long Sugar chains of two alter 0071. During sepsis the cytokine cascade is strongly acti nating Sugar derivatives, N-acetylglucosamine (NAG) and vated in response to bacterial stimuli. In the early phase the N-acetylmuramic acid (NAM), which are highly cross-linked proinflammatory cytokines (e.g. IL-6 and TNFa) are upregu by peptide subunits and bridges. The peptide subunit (or stem lated which is triggered by toxins derived from cell wall peptide) consists of alternating 1- and d-amino acids, up to components (Peptidoglycan=PG and Lipoteichoic four or five in length, and is connected to the COOH group of acids=LTA). During ongoing sepsis further inflammatory NAM. Among different bacterial species, the structure of the pathways and NO production are started, ending up with Sugar chains is highly preserved, while the composition of the multiple organ failure and death. During gram positive sepsis peptide subunits varies. The present inventors now have the main stimulator of the inflammatory response are the investigated which of the structural elements of PepG are LTAS and PG-fragments of the bacterial cell walls. Thereby, essential to synergize with LTA to cause the induction of LTA in combination of PG-fragments act synergistically and iNOS in macrophages, and shock and multiple organ failure lead to a pronounced onset of sepsis. in vivo. The results of the investigation lead to the present 0072. It has been shown, that different cell wall degrading invention. enzymes (PRF-100, Lysostaphin, Mutanolysin) exhibit dif 0065. Thus, one aspect of the present invention is directed ferent potency in eliminating bacteria from the blood stream. to a method of treatment or prophylaxis of an inflammatory Additionally, the inventors found that such enzymes neutral disease, in particular sepsis, in particular sepsis caused by ize and/or degrade the cell wall toxins of bacteria and thereby gram-positive bacteria. The method comprises the steps of Suppress the pro-inflammatory cytokine response and Subse administering to a subject in need thereofa Sufficient amount quent cascades. Surprisingly, the efficiency of the enzymes to of bacterial cell wall degrading enzymes. Said Subject may be kill the bacteria is not correlated with their ability to suppress a human Subject or an animal, in particular animals used in the proinflammatory response, in Vivo and in vitro. livestock farming and/or dairy farming such as cattle. 0073. In other words, the bacterial cell wall degrading 0066. In particular the bacterial cell wall degrading enzymes have the capability to neutralize the cell wall deg enzyme is a bacteriophage endolysin, an autolysin, lysos radation products in a way that the release of proinflammatory taphin, mutanolysin, lytic enzyme similar to lysostaphin and cytokines is significantly reduced. Neutralization in this murolytic enzymes. Said bacterial cell wall degrading respect shall mean, the binding and thereby neutralizing of enzymes may be naturally occurring enzymes which have LTA, the binding and thereby neutralizing PG and its frag US 2009/0304673 A1 Dec. 10, 2009

ments, and further hydrolysis of PG and its fragments and and the number of colony forming units determined. Deter thereby destroying the activating potential of PG. mination of cfus results in 6.5x107 cell/ml. 0074 Thus, said method of treatment may be for the treat ment or prophylaxis of inhibiting or reducing the level of Example 2 proinflammatory cytokines like TNF-a, IL-6, IL-1B. IFN-g. The reduction of the level of proinflammatory cytokines like Preparation of Cell Debris TNF-a, IL-6, IL-1 B, IFN-g is preferably about 40%, 50% or 60% and more preferably about 70%, 75%, 80%, 85%, 90% I0082 1.2x107 S. aureus cells were buffered in /2 TBS or 95%. The reduction of the level of said proinflammatory buffer and lysed with 150 ug/ml of an enzyme according to cytokines can be determined by methods well known by a SEQ ID NO:1 (designated PRF-100) or 150 ug/ml Lysos person skilled in the art and as e.g. described in the examples taphin for 3 hat 37°C. To disrupt aggregates in this solution of the present invention. the debris was sonified for 30 seconds on ice, aliquoted and 0075. Furthermore, the neutralizing capability of bacterial frozen at -80° C. cell wall degrading enzymes can be measured by reducing the stimulation of proinflammatory cytokines. Example 3 0076 Furthermore, the method of treatment or prophy Infection/Treatment of Rats with S. aureus/PRF-100 laxis comprises the steps of administering to a subject in need thereof a sufficient amount of bacterial cell wall degrading I0083 Narcotized 250 gram sprague-Dawley (CD) rats enzymes in combination with conventional antibacterial were infected with 1 ml of S. aureus solution. One minute agents, such as antibiotics. Furthermore, said enzymes may later, S. aureus infected animals were treated with an enzyme be administered in combination with other endolysins, autol according to SEQID NO:1 solution (16 mg/kg) or a corre ysins, lysostaphins, mutanolysins, lytic enzymes similar to sponding Volume of an enzyme storage buffer. lysostaphin or murolytic enzymes. Furthermore, said enzymes may be administered in combination with conven Example 4 tional antibacterial agents. Such as antibiotics and other endolysins, autolysins, lysostaphins, mutanolysins, lytic Preparation of Blood, Serum Samples and Heart Tis enzymes similar to lysostaphin or murolytic enzymes. sue of the Animals 0077. The dosage and route of administration used in a method of treatment or prophylaxis according to the present 0084. After 5, 60 and 360 minutes narcotized animals invention depends on the specific inflammatory disease to be were killed, blood samples were collected and either hepari treated. The route of administration may be for example in nised or serum samples prepared. Serum samples of the ani particular embodiments parenteral, intravenous, rectal or any mals were analysed for TNF-a and IL-6 levels corresponding other route of administration. to manufacturers instructions (Quantakine cytokine kits; 0078 For application of a bacterial cell wall degrading R&D systems). Blood samples were analysed for the number enzyme alone or in combination with other enzymes or con of living S. aureus cells (cfu/ml) by plating dilution series of ventional antibacterial agents to a site of infection (or site Proteinase K treated samples. Heart tissue of the animals, endangered to be infected) the enzymes may be formulated in infected with living S. aureus and treated with an enzyme Such manner that the enzymes are protected from environ according to SEQIDNO:1 or buffer, were prepared, homoge mental influences such as proteases, oxidation, immune nised in PBS buffer and number of living cells (cfu) were response etc. determined by plating dilution series onto LB agar plates. 0079. In a further aspect of the present invention the above mentioned bacterial cell wall degrading enzymes are a com Example 5 ponent of a pharmaceutical composition, which optionally Infection/Pre-Treatment of Rats with S. aureus/PRF comprises a carrier Substance. 100 or Lysostaphin EXAMPLES I0085 Narcotized 250 gram Sprague-Dawley (CD) rats were pretreated with 16 mg/kg of PRF-100 or 8 mg/kg of 0080 All cloning procedures were performed using stan Lysostaphin, 15 minutes before infection with S. aureus cells dard techniques according to Sambrook et al. (Molecular (1x107 cfu/ml). Ten minutes after infection blood was with cloning. A laboratory manual: 2nd ed. Cold Spring Harbor drawn, serum prepared and 100 ul plated onto LBagar plates. Laboratory Press 1989). Mutations and deletions were also After growth over night at 30° C., the number of colonies introduced using standard techniques. forming units (cfu) were determined. Example 1 Example 6 Cultivation and Preparation of S. aureus Cells Infection of Rats with S. aureus Cells and S. aureus Cells Pretreated with PRF-100 or Lysostaphin 0081. An overnight culture of Staphylococcus aureus DMSZ 11823 was diluted into fresh BHI culture medium and I0086 Narcotized 250 gram Sprague-Dawley (CD) rats grown at 37° C. to mid-log phase (OD600 nm=0.78). Then, were infected with 1x107S. aureus cells or cell debris gener cells were harvested by centrifugation and resuspended in ated by PRF-100 or Lysostaphin. Blood of animals were equal volume of PBS buffer. To control the number of viable taken 1 hour or 6 hours after injection of S. aureus cells or cells in this preparation, dilution series of the cell Suspension lysine treated cells and serum was prepared. Measurement of were plated onto LB agar plates, grown over night at 30° C. proinflammatory cytokines TNF-O, IL-6, IFN-Y and IL-1B US 2009/0304673 A1 Dec. 10, 2009

was performed using commercial QuantikinetR) ELISA kits 70 uM cell strainer, which is placed on a 50 ml Falcon tube. following the instructions of the manufacturer. Single cell Suspensions were generated by grinding the spleen against the cell Strainer with the plunger of a 5 ml syringe until Example 7 mostly fibrous tissue remains left. Singularized cells were aspirated from the cell strainer by repeatedly adding 2 ml Preparation of S. aureus Cell Walls splenocyte medium. Obtained cell Suspensions were sedi 0087 An 2 liter over night culture of S. aureus mented by centrifugation at 300xg for 5 min at room tem (DSMZ11823) was harvested by centrifugation at 4500xg perature and pelleted cells were resuspended in 5 ml/spleen and resuspended in 20 ml ofbuffer A (20 mM Tris pH 7.5:250 ACK hemolysis buffer by gently but thoroughly pipetting mM NaCl and 1 mM MgCl2). Cells were mechanically dis with a 10 mL or 25 mL plastic pipette. Then, cell suspension rupted using a Microfluidizer. After centrifugation the pellet was centrifuged at 300xg for 5 min at room temperature and was dissolved in buffer A and 0.1 mg/ml DNAse and RNAse washed three times with 10 mL/spleen splenocyte medium. (Biozyme Laboratories) were added. After 30 hours at 30°C., At each washing step the cell Suspension was separated from 1 mg/ml Trypsin (Sigma) was added and incubated for addi aggregated fibrous tissue. Finally, cells were resuspended in 5 tional 6 hours at 37° C. At least, 0.1 mg/ml Proteinase K ml/spleen splenocyte medium, counted and adjusted to a final (Applichem) was added and incubated for further 24 hours. concentration of 2x10 cells/ml in splenocyte medium and After the protease treatment the cell walls were washed twice preserved at 37 C. (not longer than 30 min) until use. with buffer A. To destroy the remaining protease activity the dissolved cell walls were heat treated for 1 h at 95°C. The Example 9 optical density at 600 nm of the cell wall solution was Determination of Cytokine Levels from Mice adjusted to 100 with buffer A. 1.5 ml aliquots of the solution Splenic Cells were lyophilized. 0090. Each 2x10 freshly isolated splenic cells obtained Sample Preparation: from 3 BALB/C mice were transferred in 1 ml splenocyte medium in 24 well cell culture plates and stimulated with 12.5 I0088 0.0710 g Lyophilized cell wall preparations of S. ug/ml S. aureus cell wall preparation, pretreated either with 5 aureus (DSMZ11823) were solubilized in 960 ul HO (en ug/ml PRF 100 (sample 1), 2.5ug/ml Lysostaphin (sample 2), dotoxin-free) resulting in a solution with a final concentration 10 g/ml Mutanolysin (sample 3), 5ug/ml PRF 100+10 g/ml of 0.05 g/ml. Cell wall solutions were diluted /100 in Y2TBS Mutanolysin (sample 4) or 2.5 Lig/ml Lysostaphin--10 ug/ml buffer (OD 0.73). Treatment with cell wall enzymes was Mutanolysin (Sample 5). Cells treated with untreated cell performed for 6 hours at 30° C. wall preparation (sample 6) or with 1 g/ml LPS or 10 ul lyophilisation buffer served as positive and negative controls. Example 8 All stimulations were performed in five independent stimu Generation of Splenic Cells of BALB/c Mice lation batches. After an incubation of cells for 6 or 24 hours at 37° C. in a humidified incubator with 5% CO, cells/cell 0089 BALB/c mice were sacrificed by cervical disloca debris were removed from the cell culture supernatants by tion and spleens were removed aseptically. Then spleens were low speed centrifugation. Obtained cell-free culture superna placed in 50 ml Falcon tubes including 10 ml splenocyte tants were frozen at -80°C. until determination of cytokines. medium/spleen and kept on room temperature (not longer Measurement of proinflammatory cytokines IL-6 and TNF-C. than 15 min). Medium was replaced by the same volume of was performed using commercial BDOptEIATM kits follow fresh splenocyte medium and spleens were transferred onto a ing the instructions of the manufacturer.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 3

<210 SEQ ID NO 1 <211 LENGTH: 497 &212> TYPE: PRT <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OO SEQUENCE: 1 Met Ala Ser Ile Ile Met Glu Val Ala Thr Met Glin Ala Lys Lieu. Thr 1. 5 1O 15

Llys Lys Glu Phe Ile Glu Trp Lieu Lys Thir Ser Glu Gly Lys Glin Phe 2O 25 3 O

Asn Val Asp Lieu. Trp Tyr Gly Phe Glin Cys Phe Asp Tyr Ala Asn Ala 35 4 O 45 Gly Trp Llys Val Lieu. Phe Gly His Thr Lieu Lys Gly Lieu. Gly Ala Lys US 2009/0304673 A1 Dec. 10, 2009

- Continued

SO 55 6 O Asp Ile Pro Phe Ala Asn. Asn. Phe Asp Gly Lieu Ala Thr Val Tyr Glin 65 70 7s 8O Asn Thr Pro Asp Phe Lieu Ala Glin Pro Gly Asp Met Val Val Phe Gly 85 90 95 Ser Asn Tyr Gly Ala Gly Tyr Gly His Val Ala Trp Val Ile Glu Ala 1OO 105 11 O Thir Lieu. Asp Tyr Ile Ile Val Tyr Glu Glin Asn Trp Lieu. Gly Gly Gly 115 12 O 125 Trp. Thir Asp Arg Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val Thr 13 O 135 14 O Arg Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro Asn 145 150 155 160 Phe Llys Ser Ala Thr Ala Pro Ala Ser Ile Glin Ser Pro Thr Glin Ala 1.65 17O 17s Ser Lys Lys Glu Thir Ala Lys Pro Glin Pro Lys Ala Val Glu Lieu Lys 18O 185 19 O Ile Ile Lys Asp Val Val Lys Gly His Asp Lieu Pro Lys Arg Gly Gly 195 2OO 2O5 Asn Pro Lys Gly Ile Val Ile His Asn Asp Ala Gly Ser Lys Gly Ala 21 O 215 22O Thr Ala Glu Ala Tyr Arg ASn Gly Lieu Val Asn Ala Pro Ser Ser Arg 225 23 O 235 24 O Lieu. Glu Ala Gly Ile Ala His Ser Tyr Val Ser Gly Asn Thr Val Trp 245 250 255 Glin Ala Lieu. Asp Glu Ser Glin Val Gly Trp His Thr Ala Asn Glin Lieu. 26 O 265 27 O Gly Asn Llys Tyr Tyr Tyr Gly Ile Glu Val Cys Glin Ser Met Gly Ala 27s 28O 285 Asp Asn Ala Thr Phe Lieu Lys Asn. Glu Glin Ala Thr Phe Glin Glu. Cys 29 O 295 3 OO Ala Arg Lieu. Lieu Lys Llys Trp Gly Lieu Pro Ala Asn Arg Asn. Thir Ile 3. OS 310 315 32O Arg Lieu. His Asn Glu Phe Thir Ser Thr Ser Cys Pro His Arg Ser Ser 3.25 330 335 Val Lieu. His Thr Gly Phe Asp Pro Val Thr Arg Gly Lieu. Leu Pro Glu 34 O 345 35. O Asp Lys Arg Lieu Gln Lieu Lys Asp Tyr Phe Ile Llys Glin Ile Arg Ala 355 360 365 Tyr Met Asp Gly Lys Ile Pro Val Ala Thr Val Ser Asn Glu Ser Ser 37 O 375 38O Ala Ser Ser Asn Thr Val Llys Pro Val Ala Glu Lieu Met Pro Pro Val 385 390 395 4 OO Pro Ala Gly Tyr Thr Lieu. Asp Lys Asn. Asn Val Pro Tyr Lys Lys Glu 4 OS 41O 415 Glin Gly Asn Tyr Thr Val Ala Asn. Wall Lys Gly Asn. Asn Val Arg Asp 42O 425 43 O Gly Tyr Ser Thr Asn Ser Arg Ile Thr Gly Val Lieu Pro Asn Asn Thr 435 44 O 445 Thir Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly Tyr Arg Trp Ile 450 45.5 460 US 2009/0304673 A1 Dec. 10, 2009

- Continued

Thir Tyr Ile Ala Asn. Ser Gly Glin Arg Arg Tyr Ile Ala Thr Gly Glu 465 470 47s 48O

Val Asp Ile Ala Gly Asn Arg Ile Ser Ser Phe Gly Llys Phe Ser Ala 485 490 495

Wall

<210 SEQ ID NO 2 <211 LENGTH: 101 &212> TYPE: PRT <213> ORGANISM: unknown &220s FEATURE: <223> OTHER INFORMATION: Prophage SA2USA

<4 OO SEQUENCE: 2

Met Pro Pro Val Pro Ala Gly Tyr Thr Lieu. Asp Lys Asn Asn Val Pro 1. 5 1O 15

Tyr Lys Lys Glu Glin Gly Asn Tyr Thr Val Ala Asn. Wall Lys Gly Asn 2O 25 3O

Asn Val Arg Asp Gly Tyr Ser Thr Asn. Ser Arg Ile Thr Gly Val Lieu 35 4 O 45

Pro Asn Asn. Thir Thr Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asin Gly SO 55 6 O

Tyr Arg Trp Ile Thr Tyr Ile Ala Asn. Ser Gly Glin Arg Arg Tyr Ile 65 70 7s 8O

Ala Thr Gly Glu Val Asp Ile Ala Gly Asn Arg Ile Ser Ser Phe Gly 85 90 95

Llys Phe Ser Ala Val 1OO

<210 SEQ ID NO 3 <211 LENGTH: 96 &212> TYPE: PRT <213> ORGANISM: unknown &220s FEATURE: &223> OTHER INFORMATION: Pitti2O

<4 OO SEQUENCE: 3

Met Lys Arg Llys Llys Pro Lys Gly Trp Ser Glu Asn Pro Tyr Gly Thr 1. 5 1O 15

Tyr Tyr Lys Llys Val Asp Llys Thr Phe Ile Val Gly Ser Glu Lys Ile 2O 25 3O

Glu Thr Arg Ile Gly Ser Pro Phe Leu Ser Ala Pro Ser Gly Gly His 35 4 O 45

Val Thr Pro Asn Gln Lys Met Thr Phe Asp Tyr Lieu Ala Glin Glin Asp SO 55 6 O

Gly Tyr Glu Trp Gly Glin Lieu. Glu Asn. Asn Arg Gly Glin Glin Glu Phe 65 70 7s 8O

Val Pro Ile Arg Pro Lieu. Ser Glin Lys Glu Tyr Trp Gly Ile Lieu Lys 85 90 95 US 2009/0304673 A1 Dec. 10, 2009

1. A method of treatment of an inflammatory disease in a 6. A pharmaceutical composition according to claim 5. human or animal Subject, comprising administering an effi wherein the bacterial cell wall degrading enzyme is an endol cient amount of bacterial cell wall degrading enzymes to said ysin, autolysin, lysostaphin, mytanolysin, lytic enzyme simi Subject. lar to lysostaphin or murolytic enzyme. 2. The method according to claim 1, wherein the bacterial 7. A pharmaceutical composition according to claim 5. cell wall degrading enzyme is an endolysin, autolysin, lyso further comprising a conventional antibacterial agent, such as staphin, mytanolysin, lytic enzyme similar to lysostaphin or an antibiotic agent. murolytic enzyme. 8. A pharmaceutical composition according to claim 5. 3. The method according to claim 1, wherein the bacterial comprising different bacterial cell wall degrading enzymes. cell wall degrading enzyme is administered in combination 9. A pharmaceutical composition according to claim 5. with conventional antibacterial agents, such as antibiotics. further comprising conventional antibacterial agents, such as 4. The method according to claim 1, wherein the bacterial antibiotics and other endolysins, autolysins, lysostaphins, cell wall degrading enzyme is administered in combination mytanolysins, lytic enzymes similar to lysostaphin or muro with conventional antibacterial agents, such as antibiotics and lytic enzymes. other endolysins, mytanolysins, autolysins, lysostaphins, 10. A pharmaceutical composition according to claim 5. lytic enzymes similar to lysostaphin or murolytic enzymes. further comprising a pharmaceutical acceptable a carrier. 5. A pharmaceutical composition comprising a bacterial cell wall degrading enzyme. c c c c c