U.S. patent application number 12/271654 was filed with the patent office on 2009-03-26 for antimicrobial polymer conjugates.
This patent application is currently assigned to BIOSYNEXUS, INCORPORATED. Invention is credited to Joseph J. Drabick, Andrew Lees, James J. Mond, Anjali G. Shah, Scott M. Walsh.
Application Number | 20090081180 12/271654 |
Document ID | / |
Family ID | 28675445 |
Filed Date | 2009-03-26 |
United States Patent
Application |
20090081180 |
Kind Code |
A1 |
Walsh; Scott M. ; et
al. |
March 26, 2009 |
ANTIMICROBIAL POLYMER CONJUGATES
Abstract
Water-soluble polymer conjugates of antimicrobial agents
retaining at least a portion of the antimicrobial activity of the
agent, pharmaceutical compositions containing the polymer
conjugates, and methods for treating microbial infections with the
pharmaceutical compositions.
Inventors: |
Walsh; Scott M.;
(Germantown, MD) ; Shah; Anjali G.; (North
Potomac, MD) ; Mond; James J.; (Silver Spring,
MD) ; Lees; Andrew; (Silver Spring, MD) ;
Drabick; Joseph J.; (Silver Spring, MD) |
Correspondence
Address: |
Casimir Jones, S.C.
440 Science Drive, Suite 203
Madison
WI
53711
US
|
Assignee: |
BIOSYNEXUS, INCORPORATED
Gaithersburg
MD
|
Family ID: |
28675445 |
Appl. No.: |
12/271654 |
Filed: |
November 14, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10403223 |
Mar 26, 2003 |
7452533 |
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12271654 |
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60368112 |
Mar 26, 2002 |
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Current U.S.
Class: |
424/94.3 ;
435/188 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 38/00 20130101; A61K 47/60 20170801; A61P 31/04 20180101 |
Class at
Publication: |
424/94.3 ;
435/188 |
International
Class: |
A61K 38/48 20060101
A61K038/48; C12N 9/52 20060101 C12N009/52 |
Claims
1. A water-soluble polymer conjugated to lysostaphin such that at
least a portion of the antimicrobial activity of said lysostaphin
is retained.
2. The polymer conjugated lysostaphin of claim 1, wherein said
lysostaphin retains enzyme activity capable of cleaving the
cross-linked polyglycine bridges in the cell wall peptidoglycan of
Staphylococci.
3. The polymer conjugated lysostaphin of claim 2, wherein said
lysostaphin is wild type lysostaphin or a lysostaphin analogue.
4. The polymer conjugated agent of claim 3, wherein said wild type
lysostaphin or lysostaphin analogue is recombinantly expressed.
5. The polymer conjugated lysostaphin of claim 1, wherein said
water-soluble polymer is selected from the group consisting of
poly(alkylene oxides), polyoxyethylated polyols and poly(vinyl
alcohols).
6. The polymer conjugated lysostaphin of claim 5, wherein said
poly(alkylene oxide) is a polyoxamer or polyoxamine.
7. The polymer conjugated lysostaphin of claim 5, wherein said
poly(alkylene oxide) is polyethylene glycol (PEG).
8. The polymer conjugated lysostaphin of claim 7, wherein said PEG
is straight-chained.
9. The polymer conjugated lysostaphin of claim 7, wherein said PEG
is branched.
10. The polymer conjugated lysostaphin of claim 1 comprising from
one to about four polymer molecules per molecule of
lysostaphin.
11. An antimicrobial pharmaceutical composition for treating an
infection comprising a water-soluble polymer conjugated to
lysostaphin such that at least a portion of the antimicrobial
activity of said lysostaphin is retained and a pharmaceutically
acceptable carrier.
12. The pharmaceutical composition of claim 11, further comprising
a non-polymer conjugated antibacterial enzyme.
13. The pharmaceutical composition of claim 12, wherein said
non-polymer conjugated antibacterial enzyme is selected from the
group consisting of lysostaphin, lysozyme, mutanolysin, cellozyl
muramidase, and combinations thereof.
14. The pharmaceutical composition of claim 11, further comprising
an antibiotic.
15. The pharmaceutical composition of claim 14, wherein said
antibiotic interferes with or inhibits bacterial cell wall
synthesis.
16. The pharmaceutical composition of claim 14, wherein said
antibiotic is selected from the group consisting of
.alpha.-lactams, cephalosporins, aminoglycosides, sulfonamides,
antifolates, macrolides, quinolones, glycopeptides, polypeptides
and combinations thereof.
17. A method for the prophylactic or therapeutic treatment of a
microbial infection in a mammal comprising administering to said
mammal an effective amount of a pharmaceutical composition
according to claim 11 for treating said infection.
18. The method of claim 17, wherein said microbial infection is a
bacterial infection caused by a staphylococcus species with
sufficient polyglycine bridge cross-linking in the cell wall
peptidoglycan for cells of the species to be lysed by contact with
a water-soluble polymer conjugate of lysostaphin.
19. The method of claim 18, wherein said staphylococcal infection
is caused by Staphylococcus aureus.
20. The method of claim 18, wherein said staphylococcal infection
is caused by Staphylococcus epidermidis.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of pending U.S. patent
application Ser. No. 10/403,223 filed Mar. 26, 2003, which claims
priority benefit under 35 U.S.C. .sctn. 119(e) to U.S. Provisional
Application Ser. No. 60/368,112 filed on Mar. 26, 2002, each of
which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the conjugation of
antimicrobial agents to water-soluble polymers to improve their
clinical properties in terms of their pharmacokinetics,
pharmacodynamics, and reduced immunogenicity. More specifically,
the present invention relates to the conjugation of antimicrobial
agents such as lysostaphin to poly(alkylene oxides), such as
poly(ethylene glycol) (PEG).
BACKGROUND ART
A. Lysostaphin
[0003] Lysostaphin is a potent antimicrobial agent first identified
in Staphylococcus simulans (formerly known as S. staphylolyticus).
Lysostaphin is a bacterial endopeptidase capable of cleaving the
specific cross-linking polyglycine bridges in the cell walls of
staphylococci, and is therefore highly lethal thereto. Expressed in
a single polypeptide chain, lysostaphin has a molecular weight of
approximately 27 kDa.
[0004] The cell wall bridges of Staphylococcus aureus, a coagulase
positive staphylococcus, contain a high proportion of glycine,
therefore lysostaphin is particularly effective in lysing S.
aureus. Lysostaphin has also demonstrated the ability to lyse
Staphylococcus epidermidis.
[0005] S. aureus is a highly virulent human pathogen. It is the
cause of a variety of human diseases, ranging from localized skin
infections to life-threatening bacteremia and infections of vital
organs. If not rapidly controlled, a S. aureus infection can spread
quickly from the initial site of infection to other organs.
Although the foci of infection may not be obvious, organs
particularly susceptible to infection include the heart valves,
kidneys, lungs, bones, meninges and the skin in burn patients.
[0006] Staphylococcal infections, such as those caused by S.
aureus, are a significant cause of morbidity and mortality,
particularly in settings such as hospitals, schools, and
infirmaries.
[0007] Patients particularly at risk include infants, the elderly,
the immunocompromised, the immunosuppressed, and those with chronic
conditions requiring frequent hospital stays. Patients at greatest
risk of acquiring staphylococcal infections, are those undergoing
inpatient or outpatient surgery, in the Intensive Case Unit (ICU),
on continuous hemodialysis, with HIV infection, with AIDS, burn
victims, people with diminished natural immunity from treatments or
disease, chronically ill or debilitated patients, geriatric
populations, infants with immature immune systems, and people with
intravascular devices.
[0008] U.S. Pat. No. 6,028,051 to Climo, et al., discloses a method
for the treatment of staphylococcal disease. Relatively high doses
of lysostaphin of at least 50 and preferably 100 milligrams of
lysostaphin per kilogram of body weight are used for treatment. The
relatively high doses of lysostaphin can be used in single dose
treatments or multiple dose treatments. The lysostaphin analog can
be used singularly or in combination with additional antibiotic
agents. The '051 patent also discloses that the cloning and
sequencing of the lysostaphin gene permits the isolation of variant
forms of lysostaphin that can have properties similar to or
different from those of wild type lysostaphin.
[0009] U.S. Pat. No. 6,315,996 to O'Callaghan, discloses a method
for using lysostaphin as an effective antibiotic for topical
treatment of staphylococcus corneal infections. U.S. Pat. No.
5,760,026 to Blackburn et al., discloses a method for using
lysostaphin to eliminate and cure staphylococcal infections
including the cure of mastitis by intramammary infusion. The method
is directed to use in dairy cows.
[0010] However, small proteins (less than about 70 kDa), such as
lysostaphin, have a relatively short half-life in blood after
intravenous injection. Lysostaphin's rapid clearance from
circulation may reduce its efficacy. At the same time, because it
is derived from a bacterial species and therefore foreign to any
mammalian species, lysostaphin is also a very immunogenic molecule,
which further stimulates its clearance from the blood stream,
especially in subjects that have had previous exposure to
lysostaphin. Thus, lysostaphin's short circulating half-life cannot
be effectively countered by increasing the amount or frequency of
dosage. There exists a need for a means by which the circulating
half-life of lysostaphin may be increased without increasing the
amount or frequency of administration. It would even be more
desirable to increase the circulating half-life of lysostaphin
while at the same time reducing the amount or frequency of
administration.
B. Polymer Conjugation
[0011] The conjunction of biologically active polypeptides with
water-soluble polymers such as PEG is well-known. PEGylation is a
process in which therapeutic polypeptides, such as enzymes and
hormones, are coupled to one or more chains of polyethylene glycol
to provide improved clinical properties in terms of
pharmacokinetics, pharmacodynamics, and immunogenicity.
[0012] PEGylation can alter the characteristics of the polypeptide
without affecting the ability of the parent molecule to function,
thereby producing a physiologically active, reduced or
non-immunogenic, water-soluble polypeptide composition. The polymer
protects the polypeptide from loss of activity by reducing its
clearance and susceptibility to enzymatic degradation, and the
composition can be injected into the mammalian circulatory system
with substantially no immunogenic response. PEGylation of enzymes
and other polypeptides is described in detail in U.S. Pat. No.
4,179,337 to Davis et al., and in Zalipsky, "Functionalized
Poly(ethylene glycol) for Preparation of Biologically Relevant
Conjugates," Bioconjugate Chem., 6, 150-165 (1995), both of which
are incorporated by reference in their entirety herein.
[0013] Davis et al. disclose that polypeptides modified with PEG
have dramatically reduced immunogenicity and antigenicity. PEG
conjugates exhibit a wide range of solubilities and low toxicity,
and have been shown to remain in the bloodstream considerably
longer than the corresponding native compounds, yet are readily
excreted. The conjugates have been shown not to interfere with the
activity of other enzymes in the bloodstream or the conformation of
polypeptides conjugated thereto.
[0014] PEG conjugation is typically accomplished by means of two
commonly used types of linkages. One type of conjugation reacts a
polypeptide amino group with a PEG molecule having an active
carbonate, ester, aldehyde or tresylate group. Another type of
conjugation reacts a polypeptide thiol group with a PEG molecule
having an active vinyl sulfone, maleimide, haloacyl or
thiorthopyridyl group, or other suitable electrophile. See, for
example, Hermanson, Bioconjugate Techniques (Academic Press, San
Diego 1966). One of the two terminal hydroxyls of the PEG is
blocked by conversion to an alkoxy group when intermolecular
cross-linking is not desired. A PEG molecule with one terminal
methoxy group is referred to as mPEG.
[0015] The PEG molecule may be linear or branched, whereby PEG
conjugates can be created by conjugating a single large PEG moiety
to a single conjugation site, a single branched (but smaller) PEG
moiety to a single conjugation site, or several small PEG moieties
to multiple conjugation sites. When multiple conjugation sites are
employed, this can result in the loss of bioactivity. In addition
to PEG homopolymers, the polymer molecule can be copolymerized with
other alkylene oxide moieties, or it can be another poly(alkylene
oxide) homopolymer or copolymer.
[0016] A number of PEG-conjugates of therapeutic proteins have been
developed exhibiting reduced immunogenicity and antigenicity and
longer clearance times, while retaining a substantial portion of
the protein's physiological activity. U.S. Pat. No. 4,261,973
describes the PEG conjugation of immunogenic allergen molecules to
reduce the immunogenicity of the allergen. U.S. Pat. No. 4,301,144
discloses that the conjugation of PEG to hemoglobin increases the
oxygen-carrying ability of the molecule. U.S. Pat. No. 4,732,863
discloses the conjugation of PEG to antibodies to reduce binding to
Fc receptors. EP 154,316 and Katre et al., Proc. Natl. Acad. Sci.,
84, 1487 (1987) disclose PEG conjugated lymphokines such as IL-2.
U.S. Pat. No. 4,847,325 discloses the selective conjugation of PEG
to Colony Stimulating Factor-1 (CSF-1).
[0017] Interferon-2 (INF-2) has been conjugated without a loss of
biological activity to the succinimidyl ester of a single, branched
PEG molecule consisting of two 20 kDa mono-methoxy PEG chains
connected through a lysine molecule via urethane bonds. This PEG
conjugate is targeted for the treatment of hepatitis C, by
affecting host immunity and enhancing immune clearance of the
virus. The administration of the INF-2 can be reduced to once
weekly from three-to-seven times a week, simplifying and improving
patient compliance. In addition, serum levels are maintained with
minimal peak-to-trough variation, toxicity is reduced, and efficacy
is increased.
[0018] FDA approved PEGylated therapeutic polypeptides in clinical
use include PEG conjugates of INF-2, adenosine deaminase and
asparaginase. PEGylated therapeutic polypeptides awaiting FDA
approval include PEG conjugates of IL-2, IL-6 and Tumor Necrosis
Factor. Each of these PEGylated products contains a polypeptide
targeted at host cell activities or cancerous host cells, but not
to microbes. EG conjugation has been disclosed of proteins such as
alpha-1-proteinase inhibitor, uricase, superoxide dismutase,
streptokinase, plasminogen activator, IgG, albumin, INF-,
lipoprotein lipase, horseradish peroxidase, catalase and arginase.
These proteins also do not target microbes. The PEG conjugation was
reported to improve circulating half-life, decrease immunogenicity,
increase solubility and, in general, increase efficacy, thereby
permitting less frequent dosing. In most cases, the proteins
required multiple PEG conjugations per molecule to improve in vivo
performance, and the activity in vitro was significantly decreased
by such modification.
[0019] WO 01/04287 published Jan. 18, 2002, discloses the use of
mutagenic processes to modify polypeptides in general, and
staphylokinase in particular, for improved performance of the PEG
conjugate thereof.
[0020] There is otherwise no disclosure of a PEG conjugation of
antimicrobial agents to optimize pharmacokinetics and
pharmacodynamics. No reference discloses the conjugation of an
antimicrobial agent, or a mutagenic modification thereof, with PEG
so as to retain its biological activity while also increasing its
circulating half-life and efficacy, and decreasing its antibody
binding and toxicity.
SUMMARY OF THE INVENTION
[0021] The foregoing limitations are overcome by the present
invention. The present invention provides for the polymer
conjugation of antimicrobial agents to increase circulating
half-life in vivo while retaining antimicrobial activity. The
antimicrobial agent so modified may thus be used to treat or
prevent infection at much reduced and/or less frequent dosages than
the unmodified agent.
[0022] In addition to increasing circulating half-life while
retaining antimicrobial activity, other advantages obtained by
polymer conjugation include decreased antibody binding and
increased killing, decreased immunogenicity and reduced binding to
circulatory system surfaces, including the surfaces of man-made
implant devices, both of which also increase circulating half-life,
independent of the increase in circulating half-life typically
obtained by the increase in molecular weight contributed by the
polymer conjugate.
[0023] More specifically, the present invention provides
water-soluble polymers conjugated to antimicrobial agents, so that
at least a portion of the antimicrobial activity of the agent is
retained. Antimicrobial agents suitable for use with the present
invention include agents such as chemicals, peptides, proteins and
lipopeptides that, upon contacting a microbe in a host, kill the
microbe by any of a variety of techniques or inhibit microbial
metabolism, without damaging host cells or tissues or eliciting a
harmful host response. Antimicrobial enzymes are among the peptides
and proteins that can be used.
[0024] While microbes are defined as including bacteria and fungi,
staphylolytically active antimicrobial agents are desirable because
of the aforementioned risks posed by staphylococcal infections.
Among the staphylolytically active antimicrobial agents are
proteins and peptides that function as staphylolytically active
enzymes, including proteins capable of cleaving the cross-linked
polyglycine bridges in the cell wall peptidoglycan of
staphylococci, such as lysostaphin and lysostaphin analogues.
[0025] Water-soluble polymers include poly(alkylene oxides),
polyoxyethylated polyols and poly(vinyl alcohols). Poly(alkylene
oxides) include PEGs, poloxamers and poloxamines. The poly(alkylene
oxide) is typically conjugated to a free amino group via an amide
linkage formed from an active ester, such as the
N-hydroxysuccinimide ester, of the poly(alkylene oxide). The
poly(alkylene oxide) can be mPEG, either straight chained or
branched, having a molecular weight between about five and about
100 kDa.
[0026] In another aspect, the invention relates to a method for the
prophylactic or therapeutic treatment of a microbial infection in a
mammal by administering to the mammal an effective amount of a
pharmaceutical preparation containing the antimicrobial conjugate
of the present invention in a pharmaceutically acceptable carrier.
When the microbial infection is caused by a staphylococcus species
with sufficient cell wall polyglycine cross-linking so that cells
of the species are lysed by lysostaphin when contacted therewith,
the lysostaphin or lysostaphin analogue conjugates of the present
invention may be used. This embodiment of the method of the present
invention is particularly effective for treating Staph. aureus
infections. According to yet another aspect of the present
invention, pharmaceutical compositions are provided for use in the
inventive treatment method that contain the antimicrobial
conjugates of the present invention in a pharmaceutically
acceptable carrier.
[0027] The foregoing and other objects, features and advantages of
the present invention are more readily apparent from the detailed
description set forth below, taken in conjunction with the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 depicts the lysis activity of lysostaphin conjugates
according to the present invention in samples of heat-killed S.
aureus type 5.
[0029] FIG. 2 depicts the killing activity of lysostaphin
conjugates according to the present invention in a high innoculum
of live S. aureus type 5.
[0030] FIG. 3 is a lysostaphin-capture immunoassay depicting the
ability of PEG to shield lysostaphin from antibodies.
[0031] FIG. 4 depicts the serum concentrations and half-life of one
lysostaphin conjugate at two different concentrations according to
the present invention, in comparison to unconjugated
lysostaphin.
[0032] FIG. 5 depicts the S. aureus type 5 killing activity in
saline of lysostaphin conjugates according to the present
invention.
[0033] FIG. 6 depicts the S. aureus type 5 killing activity in
blood of lysostaphin conjugates according to the present
invention.
[0034] FIG. 7 is an ELISA depicting the reactivity of
anti-lysostaphin antibodies to lysostaphin conjugates according to
the present invention.
[0035] FIG. 8 depicts the lysis activity of lysostaphin conjugates
according to another aspect of the present invention in samples of
heat-killed S. aureus type 5.
[0036] FIG. 9 depicts the lysis activity of lysostaphin conjugates
according to yet another aspect of the present invention in samples
of heat-killed S. aureus type 5.
[0037] FIG. 10 depicts the S. aureus type 5 killing activity in
saline of lysostaphin conjugates according to another aspect of the
present invention.
[0038] FIG. 11 is an ELISA depicting the reactivity of
anti-lysostaphin antibodies to lysostaphin conjugates according to
another aspect of the present invention.
[0039] FIG. 12 depicts serum concentrations and half-life of
lysostaphin conjugates according to another aspect of the present
invention.
[0040] FIG. 13 compares the S. aureus type 5 killing activity in
saline of two different molecular weight lysostaphin conjugates
according to the present invention; and
[0041] FIG. 14 depicts the S. aureus type 5 killing activity in
saline of a lysostaphin conjugate according to yet another aspect
of the present invention.
DETAILED DESCRIPTION
[0042] For purposes of the present invention, the term
"antimicrobial agent" is defined as including any substances
(chemical, protein, peptide or lipopeptide, including enzymes)
that, upon contact such as in a host, kill microbes or inhibit
microbe metabolism without damaging the surrounding environment,
such as host cells or tissues, or upon contact with a host, elicit
a harmful host response. This includes substances that would
without polymer conjugation otherwise damage host cells or tissues
or elicit a harmful response. The term "microbe" is defined as
protists, which include bacteria and fungi.
[0043] The term "lysostaphin" is defined as including any enzyme,
including lysostaphin (wild type), any lysostaphin mutant or
variant, any recombinant, or related enzyme, or any synthetic
version or fragment of lysostaphin that retains the proteolytic
ability, in vivo and in vitro, to cleave the cross-linked
polyglycine bridges in the cell wall peptidoglycan of
staphylococci. Variants may be generated by post-translational
processing of the protein (either by enzymes present in a producer
strain or by means of enzymes or reagents introduced at any stage
of the process) or by mutation of the structural gene. Mutations
may include site deletion, insertion, domain removal and
replacement mutations.
[0044] The term "lysostaphin analogue" is defined as including any
form of lysostaphin that is not wild-type. The lysostaphin and
lysostaphin analogues contemplated in the present invention may be
recombinantly expressed from a cell culture or higher recombinant
species such as a mouse or otherwise, expressed in mammalian cell
hosts, insects, bacteria, yeast, reptiles, fungi, etc., or
synthetically constructed. This would include the activity
retaining synthetic construction including synthetic peptides and
polypeptides or recombinant expression of portions of the
lysostaphin polypeptide responsible for its activity against
staphylococci alone, or as part of a larger protein or polypeptide,
including chimeric proteins, containing the active site(s) of one
or more other antimicrobial proteins or peptides that are active
against staphylococci, or against one or more other microbe(s) or
bacteria species to provide a broader spectrum of activity.
[0045] Lysostaphin is naturally produced by bacteria as a
pro-enzyme that is cleaved to produce the full length form of
lysostaphin. Recombinant or synthetically produced lysostaphin
preparations can be used that contain only the fully active form of
lysostaphin. The recombinant expression of homogenous lysostaphin,
and homogenous fully active lysostaphin-containing compositions
prepared from the expressed protein are disclosed in a U.S. patent
app. entitled, "Truncated Lysostaphin Molecule with Enhanced
Staphylolytic Activity" filed by Jeffery Richard Stinson, Lioubov
Grinberg, John Kokai-Kun, Andrew Lees and James Jacob Mond on Dec.
21, 2002, the disclosure of which is incorporated herein by
reference in its entirety. The application claims priority from
U.S. Provisional App. No. 60/341,804 filed Dec. 21, 2001.
[0046] Antimicrobial agents such as the lysostaphin and lysostaphin
analogue proteins described above are conjugated to water-soluble
polymers via free amino groups, either at lysine and arginine
residues or a free amino group, if any, at the N-terminus. Other
suitable antimicrobial agents include nisin, amphotericin-.alpha.,
and the like. From a minimum of one up to about twelve
water-soluble polymer molecules can be attached to each molecule of
an antimicrobial agent. Because one object of the modification is
to increase in vivo half life over the unconjugated antimicrobial
agents with reduced immunogenicity, the number of conjugated
polymers and the weight-average molecular weight of these molecules
should be selected to provide a polymer conjugate of an
antimicrobial agent with an apparent weight-average molecular
weight from about 5 to 40 kDa, up to about 200 kDa.
[0047] The poly(alkylene oxides), when used, typically have
weight-average molecular weights between about one and about 100
kDa, more typically between about two, three or four and about 50
kDa, and also between about five or ten and about 40 kDa, depending
upon the number of conjugates per lysostaphin molecule. When
conjugated to lysostaphin, from one to about ten poly(alkylene
oxide) molecules per lysostaphin molecule can be used, with from
one to about three or four being typically used, and one or two
being more typical. Lysostaphin compositions with mixed degrees of
conjugation may also be used, or the lysostaphin conjugate may be
fractionated so that a lysostaphin conjugate is obtained that
essentially consists of a fraction of lysostaphin conjugated to
essentially the same number of polymers. That is, essentially all
lysostaphin in a fractionated sample is conjugated to one, two,
three or more polymers, but not mixtures thereof.
[0048] When poly(alkylene oxides) are used, they may be straight
chained or branched. Branched poly(alkylene oxides), such as
branched PEG, because of their larger spatial volume, are believed
to be less likely to penetrate protein crevasses, which are often
the binding motifs and active sites of enzymes. Typical
poly(alkylene oxides) consist of C.sub.2-C.sub.4 alkylene oxide
groups, separately as homopolymers or in combination. This includes
PEGs, poloxamers and poloxamines. The poly(alkylene oxides) can be
substituted at one end with an alkyl group, or it may be
unsubstituted. The alkyl group, when present, can be a
C.sub.1-C.sub.4 alkyl group, and is typically a methyl group.
[0049] Suitable covalent modification reactions are well known and
essentially conventional. Generally the process involves preparing
an activated polymer and thereafter reacting the antimicrobial
agent with the activated polymer. The reaction using
N-hydroxysuccinimide activated mPEG (mPEG-NHS) described by Davis
et al. can be used. MPEG-NHS is commercially available from
Shearwater Corp. of Huntsville, Ala., now known as Nektar
Therapeutics, AL.
[0050] Typically, the reaction is carried out in a buffer of pH
about 7-8, frequently at about 10 mM Hepes pH 7.5, 100 mM NaCl. The
reaction is carried out generally at 0.degree. to about 25.degree.
C. for from about 20 minutes to about 12 hours, for example, for
25-35 minutes at about 20.degree. or three hours at 4.degree. C.
Following the conjugation, the desired product is recovered and
purified by column chromatography and the like.
[0051] The antimicrobial agent thus modified is then formulated as
either an aqueous solution, semi-solid formulation, or dry
preparation (e.g., lyophilized, crystalline or amorphous, with or
without additional solutes for osmotic balance) for reconstitution.
Formulations may be in, or reconstituted in, a non-toxic, stable,
pharmaceutically acceptable, aqueous carrier medium, at a pH of
about 3 to 8, typically 5 to 8, for administration by conventional
protocols and regimes or in a semi-solid formulation such as a
cream. Delivery can be via ophthalmic administration, intravenous
(iv), intramuscular, subcutaneous or intraperitoneal routes or
intrathecally or by inhalation or used to coat medical devices,
catheters and implantable devices, or by direct installation into
an infected site so as to permit blood and tissue levels in excess
of the minimum inhibitory concentration (MIC) of the active agent
to be attained and thus to effect a reduction in microbial titers
in order to cure or to alleviate an infection. Furthermore, the
antimicrobial agent can be formulated as a semi-solid formulation,
such as a cream, that can be used in a topical or intranasal
formulation.
[0052] Furthermore, the antimicrobial conjugate can be
coadministered, simultaneously or alternating, with other
antimicrobial agents so as to more effectively treat an infectious
disease. Formulations may be in, or be reconstituted in, semi-solid
formulations for topical, ophthalmic, or intranasal application,
liquids suitable for ophthalmic administration, bolus iv or
peripheral injection or by addition to a larger volume iv drip
solution, or may be in, or reconstituted in, a larger volume to be
administered by slow iv infusion. For example, the lysostaphin
conjugate can be administered in conjunction with antibiotics that
interfere with or inhibit cell wall synthesis, such as penicillins,
such as nafcillin, and other .alpha.-lactam antibiotics,
cephalosporins such as cephalothin, aminoglycosides, sulfonamides,
antifolates, macrolides, quinolones, glycopeptides such as
vancomycin and polypeptides. Or, the lysostaphin conjugate can be
administered in conjunction with antibiotics that inhibit protein
synthesis, for example aminoglycosides such as streptomycin,
tetracyclines, and streptogramins. The lysostaphin conjugate may
also be administered with monoclonal antibodies; other
non-conjugated antibacterial enzymes such as lysostaphin, lysozyme,
mutanolysin, and cellozyl muramidase; peptides such as defensins;
and lantibiotics such as nisin; or any other lanthione-containing
molecules, such as subtilin.
[0053] Agents to be coadministered with the lysostaphin conjugate
may be formulated together with the lysostaphin conjugate as a
fixed combination or may be used extemporaneously in whatever
formulations are available and practical and by whatever routes of
administration are known to provide adequate levels of these agents
at the sites of infection.
[0054] Conjugates according to the present invention possess at
least a portion of the anti-microbial activity of the corresponding
non-conjugated antimicrobial agent. It is not essential that
complete (or full) activity be retained because increased dosages
at less frequent intervals can be given due to the decreased
immunogenicity and increased circulating half-life produced by
PEGylation. Conjugates that retain at least 10% of the activity of
the non-conjugated antimicrobial agent are preferred, with
conjugates that retain at least 15%, 20%, 25%, 30%, 40%, etc., of
the non-conjugated antimicrobial agent activity are more
progressively preferred.
[0055] Suitable dosages and regimes of the lysostaphin conjugate
may vary with the severity of the infection and the sensitivity of
the infecting organism and, in the case of combination therapy, may
depend on the particular anti-staphylococcal agent(s) used in
combination. Dosages may range from about 0.05 to about 100
mg/kg/day, preferably from about 1 to about 40 mg/kg/day, given as
single or divided doses, or given by continuous infusion.
[0056] The present invention is further illustrated by the
following examples that teach those of ordinary skill in the art
how to practice the invention. The following examples are merely
illustrative of the invention and disclose various beneficial
properties of certain embodiments of the invention. The following
examples should not be construed as limiting the invention as
claimed.
EXAMPLES
Materials
[0057] As an example of an antimicrobial agent, lysostaphin was
employed for these studies, which can be repeated with essentially
any antimicrobial agent, as the term is defined by the present
specification. Lysostaphin (Ambicin L) was obtained from AMBI, Inc.
(now Nutrition 21). The mPEG2-NHS esters, 10 and 40 kDa were
purchased from Shearwater Corporation (Huntsville, Ala.) (now
Nektar Therapeutics, AL). Sodium Borate, DMSO, bovine serum
albumin, and extravidin-HRP were purchased from Sigma Chemical Co.
(St. Louis, Mo.). Glycine was purchased from EM Science (Gibbstown,
N.J.). The NuPage Electrophoresis System and Colloidal Blue stain
were purchased from Invitrogen (Carlsbad, Calif.). Sephacryl
S-100HR and HiTrap SP FF were purchased from Amersham-Pharmacia
(Piscataway, N.J.). Tryptic Soy Broth, TSB, and Cation-Adjusted
Mueller Hinton Broth, CAMHB, were purchased from Becton Dickinson
(Sparks, Md.). TMB Microwell and 450 STOP Reagent were purchased
from BioFX (Owings Mills, Md.).
Example 1
Study of PEGylated Lysostaphin
Lysostaphin PEGylation
[0058] Lysostaphin at 0.27, 1, or 5 mg/mL was dissolved in either
0.2M borate buffer (pH 8.5) or DMSO. The mPEG2-NHS esters were
prepared in DMSO and added to the lysostaphin solution in molar
excess at ratios of 40, 20, 10, 5 or 2.5:1. PEGylation was
performed with three different buffer conditions, all at room
temperature for 1, 2, or 3 hours: borate buffer (with <10% DMSO
contributed by adding PEG), 50% borate/50% DMSO, and 100% DMSO. All
reactions were quenched by added glycine to 25 mM and
vortexing.
[0059] PEG conjugation to lysostaphin was evaluated by SDS-PAGE
with the NuPage Electrophoresis System. Non-reduced samples (300
ng) were run on a Novex 4-12% Bis-Tris gel at 115V and stained with
colloidal blue. PEGylated lysostaphin was separated from unreached
lysostaphin by running the reaction mixture over a SEPHACRYL
S-100HR column. Purified PEG-lysostaphin was concentrated and saved
for activity assays.
[0060] Alternatively, unconjugated lysostaphin was removed from the
sample by ion-exchange chromatography. Lysostaphin, but not
PEG-lysostaphin, was bound onto a HiTrap SP FF column in 50 mM
sodium phosphate buffer, pH 7.0. The column was washed with the
same buffer until the OD280 of the eluate was reduced to background
levels. Bound lysostaphin was then removed, and the column
regenerated by washing with 50 mM sodium phosphate plus 1 M NaCl,
pH 7.0. This process was repeated several times until the
PEG-lysostaphin fraction (unbound) was at least 99% pure of
unconjugated lysostaphin.
In Vitro Activity of PEG-Lysostaphin
[0061] Lysostaphin's ability to lyse staphylococcus aureus type-5
(SA5) was determined by measuring the drop in absorbance at 650 nm
of a solution containing heat-killed SA5 (HKSA5). HKSA5 was
prepared by incubating live bacteria at 62.degree. C. for 2 hours
and then diluted such that the initial absorbance was about 1.
Lysostaphin was then added at a concentration of 32 .mu.g/mL and
absorbance readings were taken every 60 seconds for 20 minutes.
Clearance of live SA5 cultures were measured by adding lysostaphin
from 0 to 10 .mu.g/mL to an SA5 suspension in PBS (% T=40). The
samples were incubated at 37.degree. C. for 1 hour and then spread
onto blood agar plates. After overnight culture at 37.degree. C.,
colonies were counted and compared to untreated samples.
[0062] The minimum inhibitory concentration (MIC) of the conjugated
lysostaphin was determined against SA5. After an overnight culture
of SA5 in TSB, the bacteria was diluted to % T=80. 100 .mu.L of
growth media (CAMHB+1% BSA+0 to 32 .mu.g lysostaphin) was added to
each well of a 96-well culture plate. 5 .mu.L of SA5 was added to
each well and the plate incubated at 37.degree. .mu.C. and 200 rpm
for 24 hours. The absorbance at 650 nm was read and the MIC defined
as the last well where there was no SA5 growth.
Anti-Lysostaphin Binding Activity
[0063] A lysostaphin capture ELISA was performed to determine if
PEGylated lysostaphin shields the protein from antibody binding.
96-well microtiter plates were coated with a polyclonal rabbit
anti-lysostaphin antibody overnight. The wells were blocked with 1%
BSA followed by incubation with the lysostaphin samples in PBS/0.5%
Tween 20 plus 0.1% BSA. Lysostaphin binding was detected with
biotin-labeled, polyclonal rabbit anti-lysostaphin followed by
extravidin-HRP incubation and TMB calorimetric detection. The
plates were measured at an absorbance of 450 nm in a SpectraMAX
Plus plate reader (Molecular Devices; Sunnyvale, Calif.).
Serum Pharmacokinetics of PEG-Lysostaphin CF1 mice were injected in
the tail vein with S-100HR purified PEG-lysostaphin at a dose of
0.8 or 0.2 mg (4 or 1 mg/mL in 0.2 mL PBS). Control mice were
injected with 0.8 mg of unconjugated lysostaphin. Blood was
collected by orbital eye bleeding at 1, 4, 7, and 24 hours
post-administration. The blood was incubated at 37.degree. C. for
30 minutes followed by 40 for 30 minutes. Serum was then separated
by centrifugation at 1000 g for 10 minutes. The serum concentration
of lysostaphin was determined by ELISA as described above.
Results
[0064] Lysostaphin has a high net charge of +10.53 at pH 7 due to a
large number of lysine (16) and arginine (6) residues. The primary
amine groups of the side chains of these lysines are ideal targets
to covalently link PEG that has been activated with
N-hydroxysuccinimide. Branched PEG's were chosen because their
larger spatial volume makes them less likely to penetrate protein
crevasses, which are often the binding motifs and active sites of
enzymes.
[0065] The reaction conditions can be manipulated to create a
PEGylated lysostaphin molecule that has an optimal balance between
enzyme activity and enhanced properties such as reduced
immunogenicity, decreased antibody binding and toxicity and
increased serum half-life and efficacy. Unique reaction groups may
be added to lysostaphin in order to conjugate PEG in a number
controlled and site specific matter. Creating sulfhydryl groups
would be one way to achieve this goal because lysostaphin does not
contain any cysteine residues. Another way to achieve this goal
would be to introduce thiol groups into the protein by introducing
the thiol-containing amino acid cysteine into the amino acid
sequence of the protein through genetic engineering.
Enzyme Killing Activity on SA5
[0066] The in vitro killing activity of five PEGylated lysostaphin
samples was tested by measuring lysis of heat-killed SA5 (FIG. 1)
and killing of live SA5 (FIG. 2). In FIG. 1, lysostaphin conjugated
to 10 kDa PEG is represented in lines 3133A-B pH and 3133A-DMSO.
The pH designation indicates PEGylation reactions that took place
in aqueous solution. The DMSO designation designates PEGylation
reactions that were performed in 100% DMSO. All samples were
positive for enzyme activity. A reduction in activity with
increasing degrees of PEGylation suggests that the 10 kDa PEG is
small enough to gain access to lysostaphin's active site or its
peptido-glycan binding domain, thus reducing its enzyme activity.
In contrast, no reduction in enzyme activity was observed with the
40 kDa PEG samples despite the fact that no size separation was
performed on these samples. This implies that the highly PEGylated
forms retain similar activity to lightly conjugated forms and
indicates that the 40 kDa PEG cannot easily access sites important
for enzyme function.
[0067] The ability to kill live SA5 in vitro was tested with
decreasing concentrations of PEGylated lysostaphin (FIG. 2). As was
observed for the heat-killed assay, higher degrees of PEG
conjugation reduced lysostaphin activity and the 40 KDa conjugate
retained more activity the 10 kDa form. However, new properties of
PEG-lysostaphin emerged that were not apparent in the heat-killed
assay. The killing curve for unconjugated lysostaphin shows the
titration of response with decreasing enzyme concentrations, but
all of the PEGylated enzymes appear to have a flat response over
the first four dilutions before finally titrating upward. In
particular, the 40 kDa PEG at a 2:1 ratio maintains greater killing
over the lowest three concentrations, compared to unconjugated
lysostaphin, but is comparatively less active at the three highest
concentrations. This finding indicates that PEGylated lysostaphin
has modified activity or metabolism. PEG may shield the enzyme from
degradative proteases that are released from the bacteria as they
are killed, thus enabling lysostaphin to remain active for a longer
period of time at lower concentrations. Another possibility is that
PEG conjugated onto lysostaphin may alter the enzymes interaction
with the bacterial cell wall. Reduced binding affinity to its cell
wall docking site, while still enabling peptidoglycan cleavage,
would result in quicker enzyme release and speed the recycling of
lysostaphin for the next round of cleavage. Either of these
explanations, and others yet undiscovered, could explain the
observed response and each is equally encouraging for the prospect
of creating a PEGylated form of lysostaphin that is superior to the
native drug.
Inhibition of SA5 Growth
[0068] The minimum inhibitory concentration (MIC) is a quantitative
measure of a drug's activity that is typically used to examine
levels of resistance in different bacterial strains. This was used
this assay against a single strain of SA5 to measure loss of drug
activity upon lysostaphin PEGylation, as shown in Table 1. Several
formulations retained high levels of activity although none were as
high as un-conjugated lysostaphin. The pattern of activity observed
with the different PEG-lysostaphin species is consistent with that
observed in the previous killing assays. The more lightly PEGylated
lysostaphin retained greater activity than highly conjugated forms
and under the same reaction conditions, the 40 kDa PEG conjugate
was eight times more active than the 10 kDa PEG conjugate.
TABLE-US-00001 TABLE 1 MIC of PEGylated Lysostaphin Against SA5
TYPE OF LYSOSTAPHIN MIC (.quadrature.g/mL) 10K 2:1 50/50 4 10K 5:1
50/50 >32 10K 2:1 DMSO 2 10K 5:1 DMSO 16 40K 2:1 50/50 0.5 40K
2:1 50/50 >32 Unconjugated 0.13
[0069] Both of these findings support the conclusion that low
degrees of PEGylation result in an active enzyme and that the
bulkier 40 kDa PEG lysostaphin conjugate was more active than the
kDa conjugate. The small loss in activity observed with the 2:1 PEG
ratios is an acceptable trade-off for the increased serum half-life
of this conjugate and its reduced immunogenicity. The potential
benefits of these conjugates include reduced dosing frequency,
reduced ability to induce antibody, retention of activity in
patients with anti-lysostaphin lysostaphin antibodies, and reduced
toxicity associated with immunogenic reactions.
Anti-Lysostaphin Antibody Activity for PEGylated Lysostaphin
[0070] The ability of PEG to shield lysostaphin from antibodies was
tested in vitro with a lysostaphin-capture immunoassay (FIG. 3).
Unconjugated lysostaphin shows a standard response from 0.3 ng/mL
to 20 ng/mL. A heterogeneous response is seen with the binding of
PEG-lysostaphin to anti-lysostaphin antibody, but all bind less
efficiently than unconjugated lysostaphin. The best shielding
observed resulted in a greater than 10-fold reduction in antibody
affinity to the PEGylated lysostaphin. This assay environment is
different than binding in mucosal surfaces or flowing serum, but it
does prove that PEG conjugation onto the surface of lysostaphin can
at least partially shield the enzyme from antibody binding. There
does not appear to be any correlation between enzyme activity and
reduced antibody binding, but differences in antibody binding to
the 40 kDa and 10 kDa conjugates may be explained by differing
degrees of PEGylation. In general, fewer PEG molecules are attached
to lysostaphin for the 40 kDa PEG, so it may have a more open
structure that does not exclude antibodies as well as the 10 kDa
form, which may also explain why the 40 kDa conjugate has better
activity. Nevertheless, antibody activity for the 40 kDa conjugate
is reduced compared to unconjugated lysostaphin.
Prolonged Serum Half-Life of PEGylated Lysostaphin
[0071] Conjugation of PEG onto protein drugs enables them to avoid
the normal clearance mechanisms of the body and thereby leads to
increased serum half-life of the drug. The pharmacokinetic profile
of lysostaphin with a low degree of PEG modification (1 to 4 PEG's
per lysostaphin) was determined in mice and compared to clearance
of unconjugated lysostaphin (FIG. 4). Two enhancements because of
PEGylation are apparent from the graph: (1) the half-life of
lysostaphin has been dramatically increased and (2) the total serum
concentration achieved is much greater than for unconjugated
lysostaphin. The serum concentration of the PEG-lysostaphin
conjugates drops by only two- to ten-fold over 24 hours whereas
unconjugated lysostaphin falls by nearly 500-fold over the same
time period. Such a prolonged retention of lysostaphin should
reduce the dosing frequency needed to remain above therapeutically
effective concentrations of the drug.
[0072] Maintaining these levels of lysostaphin for longer periods
of time may also result in more rapid clearance of bacterial
infections and decrease the probability that lysostaphin resistance
will emerge. Total serum concentrations were also much greater with
the PEG-lysostaphin conjugates. At 24 hours post-administration,
serum concentrations of PEG-lysostaphin were more than 10 times the
concentration of native lysostaphin at just 1 hour
post-administration, even when the initial PEG-lysostaphin dose was
1/4 that of unconjugated lysostaphin. This result suggests that a
much lower dose of PEG-lysostaphin may be used to achieve the same
or better clinical benefit as conjugated lysostaphin, which could
result in lower cost of therapy and minimize potential toxic or
allergic reactions to the drug. The foregoing Example thus
illustrates the increased activity and prolonged circulating
half-life of the lysostaphin conjugates of the present
invention.
Example 2
Fractionation of 40 kD PEG Lysostaphin Conjugates
[0073] Fractionation of the various 40 kD PEG-lysostaphin conjugate
species of Example 1 was performed by ion-exchange chromatography
as a means to test enzyme activity as a function of PEG conjugation
number. Although perfect resolution was not achieved, fractions
tended to be enriched in just one specific band. The mono-PEGylated
form was purified to greater than 99% 1-mer, while the di-PEGylated
form was purified to 93% 2-mer with the remainder contributed
mostly by the 1-mer, as determined by size-exclusion chromatography
HPLC.
[0074] Killing Assay for Activity: The ability of lysostaphin to
kill SA in saline was tested with varying concentrations of the
enzyme. The bacteria were streaked onto blood agar plates after a
1-2 hour incubation with lysostaphin and surviving colonies were
counted the next day. The data is reported in FIG. 5 as surviving
colonies of SA so that the lower value on the graph, the more
effective the killing of SA by lysostaphin. The 1-mer has greater
activity than the 2-mer, but both have significantly reduced
activity compared to unconjugated lysostaphin.
[0075] Killing Activity in Blood: The ability of lysostaphin to
kill SA in whole, heparinized human blood was tested with varying
concentrations of the enzyme. The bacteria were streaked onto blood
agar plates after a 1-2 hour incubation with lysostaphin and
surviving colonies were counted the next day. The data is reported
in FIG. 6 as surviving colonies of SA so that the lower value on
the graph, the more effective the killing of SA by lysostaphin. The
40 k 1-mer BS was the Example 1 conjugate made with 50% DMSO. The
activity of the 40 k 1-mer is reduced as was observed in the
killing assay performed in saline, but the reduction in activity
appears to be even greater in blood than in saline.
[0076] There are two possible explanations for the loss of activity
with the 2-mer. Although there may be as many as 10 lysine residues
available for PEG conjugation onto lysostaphin, each will have a
different reactivity, and it is likely that only one or two lysine
residues are preferentially PEGylated for a given reaction
condition. The preferred site for conjugation of the first PEG
chain might lie in a region that is not critical for enzyme
function and may explain why there is little loss inactivity for
the 1-mer. However, the next-most-preferred lysine for PEGylation
may reside in or near the active or cell wall binding sites of
lysostaphin, and attachment of PEG to these regions may seriously
disrupt enzyme function.
[0077] Another possible explanation for the loss of activity with
the 2-mer relates to the increased spatial volume of lysostaphin
due to PEGylation. Lysostaphin does not act on a soluble,
diffusible substrate but rather must be able to penetrate the
thick, solid peptidoglycan scaffold of the bacterial cell wall.
Each successive addition of PEG raises the molecular weight of
lysostaphin, and the increase in spatial volume from the 1-mer to
the 2-mer may hinder enzyme access to the pentaglycine cross
bridges in the cell wall, thus eliminating its killing
activity.
[0078] Antibody Reactivity: Reactivity of anti-lysostaphin
antibodies to PEGylated lysostaphin was measured by ELISA (FIG. 7):
96-well plates were coated with a polyclonal anti-lysostaphin
antibody (Ab) and then incubated with lysostaphin. Bound
lysostaphin was then detected with a polyclonal, HRP-labeled,
anti-lysostaphin Ab. The binding level of lysostaphin to these
antibodies (Mean Value on y-axis of graph) was determined as a
function of enzyme concentration. Both PEG conjugates have reduced
Ab binding activity compared to unconjugated lysostaphin, but the
2-mer was much less reactive than the 1-mer.
Example 3
Fractionation of 30 kD PEG-Lysostaphin Conjugates
[0079] Example 2 was repeated substituting 30 kD PEG for 40 kD PEG
and 1-mers and 2-mers of mPEG 30 kD lysostaphin conjugates were
isolated in separate fractions having the following properties:
[0080] OD drop assay: The OD at 280 nm of a high innoculum of S.
aureus (SA, about 10.sup.9/mL) in saline is monitored over time.
When bacteria are lysed, the OD drops and thus is a measure of
lysostaphin activity. The faster the OD drops, the greater the
enzyme activity. A typical standard takes 6-7 minutes to reach 50%
of starting OD. The 1-mer has greater activity than the 2-mer, but
both have significantly reduced activity compared to unconjugated
lysostaphin (FIGS. 8 and 9).
[0081] Killing Assay for Activity: The ability of lysostaphin to
kill SA in saline was tested with varying concentrations of the
enzyme. The bacteria were streaked onto blood agar plates after a
1-2 hour incubation with lysostaphin and surviving colonies were
counted the next day. The data is reported as surviving colonies of
SA so that the lower value on the graph, the more effective the
killing of SA by lysostaphin. The 1-mer has greater activity than
the 2-mer, but both have significantly reduced activity compared to
unconjugated lysostaphin (FIG. 10).
[0082] Antibody Reactivity: Reactivity of anti-lysostaphin
antibodies to PEGylated lysostaphin was measured by ELISA. 96-well
plates were coated with a polyclonal anti-lysostaphin Ab and then
incubated with lysostaphin. Bound lysostaphin was then detected
with a polyclonal, HRP-labeled anti-lysostaphin Ab. The binding
level of lysostaphin to these antibodies (Mean Value on y-axis of
graph) was determined as a function of enzyme concentration. The
1-mer has about 7.times. less Ab binding activity and the 2-mer has
about 70.times. less Ab binding activity compared to unconjugated
lysostaphin (FIG. 11).
[0083] Serum Pharmacokinetics: Mice were injected with standard
lysostaphin or PEGylated lysostaphin (30 k 1 and 2-mers) and the
serum concentration was determined by ELISA over 24 hours. Higher
serum concentrations are achieved with PEGylated enzyme and the
half-life of the drug is dramatically increased. The 2-mer achieves
higher peak serum lysostaphin concentrations but the long-term
persistence seems comparable to that of the 1-mer (FIG. 12).
[0084] Killing Assay for Activity: The ability of lysostaphin 30 kD
and 40 kD PEG 1-mers to kill SA in saline was tested with varying
concentrations of the enzyme. The bacteria were streaked onto blood
agar plates after a 1-2 hour incubation with lysostaphin and
surviving colonies were counted the next day. The data is reported
in FIG. 13 as surviving colonies of SA so that the lower value on
the graph, the more effective the killing of SA by lysostaphin. The
40 k 1-mer has greater activity than the 30 k 1-mer, but both have
significantly reduced activity compared to unconjugated
lysostaphin.
Example 4
Recombinant Lysostaphin with a Terminal Cys for Polymer
Conjugation
[0085] A lysostaphin construct, similar to lysostaphin was prepared
except that it contained coding for the amino acids Ala-ala-Cys (in
other words, similar to "mature" lysostaphin, but containing a
terminal cysteine). The procedures described by Goodson et al., Bio
Technology, 8, 343 (1990) and Benhar et al., J. Biol Chem., 269,
13398 (1994) for the insertion of cysteine were followed.
[0086] Native lysostaphin does not contain any cysteines. Because
the two alanines are un-important to the activity of lysostaphin,
this portion of the enzyme could be modified without affecting
activity. Thus, in order to conjugate lysostaphin with PEG in a
defined and controlled manner, recombinant lysostaphin with a
terminal ala-ala-cys was produced in E. coli.
Purification of Cysteine-containing Recombinant Lysostaphin
[0087] Cells from a 250 mL culture were harvested by centrifugation
and frozen. They were thawed and lysed by extracting the pellet in
70 ml 0.1 M HCl. The extract was centrifuged at 4000 rpm and the
supernatant dialyzed overnight at 4.degree. C. against 4 liters PBS
diluted 1:2 with water. The dialysate (approximately 150 mL) was
further diluted to about 250 mL with water.
[0088] Prep 1. Approximately 200 mL of the crude solution was
pumped onto a 1 ml SP Sepharose column (Pharmacia), equilibrated in
12.5 mM sodium phosphate, pH 7. After loading, the column was
washed with the equilibration buffer and then eluted with 0.25 M
NaCl in 12.5 mM sodium phosphate, pH 7. The eluant was concentrated
using an Ultrafree 4 (10 kDa cutoff) device, (Millipore) to about
700 .mu.L. Concentration was estimated by adsorption at 280 nm,
following 1:20 dilution into PBS, using an extinction coefficient
for lysostaphin of 0.49 mg/ml/OD 280:OD
280=0.201.times.20.times.0.49 mg/ml/OD 280=2.3 mg/ml. For 27 kDa,
this corresponds to 85 .mu.M lysostaphin if 100% pure, for a
recovery of 0.7 mL.times.2.3 mg/mL, or 1.6 mg.
[0089] Prep 2. The remaining 50 mL was processed in a similar
manner. The concentration of this material was determined as 1.18
mg/mL in a volume of 0.7 L. Recovery=0.82 mg. The total estimated
recovery was 1.6+0.8 mg, or 2.4 mg.
Determination of Purity and Presence of Thiol
[0090] The free thiol (SH groups) of the recombinant lysostaphin
were determined using DTNB (Ellman's reagent) and found to be 23.6
.mu.M. Thus, at least 23.6/85=28% of the lysostaphin contains a
free thiol, assuming no other proteins are contributing. SDS PAGE
using 8-25% Phast gel (Pharmacia) indicated that a fraction was
dimerized, further increasing the percentage of
cysteine-lysostaphin obtained upon reduction of the dimer.
[0091] Labeling with iodoacetyl biotin, a reagent that reacts only
with thiols confirmed that the recombinant lysostaphin contained a
cysteine, unlike native lysostaphin. The cysteine can be reacted
with reagents such as maleimide-PEG or iodoacetyl-PEG to conjugate
lysostaphin at a unique site with PEG.
Example 5
NH.sub.2 Terminal Lysostaphin PEGylation by Site-specific Oxidative
Coupling
[0092] Lysostaphin has a threonine on its amino terminus. As has
been described by Fields et al., Biochem. J., 108, 883 (1968),
Gaertner et al., J. Biol. Chem., 269, 7224 (1994), and Geoghegan et
al., Bioconj. Chem., 3, 7224 (1992), amino terminus serine or
threonine can be oxidized to a glyoxylyl derivative under mild
conditions using sodium periodate. This group can then be reacted
with amino-oxy PEG, hydrazide PEG or hydrazine PEG to yield
lysostaphin pegylated on its amino terminus. An example of this
reaction for the pegylation of IL-8 is described in Gaertner et
al., Bioconj. Chem., 7, 38 (1996).
[0093] Amino-oxy PEG (30 kD) is prepared as described in Gaertner
et al., Bioconjugate Chemistry or purchased from Shearwater.
Lysostaphin is prepared at 20 mg/mL in 1% NH.sub.4(HCO.sub.3), pH
8.3 and a 50-fold molar excess of methionine. A 10-fold molar
excess of sodium periodate is added. After 10 min. at room
temperature in the dark, the reaction is quenched by the addition
of 1/20 volume 50% glycerol. The solution is then dialyzed in the
dark against 0.1M sodium acetate, pH 4.6. The solution of oxidized
lysostaphin is adjusted to pH 3.6 with 1N acetic acid, then reacted
with a 5-fold molar excess of an amino-oxy PEG for 20 hr at room
temp in the dark, with gentle stirring. Unreacted PEG is removed by
ion exchange chromatography, followed by hydrophobic interaction
chromatography to separate unconjugated lysostaphin.
[0094] The oxidized lysostaphin may also be functionalized with a
reagent such as a (2-thio-pyridyl-cysteine hydrazide (Zara et al,
Anal. Biochem., 194, 156 (1991), which can then be reacted with a
thiol reactive PEG such as PEG-maleimide.
[0095] Killing Assay for Activity: The ability of the N-terminal 30
kD PEGylated lysostaphin to kill SA in saline was tested with
varying concentrations of the enzyme. The bacteria were streaked
onto blood agar plates after a 1-2 hour incubation with lysostaphin
and surviving colonies were counted the next day. The data is
reported in FIG. 14 as surviving colonies of SA so that the lower
value on the graph, the more effective the killing of SA by
lysostaphin. The N-terminal 30 k 1-mer has activity but not at a
greater level than previously tested 1-mers (either 30 k or 40
K).
Examples 6 to 8
PEGylation Using Two-Step Heteroligation
[0096] Heteroligation chemistry involves labeling component A (in
this case lysostaphin) with a reactive group that is capable of
reacting only with the reactive group present on component B (in
this case, a PEG). In this Example, lysostaphin is chemically
modified on a lysine amino groups with a thiol group and then
reacted with an electrophilic PEG reagent (e.g., PEG-maleimide), as
compared to Example 4, wherein lysostaphin is genetically modified
to insert a cysteine group, (which contains a reactive thiol
group). Heteroligation chemistry is described in above-referenced
Bioconjugate Chemistry.
[0097] Lysostaphin was prepared at 20 mg/mL in 75 mM HEPES+2 mM
EDTA, pH 7.5 buffer and N-succinimidyl
3-[2-pyridylidithio]propionate (SPDP) (0.1M in DMF) was added
dropwise while mixing. The molar ratio of SPDP to lysostaphin was
varied in order to vary the degree of labeling. After 1 hr, the pH
was reduced by the addition of 1/10 volume 1M sodium acetate, pH 5
and adjusted to pH 5 with 1 N HCl. The solution was made 25 mM by
the addition of solid dithiothreitol (DTT). After 15 min., the
solution was dialyzed overnight into 10 mM sodium acetate, 2 mM
EDTA, pH 5 at 4.degree. C. to remove the DTT. The extent of
labeling was determined by the use of DTNB (Ellman's reagent) and
from the molar concentration determined by absorbance, at 280 nm
using an extinction coefficient of 0.49 mg/ml per absorbance unit
at 280 nm.
[0098] The thiolated lysostaphin was then reacted with the
electrophilic, thiol-selective mPEG-vinylsulfone (Shearwater
M-VS-5000), mPEG-maleimide (Shearwater M-MAL-5000) and
mPEG-orthopyridyl disulfide (Shearwater M-OPSS-5000). A haloacyl
PEG would also be suitable. The reactions were performed at
appropriate pH for the PEG reagents to be selective for the thiol.
For example, the vinylsulfone addition was performed about pH 7-8.
The maleimide addition and disulfide exchange were at performed at
pH 6-7.
[0099] Excess PEG was removed by ion exchange chromatography.
Hydrophobic chromatography could also be used. Lysostaphin
containing varying amounts of PEG was thereby fractionated.
[0100] The advantages of the two-step method include the ability to
limit or control the extent of PEGylation. Additionally, a long
chain thiolating reagent can be used (e.g., LC-SPDP, Pierce,
#21651). These reagents allow the thiol group to extend further
beyond the protein surface and facilitate conjugation to bulky
molecules such as PEG.
[0101] A further advantage of the above two-step method is that the
thiol will remain reactive for extended periods of time, especially
in the absence of oxygen, in EDTA containing buffers and under
acidic conditions, all of which minimize oxidation. Likewise, the
PEGS described above are all stable at a pH where the reagents are
reactive with thiols. This is in contrast to the NHS-ester PEGS
which require alkaline conditions to react with amino groups. NHS
esters are not stable in base.
[0102] Bulky reagents generally react more slowly than small
reagents. By employing the two step heteroligation method described
in this example, the reaction may be allowed to proceed for an
extended period of time and allow for more efficient coupling. This
permits less of the PEG reagent to be used, reducing costs.
Furthermore, because a higher percentage of the PEG is coupled,
purification of the desired PEG-lysostaphin conjugate may be
facilitated. These advantages are also present in Example 4.
[0103] The methods described in Examples 1-8 can be extended to
other anti-microbial proteins, such as nisin. A terminal cysteine
can be engineered into a protein as in Example 4, and then coupled
to PEG as described in Examples 6 to 8.
[0104] As will be readily appreciated, numerous variations of and
combinations of the features set forth above can be utilized
without departing from the present invention as set forth in the
claims. Such variations are not regarded as a departure from the
spirit and scope of the invention, and all such modifications are
intended to be included within the scope of the following
claims.
* * * * *