ZA200205590B - The parenteral use of bacterial phage associated lysing enzymes for the therapeutic treatment of bacterial infections. - Google Patents

Description

THE PARENTERAL USE OF BACTERIAL PHAGE ASSOCIATED LYSING

ENZYMES FOR THE THERAPEUTIC TREATMENT OF BACTERIAL

; INFECTIONS

DESCRIPTION BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention discloses a method and composition for the treatment of bacterial | infections by the parenteral introduction of lytic enzymes, holin lytic enzymes, shuffled lytic enzymes, and/or chimeric lytic enzymes, blended with an appropriate carrier into a patient. The injection can be done intramuscularly, subcutaneously, or intravenously. 2. Description of the Prior Art

In the past, antibiotics have been used to treat various infections. The work of Selman Waksman in the introduction and production of Streptomycetes, and Dr.

Fleming ’s discovery of penicillin, as well as the work of numerous others in the field of antibiotics, are well known. Over the years, there have been additions and chemical modifications to the "basic" antibiotics in attempts to make them more powerful, or to treat people allergic to these antibiotics.

Others have found new uses for these antibiotics. U.S. Patent No. 5,260,292 (Robinson et al.) discloses the topical treatment of acne with aminopenicillins. The , method and composition for topically treating acne and acneiform dermal disorders ’ includes applying an amount of an antibiotic, effective for treating acne and acneiform dermal disorders, selected from the group consisting of ampicillin, amoxicillin, other aminopenicillins, and cephalosporins, and derivatives and analogs thereof. U.S. Patent No. 5,409,917 (Robinson et al.) discloses the topical treatment of acne with cephalosporins.

However, as more antibiotics have been prescribed or used at an ever

K increasing rate for a variety of illnesses, increasing numbers of bacteria have developed a resistance to antibiotics. Larger doses of stronger antibiotics are now being used to treat ever more resistant strains of bacteria. Consequently, multiple antibiotic resistant bacteria have been developed. The use of more antibiotics and the number of bacteria showing resistance has led to increases in the amount of time that the antibiotics need to be used. Broad, non-specific antibiotics, some of which have detrimental effects on the patient, are now being used more frequently.

Additionally, the number of people showing allergic reactions to antibiotics appears to be increasing.

Consequently, other efforts have been sought to first identify and then kill bacteria..

Attempts have been made to treat bacterial diseases with the use of bacteriophages. U.S. Patent No. 5,688,501 (Merril, et al.) discloses a method for treating an infectious disease caused by bacteria in an animal with lytic or non-lytic bacteriophages that are specific for particular bacteria.

U.S. Patent No. 4,957,686 (Norris) discloses a procedure of improved dental hygiene which introduces into the mouth bacteriophages parasitic to bacteria which possess the property of readily adhering to the salivary pellicle.

It is to be noted that the direct introduction of bacteriophages into an animal to prevent or fight diseases has certain drawbacks. Specifically, the bacteria must be . in the right growth phase for the phage to attach. Both the bacteria and the phage have to be in the correct and synchronized growth cycles. Additionally, there must ’ be the right number of phages to attach to the bacteria. The phage must also be active enough. The phages are also inhibited by many substances including bacterial debris from the organism it is going to attack. Further complicating the direct use of

‘ bacteriophage to treat bacterial infections is the possibility of immunological ] reactions, rendering the phage non-functional. Another problem is the mutation of the receptor on the bacterial surface preventing bacteriophage attachment. : Consequently, others have explored the use of other safer and more effective means to treat and prevent bacterial infections.

One bacteria for which a more effective treatment has been extensively explored is Streptococcus. The genus Streptococcus is comprised of a wide variety of both pathogenic and commensal gram-positive bacteria which are found to inhabit a wide range of hosts, including humans, horses, pigs, and cows.

Within the host, streptococci are often found to colonize the mucosa surfaces of the mouth, nares and pharynx. However, in certain circumstances, they may also inhabit the skin, heart or muscle tissue.

Pathogenic streptococci of man include S. pyogenes, S. pneumoniae, and S. faecalis. While Group A streptococci may be present in the throat or on the skin and cause no symptoms of disease, they may also cause infections that range from mild to severe, and even life-threatening. Among the pathogenic hemolytic streptococci, S. pyogenes, or group A streptococci have been implicated as the etiologic agent of acute pharyngitis ("strep throat"), impetigo, rheumatic fever, scarlet fever, glomerulonephritis, and invasive fasciitis. Necrotizing fasciitis (sometimes described by the media as "the flesh-eating bacteria”) is a destructive infection of muscle and fat tissue. Invasive group A streptococcal infections occur when the bacteria get past the defenses of the person who is infected. About 10,000-15,000 cases of invasive GAS disease occur in the United States each year, ‘ resulting in over 2,000 deaths. CDC estimates that 500 to 1,500 cases of necrotizing fasciitis and 2,000 to 3,000 cases of streptococcal toxic shock syndrome occur each year in the United States. Approximately 20% of patients with necrotizing fasciitis die, and 60% of patients with streptococcal toxic shock syndrome die. About 10 to 15% of patients with other forms of invasive group A streptococcal disease die.

Additionally, Group C Streptococcus can cause cellulitis from skin breaks, } although cellulitis is normally associated with Staphylococcus aureus. Cellulitis can result in death, particularly in older individuals or in individuals who are already - weakened.

Reports have described the characteristics of an enzyme produced by the group C streptococcal organism after being infected with a particular bacteriophage identified as C1 (Maxted, W.R. "The Active Agent in Nascent Page Lywsis of

Streptococci,” J. Gen Micro., vol 16, pp585-595, 1957, Krause, R.M.,, "Studies on the Bacteriophages of Hemolytic Streptococci," J. Exp. Med, vol. 108, pp 803-821, 1958) and Fischetti, (Fischetti, V.A., et al, "Purification and Physical Properties of

Group C Streptococcal Phage Associated Lysin," J. Exp. Med, Vol 133 pp. 1105-1117, 1971) . The enzyme was given the name lysin and was found to specifically cleave the cell wall of group A, group C, and group E streptococci.

These investigators provided information on the characteristics and activities of this enzyme with regard to lysing the group A streptococci and releasing the cell wall carbohydrate.

U.S. Patent No. 5,604,109 (Fischetti et al.) relates to the rapid detection of

Group A streptococci in clinical specimens, through the enzymatic digestion by a semi-purified Group C streptococcal phage associated lysin enzyme. The present invention is based upon the discovery that phage lytic enzymes specific for bacteria infected with a specific phage can effectively and efficiently break down the cell wall of the bacterium in question. At the same time, the substrate for the enzyme is not present in mammalian tissues, and therefore is non-destructive to mammalian ; proteins and tissues when present during the digestion of the bacterial cell wall.

U.S. Patent No. 5,985,271 (Fischetti, et. al), U.S. Patent No.5,997,862 ) (Fischetti et al.), and U.S. Patent No. 6,017,528 (Fischetti et al.) disclose the compositions and use of an oral delivery mode, such as a candy, chewing gum, lozenge, troche, tablet, a powder, an aerosol, a liquid or a liquid spray, containing a lysin enzyme produced by group C streptococcal bacteria infected with a Cl bacteriophage for the prophylactic and therapeutic treatment of Streptococcal A throat infections, commonly known as strep throat. This is the lysin enzyme of U.S. ~ Patent No. 5,604,109

The same general technique used to produce and purify the lysin enzyme in

U.S. Patent 5,604,109 may be used to manufacture other lytic enzymes produced by bacteria infected with a bacteriophage specific for that bacteria. Depending on the bacteria, there may be variations in the growth media and conditions.

The use of phage associated lytic enzymes produced by the infection of a bacteria with a bacteria specific phage has numerous advantages for the treatment of diseases. Lytic enzymes have similar characteristics as their complementary phage in that both are targeted for specific bacteria and neither, when selected, interferes with the functioning of normal bacterial flora.. Also, lytic phages primarily attack cell wall structures which are not affected by plasmid variation. The actions of the lytic enzymes are fast and do not depend on bacterial growth. Additionally, lytic enzymes can be directed to the mucosal lining, where, in residence, they will be able to kill colonizing bacteria. :

U.S. Patent No. 6,056,954 (Fischetti et al.) discloses a method and composition for the prophylactic or therapeutic treatment of bacterial infections, comprising administering an effective amount of at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for the bacteria to the site of the infection. The lytic enzyme preferably comprises a carrier suitable for delivering the lytic enzyme to the site of the infection. This method and treatment may be used for ; treating upper respiratory infections, topical infections, vaginal infections, eye infections, ear infections, for parenteral treatment, and for most other bacterial infections.

U.S. Patent No. 6,056,955 (Fischetti et al.) discloses the topical treatment of streptococcal infections.

The use of phage associated lytic enzymes produced by the infection of a } bacteria with a bacteria specific phage has numerous advantages for the treatment of diseases. As the phage are targeted for specific bacteria, the lytic enzymes do not : interfere with normal flora. Also, lytic phages primarily attack cell wall structures which are not affected by plasmid variation. The actions of the lytic enzymes are fast and do not depend on bacterial growth.

However, sometimes the bacterial infections, by the time they are treated, have developed into more serious illnesses. For example, dermatological infections such as Staphylococcus aureus and Streptococcal pneumoniae can develop into cellulitis, which, unchecked, can lead to a degradation of the connective tissue, septicemia, and possibly death. Other bacterial infections can also evolve into deep tissue infections. Other infections by other bacteria, not necessarily dermatological by nature, can infect and localize in certain tissues of the body, making the infections difficult to treat.

U.S. Application No. 09/482,992 discloses a method and composition for the treatment of bacterial infections by the parenteral introduction of at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for that bacteria and an appropriate carrier for delivering the lytic enzyme into a patient. The injection can be done intramuscularly, subcutaneously, or intravenously.

SUMMARY OF THE INVENTION

The present invention discloses the extraction and parenteral use of a variety of bacterial phage associated lytic enzymes, holin lytic enzymes, chimeric lytic : enzymes, and shuffled lytic enzymes, in addition to lytic enzymes, for increased efficiency for the treatment of a wide variety of illnesses caused by bacterial infections. The lytic enzymes may be administered intravenously, subcutaneously, subdermally, or intramuscularly.

The method for obtaining and purifying the lytic enzyme produced by a bacteria infected with the bacteriophage is known in the art. Some recent evidence suggests that the phage enzyme that lyses the streptococcus organism may actually : be a bacterial enzyme that is used to construct the cell wall and the phage. While replicating in the bacterium, a phage gene product may cause the upregulation or derepression of bacterial enzyme for the purpose of releasing the bacteriophage.

These bacterial enzymes may be tightly regulated by the bacterial cell and are used by the bacteria for the construction and assembly of the cell wall.

The use of these lytic enzymes for the prophylactic and therapeutic treatment of bacterial diseases, however, has not been explored, except by the inventors of the present invention. The lytic enzymes produced by bacterial phages are specific and effective for killing select bacteria.

It is the current trend in biotechnology to do genomic sequencing of microorganisms in order, in part, to aid in the determination of designing drugs for treatment. This type of modeling is both timely and costly, and may or may not lead to new drug discoveries. The elegance of this invention resides in the fact that phage-induced lytic enzymes have evolved into extremely effective and targeted killing agents of selected bacteria. The primary structure and sequencing of purified enzymes serve as templates from which a variety of chemical procedures (such as shuffling) can be performed to optimize the enzyme’s effectiveness.

These phage-induced lytic enzymes are useful in killing a variety of bacterial pathogens including those involved in classical clinical diseases such Streptococcus,

Pseudomonas, etc. and in biowarfare agents, such as Bacillus and Yersinia, causing . anthrax & plague, respectively, among other uses.

The invention (which incorporates U.S. Patent No. 5,604,109 in its entirety by reference) uses an enzyme produced by the bacterial organism after being infected with a particular bacteriophage as a therapeutic treatment for those who have already become ill from the infection. The present invention is based upon the discovery that phage lytic enzymes specific for bacteria infected with a specific ) phage can effectively and efficiently break down the cell wall of the bacterium in question. At the same time, the semipurified enzyme is lacking in proteolytic ’ enzymatic activity and therefore non-destructive to mammalian proteins and tissues when present during the digestion of the bacterial cell wall.

The creation, purification, and isolation of chimeric, shuffled, lytic, and holin enzymes are also well known to those skilled in the art. In particular, U.S. Patent No. 6,132,970 (Stemmer) (incorporated herein by reference) discloses a number of new techniques, and modifications of more established procedures, for the creation of these enzymes. The proposed invention utilizes these techniques and applies them for the enhancement of specifically noted phage associated lytic enzymes. The technique for isolating lysin enzymes found in U.S. Patent No. 6,056,954 (also incorporated herein by reference) may be applied to other phage associated lytic enzymes. Similarly, other state of the art techniques may be used to isolate lytic enzymes.

For definitional purposes, shuffled enzymes are enzymes where more than one sequence of usually more than one particular enzyme has been cleaved in one or more locations, and reconstructed in a specific or random order, increasing their activity or specificity.

Chimeric enzymes are enzymes which are a combination of two or more enzymes having two or more active sites such that the chimeric enzyme can act independently on the same or different molecules. This will allow for potentially treating two or more different bacterial infections at the same time. : Holin enzymes produce holes in the cell membrane. More specifically, holin enzymes form lethal membrane lesions that terminate respiration. Like the lytic enzymes, the holin enzymes are coded for and carried by a phage genome. In fact, it is quite common for the genetic code for the holin enzyme to be found next to or even within the code for the lytic enzyme in the phage. Most holin sequences are short, and overall, hydrophobic in nature, with a highly hydrophilic carboxy-terminal domain. In many cases, the putative holin enzyme is encoded on a different reading frame within the enzymatically active domain of the phage. In . other cases, the holin enzyme is encoded on the DNA next to or close to the DNA coding for the phage. The holin enzyme is frequently synthesized during the late stage of phage infection and found in the cytoplasmic membrane where it causes membrane lesions.

Holin enzymes can be grouped into two general classes based on primary structure analysis. Class I holins are usually 95 residues or longer and may have three potential transmembrane domains. Class II holins are usually smaller, at approximately 65-95 residues, and the distribution of charged and hydrophobic residues indicating two TM domains (Young, et al. Trends in Microbiology v. 8, No. 4, March 2000). At least for the phages of gram-positive hosts, however, the dual-component lysis system may not be universal. Although the presence of holins has been shown or suggested for several phages, no genes have yet been found encoding putative holins for all of the phages. Holins have been shown to be present or suggested for among others, lactococcal bacteriophage Tuc2009, lactococcal

NLC3, pneumococcal bacteriophage EJ-1, Lactobacillus gasseri bacteriophage

Nadh, Staphylococcus aureus bacteriophage Twort, Listeria monocytogenes bacteriophages, pneumococcal phage Cp-1, Bacillus subtillis phage M29,

Lactobacillus delbrueckki bacteriophage LL-H lysin, and bacteriophage N11 of

Staphylococcus aureus. (Loessner, et al., Journal of Bacteriology, Aug. 1999, p. 4452-4460). . The lytic system consists of a holin and at least one peptidoglycan hydrolase, or "lysin", capable of degrading the bacterial cell wall. Lysins can be endo--N- acetylglucosaminidases or N-acetylmuramidases (lysozymes), which act on the sugar moiety, endopeptidases which act on the

R : AMENDED SHEET t ® WO 01/51073 PCT/US01/00913 peptide cross bridge, or more commonly, an N-acetylmuramoyl-L-alanine amidase, which hydrolyzes the amide bond connecting the sugar and peptide moieties.

Typically, the holin is expressed in the late stages of phage infection forming a pore in the cell membrane, allowing the lysin(s) to gain access to the cell wall peptidoglycan resulting in release of progeny phage. Significantly, exogenously added lysin can lyse the cell wall of bacterial cells, producing a phenomenon known as "lysis from without". However, in the case of gram-negative bacteria which contain an outer membrane, the presence of exogenously added holing in combination with a lytic enzyme may allow more efficient access of the lytic enzyme to the peptidoglycan enabling better lysis.

More specifically, the sequence of enzymes when purified can be determined by conventional techniques, and rearrangements of primary structures can be achieved by state of the art techniques, such as shuffling, to increase the activity and stability of the enzyme(s). Shuffling also allows for combination enzymes ("chimeric enzymes") to have more than one activity.

The use of phage associated lytic enzymes produced by the infection of a bacteria with a bacteria specific phage has numerous advantages for the treatment of diseases. As the phage are targeted for specific bacteria, the lytic enzymes do not interfere with normal flora. Also, lytic phages primarily attack cell wall structures which are not affected by plasmid variation. The actions of the lytic enzymes are fast and do not depend on bacterial growth.

A need exists to apply at least one phage associated lytic enzyme, at least one chimeric lytic enzyme, at least one shuffled lytic enzyme, at least one holin enzyme, or combinations thereof, intravenously to treat septicemia, general infections, and deep tissue infections.

A need also exists to inject into the tissue of an organism at least one phage associated lytic enzyme, at least one chimeric lytic enzyme, at least

’ AMENDED SHEET 3 ® WO 01/51073 PCT/US01/00913 one shuffled lytic enzyme, at least one holin enzyme, or combinations thereof, to treat a deep tissue infection.

In one embodiment of the invention, the treatments of a variety of illnesses caused by Streptococcus fasciae, and Staphylococcus aureus are disclosed.

In yet another embodiment of the invention, lysostaphin, the enzyme which lyses Staphylococcus aureus, can be included in the therapeutic agent.

In a further embodiment of the invention, conventional antibiotics may be included in the therapeutic agent with the lytic enzyme, and with or without the presence of lysostaphin.

In another embodiment of the invention, at least one lytic enzyme, at least one chimeric lytic enzyme, at least one shuffled lytic enzyme, at least one holin enzyme, or combinations thereof may also be included in the therapeutic agent.

The therapeutic agent may be given parenterally, by means of an intramuscular, intradermal, or subcutaneous injection, or the agent may be given intravenously.

Another need exists to apply at least one phage associated lytic enzyme, at least one chimeric lytic enzyme, at least one shuffled lytic enzyme, at least one holin enzyme, or combinations intravenously, to treat septicaemia and general infections.

A need also exists to inject a phage associated lytic enzyme into the tissue of an organism to treat a deep tissue infection.

In one embodiment of the invention, the treatments of a variety of illnesses caused by Streptococcal pneumoniae, Streptococcus fasciae, and Staphylococcus aureus are disclosed.

In yet another embodiment of the invention, lysostaphin, the enzyme which lyses Staphylococcus aureus, can be included in the therapeutic agent.

In a further embodiment of the invention, conventional antibiotics may be included in the therapeutic agent with the lytic enzyme, and with or without the presence of lysostaphin. : In another embodiment of the invention, more than one lytic enzyme may also be included in the therapeutic agent.

In a preferred embodiment of the invention, at least one lytic enzyme, at least one chimeric lytic enzyme, at least one shuffled lytic enzyme, at least one holin enzyme, or combinations are used to treat bacterial infections, thereby increasing the speed and efficiency with which the bacteria are killed.

Chimeric enzymes may also be used to treat one bacterial infection by cleaving the cell wall in more than one location.

A number of chimeric lytic enzymes have been produced and studied. Gene

E-L, a chimeric lysin constructed from bacteriophages phi X174 and MS2 lysis proteins E and L, respectively, was subjected to internal deletions to create a series of new E-L clones with altered lysis or killing properties. The lytic activities of the parental genes E, L, E-L, and the internal truncated forms of E-L were investigated in this study to characterize the different lysis mechanism, based on differences in the architecture of the different membranes spanning domains. Electron microscopy and release of marker enzymes for the cytoplasmic and periplasmic spaces revealed that two different lysis mechanisms can be distinguished depending on penetrating of the proteins of either the inner membrane or the inner and outer membranes of the

E. coli. FEMS Microbiol. Lett. 1998 Jul 1, 164(1); 159-67.

In another experiment an active chimeric cell wall lytic enzyme (TSL) has . been constructed by fusing the region coding for the N-terminal half of the : lactococcal phage Tuc2009 lysin and the region coding for the C-terminal domain of ) the major pneumococcal autolysin. The chimeric enzyme exhibited a glycosidase activity capable of hydrolysing choline-containing pneumococcal cell walls.

A preferred embodiment of this invention discloses the use of chimeric lytic enzymes to treat two infectious bacteria at the same time, or to cleave the cell wall of a bacteria in two different locations. : In another embodiment of the invention, holin enzymes are used in conjunction with the lytic enzymes to accelerate the speed and efficiency at which the bacteria are killed. Holin enzymes may also be in the form of chimeric and/or shuffled enzymes. Holin enzymes may also be used alone in the treatment of bacterial infections

In another embodiment of the invention, the holin enzymes are shuffled holin enzymes or chimeric holin enzymes, either in combination with or independent of the lytic enzymes.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 is an electron micrograph of group A streptococci treated with lysin showing the formation of a hole in the cell wall causing the cytoplasmic membrane. to extrude and the cell contents to pour out.

DETAILED DESCRIPTION OF THE INVENTION

The method for treating systemic or tissue bacterial infections comprises parenterally treating the infection with a therapeutic agent comprising an effective amount of at least one lytic enzyme, at least one chimeric lytic enzyme, at least one shuffled lytic enzyme, or combinations thereof, produced by a bacteria infected with a bacteriophage specific for the bacteria, and an appropriate carrier. In a preferred : embodiment of the invention, a holin lytic enzyme may also be used, either alone or preferably in combination with one of the other lytic enzymes that will lyse the cell wall.

The method for treating bacterial infections comprises treating the infection with a therapeutic agent comprising an effective amount of a modified version of at

Claims (1)

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