U.S. patent application number 10/105075 was filed with the patent office on 2002-08-29 for use of bacterial phage associated lysing enzymes for treating various illnesses.
Invention is credited to Fischetti, Vincent, Loomis, Lawrence.
Application Number | 20020119133 10/105075 |
Document ID | / |
Family ID | 46277038 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119133 |
Kind Code |
A1 |
Fischetti, Vincent ; et
al. |
August 29, 2002 |
Use of bacterial phage associated lysing enzymes for treating
various illnesses
Abstract
A composition for treatment of bacterial infections of the
vagina is disclosed which comprises a lytic enzyme composition
specific for the infecting bacteria, and a carrier for delivering
said lytic enzyme.. The carrier for delivering at least one lytic
enzyme to the digestive tract is selected from the group consisting
of suppositories,, syrups, or enteric coated pills.
Inventors: |
Fischetti, Vincent; (West
Hempstead, NY) ; Loomis, Lawrence; (Columbia,
MD) |
Correspondence
Address: |
JONATHAN E. GRANT
SUITE 210
2120 L STREET, N.W.
WASHINGTON
DC
20037
US
|
Family ID: |
46277038 |
Appl. No.: |
10/105075 |
Filed: |
March 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10105075 |
Mar 25, 2002 |
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09671881 |
Sep 28, 2000 |
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09671881 |
Sep 28, 2000 |
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09497495 |
Apr 18, 2000 |
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6238661 |
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09497495 |
Apr 18, 2000 |
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09395636 |
Sep 14, 1999 |
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6056954 |
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09395636 |
Sep 14, 1999 |
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08962523 |
Oct 31, 1997 |
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5997862 |
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Current U.S.
Class: |
424/94.1 |
Current CPC
Class: |
Y02A 50/30 20180101;
Y10S 514/937 20130101; Y02A 50/473 20180101; Y10S 514/948 20130101;
C12N 9/503 20130101; A61K 9/00 20130101; Y02A 50/481 20180101; A61K
38/00 20130101 |
Class at
Publication: |
424/94.1 |
International
Class: |
A61K 038/43 |
Claims
What we claim is:
1.) A composition for treating bacterial infections of the vagina,
comprising: an effective amount of at least one lytic enzyme
produced by said bacteria being infected with a bacteriophage
specific for said bacteria; and a carrier for delivering said at
least one lytic enzyme to a vagina.
2.) The composition according to claim 1, wherein said carrier is a
tampon.
3.) The composition according to claim 1, wherein said carrier is a
douche.
4.) The composition according to claim 1, wherein said carrier is a
pad.
5.) The composition according to claim 1, wherein said carrier is a
suppository.
6.) The composition according to claim 1, wherein said at least one
lytic enzyme is specific for Group B Streptococcus.
7.) The composition according to claim 1, wherein said composition
is used in prophylactic treatment of said vaginal infections.
8.) The composition according to claim 1, wherein said composition
is used in therapeutic treatment of said vaginal infections.
Description
[0001] The following application is a continuation-in-part of U.S.
patent application Ser. No. 09/497,495 filed Apr. 18, 2000 which is
a continuation of U.S. patent application Ser. No. 09/395,636 filed
Sep. 14, 2000, now U.S. Pat. No. 6,056,954 which is a
continuation-in-part of U.S. patent application Ser. No.
08/962,523, filed Oct. 31,1997, now U.S. Pat. No. 5,997,862.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention discloses a method and composition for
the treatment of bacterial infections by the use of a lysing enzyme
blended with an appropriate carrier suitable for the treatment of
the infection.
[0004] 2. Description of the Prior Art
[0005] In the past, antibiotics have been used to treat various
infections. The work of Selman Waksman in the introduction and
production of Streptomycetes, Dr. Fleming's discovery of
penicillin, are well known as well as the work of numerous others
in the field of antibiotics. 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.
[0006] 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.
[0007] 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, non-specific antibiotics, some of which have
detrimental effects on the patient, are now being used more
frequently. Also, antibiotics do not easily penetrate mucus
linings. Additionally, the number of people allergic to antibiotics
appears to be increasing.
[0008] Consequently, other efforts have been sought to first
identify and then kill bacteria..
[0009] Attempts have been made to treat bacterial diseases with by
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.
[0010] 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.
[0011] It is to be noted that the direct introduction of
bacteriophages into an animal to prevent or fight diseases has
certain drawbacks. Specifically, the bacteria must be in the right
growth phase for the phage to attach. Both the bacteria and the and
the phage have to be in the correct and synchronized growth cycles.
Additionally, there must be the right number of phages to attach to
the bacteria; if there are too many or too few phages, there will
either be no attachment or no production of the lysing enzyme. The
phage must 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
bacteriophage to treat bacterial infections is the possibility of
immunological reactions, rendering the phage non-functional.
[0012] Consequently, others have explored the use of other safer
and more effective means to treat and prevent bacterial
infections.
[0013] 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 lytic enzyme of
this patent is used in U.S. Pat. No.5,997,862(Fischetti, et. al.),
U.S. Pat. No. 5,985,271, (Fischetti et al.) and U.S. Pat. No.
6,017,528(Fischetti et al.) which disclose the use of an oral
delivery mode, such as a candy, chewing gum, lozenge, troche,
tablet, a powder, an aerosol, a liquid or a liquid spray,
containing a lysin enzyme produced by group C streptococcal
bacteria infected with a C1 bacteriophage for the prophylactic and
therapeutic treatment of Streptococcal A throat infections,
commonly known as strep throat.
[0014] U.S. Pat. No. 6,056,955(Fischetti et al.) discloses the
topical treatment of streptococcal infections.
SUMMARY OF THE INVENTION
[0015] The method for obtaining and purifying the lytic enzyme
produced by a bacteria infected with the bacteriophage is known in
the art. Some recent evidence suggests that the phage enzyme that
lyses the streptococcus organism may actually be a bacterial enzyme
that is used to construct the cell wall and the phage. While
replicating in the bacterium, a phage gene product may cause the
upregulation or derepression of bacterial enzyme for the purpose of
releasing the bacteriophage. These bacterial enzymes may be tightly
regulated by the bacterial cell and are used by the bacteria for
the construction and assembly of the cell wall.
[0016] The use of these lytic enzymes for the prophylactic and
therapeutic treatment of bacterial diseases, however, has not been
explored, except by the inventors of the present invention.
Consequently, the present invention discloses the extraction and
use of a variety of bacterial phage associated lytic enzymes for
the treatment of a wide variety of illnesses caused by bacterial
infections.
[0017] The use of phage associated lytic enzymes produced by the
infection of a bacteria with a bacteria specific phage has numerous
advantages for the treatment of diseases. As the phage are targeted
for specific bacteria, the lytic enzymes do not interfere with
normal flora. Also, lytic phages primarily attack cell wall
structures which are not affected by plasmid variation. The actions
of the lytic enzymes are fast and do not depend on bacterial
growth.
[0018] Lytic enzymes can be directed to the mucosal lining, where,
in residence, they will be able to kill colonizing bacteria.
[0019] It is an object of the invention to use phage associated
enzymes to prophylactically and therapeutically treat bacterial
diseases.
[0020] The invention (which incorporates U.S. Pat. No. 5,604,109 in
its entirety by reference) uses an enzyme produced by the bacterial
organism after being infected with a particular bacteriophage as
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, in most if not all cases, the semipurified enzyme
is lacking in mammalian cell receptors and therefore is
non-destructive to mammalian proteins and tissues when present
during the digestion of the bacterial cell wall. The same general
technique used to produce and purify the lysin enzyme 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.
[0021] 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,
gram negative bacterial infections caused by Listeria, Salmonella,
E. coli, and Campylobacter, are treated by the use of lytic
enzymes. These and other bacteria, which can infect the digestive
system, can be treated by incorporating the lytic enzymes in
suppository enemas, in syrups, or in other carriers to get directly
to the site of the infection(s).
[0022] In another embodiment of the invention, 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.
[0023] Vaginal infections caused by Group B Streptococcus can cause
premature birth and subsequent complications resulting in neonatal
sepsis. Lysin 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.
[0024] 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.
[0025] In yet another embodiment of the invention the phage
associated lytic enzyme is 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.
[0026] In another embodiment of the invention the lytic enzyme is
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.
[0027] 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 DRAWING
[0028] 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..
DETAILED DESCRIPTION OF THE INVENTION
[0029] 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.
The lytic enzyme is preferably in an environment having a pH which
allows for activity of said lytic enzyme.
[0030] The lytic enzyme 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.
[0031] 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. It is 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.
[0032] 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.
[0033] 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 Ec
67) produces a lytic enzyme capable of lysing the bacteria. The
lytic enzyme for Streptococcus pneumoniae, previously identified as
an N-acetyl-muramoyl-L-alanine amidase, is produced by the
infecting Streptococcus pneumoniae with the Pa1 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 reach 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 mucosa
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.
[0034] 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.
[0035] 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.
[0036] Means of application include, but are not limited to direct,
indirect, carrier 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 lysin enzyme may be
administered include but are not limited to lozenges, troches,
candies, injectants, chewing gums, tablets, powders, sprays,
liquids, ointments, and aerosols.
[0037] The lozenge, tablet, or gum into which the lytic enzyme is
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.
[0038] 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.
[0039] 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.
[0040] Any of the carriers for the lytic enzyme 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 enzyme is 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.
[0041] 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.
[0042] The effective dosage rates or amounts of the lytic enzyme 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 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.
[0043] While this treatment may be used in any mammalian species,
the preferred use of this product is for a human.
[0044] 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
lysin phage enzyme 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 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.
[0045] 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.
[0046] For the prophylactic and therapeutic treatment of
tuberculosis, the phage associated lytic enzyme 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.
[0047] The lozenge, tablet, or gum into which the lytic enzyme is
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.
[0048] 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. However, to tackle bacterial
infections in the lung, the use of an inhaler carrier the lytic
enzyme in a carrier is preferred.
[0049] 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 being caused by gram negative bacteria
selected from the group consisting of Listeria, Salmonella, E.
coli, and Campylobacter. However, this method and composition will
effectively treat other bacteria, when the appropriate lytic enzyme
is used.
[0050] 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. To that end, 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.
[0051] 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 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.
[0052] The lytic enzyme is 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 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.
[0053] 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.
[0054] 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 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.
[0055] The carriers of the compositions of the present invention
may comprise semi-solid 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.
[0056] 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%.
[0057] "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.
[0058] 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.
[0059] 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.
[0060] 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%.
[0061] 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.
[0062] 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.
[0063] 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
antifungals 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%.
[0064] 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 ofminoxidil 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.
[0065] 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.
[0066] 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.
[0067] 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."
[0068] 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.
[0069] 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.
[0070] 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 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.
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,
nDecyl-.beta.-D-glucopyranoside,
n-Dodecyl-.beta.-D-glucopyranoside, and biologically occurring
surfactants, e.g., fatty acids, glycerides, monoglycerides,
deoxycholate and esters of deoxycholate.
[0071] 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.
[0072] 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
[0073] In preferred embodiments of the invention, the 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.
[0074] 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
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 enzyme 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
enzyme in the vagina would best provide a prophylactic effect,
although therapeutic use would also be advisable.
[0075] To produce a pad or tampon containing the enzyme, the lytic
enzyme 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.
[0076] 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.
[0077] 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 citratephosphate buffer. As with all
compositions described in this patent, the composition may, further
include a bactericidal or bacteriostatic agent as a
preservative.
[0078] The lytic enzyme is 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.
[0079] 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.
[0080] 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.
[0081] It is also to be remembered that 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.
[0082] Lytic enzymes can also be used to fight dental caries.
Specifically, 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.
[0083] 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.
[0084] The following example illustrates the isolation of the lytic
enzyme.
[0085] Phage Associated Enzyme
[0086] The group C phage lysin enzyme is prepared as follows:
[0087] 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-BI) 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 1/300 th of the original volume in 0.1 M 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.
[0088] 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.
[0089] The enzyme may be diluted in a stabilizing buffer containing
the appropriate conditions for stability, maximum enzymatic
activity, inhibitors of 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 0.001 M to 1.0 M, preferably 0.005 M. 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 0.001 M to 1.0 M,
preferably 0.05 M. 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.
[0090] The preparation of phage stocks for lysin production is the
same procedure described above for the infection of phage and group
C streptococcus 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 also 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.
[0091] 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.
[0092] This general technique can be used to produce other lytic
enzymes for the treatment of various bacterial infections.
[0093] 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.
* * * * *