U.S. patent application number 10/855978 was filed with the patent office on 2005-06-23 for use of bacterial phage associated lysing enzymes for treating various illnesses.
This patent application is currently assigned to New Horizons Diagnostics Corporation. Invention is credited to Fischetti, Vincent, Loomis, Lawrence.
Application Number | 20050136088 10/855978 |
Document ID | / |
Family ID | 32469737 |
Filed Date | 2005-06-23 |
United States Patent
Application |
20050136088 |
Kind Code |
A1 |
Loomis, Lawrence ; et
al. |
June 23, 2005 |
Use of bacterial phage associated lysing enzymes for treating
various illnesses
Abstract
A composition and method for treating bacterial infections is
disclosed which comprises the treatment of an individual with an
effective amount of at least one lytic enzyme produced by a
bacteria infected with a bacteriophage specific for said bacteria
wherein at least one lytic enzyme is selected from the group
consisting of shuffled lytic enzymes, chimeric lytic enzymes, holin
enzymes, and combinations thereof. A carrier may be used for
delivering the lytic enzyme. This method, and composition can be
used for the treatment of upper respiratory infections, skin
infections, wounds, and burns, vaginal infections, eye infections,
intestinal disorders and dental problems.
Inventors: |
Loomis, Lawrence; (Columbia,
MD) ; Fischetti, Vincent; (West Hempstead,
NY) |
Correspondence
Address: |
Colin G. Sandercock, Esquire
Heller Ehrman White & McAuliffe LLP
1717 Rhode Island Avenue, N.W.
Washington
DC
20036
US
|
Assignee: |
New Horizons Diagnostics
Corporation
Columbia
MD
|
Family ID: |
32469737 |
Appl. No.: |
10/855978 |
Filed: |
May 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10855978 |
May 28, 2004 |
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09844435 |
Apr 30, 2001 |
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09844435 |
Apr 30, 2001 |
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09560650 |
Apr 28, 2000 |
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6752988 |
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Current U.S.
Class: |
424/405 ;
424/431; 424/439; 424/443; 424/94.1 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61K 38/47 20130101; A61K 38/162 20130101; Y02A 50/473 20180101;
Y02A 50/481 20180101; A61K 38/162 20130101; A61K 2300/00 20130101;
A61K 38/47 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/405 ;
424/094.1; 424/439; 424/443; 424/431 |
International
Class: |
A61K 038/43; A61K
009/00 |
Claims
1. A method for treating bacterial infections, comprising the
steps: a) obtaining a composition comprising an effective amount of
at least one lytic enzyme; and b) applying said composition to a
site of the infection wherein the enzyme is produced by infecting a
bacterium causing said infection with a bacteriophage specific for
said bacteria and wherein said bacteria produces said at least one
recombinant lytic enzyme selected from the group consisting of
chimeric lytic enzymes, shuffled lytic enzymes, and combinations
thereof.
2. The method according to claim 1, wherein said method for
treating bacterial infections is used for the prophylactic
treatment of infections.
3. The method according to claim 1, wherein said method for
treating bacterial infections is used for the therapeutic treatment
of infections.
4. The method according to claim 1, wherein said method further
comprises including at least one holin enzyme in said
composition.
5. (canceled)
6. The method according to claim 1, wherein said composition
further comprises at least one antibiotic that potentiates the
bactericidal activity of the lytic enzyme.
7. The method according to claim 1, comprising a first lytic enzyme
that is recombinant and a second lytic enzyme that is not
recombinant.
8. The method according to claim 1, further comprising delivering
said at least one lyticenzyme in a carrier suitable for delivering
said lytic enzyme to the site of the infection.
9. (canceled)
10. A method according to claim 1, wherein at least one lytic
enzyme is active against any gram negative or gram positive
bacteria.
11. The method according to claim 1, wherein the carrier is
selected from the group consisting of an inhalant, a topical cream,
a nasal spray, a syrup, a tablet, tampon, a suppository, an eye
drop solution, a candy, a chewing gum, a lozenge, a troche, a
powder, an aerosol, a liquid, a liquid spray, a bandage, a
toothpaste and an oral wash.
12. A method as described in claim 1, wherein the bacterial
infection is an infection of the upper respiratory tract, and the
carrier is suitable for delivery of said at least one lytic enzyme
to a mouth, throat, or nasal passage.
13. The method according to claim 12, wherein said method is used
for the prophylactictreatment of infection.
14-17. (canceled)
18. The method according to claim 10, wherein said carrier is
selected from the group consisting of a candy, chewing gum,
lozenge, troche, tablet, a powder, an aerosol, a liquid and a
liquid spray.
19-26. (canceled)
27. A composition for the treatment of a bacterial infection of an
upper respiratory tract, prepared by a process comprising the
steps: a) obtaining at least one lytic enzyme produced by infecting
a bacteria causing said bacterial infection with a bacteriophage
specific for said bacteria wherein said bacteria produces said at
least one lytic enzyme selected from the group consisting of
chimeric lytic enzymes, shuffled lytic enzymes, and combinations
thereof, and wherein said at least one lytic enzyme has the ability
to digest a cell wall of said bacteria; and b) admixing said at
least one lytic enzyme with a carrier suitable for delivery to a
mouth, throat, nasal passage, or digestive tract.
28-30. (canceled)
31. The composition according to claim 27, wherein said carrier is
selected from the group consisting of a candy, chewing gum,
lozenge, troche, tablet, a powder, an aerosol, a liquid and a
liquid spray, suppository, enema, syrup or enteric coated pill.
32-46. (canceled)
47. A composition for the therapeutic or prophylactic treatment of
bacterial infections of burns and wounds of the skin, comprising:
1) obtaining at least one lytic enzyme wherein said at least one
lytic enzyme is produced by infecting a bacteria causing said
bacterial infection with a bacteriophage specific for said bacteria
wherein said bacteria produces said at least one lytic enzyme
selected from the group consisting of chimeric lytic enzymes,
shuffled lytic enzymes, and combinations thereof, and wherein said
at least one lytic enzyme has the ability to digest a cell wall of
said bacteria, and 2) admixing said at least one lytic enzyme with
a carrier suitable for delivery of said at least one lytic enzyme,
and a carrier for suitable for delivering said at least one lytic
enzyme to the skin.
48-50. (canceled)
51. The composition according to claim 47, wherein said carrier is
a bandage.
52-67. (canceled)
68. A method for the prophylactic and therapeutic treatment of
vaginal infections, comprising: obtaining a composition comprising
an effective amount of at least one lytic enzyme, wherein said
composition is prepared by the steps of: a) obtaining a composition
comprising an effective amount of at least one lytic enzyme,
wherein said composition is prepared by the steps of: 1) obtaining
at least one lytic enzyme wherein said at least one lytic enzyme is
produced by infecting a bacteria causing said bacterial infection
with a bacteriophage specific for said bacteria wherein said
bacteria produces said at least one lytic enzyme selected from the
group consisting of chimeric lytic enzymes, shuffled lytic enzymes,
and combinations thereof, and wherein said at least one lytic
enzyme has the ability to digest a cell wall of said bacteria, and
2) admixing said at least one lytic enzyme with a carrier suitable
for delivery of said at least one lytic enzyme, and b) applying
said composition to the vagina.
69-80. (canceled)
81. The composition according to claim 68, wherein said carrier is
a tampon.
82-85. (canceled)
86. A method for treating bacterial infections of an eye comprising
the steps of: a) obtaining a composition comprising an effective
amount of at least one lytic enzyme, wherein said composition is
prepared by the steps of 1) obtaining at least one lytic enzyme
wherein said at least one lytic enzyme is produced by infecting a
bacteria causing said bacterial infection with a bacteriophage
specific for said bacteria wherein said bacteria produces said at
least one lytic enzyme selected from the group consisting of
chimeric lytic enzymes, shuffled lytic enzymes, and combinations
thereof, and wherein said at least one lytic enzyme has the ability
to digest a cell wall of said bacteria, and 2) admixing said at
least one lytic enzyme with a carrier suitable for delivery of said
at least one lytic enzyme, and a carrier for suitable for
delivering said at least one lytic enzyme to said eye, and b)
applying said composition to said eye.
87-91. (canceled)
92. The method according to claim 86, wherein the carrier is an eye
drop solution.
93-102. (canceled)
103. A method for the prophylactic or therapeutic treatment of
dermatological infections comprising: a) obtaining a composition
comprising an effective amount of at least one lytic enzyme,
wherein said composition is prepared by the steps of 1) obtaining
at least one lytic enzyme wherein said at least one lytic enzyme is
produced by infecting a bacteria causing said bacterial infection
with a bacteriophage specific said bacteria produces said at least
one lytic enzyme selected from the group consisting of chimeric
lytic enzymes, shuffled lytic enzymes, and combinations thereof,
and wherein said at least one lytic enzyme has the ability to
digest a cell wall of said bacteria, and 2) admixing said at least
one lytic enzyme with a carrier suitable for delivery of said at
least one lytic enzyme, and b) topically applying said composition
to the skin.
104-106. (canceled)
107. The method according to claim 103, wherein said carrier is
selected from the group consisting of an aqueous liquid, an alcohol
base, a water soluble gel, a lotion, an ointment, a nonaqueous
liquid base, a mineral oil base, a blend of mineral oil and
petrolatum, lanolin, liposomes, hydrophilic gelling agents,
cross-linked acrylic acid polymers (carbomer), cellulose polymers,
hydroxy ethyl cellulose, cellulose gum, MVE/MA decadiene
crosspolymers, PVM/MA copolymers, and any combinations thereof.
108-111. (canceled)
112. The method according to claim 103, wherein the composition
further comprises at least one complementary agent which
potentiates the bactericidal activity of the lytic enzyme, said
complementary agent being selected from the group consisting of
penicillin, synthetic penicillins bacitracin, methicillin,
cephalosporin, polymyxin, cefaclor. Cefadroxil, cefamandole nafate,
cefazolin, cefixime, cefmetazole, cefonioid, cefoperazone,
ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin,
cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone,
cefriaxone moxalactam, cefuroxime, cephalexin, cephalosporin C,
cephalosporin C sodium salt, cephalothin, cephalothin sodium salt,
cephapirin, cephradine, cefuroximeaxetil, dihydratecephalothin,
moxalactam, loracarbef. mafate and chelating agents in an amount
effective to synergistically enhance effects of the lytic
enzyme.
113-114. (canceled)
115. A composition for the treatment of bacterial infections of the
mouth or teeth, comprising prepared by a process comprising the
steps of: 1) obtaining at least one lytic enzyme wherein said at
least one lytic enzyme is produced by infecting a bacteria causing
said bacterial infection with a bacteriophage specific for said
bacteria wherein said bacteria produces said at least one lytic
enzyme selected from the is lytic enzymes, shuffled lytic enzymes,
and combinations thereof, and wherein said at least one lytic
enzyme has the ability to digest a cell wall of said bacteria, and
2) admixing said at least one lytic enzyme with a carrier suitable
for delivery of said at least one lytic enzyme, and a carrier for
suitable for delivering said at least one lytic enzyme to said
mouth or teeth.
116-120. (canceled)
121. The composition according to claim 115, wherein said carrier
is selected from the group consisting of a toothpaste, an oral
wash, a chewing gum and a lozenge.
122-124. (canceled)
125. A method for parenterally treating bacterial infections,
comprising the steps of: a) obtaining a composition comprising an
effective amount of at least one lytic enzyme, wherein said
composition is prepared by the steps of 1) obtaining at least one
lytic enzyme wherein said at least one lytic enzyme is produced by
infecting a bacteria causing said bacterial infection with a
bacteriophage specific for said bacteria wherein said bacteria
produces said at least one lytic enzyme selected from the group
consisting of chimeric lytic enzymes, shuffled lytic enzymes, and
combinations thereof, and wherein said at least one lytic enzyme
has the ability to digest a cell wall of said bacteria, and 2)
admixing said at least one lytic enzyme with a carrier suitable for
parenterally delivering said at least one lytic enzyme, and b)
parenterally administering said composition to a site of the
infection.
126-139. (canceled)
140. The method according to claim 125, wherein said composition is
administered intravenously, intramuscularly or subcutaneously.
141. The method according to claim 125, wherein said composition
further comprises at least one complementary agent which
potentiates the bactericidal activity of the lysine enzyme, said
complementary agent being selected from the group consisting of
penicillin, synthetic penicillins bacitracin, methicillin,
cephalosporin, polymyxin, cefaclor. Cefadroxil, cefamandole nafate,
cefazolin, cefixime, cefmetazole, cefonioid, cefoperazone,
ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin,
cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone,
cefriaxone moxalactam, cefuroxime, cephalexin, cephalosporin C,
cephalosporin C sodium salt, cephalothin, cephalothin sodium salt,
cephapirin, cephradine, cefuroximeaxetil, dihydratecephalothin,
moxalactam, loracarbef mafate and chelating agents in an amount
effective to synergistically enhance the therapeutic effect of the
lysin enzyme.
142-151. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention discloses methods and compositions for
the treatment of bacterial infections by the use of lytic enzymes,
modified lytic enzymes such as shuffled lytic enzymes, and chimeric
lytic enzymes, and optionally, holin enzymes blended with an
appropriate carrier suitable for the treatment of the
infection.
[0003] 2. Description of the Prior Art
[0004] 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.
[0005] Additionally, others have found new uses for these
antibiotics. U.S. Pat. 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 selected
from the group consisting of ampicillin, amoxicillin, other
aminopenicillins, and cephalosporins, and derivatives and analogs
thereof, effective to treat the acne and acneiform dermal
disorders. U.S. Pat. No. 5,409,917 (Robinson et al.) discloses the
topical treatment of acne with cephalosporins.
[0006] However, as more antibiotics have been prescribed or used at
an ever 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. Multiple antibiotic
resistant bacteria have consequently developed. The use of more
antibiotics and the number of bacteria showing resistance has led
to increasing the amount of time that the antibiotics need to be
used. Broad, nonspecific antibiotics, some of which have
detrimental effects on the patient, are now being used more
frequently. Also, antibiotics do not easily penetrate mucus
linings.
[0007] Additionally, the number of people allergic to antibiotics
appears to be increasing. Consequently, other efforts have been
sought to first identify and then kill bacteria.
[0008] Attempts have been made to treat bacterial diseases with the
use of bacteriophages. U.S. Pat. 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.
[0009] U.S. Pat. No. 4,957,686 (Norris) discloses a procedure of
improved dental hygiene which comprises introducing into the mouth
bacteriophages parasitic to bacteria which possess the property of
readily adhering to the salivary pellicle.
[0010] It is to be noted that the direct introduction of
bacteriophages into an animal to prevent or fight diseases has
certain drawbacks. Typically, the bacteria should be in the right
growth phase for the phage to attach. Both the bacteria and the
phage should be in the correct and synchronized growth cycles.
Additionally, there should be the right number of phages to attach
to the bacteria; if there are too many or too few phages, there
will be either no attachment or no production of the lysing enzyme.
The phage should also be active enough. The phages are also
inhibited by many things including bacterial debris from the
organism it is going to attack. Further complicating the direct use
of bacteriophages to treat bacterial infections is the possibility
of immunological reactions, rendering the phage nonfunctional.
[0011] Consequently, others have explored the use of safer and more
effective means to treat and prevent bacterial infections.
[0012] U.S. Pat. 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 associated 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, in most if not all cases,
the semipurified enzyme is lacking in mammalian cell receptors and
therefore tends to be less destructive to mammalian proteins and
tissues when present during the digestion of the bacterial cell
wall.
[0013] U.S. Pat. No. 5,985,271 (Fischetti, et. al.), U.S. Pat. No.
5,997,862 (Fischetti et al.), and U.S. Pat. No. 6,017,528
(Fischetti et al.) disclose the compositions and their use in an
oral delivery mode, such as a candy, chewing gum, lozenge, troche,
tablet, a powder, an aerosol, a liquid or a liquid spray that
contains a lysin enzyme produced by group C streptococcal bacteria
infected with a C1 bacteriophage for the prophylactic and
therapeutic treatment of Streptococcal A throat infections,
commonly known as strep throat. This lysin enzyme is described in
U.S. Pat. No. 5,604,109
[0014] The same general technique used to produce and purify a
lysin enzyme shown in U.S. Pat. No. 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.
[0015] 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 generally 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. Additionally, lytic enzymes can be directed to
the mucosal lining, where, in residence, they will be able to kill
colonizing bacteria.
[0016] U.S. Pat. No. 6,056,954 (Fischetti et al.) discloses a
method and composition for the prophylactic and/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.
[0017] U.S. Pat. No. 6,056,955 (Fischetti et al.) discloses the
topical treatment of streptococcal infections.
SUMMARY OF THE INVENTION
[0018] Methods for obtaining and purifying the lytic enzyme
produced by a bacterium infected with the bacteriophage are known.
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.
[0019] The use of these lytic enzymes for the prophylactic and
therapeutic treatment of bacterial diseases, however, has not been
explored in a sufficient manner, except by the inventors of the
present invention. The lytic enzymes produced by bacterial phages
generally are specific and effective for killing select
bacteria.
[0020] The present invention discloses the extraction and use of a
variety of bacterial phage associated 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. More
specifically, the present invention provides a pharmaceutical
composition comprising at least one bacteria-associated phage
enzyme that is isolated from one or more bacteria species and
includes phage lytic and/or holin enzymes. In one embodiment, the
lytic or holin enzymes, including their isozymes, analogs, or
variants, are used in a modified form. In another embodiment the
lytic or holin enzymes, including their isozymes, analogs, or
variants, are used in a combination of natural and modified forms.
The modified forms of lytic and holin enzymes are made
synthetically by chemical synthesis and/or DNA recombinant
techniques. and, more preferably, the enzymes are made
synthetically by chimerization and/or shuffling.
[0021] According to one embodiment, the pharmaceutical composition
includes one or more natural lytic enzyme produced by the bacterial
organism, after being infected with a particular bacteriophage, for
prophylactic or therapeutic treatment. Preferably, the
pharmaceutical composition contains combinations of one or more
natural lytic enzyme and one or more chimeric or shuffled lytic
enzymes.
[0022] 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.
[0023] Holin enzymes produce holes in the cell membrane. More
specifically, holins form lethal membrane lesions that terminate
respiration. Like the lytic enzymes, many holin enzymes are coded
for and carried by a phage. In fact, it is quite common for the
genetic code of 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 is encoded on a different reading frame within the
enzymatically active domain of the phage. In other cases, the holin
is encoded on the DNA next or close to the DNA coding for the
phage. The holin is frequently synthesized during the late stage of
phage infection and found in the cytoplasmic membrane where it
causes membrane lesions.
[0024] 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, August 1999, p. 4452-4460).
[0025] Shuffled enzymes are enzymes in which the genes, gene
products, or peptides for more than one related phage enzyme have
been randomly cleaved and reassembled into a more active or
specific enzyme. Shuffled oligonucleotides, peptides or peptide
fragment molecules are then selected or screened to identify a
molecule having a desired functional property. This method is
described, for example, in Stemmer, U.S. Pat. No. 6,132,970.
(Method of shuffling polynucleotides); Kauffman, U.S. Pat. No. 5,
976,862 (Evolution via Condon-based Synthesis) and Huse, U.S. Pat.
No. 5,808,022 (Direct Codon Synthesis). The contents of these
patents are incorporated herein by reference.
[0026] Shuffling is used to create an enzyme 10 to 100 fold more
active than the template. The template enzyme is selected among
different varieties of lysin or holin enzymes. The shuffled enzyme
constitutes, for example, one or more binding domains and one or
more catalytic domains. Each of the binding or catalytic domains is
derived from the same or different phage or phage enzyme. The
shuffled domains are either oligonucleotide based molecules, as
gene or gene products, that either alone or in combination with
other genes or gene products are translatable into a peptide
fragment, or they are peptide based molecules. Gene fragments
include any molecules of DNA, RNA, DNA-RNA hybrid, antisense RNA,
Ribozymes, ESTs, SNIPs and other oligonucleotide-based molecules
that either alone or in combination with other molecules produce an
oligonucleotide molecule capable of translation into a peptide.
[0027] All isozymes, variants or analogs of the
bacterial-associated phage enzymes of the invention, whether
natural or modified, are encompassed and included within the scope
of the invention.
[0028] 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.
[0029] The creation, purification, and isolation of chimeric,
shuffled, and holin enzymes are well known to those skilled in the
art. In particular, U.S. Pat. No. 6,132,970 (Stemmer) 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. Pat. 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.
[0030] In a preferred embodiment of the invention, shuffled enzymes
are used to treat bacterial infections, thereby increasing the
speed and efficiency with which the bacteria are killed.
[0031] Chimeric enzymes may also be used to treat one bacterial
infection by cleaving the cell wall of the bacteria in more than
one location.
[0032] A number of chimeric lytic enzymes have been produced and
studied. Gene E-L, a chimeric lysis 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. Jul. 1, 1998
164(1); 159-67.
[0033] 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.
[0034] 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.
[0035] 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
[0036] It is an object of the invention to use phage associated
lytic enzymes in combination with chimeric or shuffled lytic
enzymes to prophylactically and therapeutically treat bacterial
diseases.
[0037] In another embodiment of the invention, chimeric lytic
enzymes are used to prophylactically and therapeutically treat
bacterial diseases.
[0038] In yet another embodiment of the invention, shuffled lytic
enzymes are used to prophylactically and therapeutically treat
bacterial infections.
[0039] In yet another embodiment of the invention, holin enzymes
are used in conjunction with phage associated lytic enzymes to
prophylactically and therapeutically treat bacterial
infections.
[0040] In another embodiment of the invention, holin enzymes alone
are used to prophylactically and therapeutically treat bacterial
infections.
[0041] In another embodiment of the invention, the holin enzymes
are shuffled holin enzymes or chimeric holin enzymes, in either
combination with or independent of the lytic enzymes.
[0042] The invention (which incorporates U.S. Pat. No. 5,604,109 in
its entirety by reference) uses a lytic enzyme produced by the
bacterial organism after being infected with a particular
bacteriophage as either a prophylactic treatment for preventing
those who have been exposed to others who have the symptoms of an
infection from getting sick, or 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
nondestructive to mammalian proteins and tissues when present
during the digestion of the bacterial cell wall. As discussed
above, the lytic enzymes may be chimeric, shuffled or "natural,"
and may be in combination with at least one holin enzyme, which may
also be chimeric, shuffled, or "natural."
[0043] In one embodiment of the invention, the prophylactic and
therapeutic treatment of a variety of illnesses caused by
Streptococcal pneumoniae, Streptococcus fasciae, and Hemophilus
influenza are disclosed. In another embodiment of the invention,
infections caused by Listeria, Salmonella, E. coli, and
Campylobacter, are treated by the use of other shuffled and/or
lytic enzymes, possibly in combination with holin and other lytic
enzymes. The bacteria infecting the digestive system can be treated
by incorporating the enzymes in suppository enemas, in syrups, or
in other carriers to get directly to the site of the
infection(s).
[0044] In another embodiment of the invention, lytic enzymes,
modified lytic enzymes such as shuffled lytic enzymes and/or
chimeric lytic enzymes are incorporated into bandages to prevent or
treat infections of burns and wounds. In yet another embodiment of
the invention, the lytic enzymes of phage associated with
Staphylococcus or Pseudomonas are incorporated into bandages to
prevent or treat infections of burns and wounds. Staphylococcus,
Pseudomonas, and Streptococcus are frequently found in
dermatologiical infections. Similarly, holin and other lytic
enzymes may be used in combination with the chimeric and/or
shuffled enzymes.
[0045] Vaginal infections caused by Group B Streptococcus can cause
premature birth and subsequent complications resulting in neonatal
sepsis. Chimeric lytic enzymes, shuffled lytic enzymes, lytic
enzymes, alone or in combination with holin lytic enzymes and other
lytic enzymes, incorporated into tampons specific for group B strep
would prevent infection of the neonate during birth without
disturbing normal vaginal flora so that women would not be overcome
by yeast infection as a result of antibiotic therapy.
[0046] In another embodiment of the invention, eye drops containing
lytic enzymes of Hemophilus, Pseudomonas, and/or Staphylococcus can
be used to directly treat eye infections. Treatment with lytic
enzymes are faster and more expedient than with antibiotics.
[0047] In yet another embodiment of the invention the phage
associated lytic enzyme(s) is (are) put into a carrier which is
placed in an inhaler to treat or prevent the spread of diseases
localized in the mucus lining of the oral cavity and lungs.
Specific lytic enzymes for tuberculosis have been isolated and can
be used.
[0048] In another embodiment of the invention the lytic enzymes,
shuffled lytic enzymes, and/or chimeric lytic enzymes, possibly
with holin lytic enzymes, are administered in the form of a candy,
chewing gum, lozenge, troche, tablet, a powder, an aerosol, a
liquid, a liquid spray, or toothpaste for the prevention or
treatment of bacterial infections associated with upper respiratory
tract illnesses.
[0049] In another embodiment of the invention, species specific
lytic enzymes can be used in the treatment of bacterial infections
associated with topical or dermatological infections, administered
in the form of a topical ointment or cream. In another embodiment
of the invention, the lytic enzyme would be administered in an
aqueous form. 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. More than one lytic enzyme may also be
included in the prophylactic or therapeutic agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1 is an electron micrograph of group A streptococci
treated with lysin showing the collapse of the cell wall and the
cell contents pouring out;
[0051] FIG. 2 is a graph for the killing of S. pneumoniae (#DCC
1490) serotype 14 with PAL at various dilutions;
[0052] FIG. 3 is a graph showing the the decrease of bacterial
titer within 30 seconds after addition of 100 U Pal phage
enzyme;
[0053] FIG. 4 is a series of graphs showing the decrease of the
Bacterial titer with 30 seconds after the addition of 100, 1,000,
and 10,000 U Pal Lytic Enzyme; and
[0054] FIG. 5 is a series of graphs showing the decrease of
bacterial titer within 30 seconds after addition of different
amounts of U Pal.
DETAILED DESCRIPTION OF THE INVENTION
[0055] The method for treating bacterial infections comprises
treating the infection with a therapeutic agent comprising an
effective amount of at least one lytic enzyme produced by a
bacteria infected with a bacteriophage specific for the bacteria
wherein at least one lytic enzyme is selected from the group
consisting of shuffled lytic enzymes, chimeric lytic enzymes, and
combinations thereof. The lytic enzyme is preferably in an
environment having a pH which allows for activity of said lytic
enzyme. A holin enzyme may be used in conjunction with the
administration of the modified lytic enzyme. The holin enzyme may
be in its "natural" state, may be shuffled holin enzymes or may be
chimeric lytic enzymes.
[0056] Additionally, therapeutic compositions of this invention
include one or more bacteria-associated phage enzymes, including
isozymes, analogs, or variants thereof, in a natural or modified
form. The modified form of the enzyme, for example, shuffled and/or
chimeric enzymes, is produced enzymatically by chemical synthesis
and/or DNA recombination technology.
[0057] The invention features the use of the chimeric and shuffled
lytic and holin enzymes, as examples of bacteria-associated phage
enzymes, in the therapeutic compositions and methods disclosed.
These enzymes are used, for example, in the treatment or prevention
of, for example, Streptococcal pygenes, Hemophilus influenza,
Pseudomonas, Streptococcus pneumoniae, Streptococcus fasciae,
Streptococcus group B, Listeria, Salmonella, E. coli,
Campylobacter, Mycobacteria tuberculosis Staphylococcu,
Helicobacter pylori or combinations thereof.
[0058] The lytic enzymes, shuffled lytic enzymes, chimeric lytic
enzymes, as well as, or in conjunction with holin lytic enzymes,
can be used for the treatment or prevention of Hemophilus
influenza, Pseudomonas, Streptococcus pneumoniae, Streptococcus
fasciae, Streptococcus group B, Listeria, Salmonella, E. coli,
Campylobacter, and other bacteria, and any combination thereof.
This lytic enzyme(s) may be either supplemented by chimeric and/or
shuffled lytic enzymes, or may be itself a chimeric and/or shuffled
lytic enzyme. Similarly, a holin enzyme may be included, which may
also be a chimeric and/or shuffled lytic enzyme.
[0059] It should be understood that bacteriophage lytic enzyme are
enzymes that specifically cleave bonds that are present in the
peptidoglycan of bacterial cells. Since the bacterial cell wall
peptiodglycan is highly conserved among all bacteria, there are
only a few bonds to be cleaved to disrupt the cell wall. Enzymes
that cleave these bonds are muramidases, glucosaminidases,
endopeptidases, or N-acetyl-muramoyl L alanine amidases
(hereinafter referred to as amidases). The majority of reported
phage enzymes are either muramidases or amidases, and there have
been no reports of bacteriophage glucosaminidases. Fischetti et al
(1974) reported that the C1 streptococcal phage lysin enzyme was an
amidase. Garcia et al (1987, 1990) reported that the Cp-1 lysin
from a S pneumoniae phage was a muramidase. Caldentey and Bamford
(1992) reported that a lytic enzyme from the phi 6 Pseudomonas
phage was an endopeptidase, splitting the peptide bridge formed by
meso-diaminopimilic acid and D-alanine. The E. coli T1 and T6 phage
lytic enzymes are amidases as is the lytic enzyme from Listeria
phage (ply) (Loessner et al, 1996).
[0060] There are a large number of phages which will attach to
specific bacteria and produce enzymes which will lyse that
particular bacteria. The following are a list of bacteriophages and
bacteria for which they are specific:
[0061] Streptococci
[0062] Pseudomonas
[0063] Pneumococci
[0064] Salmonella
[0065] Staphylococci
[0066] Shigella
[0067] Haemophilus
[0068] Listeria
[0069] Mycobacteria
[0070] Vibrio
[0071] Corynebacteria
[0072] Bacillus
[0073] Spirochete
[0074] Myxococcus
[0075] Burkholderia
[0076] Brucella
[0077] Yersinia
[0078] Clostridium
[0079] Campylobacter
[0080] Neisseria
[0081] Actinomycetes
[0082] Agrobacterium
[0083] Alcaligenes
[0084] Clostridium
[0085] Coryneforms
[0086] Cyanobacteria
[0087] Enterobacteria
[0088] Lactobacillus
[0089] Lactoctococcus
[0090] Micrococcus
[0091] Pasteurella
[0092] Rhizobium
[0093] Xanthomonas
[0094] Bdellovibrio
[0095] mollicutes
[0096] Chlamydia
[0097] Spiroplasma
[0098] Caulobacter
[0099] Various phages which can be used to infect these bacteria
and create the lytic enzyme include:
1 BACTERIA PHAGE(S) Actinomycetes A1-Dat, Bir, M1, MSP8, P-a-1, R1,
R2, SV2, VP5, PhiC, .perp.31C, .perp.UW21, .perp.115-A, .perp.150A,
119, SK1, 108/016 Aeromonas 29, 37, 43, 51, 59.1 Altermonas PM2
Bacillus AP5, .perp.NS11, BLE, Ipy-1, MP15, mor1, PBP1, SPP1, Spbb,
type F, alpha, .perp.105, 1A, II, Spy-2, SST, G, MP13, PBS1, SP3,
SP8, SP10, SP15, SP50 Bdellovibrio MAC-1, MAC-1', MAC-2, MAC-4,
MAC-4', MAC-5, MAC-7 Caulobacter .perp.Cb2, .perp.Cb4, .perp.Cb5,
.perp.Cb8r, .perp.Cb9, .perp.CB12r, .perp.Cb23r, .perp.CP2,
.perp.CP18, .perp.Cr14, .perp.Cr28, PP7, .perp.Cb2, .perp.Cb4,
.perp.Cb5, .perp.Cb8r, .perp.Cb9, .perp.CB12r, .perp.Cb23r,
.perp.CP2, .perp.CP18, .perp.Cr14, .perp.Cr28, PP7 Chlamydia Chp-1
Clostridium F1, HM7, HM3, CEB, Coliform AE2, dA, Ec9, f1, fd, HR,
M13, ZG/2, ZJ/2 Coryneforms Arp, BL3, CONX, MT, Beta, A8010, A19
Cyanobacteria S-2L, S-4L, N1, AS-1, S-6(L) Enterobacter C-2, If1,
If2, Ike, I2-2, PR64FS, SF, tf-1, PRD1, H-19J, B6, B7, C-1, C2,
Jersey, ZG/3A, T5, ViII, b4, chi, Beccles, tu, PRR1, 7s, C-1, c2,
fcan, folac, Ialpha, M, pilhalpha, R23, R34, ZG/1, ZIK/1, ZJ/1,
ZL/3, ZS/3, alpha15, f2, fr, FC3-9, K19, Mu, 01, P2, ViI, .perp.92,
121, 16-19, 9266, C16, DdVI, PST, SMB, SMP2, a1, 3, 3T+, 9/0, 11F,
50, 66F, 5845, 8893, M11, QB, ST, TW18, VK, FI, ID2, fr, f2,
Listeria H387, 2389, 2671, 2685, 4211 Micrococcus N1, N5
Mycobacterium Lacticola, Leo, R1-Myb, 13 Pasteurella C-2, 32, AU
Pseudomonas Phi6, Pf1, Pf2, Pf3, D3, Kf1, M6, PS4, SD1, PB-1, PP8,
PS17, nKZ, nW-14, n1, 12S, Staphyloccous 3A, B11-M15, 77, 107, 187,
2848A, Twort Streptococcus A25, A25 PE1, A25 VD13, A25 omega8, A25
24 Steptococcus A OXN-52P, VP-3, VP5, VP11, alpha3alpha, IV, kappa,
Vibrio 06N- 22-P, VP1, x29, II, nt-1, Xanthomonas Cf, Cf1t, Xf,
Xf2, XP5
[0100] There are numerous other phages infecting these and other
bacteria. The bacteriophages are normally grouped into family,
genus and species, including Genus Chlamydiamicrovirus, Genus
Bdellomicrovirus, Genus Spiromicrovirus, Genus Microvirus, Genus
Microvirus, Genus Levivirus, Genus Allolevivirus, and other
genuses.
[0101] The DNA coding of these phages and other phages may be
altered to allow a recombinant enzyme to attack one cell wall at
more than two locations, to allow the recombinant enzyme to cleave
the cell wall of more than one species of bacteria, to allow the
recombinant enzyme to attack other bacteria, or any combinations
thereof. The type and number of alterations to a recombinant
bacteriophage produced enzyme are incalculable
[0102] For example, if there is a bacterial infection of the upper
respiratory tract, the infection can be prophylactically or
therapeutically treated with a composition comprising an effective
amount of at least one lytic enzyme produced by a bacteria being
infected with a bacteriophage specific for that bacteria, and a
carrier for delivering the lytic enzyme to a mouth, throat, or
nasal passage. The lytic enzyme is preferably a chimeric and/or
shuffled lytic enzyme which may be used in conjunction with a holin
enzyme or modified or unmodified phage associated lytic enzyme. It
is also preferred that the lytic enzyme is in an environment having
a pH which allows for activity of the lytic enzyme. If an
individual has been exposed to someone with the upper respiratory
disorder, the lytic enzyme will reside in the mucosal lining and
prevent any colonization of the infecting bacteria.
[0103] Two examples of bacteria which infect the upper respiratory
system are Streptococcus pneumoniae and Hemophilus influenzae. In
recent years, there has been an increase in the number of people,
particularly children and the elderly, that are infected or are
carriers of penicillin resistant Streptococcus pneumoniae and
Hemophilus. While these bacteria are normally harmless residents of
the host, they are opportunistic organisms that are able to cause
infections when the resistance of the host has been compromised. By
eliminating or reducing the number of these organisms in the upper
respiratory tract, there will be a commensurate reduction in the
number of infections by these bacteria.
[0104] Infection of the Hemophilus bacteria by Bacteriophage HP1 (a
member of the P2-like phage family with strong similarities to
coliphages P2 and 186, and some similarity to the retronphage Ec67)
produces a lytic enzyme capable of lysing the bacteria. The lytic
enzyme for Streptococcus pneumoniae, previously identified as a
-acetyl-muramoyl-L-alanine amidase, is produced by the infecting
Streptococcus pneumoniae with the Pal bacteriophage. The
therapeutic agent can contain either or both of the lytic enzymes
produced by these two bacteria, and may contain other lytic enzymes
for other bacteria. The composition which may be used for the
prophylactic and therapeutic treatment of a strep infection
includes the lysin enzyme and a means of application, (such as a
carrier system or an oral delivery mode), to the mucosal lining of
the oral and nasal cavity, such that the enzyme is put in the
carrier system or oral delivery mode to reach the mucosal lining.
Another infection which can be treated prophylactically is
Streptococcus group A, which can produce what is commonly known as
"strep" throat. When group C Streptococci are infected with a C1
bacteriophage, a lysin enzyme is produced specific for the lysing
of Streptococcus group A.
[0105] While "nonrecombinant" or "unmodified" phage associated
lytic enzymes may be used for treatment of the Streptococcus, it is
preferred that a shuffled or chimeric lytic enzyme be used,
possibly with a holin enzyme.
[0106] Prior to, or at the time the lysin enzyme is put in the
carrier system or oral delivery mode, it is preferred that the
enzyme be in a stabilizing buffer environment for maintaining a pH
range between about 4.0 and about 9.0, more preferably between
about 5.5 and about 7.5 and most preferably at about 6.1.
[0107] The stabilizing buffer should allow for the optimum activity
of the lysin enzyme. The buffer may be a reducing reagent, such as
dithiothreitol. The stabilizing buffer may also be or include a
metal chelating reagent, such as ethylenediaminetetracetic acid
disodium salt, or it may also contain a phosphate or
citrate-phosphate buffer.
[0108] Means of application include, but are not limited to direct,
indirect, carrier and special means or any combination of means.
Direct application of the enzyme may be by nasal sprays, nasal
drops, nasal ointments, nasal washes, nasal injections, nasal
packings, bronchial sprays and inhalers, or indirectly through use
of throat lozenges, or through use of mouthwashes or gargles, or
through the use of ointments applied to the nasal nares, the bridge
of the nose, or the face or any combination of these and similar
methods of application. The forms in which the lysin enzyme may be
administered include but are not limited to lozenges, troches,
candies, injectants, chewing gums, tablets, powders, sprays,
liquids, ointments, and aerosols.
[0109] The lozenge, tablet, or gum into which the enzymes are added
may contain sugar, corn syrup, a variety of dyes, non-sugar
sweeteners, flavorings, any binders, or combinations thereof.
Similarly, any gum based products may contain acacia, carnauba wax,
citric acid, corn starch, food colorings, flavorings, non-sugar
sweeteners, gelatin, glucose, glycerin, gum base, shellac, sodium
saccharin, sugar, water, white wax, cellulose, other binders, and
combinations thereof.
[0110] Lozenges may further contain sucrose, corn starch, acacia,
gum tragacanth, anethole, linseed, oleoresin, mineral oil, and
cellulose, other binders, and combinations thereof. In another
embodiment of the invention, sugar substitutes are used in place of
dextrose, sucrose, or other sugars.
[0111] The enzyme may also be placed in a nasal spray, wherein the
nasal spray is the carrier. The nasal spray can be a long acting or
timed release spray, and can be manufactured by means well known in
the art. An inhalant may also be used, so that the phage enzyme may
reach further down into the bronchial tract, including into the
lungs.
[0112] Any of the carriers for the lytic enzymes may be
manufactured by conventional means. However, it is preferred that
any mouthwash or similar type products not contain alcohol to
prevent denaturing of the enzyme. Similarly, when the lytic enzymes
are being placed in a cough drop, gum, candy or lozenge during the
manufacturing process, such placement should be made prior to the
hardening of the lozenge or candy but after the cough drop or candy
has cooled somewhat, to avoid heat denaturation of the enzyme.
[0113] The enzyme may be added to these substances in a liquid form
or in a lyophilized state, whereupon it will be solubilized when it
meets body fluids such as saliva. The enzyme may also be in a
micelle or liposome.
[0114] The effective dosage rates or amounts of the lytic enzyme(s)
to treat the infection will depend in part on whether the lytic
will be used therapeutically or prophylactically, the duration of
exposure of the recipient to the infectious bacteria, the size and
weight of the individual, etc. The duration for use of the
composition containing the enzyme also depends on whether the use
is for prophylactic purposes, wherein the use may be hourly, daily
or weekly, for a short time period, or whether the use will be for
therapeutic purposes wherein a more intensive regimen of the use of
the composition may be needed, such that usage may last for hours,
days or weeks, and/or on a daily basis, or at timed intervals
during the day. Any dosage form employed should provide for a
minimum number of units for a minimum amount of time. The
concentration of the active units of enzyme(s) believed to provide
for an effective amount or dosage of enzyme may be in the range of
about 100 units/ml to about 100,000 units/ml of fluid in the wet or
damp environment of the nasal and oral passages, and possibly in
the range of about 100 units/ml to about 10,000 units/ml. More
specifically, time exposure to the active enzyme units may
influence the desired concentration of active enzyme units per ml.
It should be noted that carriers that are classified as "long" or
"slow" release carriers (such as, for example, certain nasal sprays
or lozenges) could possess or provide a lower concentration of
active (enzyme) units per ml, but over a longer period of time,
whereas a "short" or "fast" release carrier (such as, for example,
a gargle) could possess or provide a high concentration of active
(enzyme) units per ml, but over a shorter period of time. The
amount of active units per ml and the duration of time of exposure
depends on the nature of infection, whether treatment is to be
prophylactic or therapeutic, and other variables.
[0115] While this treatment may be used in any mammalian species,
the preferred use of this product is for a human.
[0116] This composition and method may also be used for the
treatment of Streptococcus A infections of the respiratory tract.
When using this composition for a Streptococcus A infection, the
chimeric and/or shuffled lytic enzymes should be used for the
prophylactic prevention of Streptococcus infections. Similarly, in
another embodiment of the invention, this method may be used for
the therapeutic and, preferably, the prophylactic treatment of
tuberculosis. In a preferred embodiment of the invention, the phage
associated lysing enzyme for Mycobacteria tuberculosis is placed in
a carrier in an inhaler. The carrier may be sterile water or a
water base, or any other carrier used in an inhaler for dispersing
drugs into the bronchial tract. The phage associated chimeric
and/or shuffled lytic enzyme specific for tuberculosis is subject
to the same conditions as the phage associated lytic enzyme for
other lytic enzymes. Specifically, prior to, or at the time the
enzyme is put in the carrier system or oral delivery mode, it is
preferred that the enzyme be in a stabilizing buffer environment
for maintaining a pH range between about 4.0 and about 9.0.
[0117] The stabilizing buffer should allow for the optimum activity
of the lytic enzyme. The buffer may be a reducing reagent, such as
dithiothreitol. The stabilizing buffer may also be or include a
metal chelating reagent, such as ethylenediaminetetracetic acid
disodium salt, or it may also contain a phosphate or
citrate-phosphate buffer.
[0118] For the prophylactic and therapeutic treatment of
tuberculosis, the phage associated chimeric and/or shuffled lytic
enzymes associated with tuberculosis may also be applied by direct,
indirect, carriers and special means or any combination of means.
Direct application of the lytic enzyme may be by nasal sprays,
nasal drops, nasal ointments, nasal washes, nasal injections, nasal
packings, bronchial sprays and inhalers, or indirectly through use
of throat lozenges, or through use of mouthwashes or gargles, or
through the use of ointments applied to the nasal nares, the bridge
of the nose, or the face or any combination of these and similar
methods of application. The forms in which the lytic enzyme may be
administered include but are not limited to lozenges, troches,
candies, injectants, chewing gums, tablets, powders, sprays,
liquids, ointments, and aerosols. For the therapeutic treatment of
tuberculosis, the bronchial sprays and aerosols are most
beneficial, as these carriers, or means of distributing the
composition, allow the lytic enzyme to reach the bronchial tubes
and the lungs. An appropriate transport carrier may be attached to
the enzyme to transport the enzyme across the cell membrane to the
site of the bacteria. The chimeric and/or shuffled lytic enzymes
may be used in combination with other chimeric and shuffled lytic
enzymes, holin enzymes, other lytic enzymes, and other phage
associated lytic enzymes which have not been modified or which are
not "recombinant."
[0119] Another use of a lytic enzyme is for the treatment of
bacterial infections of the digestive tract. The method for
treating a bacterial infection of the digestive tract comprises
treating the bacterial infection with composition comprising an
effective amount of at least one lytic enzyme produced by a
bacteria infected with a bacteriophage specific for the bacteria,
and a carrier for delivering said lytic enzyme to the digestive
tract. In a preferred embodiment of the invention, the bacterial
infections being treated are selected from the group consisting of
H., pyogenes, Listeria, Salmonella, E. coli, and Campylobacter.
However, this method and composition will effectively treat other
bacteria, when the appropriate lytic enzyme is used. The lytic
enzymes used in the digestive tract may be either supplemented by
chimeric and/or shuffled lytic enzymes, or may be themselves
chimeric and/or shuffled lytic enzymes. Similarly, a holin enzyme
may be included, which may also be a chimeric and/or shuffled lytic
enzyme.
[0120] In a preferred embodiment of the invention, the carrier is
selected from the group consisting of suppository enemas, syrups,
or enteric coated pills. These proposed carriers can be made by
conventional methods. However, the only difference in their
manufacture is that the enzyme being placed in the carrier must not
be allowed to denature. The enzyme should be incorporated into a
carrier which does not contain alcohol, and which has been cooled
to a temperature that will not cause the denaturing of the enzyme.
The enzyme may be incorporated in a lyophilized state, or may be
incorporated in a liposome before being placed in the suppository,
syrup or enteric coated pill. The enzyme placed in the composition
or carrier should be in an environment having a pH which allows for
activity of the lytic enzyme. To this end, the pH of the
composition is preferably kept in a range of between about 2 and
about 11, more preferably in a range of between about between about
4.0 and about 9.0, and even more preferably at a pH range of
between about 5.5 and about 7.5. As described above with the other
lytic enzyme, the pH can be moderated by the use of a buffer. The
buffer may contain a reducing agent, and more specifically
dithiothreitol. The buffer may also be a metal chelating reagent,
such as ethylenediaminetetracetic disodium salt or the buffer may
contain a citrate-phosphate buffer. As with all compositions
described in this patent, the composition may, further include a
bactericidal or bacteriostatic agent as a preservative.
[0121] The lytic enzyme(s) preferably are present in a
concentration of about 100 to about 500,000 active enzyme units per
milliliter of fluid in the wet environment of the gastrointestinal
tract, preferably about 100 to about 100,000 active enzyme units
per milliliter of fluid, and preferably present in a concentration
of about 100 to about 10,000 active enzyme units per milliliter of
fluid in the wet environment of the gastrointestinal tract.
[0122] The suppository is known in the art, and is made of
glycerin, fatty acids, and similar type substances that dissolve at
body temperature. As the suppository dissolves, the phage
associated lytic enzyme will be released.
[0123] Another composition and use of the lytic enzyme is for the
therapeutic or prophylactic treatment of bacterial infections of
burns and wounds of the skin. The composition comprises an
effective amount of at least one lytic enzyme produced by a
bacteria infected with a bacteriophage specific for the bacteria
and a carrier for delivering at least one lytic enzyme to the
wounded skin. The lytic enzyme(s) used for the topical treatment of
burns may be either supplemented by chimeric and/or shuffled lytic
enzymes, or may themselves be chimeric and/or shuffled lytic
enzymes. Similarly, a holin enzyme may be included, which may also
be a chimeric and/or shuffled lytic enzyme. The mode of application
for the lytic enzyme includes a number of different types and
combinations of carriers which include, but are not limited to an
aqueous liquid, an alcohol base liquid, a water soluble gel, a
lotion, an ointment, a nonaqueous liquid base, a mineral oil base,
a blend of mineral oil and petrolatum, lanolin, liposomes, protein
carriers such as serum albumin or gelatin, powdered cellulose
carmel, and combinations thereof. A mode of delivery of the carrier
containing the therapeutic agent includes but is not limited to a
smear, spray, a time-release patch, a liquid absorbed wipe, and
combinations thereof. The lytic enzyme may be applied to a bandage
either directly or in one of the other carriers. The bandages may
be sold damp or dry, wherein the enzyme is in a lyophilized form on
the bandage. This method of application is most effective for the
treatment of burns.
[0124] The carriers of the compositions of the present invention
may comprise semisolid and gel-like vehicles that include a polymer
thickener, water, preservatives, active surfactants or emulsifiers,
antioxidants, sun screens, and a solvent or mixed solvent system.
U.S. Pat. No. 5,863,560 (Osborne) discusses a number of different
carrier combinations which can aid in the exposure of the skin to a
medicament.
[0125] Polymer thickeners that may be used include those known to
one skilled in the art, such as hydrophilic and hydroalcoholic
gelling agents frequently used in the cosmetic and pharmaceutical
industries. Preferably, the hydrophilic or hydroalcoholic gelling
agent comprises "CARBOPOL.RTM." (B. F. Goodrich, Cleveland, Ohio),
"HYPAN.RTM." (Kingston Technologies, Dayton, N.J.), "NATROSOL.RTM."
(Aqualon, Wilmington, Del.), "KLUCEL.RTM." (Aqualon, Wilmington,
Del.), or "STABILEZE.RTM." (ISP Technologies, Wayne, N.J.).
Preferably, the gelling agent comprises between about 0.2% to about
4% by weight of the composition. More particularly, the preferred
compositional weight percent range for "CARBOPOL.RTM." is between
about 0.5% to about 2%, while the preferred weight percent range
for "NATROSOL.RTM." and "KLUCEL.RTM." is between about 0.5% to
about 4%. The preferred compositional weight percent range for both
"HYPAN.RTM." and "STABILEZE.RTM." is between about 0.5% to about
4%. CARBOPOL.RTM." is one of numerous cross-linked acrylic acid
polymers that are given the general adopted name carbomer. These
polymers dissolve in water and form a clear or slightly hazy gel
upon neutralization with a caustic material such as sodium
hydroxide, potassium hydroxide, triethanolamine, or other amine
bases. "KLUCEL.RTM." is a cellulose polymer that is dispersed in
water and forms a uniform gel upon complete hydration. Other
preferred gelling polymers include hydroxyethylcellulose, cellulose
gum, MVE/MA decadiene crosspolymer, PVM/MA copolymer, or a
combination thereof.
[0126] Preservatives may also be used in this invention and
preferably comprise about 0.05% to 0.5% by weight of the total
composition. The use of preservatives assures that if the product
is microbially contaminated, the formulation will prevent or
diminish microorganism growth. Some preservatives useful in this
invention include methylparaben, propylparaben, butylparaben,
chloroxylenol, sodium benzoate, DMDM Hydantoin,
3-Iodo-2-Propylbutyl carbamate, potassium sorbate, chlorhexidine
digluconate, or a combination thereof.
[0127] Titanium dioxide may be used as a sunscreen to serve as
prophylaxis against photosensitization. Alternative sun screens
include methyl cinnamate. Moreover, BHA may be used as an
antioxidant, as well as to protect ethoxydiglycol and/or dapsone
from discoloration due to oxidation. An alternate antioxidant is
BHT.
[0128] Pharmaceuticals for use in all embodiments of the invention
include antimicrobial agents, anti-inflammatory agents, antiviral
agents, local anesthetic agents, corticosteroids, destructive
therapy agents, antifungals, and antiandrogens. In the treatment of
acne, active pharmaceuticals that may be used include antimicrobial
agents, especially those having anti-inflammatory properties such
as dapsone, erythromycin, minocycline, tetracycline, clindamycin,
and other antimicrobials. The preferred weight percentages for the
antimicrobials are 0.5% to 10%. Local anesthetics include
tetracaine, tetracaine hydrochloride, lidocaine, lidocaine
hydrochloride, dyclonine, dyclonine hydrochloride, dimethisoquin
hydrochloride, dibucaine, dibucaine hydrochloride, butambenpicrate,
and pramoxine hydrochloride. A preferred concentration for local
anesthetics is about 0.025% to 5% by weight of the total
composition. Anesthetics such as benzocaine may also be used at a
preferred concentration of about 2% to 25% by weight.
[0129] Corticosteroids that may be used include betamethasone
dipropionate, fluocinolone actinide, betamethasone valerate,
triamcinolone actinide, clobetasol propionate, desoximetasone,
diflorasone diacetate, amcinonide, flurandrenolide, hydrocortisone
valerate, hydrocortisone butyrate, and desonide are recommended at
concentrations of about 0.01% to 1.0% by weight. Preferred
concentrations for corticosteroids such as hydrocortisone or
methylprednisolone acetate are from about 0.2% to about 5.0% by
weight.
[0130] Destructive therapy agents such as salicylic acid or lactic
acid may also be used. A concentration of about 2% to about 40% by
weight is preferred. Cantharidin is preferably utilized in a
concentration of about 5% to about 30% by weight. Typical
antifingals that may be used in this invention and their preferred
weight concentrations include: oxiconazole nitrate (0.1% to 5.0%),
ciclopirox olamine (0.1% to 5.0%), ketoconazole (0.1% to 5.0%),
miconazole nitrate (0.1% to 5.0%), and butoconazole nitrate (0.1%
to 5.0%). For the topical treatment of seborrheic dermatitis,
hirsutism, acne, and alopecia, the active pharmaceutical may
include an antiandrogen such as flutamide or finasteride in
preferred weight percentages of about 0.5% to 10%.
[0131] Typically, treatments using a combination of drugs include
antibiotics in combination with local anesthetics such as polymycin
B sulfate and neomycin sulfate in combination with tetracaine for
topical antibiotic gels to provide prophylaxis against infection
and relief of pain. Another example is the use of minoxidil in
combination with a corticosteroid such as betamethasone
diproprionate for the treatment of alopecia ereata. The combination
of an anti-inflammatory such as cortisone with an antifungal such
as ketoconazole for the treatment of tinea infections is also an
example.
[0132] In one embodiment, the invention comprises a dermatological
composition having about 0.5% to 10% carbomer and about 0.5% to 10%
of a pharmaceutical that exists in both a dissolved state and a
micro particulate state. The dissolved pharmaceutical has the
capacity to cross the stratum corneum, whereas the micro
particulate pharmaceutical does not. Addition of an amine base,
potassium, hydroxide solution, or sodium hydroxide solution
completes the formation of the gel. More particularly, the
pharmaceutical may include dapsone, an antimicrobial agent having
anti-inflammatory properties. A preferred ratio of micro
particulate to dissolved dapsone is five or less.
[0133] In another embodiment, the invention comprises about 1%
carbomer, about 80-90% water, about 10% ethoxydiglycol, about 0.2%
methylparaben, about 0.3% to 3.0% dapsone including both micro
particulate dapsone and dissolved dapsone, and about 2% caustic
material. More particularly, the carbomer may include
"CARBOPOL.RTM. 980" and the caustic material may include sodium
hydroxide solution.
[0134] In a preferred embodiment, the composition comprises dapsone
and ethoxydiglycol, which allows for an optimized ratio of micro
particulate drug to dissolved drug. This ratio determines the
amount of drug delivered, compared to the amount of drug retained
in or above the stratum corneum to function in the supracorneum
domain. The system of dapsone and ethoxydiglycol may include
purified water combined with "CARBOPOL.RTM." gelling polymer,
methylparaben, propylparaben, titanium dioxide, BHA, and a caustic
material to neutralize the "CARBOPOL.RTM."
[0135] Any of the carriers for the lytic enzyme may be manufactured
by conventional means. However, if alcohol is used in the carrier,
the enzyme should be in a micelle, liposome, or a "reverse"
liposome, to prevent denaturing of the enzyme. Similarly, when the
lytic enzyme is being placed in the carrier, and the carrier is, or
has been heated, such placement should be made after the carrier
has cooled somewhat, to avoid heat denaturation of the enzyme. In a
preferred embodiment of the invention, the carrier is sterile.
[0136] The enzyme may be added to these substances in a liquid form
or in a lyophilized state, whereupon it will be solubilized when it
meets a liquid body.
[0137] The effective dosage rates or amounts of the lytic enzyme to
treat the infection, and the duration of treatment will depend in
part on the seriousness of the infection, the duration of exposure
of the recipient to the infectious bacteria, the number of square
centimeters of skin or tissue which are infected, the depth of the
infection, the seriousness of the infection, and a variety of a
number of other variables. The composition may be applied anywhere
from once to several times a day, and may be applied for a short or
long term period. The usage may last for days or weeks. Any dosage
form employed should provide for a minimum number of units for a
minimum amount of time. The concentration of the active units of
enzyme believed to provide for an effective amount or dosage of
enzyme may be in the range of about 100 units/ml to about 500,000
units/ml of composition, preferably in the range of about 1000
units/ml to about 100,000 units/ml, and most preferably from about
10,000 to 100,000 units/ml. The amount of active units per ml and
the duration of time of exposure depends on the nature of
infection, and the amount of contact the carrier allows the lytic
enzyme(s) to have. It is to be remembered that the enzyme works
best when in a fluid environment. Hence, effectiveness of the
enzyme(s) is in part related to the amount of moisture trapped by
the carrier. In another preferred embodiment, a mild surfactant in
an amount effective to potentiate the therapeutic effect of the
lytic enzyme. Suitable mild surfactants include, inter alia, esters
of polyoxyethylene sorbitan and fatty acids (Tween series),
octylphenoxy polyethoxy ethanol (Triton-X series),
n-Octyl-.beta.-D-glucopyranoside,
n-Octyl-.beta.-D-thioglucopyranoside,
n-Decyl-.beta.-D-glucopyranoside,
n-Dodecyl-.beta.-D-glucopyranoside, and biologically occurring
surfactants, e.g., fatty acids, glycerides, monoglycerides,
deoxycholate and esters of deoxycholate.
[0138] In order to accelerate treatment of the infection, the
therapeutic agent may further include at least one complementary
agent which can also potentiate the bactericidal activity of the
lytic enzyme. The complementary agent can be penicillin, synthetic
penicillins bacitracin, methicillin, cephalosporin, polymyxin,
cefaclor. Cefadroxil, cefamandole nafate, cefazolin, cefixime,
cefmetazole, cefonioid, cefoperazone, ceforanide, cefotanme,
cefotaxime, cefotetan, cefoxitin, cefpodoxime proxetil,
ceftazidime, ceftizoxime, ceftriaxone, cefriaxone moxalactam,
cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium
salt, cephalothin, cephalothin sodium salt, cephapirin, cephradine,
cefuroximeaxetil, dihydratecephalothin, moxalactam, loracarbef.
mafate, chelating agents, streptomycin, erythromycin,
chloramphenicol, numerous other antibiotics, and any combinations
thereof in amounts which are effective to synergistically enhance
the therapeutic effect of the lytic enzyme. It should be noted that
virtually any antibiotic may be used as complementary agents for or
with any use of the recombinant lytic enzymes.
[0139] Additionally, the therapeutic agent may further comprise the
enzyme lysostaphin for the treatment of any Staphylococcus aureus
bacteria. Mucolytic peptides, such as lysostaphin, have been
suggested to be efficacious in the treatment of S. aureus
infections of humans (Schaffner et al., Yale J. Biol. & Med.,
39:230 (1967) and bovine mastitis caused by S. aureus (Sears et
al., J. Dairy Science, 71 (Suppl. 1): 244(1988)). Lysostaphin, a
gene product of Staphylococcus simulans, exerts a bacteriostatic
and bactericidal effect upon S. aureus by enzymatically degrading
the polyglycine crosslinks of the cell wall (Browder et al., Res.
Comm., 19: 393-400 (1965)). U.S. Pat. No. 3,278,378 describes
fermentation methods for producing lysostaphin from culture media
of S. staphylolyticus, later renamed S. simulans. Other methods for
producing lysostaphin are further described in U.S. Pat. Nos.
3,398,056 and 3,594,284. The gene for lysostaphin has subsequently
been cloned and sequenced (Recsei et al., Proc. Natl. Acad. Sci.
USA, 84: 1127-1131 (1987)). The recombinant mucolytic bactericidal
protein, such as r-lysostaphin, can potentially circumvent problems
associated with current antibiotic therapy because of its targeted
specificity, low toxicity and possible reduction of biologically
active residues. Furthermore, lysostaphin is also active against
non-dividing cells, while most antibiotics require actively
dividing cells to mediate their effects (Dixon et al., Yale J.
Biology and Medicine, 41: 62-68 (1968)). Lysostaphin, in
combination with the lysin enzyme, can be used in the presence or
absence of the listed antibiotics. There is a degree of added
importance in using both lysostaphin and the lysin enzyme in the
same therapeutic agent. Frequently, when a body has a bacterial
infection, the infection by one genus of bacteria weakens the body
or changes the bacterial flora of the body, allowing other
potentially pathogenic bacteria to infect the body. One of the
bacteria that sometimes co-infects a body is Staphylococcus aureus.
Many strains of Staphylococcus aureus produce penicillinase, such
that Staphylococcus, Streptococcus, and other gram positive
bacterial strains will not be killed by standard antibiotics.
Consequently, the use of the lysin and lysostaphin, possibly in
combination with antibiotics, can serve as the most rapid and
effective treatment of bacterial infections. In yet another
preferred embodiment, the invention may include mutanolysin, and
lysozyme
[0140] In preferred embodiments of the invention, the chimeric
and/or shuffled lytic enzymes for Pseudomonas, Staphylococcus, and
Streptococcus, jointly or individually, may be incorporated into
the carrier, or into a bandage to be used on burn patients, or in a
solution or cream carrier. These enzymes may be used in combination
with holin and other lytic enzymes.
[0141] Yet another use of lytic enzymes is for the prophylactic or
therapeutic treatment of vaginal infections. This treatment
comprises treating the vaginal infection with an effective amount
of at least one lytic enzyme produced by a bacteria being infected
with a bacteriophage specific for that bacteria, wherein that lytic
enzyme is incorporated in a carrier to be placed in a vagina. The
lytic enzyme(s) used to treat bacterial infections of the vagina
may be either supplemented by chimeric and/or shuffled lytic
enzymes, or may be itself a chimeric and/or shuffled lytic enzyme.
Similarly, a holin enzyme may be included, which may also be a
chimeric and/or shuffled lytic enzyme. The preferred carrier is a
tampon, or vaginal douche. A pad may also be used as a carrier,
although it is not as effective. While any number of bacteria could
be treated using this composition and method, it is believed that
the most optimum use of this treatment composition and method would
be for the treatment of an E. coli and Streptococcus B infection.
Vaginal infections caused by Group B Streptococcus can cause
neonatal meningitis resulting in brain damage and premature death.
Lytic enzymes incorporated into tampon specific for group B Strep
would eliminate the group B organisms without disturbing normal
flora so that woman would not be overcome by yeast infection post
antibiotic therapy. The use of the lytic enzymes in the vagina
would best provide a prophylactic effect, although therapeutic use
would also be advisable.
[0142] To produce a pad or tampon containing the enzyme, the lytic
enzymes can be applied in a solution to the tampon, and allowed to
dry. The lytic enzyme may be incorporated into the pad or tampon by
any other means known in the art, including lyophilization,
spraying, etc. The tampons and pads may also be kept slightly
moist, and in a sealed wrapper until ready for use. In that case,
bactericide and bacteriostatic compounds and inhibitors should be
present in the tampons and pads. The method to be used for
incorporating the lytic enzyme into the tampon or pad can be one of
the methods known in the art for incorporating a pharmaceutical
product. In another embodiment of the invention, the lytic enzyme
is incorporated into a vaginal suppository. The vaginal suppository
into which the lytic enzyme is being incorporated may be a standard
vaginal suppository, comprised of glyceride, alginate, starch,
other standard binders and any combinations thereof.
[0143] When using a tampon as the carrier, it is best to insert the
tampon in the vagina and leave it in for up to 12 hours to
distribute the enzyme vaginally.
[0144] As with other lytic enzymes, it is preferable that the pH be
kept in a range of about 4.0 and about 9.0 even more preferably at
a pH range of between about 5.5 and about 7.5. As described above
with the other lytic enzyme, the pH can be moderated by the use of
a buffer. The buffer may contain a reducing agent, and more
specifically dithiothreitol. The buffer may also contain a metal
chelating reagent, such as ethylenediaminetetracetic disodium salt
or the buffer may be a citrate-phosphate buffer. As with all
compositions described in this patent, the composition may, further
include a bactericidal or bacteriostatic agent as a
preservative.
[0145] The lytic enzyme(s) are preferably present in a
concentration of about 100 to about 500,000 active enzyme units per
milliliter of fluid in the wet environment of the vaginal tract,
preferably about 100 to about 100,000 active enzyme units per
milliliter of fluid, and preferably present in a concentration of
about 100 to about 10,000 active enzyme units per milliliter of
fluid in the wet environment of the vaginal tract.
[0146] Another use of the invention is for the prophylactic and
therapeutic treatment of eye infections. The method of treatment
comprises administering eye drops which comprise an effective
amount of at least one lytic enzyme produced by the bacteria being
infected with a bacteriophage specific for the bacteria and a
carrier capable of being safely applied to an eye, with the carrier
containing the lytic enzyme. In a preferred embodiment of the
invention, the bacteria being treated is Hemophilus or
Staphylococcus The eye drops are in the form of an isotonic
solution. The pH of the solution should be adjusted so that there
is no irritation of the eye, which in turn would lead to possibly
infection by other organisms, and possibly to damage to the eye.
While the pH range should be in the same range as for other lytic
enzymes, the most optimal pH will be in the range of from 6.0 to
7.5. Similarly, buffers of the sort described above for the other
lytic enzymes should also be used. Other antibiotics which are
suitable for use in eye drops may be added to the composition
containing the lytic enzymes. Bactericides and bacteriostatic
compounds may also be added. As stated above, this lytic enzyme may
be either supplemented by chimeric and/or shuffled lytic enzymes,
or may be itself a chimeric and/or shuffled lytic enzyme.
Similarly, a holin enzyme may be included, which may also be a
chimeric and/or shuffled lytic enzyme.
[0147] It is to be remembered that all of the enzymes can be used
for prophylactic and therapeutic treatments of the bacteria for
which the enzymes are specific.
[0148] Additionally, a carrier may have more than one lytic enzyme.
For instance, a throat lozenge may comprise just a lysin enzyme
(which lyses the Streptococcus A strain causing "strep" throat) or
it may also include the lytic enzymes for Hemophilus. Similarly,
the carrier for treating burns and wounds, or infections of the
skin, may contain just one lytic enzyme, or a combination of lytic
enzymes, for the treatment of Pseudomonas, Streptococcus,
Staphylococcus, or any other of a number of bacteria. The carrier
may include any combination of lytic enzymes, shuffled lytic
enzymes, chimeric lytic enzymes, and holin enzymes,
[0149] Lytic enzymes can also be used to fight dental caries. See,
for example, a lytic enzyme specific for Streptococcus mutans may
be incorporated in a toothpaste or oral wash. Similarly, this lytic
enzyme may also be incorporated into a chewing gum or lozenge. Any
other carrier can be used that allows for the exposure of the
mouth, gums, and teeth to the lytic enzyme.
[0150] The lytic enzyme may also be incorporated in a lyophilized
or dried form in tooth powder. If the lytic enzyme is to be used in
an oral wash, it is preferred that the oral wash not contain any
alcohol, so as to not denature the enzyme. The enzyme can also be
in a liposome when mixed in with the toothpaste or oral wash. The
concentrations of the enzyme units per ml of toothpaste or mouth
wash can be in the range of from about 100 units/ml to about
500,000 units/ml of composition, preferably in the range of about
1000 units/ml to about 100,000 units/ml, and most preferably from
about 10,000 to 100,000 units/ml. The pH of the toothpaste or oral
wash should be in a range that allows for the optimum performance
of the enzyme, while not causing any discomfort to the user of the
toothpaste or oral wash. Again, as with the other uses of lytic
enzymes, the lytic enzyme use to treat dental caries may be either
supplemented by chimeric and/or shuffled lytic enzymes, or may be
itself a chimeric and/or shuffled lytic enzyme. Similarly, a holin
enzyme may be included, which may also be a chimeric and/or
shuffled lytic enzyme.
[0151] The lytic enzymes may also be administered parenterally. The
lytic enzyme, holin lytic enzyme, chimeric enzyme, shuffled enzyme,
and combinations thereof may be administered parenterally using an
effective amount of a therapeutic agent, the therapeutic agent
comprising at least one lytic enzyme produced by a bacteria
infected with a bacteriophage specific for said bacteria selected
from the group consisting of holin lytic enzymes, chimeric lytic
enzymes, shuffled lytic enzymes, and combinations thereof, and a
carrier for delivering the lytic enzyme to the site of the
infection.
[0152] The composition may be used for the therapeutic treatment of
Pseudomonas, Clostridium, Staphylococcus infections, among
others.
[0153] A number of different bacteria may be treated. Among the
bacteria which most often infect deep tissues, and, more
specifically connective tissues, are Group A Streptococcus,
Staphylococcus, Pseudomonas, and Clostridium. More than one lytic
enzyme may be introduced into the infected body at a time.
[0154] A number of different methods may be used to introduce the
lytic enzyme(s). These methods include introducing the lytic enzyme
intravenously, intramuscularly, subcutaneously, and
subdermally.
[0155] In one preferred embodiment of the invention, a deep tissue
infection may be treated by injecting into the infected tissue of
the patient a therapeutic agent comprising the appropriate lytic
enzyme(s) (holin lytic enzyme, chimeric lytic enzyme and/or
shuffled lytic enzyme) and a carrier for the enzyme. The carrier
may be comprised of distilled water, a saline solution, albumin, a
serum, or any combinations thereof. More specifically, solutions
for infusion or injection may be prepared in a conventional manner,
e.g. with the addition of preservatives such as p-hydroxybenzoates
or stabilizers such as alkali metal salts of ethylene-diamine
tetraacetic acid, which may then be transferred into fusion
vessels, injection vials or ampules. Alternatively, the compound
for injection may be lyophilized either with or without the other
ingredients and be solubilized in a buffered solution or distilled
water, as appropriate, at the time of use. Non-aqueous vehicles
such as fixed oils and ethyl oleate are also useful herein.
[0156] In cases where intramuscular injection is the chosen mode of
administration, an isotonic formulation is preferably used.
Generally, additives for isotonicity can include sodium chloride,
dextrose, mannitol, sorbitol and lactose. In some cases, isotonic
solutions such as phosphate buffered saline are preferred.
Stabilizers include gelatin and albumin. In some embodiments, a
vasoconstriction agent is added to the formulation. The
pharmaceutical preparations according to the present invention are
provided sterile and pyrogen free.
[0157] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
glycine; amino acids such as glutamic acid, aspartic acid,
histidine, or arginine; monosaccharides, disaccharides, and other
carbohydrates including cellulose or its derivatives, glucose,
mannose, trehalose, or dextrins; chelating agents such as EDTA;
sugar alcohols such as mannitol or sorbitol; counter-ions such as
sodium; non-ionic surfactants such as polysorbates, poloxamers, or
polyethylene glycol (PEG); and/or neutral salts, e.g., NaCl, KCl,
MgCl.sub.2, CaCl.sub.2, etc.
[0158] Glycerin or glycerol (1,2,3-propanetriol) is commercially
available for pharmaceutical use. It may be diluted in sterile
water for injection, or sodium chloride injection, or other
pharmaceutically acceptable aqueous injection fluid, and used in
concentrations of 0.1 to 100% (v/v), preferably 1.0 to 50% and
more, but preferably about 20%.
[0159] DMSO, an aprotic solvent with a remarkable ability to
enhance penetration of many locally applied drugs, may be diluted
in sterile water for injection, or sodium chloride injection, or
other pharmaceutically acceptable aqueous injection fluid, and used
in concentrations of 0.1 to 100% (v/v).
[0160] The carrier vehicle may also include Ringer's solution, a
buffered solution, and dextrose solution, particularly when an
intravenous solution is prepared.
[0161] Prior to, or at the time the lytic enzyme is put in the
carrier system or oral delivery mode, it is preferred that the
enzyme be in a stabilizing buffer environment for maintaining a pH
range between about 4.0 and about 9.0, more preferably between
about 5.5 and about 7.5 and most preferably at about 6.1. This is
pH range is most suitable for the lysin enzyme for
Streptococcus.
[0162] The stabilizing buffer should allow for the optimum activity
of the lysin enzyme. The buffer may be a reducing reagent, such as
dithiothreitol. The stabilizing buffer may also be or include a
metal chelating reagent, such as ethylenediaminetetracetic acid
disodium salt, or it may also contain a phosphate or
citrate-phosphate buffer. The buffers found in the carrier can
serve to stabilize the environment for the lytic enzymes.
[0163] The effective dosage rates or amounts of the chimeric and/or
shuffled lytic enzymes to treat the infection, and the duration of
treatment will depend in part on the seriousness of the infection,
the duration of exposure of the recipient to the infectious
bacteria, the number of square centimeters of skin or tissue which
are infected, the depth of the infection, the seriousness of the
infection, and a variety of a number of other variables. The
composition may be applied anywhere from once to several times a
day, and may be applied for a short or long term period. The usage
may last for days or weeks. Any dosage form employed should provide
for a minimum number of units for a minimum amount of time. The
concentration of the active units of enzyme believed to provide for
an effective amount or dosage of enzyme may be in the range of
about 100 units/ml to about 500,000 units/ml of composition,
preferably in the range of about 1000 units/ml to about 100,000
units/ml, and most preferably from about 10,000 to 100,000
units/ml. The amount of active units per ml and the duration of
time of exposure depends on the nature of infection, and the amount
of contact the carrier allows the lytic enzyme to have. It is to be
remembered that the enzyme works best when in a fluid environment.
Hence, effectiveness of the enzyme is in part related to the amount
of moisture trapped by the carrier. For the treatment of
septicemia, there should be a continuous intravenous flow of
therapeutic agent into the blood stream. The concentration of lytic
enzymes for the treatment of septicemia is dependent upon the
seriousness of the infection.
[0164] In order to accelerate treatment of the infection, the
therapeutic agent may further include at least one complementary
agent which can also potentiate the bactericidal activity of the
lytic enzyme. The complementary agent can be penicillin, synthetic
penicillins bacitracin, methicillin, cephalosporin, polymyxin,
cefaclor. Cefadroxil, cefamandole nafate, cefazolin, cefixime,
cefmetazole, cefonioid, cefoperazone, ceforanide, cefotanme,
cefotaxime, cefotetan, cefoxitin, cefpodoxime proxetil,
ceftazidime, ceftizoxime, ceftriaxone, cefriaxone moxalactam,
cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium
salt, cephalothin, cephalothin sodium salt, cephapirin, cephradine,
cefuroximeaxetil, dihydratecephalothin, moxalactam, loracarbef.
mafate, chelating agents and any combinations thereof in amounts
which are effective to synergistically enhance the therapeutic
effect of the lytic enzyme. As previously noted, virtually any
antibiotic may be used with the the various lytic enzymes, which
include the shuffled and/or chimeric lytic enzymes, the holin
enzymes, etc.
[0165] Additionally, the therapeutic agent may further comprise the
enzyme lysostaphin for the treatment of any Staphylococcus aureus
bacteria. In yet another preferred embodiment, the invention may
include mutanolysin, and lysozyme
[0166] The use of lytic enzymes, including but not limited to holin
lytic enzymes, chimeric lytic enzymes, shuffled lytic enzymes, and
combinations thereof, rapidly lyse the bacterial cell. The thin
section electron micrograph of FIG. 1 shows the results of a group
A streptococci 1 treated for 15 seconds with lysin. The micrograph
(25,000.times. magnification) shows the cell contents 2 pouring out
through a hole 3 created in the cell wall 4 by the lysin
enzyme.
[0167] As noted above, the use of the holin lytic enzyme, the
chimeric lytic enzyme, and/or the shuffled lytic enzyme, may be
accompanied by the use of a "natural" lytic enzyme, which has not
been modified by the methods cited in U.S. Pat. No. 6,132,970, or
by similar state of the art methods. The phage associated lytic
enzyme may be prepared as shown in the following example:
EXAMPLE 1
[0168] Harvesting Phage Associated Lytic Enzyme
[0169] Group C streptococcal strain 26RP66 (ATCC #21597) or any
other group C streptococcal strain is grown in Todd Hewitt medium
at 37.degree. C. to an OD of 0.23 at 650 nm in an 18 mm tube. Group
C bacteriophage (C1) (ATCC #21597-B1) at a titer of
5.times.10.sup.6 is added at a ratio of 1 part phage to 4 parts
cells. The mixture is allowed to remain at 37.degree. C. for 18 min
at which time the infected cells are poured over ice cubes to
reduce the temperature of the solution to below 15.degree. C. The
infected cells are then harvested in a refrigerated centrifuge and
suspended in {fraction (1/300)}th of the original volume in 0.1M
phosphate buffer, pH 6.1 containing 5.times.10.sup.-3 M
dithiothreitol and 10 ug of DNAase. The cells will lyse releasing
phage and the lysin enzyme. After centrifugation at 100,000.times.
g for 5 hrs to remove most of the cell debris and phage, the enzyme
solution is aliquoted and tested for its ability to lyse Group A
Streptococci.
[0170] The number of units/ml in a lot of enzyme is determined to
be the reciprocal of the highest dilution of enzyme required to
reduce the OD650 of a suspension of group A streptococci at an OD
of 0.3 to 0.15 in 15 minutes. In a typical preparation of enzyme
4.times.10.sup.5 to 4.times.10.sup.6 units are produced in a single
12 liter batch.
[0171] Use of the enzyme in an immunodiagnostic assay requires a
minimum number of units of lysin enzyme per test depending on the
incubation times required. The enzyme is diluted in a stabilizing
buffer maintaining the appropriate conditions for stability and
maximum enzymatic activity, inhibiting nonspecific reactions, and
in some configurations contains specific antibodies to the Group A
carbohydrate. The preferred embodiment is to use a lyophilized
reagent which can be reconstituted with water. The stabilizing
buffer can comprise a reducing reagent, which can be dithiothreitol
in a concentration from 0.001M to 1.0M, preferably 0.005M. The
stabilizing buffer can comprise an immunoglobulin or immunoglobulin
fragments in a concentration of 0.001 percent to 10 percent,
preferably 0.1 percent. The stabilizing buffer can comprise a
citrate-phosphate buffer in a concentration from 0.001M to 1.0M,
preferably 0.05M. The stabilizing buffer can have a pH value in the
range from 5.0 to 9.0. The stabilizing buffer can comprise a
bactericidal or bacteriostatic reagent as a preservative. Such
preservative can be sodium azide in a concentration from 0.001
percent to 0.1 percent, preferably 0.02 percent.
[0172] The preparation of phage stocks for lysin production is the
same procedure described above for the infection of group C
streptococcus by phage in the preparation of the lysin enzyme.
However, instead of pouring the infected cells over ice, the
incubation at 37.degree. C. is continued for a total of 1 hour to
allow lysis and release of the phage and the enzyme in the total
volume. In order for the phage to be used for subsequent lysin
production the residual enzyme must be inactivated or removed to
prevent lysis from without of the group C cells rather than phage
infection.
[0173] The use of chimeric or shuffled enzymes shows a great
improvement as to the properties of the enzyme, as illustrated by
the following examples:
EXAMPLE 2
[0174] A number of chimeric lytic enzymes have been produced and
studied. Gene E-L, a chimeric lysis 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. Jul. 1, 1998
164(1); 159-67.
[0175] Also, an active chimeric cell wall lytic enzyme (TSL) is
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 pneumoccal cell walls.
EXAMPLE 3
[0176] Isolation of the Pal Lytic Enzyme:
[0177] Recombinant E. coli DH5 (pMSP11) containing the pal lytic
enzyme gene were grown overnight, induced with lactose, pelleted,
resupended in phosphate buffer, broken by sonication. After
centrifugation, the Pal enzyme in the supernatant was purified in a
single step using a DEAE-cellulose column and elution with choline.
Protein content was analyzed with the Bradford method. Using this
method, a single protein band was identified by SDS-PAGE.
EXAMPLE 4
[0178] Killing Assay:
[0179] S. pneumoniae of various serotypes and 8 different viridans
streptococi were grown overnight and for most assays diluted and
re-grown for 6 h to log phase of growth, pelleted and resupended in
0.9% saline to an OD @ 620 nm of 1.0. In some experiments,
stationary phase organisms were used. Killing assays were performed
by adding 100, 1,000 or 10,000 U/mL of Pal to an equal volume of
the bacterial suspension and incubating for 15 minutes at 37 C.
Phosphate buffer served as control in place of enzyme. Bacterial
counts before and after Pal or control phosphate buffer treatment
were assessed by serial 10-fold dilutions at various time points
and plated to determine colony forming units.
[0180] One unit (U) of Pal was defined as the highest dilution at
which Pal decreased the OD of a pneumococcal strain by half in 15
minutes.
EXAMPLE 5
[0181] Production of Chimeric Lytic Enzymes
[0182] A number of chimeric lytic enzymes have been produced and
studied. Gene E-L, a chimeric lysis 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. Jul. 1, 1998
164(1); 159-67.
[0183] Also, an active chimeric cell wall lytic enzyme (TSL) is
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 pneumoccal cell walls.
EXAMPLE 6
[0184] Isolation of the Pal Lytic Enzyme
[0185] Recombinant E.coli DH5 (pMSP11) containing the pal lytic
enzyme gene were grown overnight, induced with lactose, pelleted,
resupended in phosphate buffer, broken by sonication. After
centrifugation, the Pal enzyme in the supernatant was purified in a
single step using a DEAE-cellulose column and elution with choline.
Protein content was analyzed with the Bradford method. Using this
method, a single protein band was identified by SDS-PAGE.
EXAMPLE 7
[0186] Killing Assay
[0187] S. pneumoniae of various serotypes and 8 different viridans
streptococi were grown overnight and for most assays diluted and
re-grown for 6 h to log phase of growth, pelleted and resupended in
0.9% saline to an OD @ 620 nm of 1.0. In some experiments,
stationary phase organisms were used. Killing assays were performed
by adding 100, 1,000 or 10,000 U/mL of Pal to an equal volume of
the bacterial suspension and incubating for 15 minutes at 37 C.
Phosphate buffer served as control in place of enzyme. Bacterial
counts before and after Pal or control phosphate buffer treatment
were assessed by serial 10-fold dilutions at various time points
and plated to determine colony forming units. One unit (U) of Pal
was defined as the highest dilution at which Pal decreased the OD
of a pneumococcal strain by half in 15 minutes. The results, (see
FIG. 2) show that the viability of Pneumococci dropped more than 8
logs in five seconds after adding the Pal enzyme.
EXAMPLE 8
[0188] Susceptability of Oral Streptoccocci to Pal Enzyme
[0189] Various serotypes of oral streptoccoci were tested against
bacteria-associated lytic enzymes, in particular, the Pal enzyme. A
variety of S. pneumoniae type bacteria was also included in the
test. Pal enzyme were used at a concentration of 100 U of the
purified enzyme. As can be seen in FIG. 3 all S. pneumoniae
serotypes are killed (.about.4 logs) within the 30 seconds of
exposure. Of the oral streptococci tested, only S. oralis and S.
mitis show low sensitivity to the Pal enzyme.
EXAMPLE 9
[0190] Susceptability of Stationary Phase bacteria to Lytic
Enzyme
[0191] In order to confirm that activity of lytic enzymes are
independent of the bacterial grwoth, several serotypes of serotypes
of S.pneumoniae at stationary phase of growth were tested against
lytic enzymes. In particular, 3 strains of Pal lytic enzyme were
used against 3 sereotypes of S. pneumoniae. The results show that
that all bacterial strains tested against Pal enzyme were killed in
30 seconds (see FIG. 4). An approximately 2-log drop in viability
of the bacteria occurred with 1,000 U of enzyme, as opposed to
about 3-4 log drop in the viability with 10,000 units.
EXAMPLE 10
[0192] Effect of Pal Lytic Enzyme on Log-Phase and Stationary Phase
Oral Streptococci.
[0193] Streptococci oralis and Streptococci.mitis in log or
stationary phases of growth were treated with different
concentrations of the Pal lytic enzyme. Viability was measured
after 30 seconds. Results, as shown in FIG. 5, indicate that both
bacterial species were equally sensitive to the Pal enzyme in both
log or stationary phases of growth.
[0194] Many modifications and variations of the present invention
are possible in light of the above teachings. It is, therefore, to
be understood within the scope of the appended claims the invention
may be protected otherwise than as specifically described.
[0195] Each publication cited herein is incorporated by reference
in its entirety.
* * * * *