U.S. patent application number 16/658861 was filed with the patent office on 2020-09-17 for composition with high antimicrobial activity and low toxicity.
The applicant listed for this patent is Amicrobe, Inc., The Regents of the University of California. Invention is credited to Diego Benitez, Michael P. Bevilacqua, Timothy J. Deming, Jarrod A. Hanson, Lucas Koziol.
Application Number | 20200288709 16/658861 |
Document ID | / |
Family ID | 1000004869743 |
Filed Date | 2020-09-17 |
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United States Patent
Application |
20200288709 |
Kind Code |
A1 |
Bevilacqua; Michael P. ; et
al. |
September 17, 2020 |
COMPOSITION WITH HIGH ANTIMICROBIAL ACTIVITY AND LOW TOXICITY
Abstract
Improved synthetic copolypeptide antimicrobials contain cationic
amino acid residues and may be based on a blocky sequence. These
antimicrobials show low mammalian toxicity and may undergo directed
self-assembly. The inventive synthetic copolypeptides are useful in
treatment of wounds and other infections.
Inventors: |
Bevilacqua; Michael P.;
(Boulder, CO) ; Benitez; Diego; (Los Angeles,
CA) ; Deming; Timothy J.; (Los Angeles, CA) ;
Hanson; Jarrod A.; (Los Angeles, CA) ; Koziol;
Lucas; (Penngrove, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California
Amicrobe, Inc. |
Oakland
Pasadena |
CA
CA |
US
US |
|
|
Family ID: |
1000004869743 |
Appl. No.: |
16/658861 |
Filed: |
October 21, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14693601 |
Apr 22, 2015 |
10448634 |
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16658861 |
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13776221 |
Feb 25, 2013 |
9017730 |
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14693601 |
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PCT/US2011/048869 |
Aug 23, 2011 |
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13776221 |
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61376195 |
Aug 23, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 37/18 20130101;
A23L 3/3526 20130101; A61K 38/02 20130101; A61K 45/06 20130101;
A61K 9/0019 20130101; C07K 14/001 20130101; A61K 9/107 20130101;
A61K 38/16 20130101; A61K 47/02 20130101; Y02A 50/30 20180101 |
International
Class: |
A01N 37/18 20060101
A01N037/18; A23L 3/3526 20060101 A23L003/3526; A61K 38/02 20060101
A61K038/02; A61K 9/00 20060101 A61K009/00; A61K 47/02 20060101
A61K047/02; A61K 9/107 20060101 A61K009/107; A61K 38/16 20060101
A61K038/16; A61K 45/06 20060101 A61K045/06; C07K 14/00 20060101
C07K014/00 |
Claims
1. An antimicrobial composition comprising: water and one or more
hierarchical structures comprising at least one species of
synthetic polypeptide, wherein: said at least one species of
synthetic polypeptide comprises at least forty amino acid residues;
said at least one species of synthetic polypeptide comprises a
plurality of cationic amino acid residues and a plurality of
hydrophobic amino acid residues; said at least one species of
synthetic polypeptide has a net positive charge at neutral pH; said
at least one species of synthetic polypeptide demonstrates a
critical aggregation concentration below that of a random-sequence
polypeptide of the same amino acid composition; said at least one
species of synthetic polypeptide inhibits or kills microbes; and
said antimicrobial composition inhibits or kills microbes.
2-20. (canceled)
21. The antimicrobial composition of claim 1, wherein said one or
more hierarchical structures is selected from multimers, micelles,
fibrils, sheets, and vesicles, or mixtures thereof.
22. The antimicrobial composition of claim 1, wherein said
plurality of cationic amino acid residues comprises amino acid
residues selected from lysine, arginine, homoarginine and
ornithine.
23. The antimicrobial composition of claim 1, wherein said
plurality of hydrophobic amino acid residues are selected from
leucine, valine, phenylalanine, isoleucine and alanine.
24. The antimicrobial composition of claim 1, wherein the molar
fraction of hydrophobic amino acid residues in said at least one
species of synthetic polypeptide is 40% or less.
25. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide comprises at least five
cationic amino acid residues.
26. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide has a critical aggregation
concentration that is at least 20% lower than that of a random
sequence polypeptide having the same amino acid residue composition
as said at least one species of synthetic polypeptide.
27. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide has a critical aggregation
concentration that is at least 1 log lower than that of a random
sequence polypeptide having the same amino acid residue composition
as said at least one species of synthetic polypeptide.
28. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide demonstrates a critical
aggregation concentration in water of less than 160 .mu.M.
29. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide is a surfactant.
30. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide is a surfactant, as measured
by a decrease in surface tension.
31. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide forms emulsions when mixed
with oil and water.
32. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide forms a hydrogel in water at a
concentration of 40 mg/mL or less.
33. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide comprises L-amino acid
residues, D-amino acid residues, a racemic mixture of L- and
D-amino acid residues, or a mixture of varying optical purity of
amino acid residues.
34. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide kills or inhibits microbes in
vitro at a lower concentration than the synthetic polypeptide kills
mammalian cells in vitro.
35. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide kills or inhibits microbes in
vitro at concentrations below 0.1% (w/w) of the synthetic
polypeptide in water.
36. The antimicrobial composition of claim 1, wherein said at least
one species of synthetic polypeptide kills or inhibits microbes in
vitro as measured by greater than 3 logs killing of Staphylococcus
epidermidis and Escherichia coli in standard 60 minute time-kill
assays at synthetic polypeptide concentrations of 100 .mu.g/mL or
less in water.
37. The antimicrobial composition of claim 1, wherein the microbes
are selected from bacteria, viruses, fungi and protozoans.
38. The antimicrobial composition of claim 1, wherein the
antimicrobial composition further comprises an added active
pharmaceutical ingredient selected from a steroid, a
pro-inflammatory agent, an anti-inflammatory agent, an antiacne
agent, a preservative, a hemostatic agent, an angiogenic agent, a
wound healing agent, an antibiotic, an antibody, and an anti-cancer
agent.
39. The antimicrobial composition of claim 1, wherein the
antimicrobial composition is formulated as a solution, a gel, a
cream, a foam or a dressing.
40. The antimicrobial composition of claim 1, wherein the
antimicrobial composition promotes platelet aggregation.
41. The antimicrobial composition of claim 1, wherein the
antimicrobial composition inhibits fibrinolysis.
42. A method of preventing or treating an infection comprising:
administering or applying to a site the antimicrobial composition
of claim 1; wherein the infection is selected from topical
infection, microbial colonization, wound infection, surgical site
infection, trauma infection, burn infection, diabetic foot ulcers,
eye infection, vaginal infections, or urinary tract infections.
Description
CROSS-REFERENCE TO PRIOR APPLICATIONS
[0001] This application claims the benefit and priority of U.S.
Provisional Patent Application Ser. No. 61/376,195, filed 23 Aug.
2010, which is incorporated herein by reference to the extent
permitted by applicable law.
U.S. GOVERNMENT SUPPORT
[0002] Not Applicable.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The current invention relates to compositions of matter that
are able to kill (or inhibit) microbes, and have low mammalian
toxicity. The current invention also relates to certain
compositions and their uses in a variety of settings including but
not limited to preservatives, antiseptics, and the prevention and
treatment of wound infections, as well as other infectious
diseases.
Discussion of Related Art
[0004] Cationic antimicrobials have demonstrated utility; toxicity
is a problem. For over half a century, cationic (positively
charged) antimicrobials have been used in a variety of medical and
non-medical settings, ranging from systemic antibiotics to
industrial cleansers. Cationic antimicrobials bind preferentially
to bacterial membranes, which typically display more negative
charge than mammalian membranes. This interaction can disrupt
membrane function and potentially lead to bacterial cell death.
Cationic antimicrobial compounds include certain antibiotics (e.g.,
polymyxins), bisbiguanides (e.g., chlorhexidine), polymeric
biguanides (e.g., polyhexamethylene biguanide), and quaternary
ammonium compounds (QAC) (e.g., benzalkonium chloride), as well as
natural antimicrobial peptides (AMPs) (e.g., defensins). While each
class of cationic antimicrobial compounds has demonstrated
antimicrobial activity in one or more settings, toxicity has been a
consistent problem.[1-12]
[0005] Polymyxins, produced by Bacillus polymyxa, are cyclic
peptides with hydrophobic tails.[6, 7] The cyclic peptide portion
(approx. 10 amino acid residues; positively charged) interacts
strongly with negatively charged lipopolysaccharide (LPS) found on
the outer membrane of Gram-negative bacteria. The hydrophobic tail
is thought to interact with, and in some cases, disrupt the
bacterial membrane. Polymyxins have antimicrobial activity against
many Gram-negative bacteria, including Pseudomonas aeruginosa (P.
aeruginosa), Escherichia coli (E. coli), and Enterobacter species,
but have limited activity against Proteus, most Serratia, or
Gram-positive bacteria [7]. Significant neurotoxicity and
nephrotoxicity have contributed to their limited use as systemic
antibiotics [13]. Today, Polymyxins are sometimes used as a last
resort for Gram-negative infections that are highly antibiotic
resistant, such as those caused by multi-drug resistant P.
aeruginosa. They are also used as topical antimicrobial agents for
small cuts and scrapes of the skin.
[0006] Chlorhexidine is widely used in the pre-operative surgical
setting as an antiseptic cleanser for general skin cleaning,
preoperative bathing, and surgical site preparation [7].
Chlorhexidine is active against a wide range of Gram-positive and
Gram-negative bacteria, although resistance by some Gram-negative
bacteria (e.g., P. aeruginosa, Providentia species) has been
reported [5, 10]. Formulations containing 2-4% chlorhexidine appear
to be most effective as antimicrobials, but can cause skin
irritation. Overall, chlorhexidine is relatively safe when applied
to intact skin because minimal amounts of the compound are
absorbed. However, due to irritation and toxicity, chlorhexidine is
contraindicated for use near the eyes, ears, brain tissues, and
meninges [2]. Low concentrations (e.g., 0.05% to 0.12%) are
sometimes used as wound washes and oral rinses. Activity is pH
dependent, as low pH environments reduce activity. In addition,
chlorhexidine is not compatible with anionic compounds (e.g., hard
water, soap, alginate) and shows reduced activity in the presence
of organic materials (e.g., blood).
[0007] Polyhexamethylene biguanide (PHMB) has been used in diverse
consumer applications for over 40 years. PHMB is used in swimming
pool sanitizers, preservatives of plasticized PVC, and
general-purpose environmental biocides [1]. Early production of
PHMB resulted in highly polydisperse oligomers with molecular
weights ranging from 500-6,000 g/mol. Limited chemical
characterization largely precluded early PHMB use in pharmaceutical
products. Recent PHMB formulations have been able to address
polydispersity. Similar to chlorhexidine, use of PHMB is
contraindicated for eyes, ears, brain tissues, meninges, and joints
[4].
[0008] Quaternary ammonium compounds (QACs) are amphoteric
surfactants, typically containing one nitrogen atom linked directly
to four alkyl groups, which may vary in hydrophobic structure [1,
2]. QACs are primarily bacteriostatic, but at higher concentrations
can be bacteriocidal against certain organisms. QACs are
antimicrobial against Gram-positive bacteria, but are less
effective against Gram-negative bacteria (e.g., P. aeruginosa).
Because of weak activity against Gram-negatives, QACs are generally
not used in health-care settings for hand antisepsis. Several
outbreaks of infection have been traced to QAC compounds
contaminated with Gram-negative bacilli [8]. QACs appear to be more
susceptible to resistance mechanisms mediated through multidrug
efflux pumps. Activity is also greatly reduced in the presence of
organic matter.
[0009] Natural antimicrobial peptides (AMPS) are often cationic.
Natural antimicrobial peptides (AMPs) (typically, less than 50
amino acids) are widely distributed in most species from insects to
mammals, and are thought to play key roles in innate immunity [14].
AMPS have demonstrated potent killing inhibition of bacteria,
viruses, fungi and parasites [15]. AMPs are thought to be important
in preventing and controlling infections. AMPs are heavily
deposited at interfaces such as the skin, respiratory tract, and
gastrointestinal lining, and are released by white blood cells at
sites of inflammation. White blood cells use AMPs as part of their
direct killing mechanisms in phagolysosomes. Certain AMPs
contribute to the regulation of inflammation and adaptive immunity
[15]. In addition, AMPs have demonstrated inhibitory activity
against spermatozoa and cancer cells.
[0010] Most AMPs share structural characteristics leading to
physical, receptor-independent modes of killing [9]. A widely
accepted mechanism of action of AMPs is microbial membrane
disruption or perturbation (followed sometimes by pore formation)
leading to cell death. Typically, AMPs contain positively charged
and hydrophobic domains that are spatially segregated cationic
amphiphiles. Substantial hydrophobic content of AMPS (typically, 30
to 60% mole fraction) is an important feature for antimicrobial
activity as it "governs the extent to which a peptide can partition
into the lipid bilayer" [16]. AMPs that form alpha-helices
"frequently exist as extended or unstructured conformers in
solution" and become helical "upon interaction with amphipathic
phospholipid membranes" [16]. This suggests that the "local
environment at the bacterial outer surface and membranes is
important and can induce antimicrobial peptide conformational
changes that are necessary for peptide attachment to and insertion
into the membrane" [3].
[0011] Nisin (a bacterially-derived AMP that has been used as a
food preservative) was shown to be a weak emulsifying agent for
oil-water mixtures, the process being significantly pH- and
temperature-dependent [17].
[0012] Several natural AMPs and related technologies have been
patented. Lehrer and Selsted disclosed AMP sequences analogous to
those of defensins isolated from macrophages (U.S. Pat. No.
4,543,252). The magainin class of AMPs, first isolated from the
skin of certain frogs, has been described by Zasloff (U.S. Pat. No.
4,810,777).
[0013] Modified magainins, particularly sequence deletions or
substitutions, have also been described (e.g., U.S. Pat. Nos.
4,962,277; 5,221,732; 5,912,231; and 5,792,831). Selsted and Cullor
disclosed bovine indolicidin AMP as a broad-spectrum antimicrobial
compound (U.S. Pat. No. 5,324,716).
[0014] Synthetic peptide-based cationic oligomers may function as
antimicrobials. Salick and colleagues have disclosed a
sequence-specific beta-hairpin peptide (20-mer) which can form an
antimicrobial hydrogel in the presence of sufficient salt
concentration (US Published Patent Application No. 2011/0171304).
When the peptide is "dissolved in water, it remains unfolded and
soluble due to the charge repulsion between positively charged side
chains." The addition of salt is thought to "screen the side
chain-derived charge and allow the peptide to fold" into a
beta-hairpin which may "assemble into a network of beta-sheet rich
fibrils." The peptide consists of 60% hydrophobic content and
contains two arginine residues that seem to be important for
effective antimicrobial activity against methicillin-resistant
Staphylococcus aureus (MRSA). The peptides themselves do not appear
to be inherently antimicrobial, as the inventors have reported that
"peptide diffusing from the gel is not the active agent." When S.
aureus was subjected to 100 .mu.M (approx. 230 .mu.g/ml) aqueous
solutions (i.e., not hydrogels) of peptide, "bacterial
proliferation was minimally affected." Thus, for antimicrobial
activity, bacteria must directly contact the hydrogel surface;
"folded but not gelled" peptide does not inhibit bacterial
proliferation. Similar findings were reported for other
closely-related beta-hairpin peptides [18].
[0015] Gellman and coworkers have disclosed antimicrobial
compositions containing beta-amino acid oligomers (U.S. Pat. Nos.
6,060,585; 6,683,154; US Published Patent Application Nos.
2007/0087404; 2008/0166388) with well-defined secondary structures.
The beta-peptides contain ring structures in the peptide backbone
which limit conformational flexibility. DeGrado and coworkers have
also described antibacterial beta-peptides, containing oligomers
(7-mer or shorter) of a tri-beta-peptide (U.S. Pat. No.
6,677,431).
[0016] Other synthetic peptide-based compounds that may mimic
overall structure of natural AMPs have been described. DeGrado
reported amphiphilic sequence-random beta-peptides based on
structural properties of the natural AMPs magainin and cecropin
[19]. Gellman and coworkers have described a random-sequence,
beta-peptide oligomer with an average length of 21 residues,
potycispersity index (Mn/Mw) of 1.4, and 40% hydrophobic residues
[20]. In other studies, Gellman identified helical beta-peptides
[19]. A 60% "hydrophobic face" along the helical cylinder was found
to have optimal antimicrobial activity, while a 40% face displayed
low activity.
[0017] Synthetic cationic polymers comprised of non-natural
building blocks may function as antimicrobials. Several classes of
synthetic antimicrobial polymers with non-natural building blocks
or repeat-units have been described; they are the subject of a 2007
review by Tew [22]. These polymers are comprised of
structures/monomeric units that are not found in nature. These
non-natural polymers often feature easy and cost-efficient
syntheses, and stability against enzymatic degradation. However,
limitations of these and other non-natural polymers may include
limited antimicrobial activity, as well as a lack of
biocompatibility and biodegradability. Materials in this class are
comprised of unnatural building blocks (e.g. aryl amides, highly
conjugated aromatic groups) and are considered outside the scope of
this invention [21-25]. (For examples, see U.S. Pat. No. 7,173,102;
US Published Patent Application Nos. 2008/0176807;
2010/0105703).
[0018] Antimicrobial peptoids (N-substituted glycines) have been
described by Winter and coworkers [28]. A series of short
(3-monomer) peptoids were tested against a broad spectrum of
Gram-positive and Gram-negative bacteria, and hemolytic activity
(HC50) was lower than antimicrobial activity (minimum inhibitory
concentrations, MICs). A representative tri-peptoid protected S.
aureus-infected mice in vivo in a simple infection model.
[0019] Synthetic methodologies for copolypeptides (Deming method).
Traditional synthetic methodologies have precluded the efficient
synthesis of oligopeptide libraries with orthogonal (or
semi-orthogonal) modification of multiple properties. Important
properties to be modified include amino acid sequence, overall
chain length, and ratio of cationic to hydrophobic amino acids.
Moreover, the practical, cost-effective synthesis of low
polydispersity (PDI between 1.0 and 1.4) copolypeptide mixtures has
also not been easily accessible [25].
[0020] Control over multiple properties, and the ability to create
low polydispersity compounds, would allow optimization of multiple
structure-function relationships. A major challenge in synthetic
polypeptide AMP research is prohibitive production costs in
solid-phase synthesis. In addition, significant chemical
limitations of both solid-phase and solution-phase synthetic
methods include lack of control over chain growth. This leads to
chain branching, polydispersity and low product yields.
[0021] In 1997, Deming developed well-defined initiators to
polymerize amino acid derivatives into oligopeptide chains [25,
26]. This methodology added amino acid monomers to a growing chain
in batches. The initiators were transition-metal complexes that
allowed controlled synthesis to yield high molecular weight,
narrowly-distributed, multi-block polypeptide formulations. The
initiators and synthetic methods are well described in the
literature and in several patents (U.S. Pat. Nos. 6,680,365;
6,632,922; 6,686,448; 6,818,732; 7,329,727; US Published Patent
Application No. 200810125581).
[0022] Typically, the synthetic polypeptides have a simple binary
composition (e.g., lysine (K), leucine (L) copolymers). Amphiphilic
polypeptides contain ionic amino acid monomers (e.g., lysine,
arginine (R), glutamate (E)) co-polymerized with neutral
hydrophobic amino acids (e.g., leucine, alanine (A)). By variation
of method of monomer addition, copolymerizations may be conducted
to obtain sequences of amino acid residues along the copolymer
chain that are blocky, random, or a combination of both (i.e.
blocks of random sequences).
[0023] Random synthetic copolypeptides in solution demonstrate
antimicrobial activity. The Deming laboratory has observed
antimicrobial activity for a series of water-soluble copolypeptides
containing varying ratios of cationic (lysine, (K)) and hydrophobic
(leucine (L), isoleucine (I), valine (V), phenylalanine (F), or
alanine (A)) amino acids that were randomly arranged [27].
Copolypeptides demonstrated varying antimicrobial activity against
S. aureus (Gram-positive), P. aeruginosa (Gram-negative), and E.
coli (Gram-negative) in suspension growth assays. Lysine-alanine
copolypeptides demonstrated a broad "toxic effect on all three
species of bacteria studied" and were concluded to be the "most
effective antimicrobial copolymer combination." Circular dichroism
spectra of lysine-alanine and lysine-leucine copolypeptides showed
"unambiguous random coil conformations when free in solution." This
work did not examine the antimicrobial activity of synthetic block
sequence copolypeptides or synthetic copolypeptides deliberately
formulated as micelles, or incorporated into
emulsions/nanoemulsions (also see [28, 29]).
[0024] Using Deming synthesis methods, Chan-Park and colleagues
recently studied the antimicrobial activity of soluble,
random-sequence copolypeptides containing 2-3 different amino acids
[26]. Random 25-mer copolypeptides, comprised of
lysine-phenylalanine or lysine-phenylalanine-leucine, demonstrated
the broadest activity against five microbes and had the lowest
MICs. The effects of total peptide length and hydrophobic content
on antimicrobial activity were investigated. Lysine-phenylalanine
copolypeptide was reported to have "broader antibacterial activity
when it is 25 residues long than at shorter or longer length."
Optimum hydrophobic content for lysine-phenylalanine compounds (and
other random copolypeptides) was found to be about 60%. However,
optimized lysine-phenylalanine and lysine-phenylalanine-leucine
compounds showed high hemolytic activity compared to other natural
and synthetic peptides. The authors suggested that the compounds'
"high hydrophobicity (60%) or more hydrophobic species present may
have resulted in high toxicity to mammalian red blood cells." In
addition, lysine-alanine and lysine-leucine random copolypeptides
showed no significant activity against the fungal organism Candida
albicans. Circular dichroism analysis indicated that
lysine-phenylalanine and lysine-phenylalanine-leucine random
copolypeptides show "lack of a distinct secondary structure" and do
not form alpha-helices or beta-sheets.
[0025] Synthetic copolypeptides can be formulated to achieve
hierarchical structures. The presence of both polyelectrolyte and
hydrophobic domains leads to microphase segregated materials.
Resulting superstructures can include multimers in solution,
micelles, emulsions (with oil), sheets, vesicles and fibrils that
form hydrogels. Self-assembly into different hierarchical
structures can be controlled by: varying composition and chain
length; varying concentration; presence of L-, D-, or racemic amino
acids; and modification of side-chains and chain-termini (e.g.
polyethylene glycol (PEG)). Secondary structure of hydrophobic
domains (i.e. random coil vs. alpha-helix) plays an important role
in superstructure formation. The nature of the hydrophobic domain
or polymer segments determines the type of intermolecular
interactions that are established between chains. These attractive
interactions are balanced by the interactions with the solvent.
There exists an equilibrium between the free energy of
self-association with the free energy of hydration for each
molecule and for each fragment of the supermolecule.
[0026] Synthetic copolypeptides can also be designed to form
hydrogels. Certain characteristics, such as long-hydrophilic blocks
(cationic or anionic) and ordered hydrophobic blocks (e.g.,
alpha-helical) were shown to favor hydrogel formation. Studies
suggest that several synthetic copolypeptide-based hydrogels,
including K.sub.180L.sub.20 (and other K.sub.xL.sub.y) block
copolypeptides, are biocompatible in vivo. Deming et al. previously
reported that block copolypeptide hydrogels can serve as tissue
scaffolds in the murine central nervous system (CNS) [27].
Hydrogels were injected into mouse forebrain and created 3D gel
deposits in vivo. Toxicity, inflammation and gliosis were minimal
and similar to saline controls. After 8 weeks, in many cases,
copolypeptide deposits were vascularized with cell density similar
to adjacent tissue, suggesting hydrogels are supportive of cellular
migration and proliferation.
[0027] Deming (PCT publication WO 2009/025802) disclosed
nanoemulsions and double nanoemulsions stabilized by synthetic
block copolypeptides [27]. Antimicrobial activity of the emulsified
copolypeptides was not disclosed therein.
[0028] Nanoemulsions prepared without copolypeptides can display
some antimicrobial activity. Baker and coworkers have focused on
the use of nanoemulsions as antimicrobial agents. They reported
antimicrobial emulsions stabilized by phosphate-based or other
small molecule surfactants (U.S. Pat. Nos. 6,015,832; 6,506,803;
6,559,189; 6,635,676; 5,618,840; 5,547,677; and 5,549,901).
[0029] Potential relationships between antimicrobial activity
and/or mammalian cell toxicity of cationic amphiphiles and their
assembly into higher-order structures are not well understood.
Limited relevant information has been reported. For example, the
antimicrobial activity of epsilon-poly-lysine (EPL) was slightly
reduced by coordination to a lipid and emulsification, relative to
free EPL in solution [33].
SUMMARY OF THE INVENTION
[0030] The present invention describes compositions of matter and
uses of synthetic copolypeptides with high antimicrobial activity
(in vitro or in vivo) and low mammalian toxicity. Notably, cationic
(positively charged) antimicrobials have been used for more than
fifty years in a variety of medical and non-medical settings,
ranging from systemic antibiotics to industrial cleansers. Despite
substantial efficacy, their use in many medical settings has been
limited due to substantial toxicities. This invention overcomes the
limitation of the inherent toxicity of cationic antimicrobials.
Simply stated, by controlling the relationship between cationic
elements and hydrophobic elements, we design materials with high
antimicrobial activity and low mammalian toxicity, often taking
advantage of unique hierarchical structures. This invention
includes the grouping of hydrophilic and/or hydrophobic amino acid
residues along a copolypeptide chain into blocky sequences to
achieve block amphiphilicity. This differs from facial
amphiphilicity that characterizes many natural AMPs, as well as
random-sequence and alternating-sequence and specific-sequence
synthetic copolypeptides and peptides. For the purposes of this
invention, blocky or block-sequence copolypeptides are
characterized as copolypeptides consisting of one or more different
domains that each contain a contiguous repeat of at least 5
residues of a single amino acid (e.g. lysine or leucine) or amino
acid type (cationic or hydrophobic). By contrast, random
copolypeptides are characterized as copolypeptides consisting of
non-ordered, statistical distributions of two or more different
amino acid residues (or amino acid types) within a sequence.
[0031] The synthetic copolypeptides of the present invention
possess one or more of the following molecular characteristics that
distinguish them from previously described natural and synthetic
antimicrobials. First, relatively high overall chain length (40 to
250 or more amino acid residues per chain); second, multimeric
display of the hydrophilic (typically, cationic) domains; third,
relatively low hydrophobic residue content (typically, 40% mole
fraction or less); and fourth, self-association/self-assembly
through interactions of the hydrophobic domains (often based on
block sequence). By way of explanation, without limiting the scope
of this invention, it is thought that high antimicrobial activity
results from the display of long hydrophilic (cationic) segments,
multimeric hydrophilic (cationic) segments, or both, which interact
very effectively with anionic (negative) charges at the surface of
microbes. Further, by way of explanation without limiting the scope
of this invention, it is thought that the relatively low hydrophobe
content, the self-associating nature of the hydrophobic domains
(often based on block sequence), or both serves to limit tissue
exposure to high hydrophobic or high amphipathic material
concentrations, thereby decreasing mammalian toxicity. In certain
cases, this limited hydrophobe or amphipathic exposure may allow
administration of larger quantities of antimicrobial material in
vivo, with potential for depot, slow-release effects and greater
antimicrobial activity (with less mammalian toxicity) over
time.
[0032] Without limiting the scope of the present invention, it is
recognized that achieving high antimicrobial activity (in vitro or
in vivo) and low toxicity may depend on one or more factors,
including the following: monomer selection (e.g., specific cations
and hydrophobes); spatial distribution of monomers (e.g., blocky
vs. random sequences); mole fraction of hydrophobic monomers;
optical purity of monomers, ordered vs. disordered hydrophobic
domains (e.g., alpha-helical vs. random coil), chemical
modification of monomers/residues; hybrid compositions (e.g.,
copolypeptide-polymer conjugates).
[0033] These synthetic copolypeptides can be designed to
self-associate/self-assemble, in part, through interactions of
poorly solvated hydrophobic regions, that are stabilized by fully
dissolved hydrophilic (typically, cationic) domains. Specific
examples include preparations involving multimers in solution,
micelles, sheets, vesicles, and fibrils that form hydrogels, as
well as emulsions upon mixture with oils. By example, we have
developed antimicrobial wash solutions, antimicrobial hydrogels and
antimicrobial emulsions. All of these preparations can be applied
to wounds, other tissues or other various surfaces. The directed
molecular self-assembly of this invention determines chemical and
biological characteristics, including hierarchical structure. It
differs from the self-association of various random-sequence
synthetic copolypeptides, which is based on non-uniform
distribution of hydrophilic and hydrophobic residues, and typically
results in irregular and ill-defined materials.
[0034] Preferred embodiments may also consider certain qualities
that can impact the overall efficacy and toxicity in human or
animal disease, including but not limited to the prevention and
treatment of wound infections or other infectious diseases. These
characteristics include, but are not limited to, fluidity (enabling
ease of application), tissue coverage, duration of antimicrobial
bioactivity, biocompatibility, degradation, biodistribution, and
effects on inflammatory response, tissue repair, angiogenesis,
hemostasis, immunogenicity and other. In certain medical settings
(e.g., surgical or traumatic wounds), efficacy and toxicity may
depend substantially on interactions of the synthetic
copolypeptides with tissues. Certain advantages may be derived from
synthetic copolypeptides that easily precipitate onto and/or
directly bind to damaged tissues where they may provide a local,
concentrated antimicrobial activity. Overall efficacy and safety in
human or animal diseases will depend on the specific disease and
the general condition of the patient. It is anticipated that in
vivo bioactivities will depend substantially on formulation and
hierarchical structure and that in vivo activity may not be fully
revealed by in vitro testing.
DESCRIPTION OF THE FIGURES
[0035] FIG. 1 is a diagram showing the variety of molecular
building blocks that can be used to construct copolypeptides;
[0036] FIG. 2 is .sup.1H-NMR of K.sub.55(rac-L).sub.20 block
copolypeptide in d-TFA;
[0037] FIG. 3 is a diagram showing the structures of selected
antimicrobial block copolypeptides: A) K.sub.x(rac-L).sub.y; B)
random K.sub.55(rac-L).sub.20; C) K.sub.55(rac-A).sub.20; D)
K.sub.55(rac-V).sub.20; E) K.sub.55(rac-V).sub.20; F)
K.sub.55(rac-L/F).sub.20; G) R.sup.H.sub.55(rac-L).sub.20;
E.sub.64(rac-L).sub.20; I) PEG.sub.205(rac-L).sub.20; and J)
K.sub.50-20;
[0038] FIG. 4 shows the antimicrobial activity of
K.sub.55(rac-L).sub.20 block copolypeptide against S. aureus, S.
epidermidis, E. coli, and P. aeruginosa; K.sub.55(rac-L).sub.20 was
incubated with bacteria for 30 min prior to plating for growth;
[0039] FIG. 5 shows the antimicrobial activity against S. aureus
and E coil, of copolypeptides with varying content of hydrophobic
amino acid residues;
[0040] FIG. 6 shows the antimicrobial activity against C. albicans
of copolypeptides at concentration of 100 .mu.g/mL;
[0041] FIG. 7 shows the antimicrobial activity of
K.sub.55(rac-L).sub.20 block copolypeptide against S. aureus and
Propionibacterium acnes (P. acnes); K.sub.55(rac-L).sub.20 was
incubated with bacteria for 30 min prior to plating for growth;
[0042] FIG. 8 show the antimicrobial activity against S. aureus and
E. coli, of copolypeptides with varying sizes of block hydrophobic
domains at peptide concentration of 10 .mu.g/mL;
[0043] FIG. 9 shows the antimicrobial activity against P. acnes, of
copolypeptides with varying sizes of hydrophobic domains at peptide
concentration of 10 .mu.g/mL;
[0044] FIG. 10 shows the antimicrobial activity against S. aureus
and E. coli, of copolypeptides formulated with blocky or random
spatial distribution of monomers at peptide concentration of 10
ug/mL;
[0045] FIG. 11 shows the antimicrobial activity of
K.sub.55(rac-L).sub.20 in a rodent model; a polypropylene mesh
pre-soaked with PBS or K.sub.55(rac-L).sub.20 was inserted
subcutaneously in rats, with additional copolypeptide, and an
inoculum of either 10.sup.6 S. aureus 6538 or P. aeruginosa
(Clinical Pig Isolate) was added; after two days, the implanted
mesh was plated for bacterial enumeration;
[0046] FIG. 12 shows the antimicrobial activity of
K.sub.55(rac-L).sub.20 in a rodent model; a polypropylene mesh
pre-soaked with PBS or K.sub.55(rac-L).sub.20 was inserted
subcutaneously in rats, with additional copolypeptide, and an
inoculum of either 10.sup.6 S. aureus 6538 or P. aeruginosa
(Clinical Pig Isolate) was added; at various timepoints, the
implanted mesh was plated for bacterial enumeration;
[0047] FIG. 13 shows the antimicrobial activity of
K.sub.55(rac-L).sub.20 in a rodent model; a polypropylene mesh
pre-soaked with PBS or 2 mg/ml K.sub.55(rac-L).sub.20 was inserted
subcutaneously in rats, with additional copolypeptide, and a
inoculum of either 10.sup.6 S. aureus 6538 or P. aeruginosa
(Clinical Pig Isolate) was added; after two days, the surrounding
tissue was plated for bacterial enumeration.
[0048] FIG. 14 shows the results of assaying inflammation in a
rodent model; a polypropylene mesh pre-soaked with
K.sub.55(rac-L).sub.20 copolypeptide was inserted subcutaneously in
rats, with additional copolypeptide, and an inoculum of 10.sup.6 S.
aureus 6538 was added; after 48 hrs, tissue was analyzed by
histology for inflammation: 0=normal, 1=mild, 2=moderate,
3=severe;
[0049] FIG. 15 shows the antimicrobial activity of
K.sub.55(rac-L).sub.20 in a porcine model; K.sub.55(rac-L).sub.20
(10 mg/mL) was applied to wounds, and after four hrs, remaining
material was aspirated and 10.sup.7 S. aureus 6538 was added to
wounds; after 48 hrs, bacterial counts were assessed;
[0050] FIG. 16 shows the result of assaying for inflammation in a
porcine model; K.sub.55(rac-L).sub.20 (10 mg/mL) was applied to
wounds, an after 30 mins, 10.sup.4 or 10.sup.7 S. aureus or P.
aeruginosa was added to wounds; after 48 hrs, tissues were analyzed
by histology for inflammation (including cell infiltration and
necrosis);
[0051] FIG. 17 shows wound healing in a porcine model in which
wounds were treated with 500 .mu.g/mL of K.sub.55(rac-L).sub.20 and
monitored over a 21 day period;
[0052] FIG. 18 shows antimicrobial activity against S. aureus and
E. coli of K.sub.55(rac-L).sub.20 block copolypeptides formulated
as solutions or emulsions;
[0053] FIG. 19 shows antimicrobial activity against S. aureus, of
copolypeptides formulated as either solutions or emulsions with
varying sizes of hydrophobic domains at peptide concentration of 10
.mu.g/mL;
[0054] FIG. 20 shows the in vivo antimicrobial activity against S.
aureus of K.sub.55(rac-L).sub.20 copolypeptide formulated as an
emulsion; a polypropylene mesh pre-soaked with copolypeptide was
inserted subcutaneously in rats, with additional copolypeptide, and
an inoculum of 10.sup.6 S. aureus 6538 was added; after 2 days, the
implanted mesh was plated for bacterial enumeration;
[0055] FIG. 21 show the results of assaying for inflammation in a
rodent model; K.sub.55(rac-L).sub.20 copolypeptide was formulated
as an emulsion, and a polypropylene mesh pre-soaked with
copolypeptide was inserted subcutaneously in rats, with additional
copolypeptide; an inoculum of 10.sup.6 S. aureus 6538 was added,
and after 48 hrs, tissue was analyzed by histology for
inflammation: 0=normal, 1 mild, 2=moderate, 3=severe;
[0056] FIG. 22 shows wound healing in a porcine model in which
wounds were treated with 500 .mu.g/mL of K.sub.55(rac-L).sub.20
formulated as an emulsion and monitored over a 21 day period;
[0057] FIG. 23' shows the antimicrobial activity of
K.sub.180L.sub.20 block copolypeptides. K.sub.180L.sub.20 was
incubated with bacteria for 30 min prior to plating for growth;
[0058] FIG. 24 shows the antimicrobial activity of
K.sub.160L.sub.20, in a rodent model; a polypropylene mesh
pre-soaked with PBS or K.sub.190L.sub.20, was inserted
subcutaneously in rats, with additional copolypeptide; an inoculum
of either 10.sup.6 S. aureus 6538 or P. aeruginosa (Clinical Pig
Isolate) was added; after 48 hrs, the implanted mesh and
surrounding tissue were plated for bacterial enumeration;
[0059] FIG. 25 shows the results of assaying inflammation in a
rodent model; a polypropylene mesh pre-soaked with PBS or
K.sub.180L.sub.20 copolypeptide was inserted subcutaneously in
rats, with additional copolypeptide, and an inoculum of 10.sup.6 S.
aureus 6538 was added; after 48 hrs, the surrounding tissue was
analyzed by histology for inflammation (including cell infiltration
and necrosis);
[0060] FIG. 26 shows the antimicrobial activity of
K.sub.180L.sub.20 in a porcine model; K.sub.180L.sub.20 (40 mg/mL)
was applied to wounds, and after 4 hrs, 10.sup.7 S. aureus 6538 was
added to wounds; after 48 hrs, final bacterial counts were
assessed.
[0061] FIG. 27 show the effect of copolypeptides on clotting time
of whole blood, at copolypeptide concentration of 10 .mu.g/mL;
[0062] FIG. 28 shows the results of a thromboelastography (TEG)
assay to measure effects of copolypeptides on blood clotting at
copolypeptide concentration of 10 .mu.g/mL; R time is latency time
between placement of blood in TEG apparatus and initial increase in
viscosity (measured by trace increase from 0-2 mm); R time
corresponds to enzymatic activity of coagulation factors prior to
ramp-up of cross-linking; K time corresponds to the amplitude
increasing from 2-20 mm; alpha angle is the slope of the TEG
tracing between R and the K times; alpha angle measures speed of
clot development, and maximum amplitude (MA) is the highest trace
and provides an absolute measure of clot strength;
[0063] FIG. 29 shows the effect of copolypeptides on platelet
aggregation in platelet-rich plasma with a copolypeptide
concentration of 100 .mu.g/mL;
[0064] FIG. 30 show the effect of copolypeptides on platelet
aggregation;
[0065] FIG. 31 shows a fibrin gel plate assay used to measure
effects on fibrinolysis of R.sup.H.sub.55(rac-L).sub.20
copolypeptide at concentrations of 100, 1000 .mu.g/ml and 1000
.mu.g/ml with 1 mg/ml albumin;
[0066] FIG. 32 shows images from porcine venous bleeding depicting
15 mm wounds at 5 min filled with PEG-based gels containing
copolypeptides; and
[0067] FIG. 33 is a table (Table 1) of polypeptide synthetic data
where .sup.a=M.sub.n and PDI is determined using gel permeation
chromatography (GPC) of the first segment,
poly(N.sub..epsilon.-CBZ-L-lysine); compositions were calculated
using: .sup.b=GPC and .sup.1H-NMR or .sup.c=.sup.1H-NMR in d-TFA.
.sup.d=Synthesized by guanylation of K.sub.55(rac-L).sub.20;
[0068] FIG. 34 is a table (Table 2) of minimum contact time (min.)
for 99.99% growth inhibition of E. coli 11229 and E. coli O157:H7,
at copolypeptide concentration of 100 .mu.g/mL;
[0069] FIG. 35 is a table (Table 3) showing minimum inhibitory
concentration (MIC) of copolypeptides against various microbes
including food-related microbes
[0070] FIG. 38 is a table (Table 4) showing log reduction against
Influenza A (enveloped virus) by copolypeptides at 1 mg/ml
concentration after 30 sec of contact time;
[0071] FIG. 37 is a table (Table 5) showing minimum inhibitory
concentration (MIC) of copolypeptides formulated as emulsions
against B. subtilis endospores;
[0072] FIG. 38 is a table (Table 6) showing in vitro cytotoxicity
in human keratinocytes, of copolypeptides formulated as solutions
or emulsions, at concentration of 100 .mu.g/ml; and
[0073] FIG. 39 is a table (Table 7) showing thromboelastography
(TEG) parameters for copolypeptides at concentration of 10
.mu.g/mL; *Values were significantly different (p<0.05) than
untreated controls.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The following description is provided to enable any person
skilled in the art to make and use the invention and sets forth the
best modes contemplated by the inventor of carrying out his
invention. Various modifications, however, will remain readily
apparent to those skilled in the art, since the general principles
of the present invention have been defined herein specifically to
provide synthetic copolypeptides with high antimicrobial activity
and low toxicity.
[0075] Antimicrobial copolypeptide compositions of this invention
may contain one or more cationic amino acids (e.g. lysine,
arginine, homoarginine, omithine) and one or more hydrophobic amino
acids (e.g. leucine, valine, isoleucine, phenylalanine, alanine)
arranged in blocks (FIGS. 1-3, FIG. 33 (Table 1)). Polycationic
amphiphilic polypeptides (e.g., containing amine groups that are
protonated at neutral pH, peralkylated ammoniums, or guanidiniums)
display high antimicrobial activity. For example, as depicted in
FIG. 4, we have demonstrated that a synthetic copolypeptide
consisting of a block of 55 lysines followed by a block of 20 D and
L (racemic) leucines (K.sub.55(rac-L).sub.20) has substantial
antimicrobial activity against S. aureus (Gram-positive), S.
epidermidis (Gram-positive), E. coli (Gram-negative) and P.
aeruginosa (Gram-negative). We have also demonstrated activity
against several other bacterial and fungal organisms (see below).
Multiple other synthetic copolypeptides have been synthesized (FIG.
33 (Table 1)) and show substantial antimicrobial activity. By
contrast, at neutral pH (.about.7) polyanionic polypeptides (e.g.
E.sub.54(rac-L).sub.20) display low antimicrobial activity.
[0076] As depicted in FIG. 5, diblock synthetic copolypeptides
based on cationic amino acid lysine and other hydrophobic amino
acids demonstrate strong antimicrobial activity. In other studies,
we demonstrated that partial guanylation of lysine residues
resulted in high antimicrobial activity, for example
X.sub.55(rac-L).sub.20 for X=K/R.sup.H (homo-arginine) achieved
high antimicrobial activity. Varying the hydrophobic amino acid
composition, while keeping all other properties constant, also
maintained high in vitro antimicrobial activity (FIG. 5).
Specifically,
poly(L-lysine-HCl).sub.55-bock-poly(racemic-hydrophobic amino
acid).sub.20, K.sub.55(rac-X).sub.20, for X=Alanine (A), Isoleucine
(I), Leucine/Phenylalanine (UF), or Valine (V), at very low
concentration (10 .mu.g/ml), achieved maximum observable (6-log)
reduction of bacterial counts for both a Gram-positive (S. aureus)
and a Gram-negative (E. coli) bacteria. Selected copolypeptides
were also shown to be quite effective against other microbes
including E. coli O157:H7, as well as other food-borne pathogens,
and even against certain endospore forms of microbes (FIGS. 34 and
35 (Tables 2 and 3)). These compounds were also shown to be
effective against certain fungal organisms as depicted for Candida
albicans in FIG. 6. As depicted in FIG. 7, certain microbial
organisms (e.g., P. acnes) may be less sensitive to certain
copolypeptides than other microorganisms (e.g., S. aureus).
Solution phase copolypeptides also demonstrated antiviral activity
against H1N1 influenza virus (FIG. 36 (Table 4)). In this
experiment, it was noted that the R.sup.H/K (partially guanylated
lysine) diblock copolypeptide were particularly active.
[0077] In these block copolypeptides, we also demonstrated high
antimicrobial activity when varying the length of the hydrophobic
block (FIGS. 8 and 9). Unexpectedly, we demonstrated high
antimicrobial activity in several series of synthetic block
copolypeptides, including block copolypeptides with hydrophobe
content below 40%. Even molecules with a block of as few as 5 or 10
hydrophobic leucine amino acids demonstrated good antimicrobial
activity when constructed with a block of 55 cationic lysine amino
acids.
[0078] In separate studies we demonstrated that blocky
copolypeptides with long hydrophilic blocks (i.e. longer than K90)
were effective as antimicrobials (FIG. 10). In addition, we
demonstrated that random synthetic copolypeptides of longer length
(greater than 100 amino acid residues) were very effective
antimicrobial agents. This was true for compounds of varying
hydrophobe content.
[0079] In separate in vitro studies, we demonstrated that
block-sequence copolypeptides in solution were less cytotoxic than
random-sequence copolypeptides of similar composition. For example,
we found that a blocky sequence K.sub.55L.sub.20 in solution
decreased cell viability of mouse keratinocytes by 50% (EC.sub.50),
at 47.4 ug/ml, whereas a synthetic copolypeptide of similar
composition in random sequence had an EC.sub.50 of 21.0 ug/ml in
solution. Similarly, block-sequence K.sub.55(rac-L).sub.20 in
solution was found to be less cytotoxic than random-sequence
K.sub.55(rac-L).sub.20, in solution. As described below, a variety
of synthetic copolypeptides were found to be antimicrobial in
emulsion preparations. In these preparations, block sequence
synthetic copolypeptides were also found to be less cytotoxic
(lower EC.sub.50) than random sequence copolypeptides, even though,
the block sequence copolypeptide stabilized emulsions typically
demonstrated equivalent (and sometimes higher) antimicrobial
activity.
[0080] A solution phase block-sequence synthetic copolypeptide
K.sub.55 (rac-L).sub.20 was also shown to be effective in a rodent
model of prevention of wound infection (FIGS. 11-13). We have
demonstrated reductions in bacterial populations in an infection
prevention model against S. aureus and P. aeruginosa. Consistent,
concentration-dependent reductions were observed typically, 1-3 log
reduction at 20 .mu.g/ml of copolypeptide, K.sub.55(rac-L).sub.20,
and complete (or near complete) reduction at 2 mg/ml. These studies
indicate that copolypeptide formulations remain active when exposed
to complex biological fluid. Notably, copolypeptides could be
formulated as either aqueous suspensions or mixed with oil and
water and self-assembled into nanoemulsions; certain antimicrobial
copolypeptides are effective surfactants (see below for
emulsions).
[0081] Importantly, the block-sequence synthetic copolypeptides
K.sub.55(rac-L).sub.20 in solution did not appear to be irritating
to open wounds. As depicted in FIG. 14, histopathological evidence
suggested that inflammation was at or below the level of control
treatments.
[0082] Solution phase antimicrobial copolypeptides were also found
to be highly effective in a porcine infection prevention model. As
depicted in FIG. 15, K.sub.55(rac-L).sub.20 solution applied to an
open wound prior to inoculation with S. aureus fully prevented
microbial infection. In separate studies, copolypeptide
K.sub.55(rac-L).sub.20, where the hydrophobic block is racemic
poly-D/L-leucine, exhibited excellent tissue biocompatibility in
animal models. For example, in a two-day porcine open-wound study
(FIG. 16), histological analysis (by a veterinary pathologist)
showed "serocellular exudates and neutrophilic inflammation were
mildly and minimally less severe, respectively," in
K.sub.55(rac-L).sub.2-treated animals versus controls. No
differences were observed in mononuclear inflammation, edema, or
hemorrhage. In a 21-day porcine wound healing study (non-infected),
K.sub.55(rac-L).sub.20-treated and control-treated wounds were
found to be similar in inflammation, necrosis, and epithelial
coverage by a veterinary pathologist (FIG. 17).
[0083] Antimicrobial emulsions based on synthetic copolypeptides.
These synthetic copolypeptides can be designed to be effective
surfactants that may stabilize (and/or be displayed on) emulsions.
We have demonstrated that a variety of synthetic
copolypeptide-emulsion preparations are effective antibacterials in
vitro (FIGS. 18 and 19). Notably, these antimicrobial emulsions
were found to be active against B. subtilis endospores (FIG. 37
(Table 5)). As described above for solution phase copolypeptides,
emulsion preparations demonstrated antiviral activity against H1N1
influenza virus (FIG. 36 (Table 4)), as well as against a
non-enveloped bacteriophage.
[0084] Antimicrobial emulsions based on synthetic copolypeptides
were also found to be effective in an infection prevention model in
rodents (FIG. 20). We have demonstrated reductions in bacterial
populations in an infection prevention model against S. aureus.
Consistent, concentration-dependent reductions were observed
typically, 1-4 log reduction at 20 .mu.g/ml of copolypeptide,
K.sub.55(rac-L).sub.20, based emulsions and complete (or near
complete) reduction at 2 mg/ml. These studies indicate that
copolypeptide emulsion formulations remain active when exposed to
complex biological fluid. These antimicrobial emulsions appear to
be well tolerated in wounds and did not result in increased
inflammation over control treatments, as assessed by histological
examination (FIG. 21). In addition, these antimicrobial emulsions
were found to be well tolerated in a 21-day porcine model of wound
healing (non-infected) (FIG. 22).
[0085] Further studies suggested that antimicrobial synthetic
copolypeptide emulsions have less cytotoxicity in vitro (FIG. 38
(Table 6)). In other studies, this observation was consistent
across multiple synthetic copolypeptides including
K.sub.55(rac-L).sub.20, K.sub.55L.sub.20, K.sub.55(rac-L/F).sub.20.
Taken together, these data indicate that the arrangement of
synthetic block-sequence copolypeptides into the hierarchical
structures of emulsions and nanoemulsions may improve antimicrobial
activity, reduce mammalian toxicity, or both.
[0086] Antimicrobial hydrogels based on synthetic copolypeptides.
This invention also describes block copolypeptides that
self-assemble into fibrils that form antimicrobial hydrogels. As
described below, K.sub.180L.sub.20, is a hydrogel-former and has
demonstrated strong antimicrobial activity in vitro and effective
prevention of microbial growth in studies in vivo. As depicted in
FIG. 23, K.sub.180L.sub.20 demonstrated potent antimicrobial
activity in vitro (5+ log reduction at 6.3 .mu.g/mL) against
Gram-positive (S. aureus, S. epidermidis) and Gram-negative (E.
coli, P. aeruginosa) bacteria that are known to be important in
wound infection. In time kill assays, K.sub.180L.sub.20 at 100
.mu.g/mL showed more than 3 log reduction in 5 min against S.
epidermidis, E. coli, and P. aeruginosa.
[0087] Other studies demonstrated that K.sub.180L.sub.20 block
copolypeptides are antimicrobial in vivo. As depicted in FIG. 24,
K.sub.180L.sub.20 was effective in inhibiting microbial growth in a
rodent closed-wound model with foreign body. In this model, a mesh
pre-soaked with phosphate buffered saline (PBS) or
K.sub.180L.sub.20 was inserted subcutaneously into the dorsal
cervical region of Sprague-Dawley rats, followed by 10 S. aureus or
P. aeruginosa. Additional PBS or K.sub.180L.sub.20 was added,
wounds closed, and animals returned to cages for 48 hr.
K.sub.180L.sub.20 (2 mg/ml and 20 mg/ml) substantially decreased
the number of bacteria (both S. aureus and P. aeruginosa) cultured
from the mesh and adjacent tissue. No enhanced inflammation was
observed with this antimicrobial hydrogel in the rodent model of
infection (FIG. 25).
[0088] In a separate study, the hydrogel based on block-sequence
copolypeptide K.sub.180L.sub.20 was effective in inhibiting S.
aureus in a porcine open-wound model (FIG. 26). Full-thickness 1 cm
diameter wounds were made in the dorsal and lateral thorax of a
25-35 kg Yorkshire-cross pig. K.sub.180L.sub.20 hydrogel (or
control buffer) was applied, and after four hr, wounds were
inoculated with S. aureus. Wounds were assessed after 48 hr for
bacterial counts by standard microbiology methods. As depicted in
FIG. 26, K.sub.180L.sub.20 hydrogel fully reduced S. aureus
counts.
[0089] Block-sequence structure. In certain embodiments, these
antimicrobial, copolypeptide compositions may have a block-sequence
structure, including one or more blocks containing segments of 2 or
more consecutive cationic amino acids/monomer (e.g., lysine,
arginine), or segments of 2 or more consecutive hydrophobic amino
acids/monomer (e.g., leucine, isoleucine, valine, alanine,
phenylalanine). In certain cases, triblock or multiblock compounds
(i.e., several blocks of distinct amino acids, monomers and/or
other polymer blocks) may be particularly effective. Blocks of
alternating amino acids or monomers may also be effective, while
blocks of random sequences may also be advantageous in certain
settings. Other embodiments may also feature a copolypeptide block
or segment of the same amino acid/monomer or different amino
acids/monomers that are chemically attached to a different polymer.
It is also anticipated that the bioactivity and chemical
composition of block copolypeptides/copolymers may be more
reproducible from batch to batch than that of random
copolypeptides/copolymers. It is also anticipated that block
copolypeptides may be less immunogenic than random copolypeptides.
Blocks may be composed of natural and/or unnatural amino acids that
display different degrees of hydrophilicity or hydrophobicity.
Natural amino acids (hydrophobic, such as but not limited to
alanine, glycine, isoleucine, leucine, phenylalanine, valine, and
hydrophilic, such as but not limited to arginine, esparto acid,
asparagine, glutamic acid, glutamine, lysine, serine, tyrosine, or
threonine) or unnatural amino acids, such as but not limited to
fluorinated or unsaturated hydrocarbons can be used, as well as
enantiopure or racemic mixtures. In addition to polypeptidic
materials or hybrids containing synthetic polymers and peptidic
segments or blocks, may also display increased antimicrobial
activity, decreased mammalian toxicity, or both. For example, a
hydrophobic polypeptide may be conjugated to a hydrophilic polymer
or oligomer, or a hydrophobic synthetic polymer or oligomer may be
conjugated to a hydrophilic peptide and display similar
characteristics than a material composed entirely of linked amino
acids. A peptidic segment, block or domain can also be replaced by
a synthetic oligomeric or polymeric segment, including direct
incorporation into the polymer backbone, or as a graft.
[0090] We have demonstrated that block-sequence structure can be
used to direct molecular self-association or self-assembly. For
example, we demonstrated by determining the critical aggregation
concentration (CAC) that block-sequence copolypeptide
K.sub.55L.sub.20 exhibits a substantially stronger self-association
(CAC=0.33 uM) than random-sequence K.sub.55L.sub.20 (CAC=160 uM).
This molecular design element is important in preferred embodiments
of our invention that involve designed hierarchical structures.
[0091] Designed hierarchical structures. These compositions may be
formulated as hierarchical structures, such as multimers, micelles,
hydrogels, or vesicles, or mixtures thereof. Enhanced antimicrobial
activity, or decreased mammalian toxicity, or both may be derived
from the organization of the antimicrobial elements into high order
structures that either display the actives in a more efficient way
or with a higher local concentration. For example, the higher
density of cationic charge at the hydrophilic sections of the
liquid interface of an emulsion may lead to better interaction with
microbial organisms. In a similar way, other high order structures
such as vesicles, micelles, lamella, or hydrogels may be able to
deliver the antimicrobial elements more effectively than an
isolated antimicrobial element alone. On the other hand, the
secondary interactions present, and sometimes responsible for the
higher ordered structures of the hydrophobic segments in
amphiphilic polymers, may be responsible for the reduced mammalian
toxicity.
[0092] These designed synthetic copolypeptides may self-assemble
into hierarchical structures (e.g., multimers, micelles, emulsions,
hydrogels, vesicles) thereby enhancing antimicrobial activity (in
vitro or in vivo), decreasing toxicity, or both. Moreover, these
compounds may easily precipitate onto and/or directly bind to
damaged tissues where they may provide a local, concentrated
antimicrobial activity.
[0093] In certain embodiments, these compositions may be formulated
as, or mixed into, emulsions, micro-emulsions or nanoemulsions. In
particular, these emulsions may be designed to have high
antimicrobial activity, low mammalian toxicity, or both. It is
recognized that these activities may depend on one or more
additional factors, such as the composition of the oil phase, or
droplet size.
[0094] In certain embodiments, these antimicrobial copolypeptides
may be formulated as hydrogels. These antimicrobial molecules would
self-assemble into hydrogels. It is anticipated that there would be
advantages to physical hydrogels, which are inherently
antimicrobial that may be able to pass through small bore openings
(e.g., 20 gauge needles) or into small tissue spaces and then
rapidly re-gel. These hydrogel forming antimicrobial copolypeptides
may be designed to be mildly tissue adherent and optically clear.
It is anticipated that they will provide localized, concentrated
antimicrobial activity, as well as the benefits of standard
hydrogels (e.g., fluid retention). The antimicrobial properties of
the copolypeptides that self-assemble into fibrils that form
hydrogels have been demonstrated at concentrations well below the
gelation concentration. For example K.sub.180L.sub.20 has been
shown to be a potent antimicrobial at concentrations of 10 ug/ml,
while its gelation concentration is approx. 10 mg/ml. This
establishes that the material is inherently antimicrobial, while at
the same time can self-associate to hierarchical structures that
provide macroscopic properties to the preparations. Also,
K.sub.180L.sub.20 at hydrogel forming concentrations (e.g., 20
mg/ml) has been shown to be an effective antimicrobial in infection
prevention model in vivo, as well as to have low toxicity in
several models in vivo.
[0095] Long chain length. In certain embodiments, these
antimicrobial copolypeptide compositions may have a relatively long
chain length (e.g., over 100 amino acids). It is anticipated that
synthetic copolypeptides with longer chain length can be optimized
to display increased efficacy, decreased mammalian toxicity or both
in certain settings. Notably, they may display multiple active
sites, conformations, domains, or fragments more effectively and
therefore could continue to display antimicrobial activity even
after partial complexation or degradation. Long-chain
copolypeptides may interact more effectively with microbial
surfaces, and interact with more than one microbe at a time. Longer
polypeptides may be able to disrupt bacterial membranes more
effectively by cross-linking of the negative components of the
bacterial membrane. They may also be able to interact with certain
soluble biomolecules or tissue components, while leaving a
molecular segment free to interact with microbes.
[0096] Low hydrophobe content. These compositions may have low
molar fractions of hydrophobic monomer (e.g., leucine, isoleucine,
valine, alanine, phenylalanine, or non-peptidic hydrophobic
monomer) by comparison to other antimicrobial peptides, for example
35% or less. In the present invention, we recognize that block
copolypeptides with a low molar fraction of hydrophobic monomers
(e.g., f.sub.HM=8%, 18%, 25%, 35%) can yield high antimicrobial
activity and low mammalian toxicity. Such compounds may overcome
specific limitations inherent to copolymers with high f.sub.HM,
Amphiphilic copolymers with low f.sub.HM offer several distinct
advantages. For example, it is anticipated that reduced hydrophobic
content decreases mammalian toxicity. It has been reported that
increased hydrophobic content in antimicrobial peptides increases
hemolytic activity, possibly by reducing selectivity for bacterial
over mammalian cell membranes [22]. Other advantages may include
improved solubility in aqueous solution. Some compositions of the
present invention incorporate low f.sub.HM. Specifically, we have
demonstrated high antimicrobial activity with mole fraction of
hydrophobic monomers as low as about 8%. Furthermore, we have shown
that high antimicrobial activity can be attained by either
decreasing the hydrophobic content or by increasing the hydrophilic
content.
[0097] Enantiopurity influences secondary structure. In certain
embodiments, the enantiopurity of the amino acids (especially in
the hydrophobic domain) can be used to control self-assembly
characteristics. By example, we demonstrated that K.sub.55L.sub.20
and K.sub.55(rac-L).sub.20 both achieve reduction of bacteria, for
both a Gram-positive (S. aureus) and Gram-negative (E. coli, P.
aeruginosa) strains at a very low concentration (10 .mu.g/ml).
Racemic mixtures, or mixtures with varying optical purity, may
offer improved solubility and reduced aggregation. Importantly,
incorporation of a fraction of D-amino acids may have particular
advantages in therapeutic applications against biofilms [38].
Moreover, decreasing optical purity removes ordered secondary
structure, which influences self-association and/or self assembly
characteristics. For example, we demonstrated by determining the
critical aggregation concentration (CAC) that block-sequence
copolypeptide K.sub.55L.sub.20 exhibits a stronger association
(CAC=0.33 uM) than K.sub.55(rac-L).sub.20 (CAC=8.1 uM).
[0098] Solution Metastability. In certain embodiments, these
antimicrobial, copolypeptide compositions can be designed with
relatively low solution stability. Moreover, these materials can be
designed to bind to/precipitate at sites where they interact with
negatively charged elements found commonly on microbes (e.g.,
bacterial micro-colonies and biofilms) and at sites of tissue
damage. These solution "metastable" antimicrobial molecules may
easily precipitate (for example, when interacting with microbes or
mammalian tissue materials of opposite charge). Certain advantages
may be derived from synthetic copolypeptides that easily
precipitate onto and/or directly bind to damaged tissues where they
may provide a local, concentrated antimicrobial activity. Moreover,
antimicrobial copolypeptides (or other antimicrobial materials) may
be made more effective in certain settings by binding
to/precipitating at sites of microbes (e.g., bacterial
micro-colonies and biofilms). Certain design elements may be
incorporated so that synthetic copolypeptide hierarchical
structures remain completely solvated in the absence of biological
materials (e.g., serum, wound fluids, damaged tissues, bacterial
biofilms), but become metastable upon binding biological materials.
Once the antimicrobial materials become metastable, they may settle
on tissues or bacterial colonies, thus dramatically increasing the
local concentration acting as an antimicrobial agent and/or as an
antimicrobial barrier.
[0099] Multivalency. In certain embodiments, these compositions may
be engineered to include multiple antimicrobial sites. These
antimicrobial sites may include local regions of cationic charge
and/or local regions of hydrophobicity. Therefore, a single
material could have several different active sites capable of
killing 1 inhibiting microbes. In this way, a single supramolecular
construct could effect a "multi-hit" approach, providing greater
effectiveness and further decreasing the likelihood of microbial
resistance. In addition, additive or synergistic activity may be
observed. In addition, the material may release antimicrobial
fragments as it is degraded.
[0100] Microbe selectivity. These compositions can be engineered to
preferentially target certain microbes over others. Notably,
targeting traditionally pathogenic organisms (e.g., S. aureus,
methicillin-resistant S. aureus (MRSA)) over traditionally normal
flora (e.g., P. acnes), may be of particular benefit. Furthermore,
targeting of selected viruses, bacteria or fungi may be relevant to
particular clinical settings, such as use in a hand sanitizer or in
prevention of wound infections. We have developed multiple
synthetic copolypeptides that have shown higher activity against S.
aureus than against P. acnes in vitro.
[0101] Mixtures. In certain embodiments, these compositions may be
formulated with two or more distinct antimicrobial
copolypeptides/copolymers. In this way, a composition could affect
a "two-hit" approach, providing greater effectiveness and further
decreasing the development of microbial resistance. In addition,
additive or synergistic activity may be observed.
[0102] In certain embodiments, these compositions may be
synthesized with chemical modification of monomer amino acids or
residues, for example, conversion of a primary amine (e.g., of
lysine monomer) to a guanidinium group. Other modifications may
include alkylation, acylation, amidation, halogenation,
transesterification, reductive amination or other chemical
transformations which add functionality or modifies existing
functionality of the monomer amino acids or residues.
[0103] In certain embodiments, these compositions may be formulated
with different classes of other antimicrobial agents (e.g. alcohol,
chlorine-based compounds, quaternary ammonium compounds, phenolic
compounds, chlorhexidine, antibiotics, antibodies). This may
include mixing in the compositions of the invention with known
antimicrobial agents. It may include formulating synthetic
copolypeptides/copolymers as a type of delivery agent or depot
(e.g., emulsion, double nanoemulsion, vesicle, hydrogel) and
incorporating one or more additional antimicrobial substances.
[0104] In certain embodiments, these compositions may be formulated
with bioactive materials or other active pharmaceutical ingredients
(APIs). In this way, the formulations could provide antimicrobial
activity, as well as a second or third function. Possibilities
include, but are not limited to hemostatic materials, growth
factors to support wound healing, pro- or anti-inflammatory agents,
and immune modulators.
[0105] In certain embodiments, the synthetic antimicrobial
copolypeptides/copolymers may be designed to contain other
bioactive elements (e.g., specific sequences, blocks, hierarchical
structures or chemical modifications). For example, they may
contain elements that would promote hemostasis by one or more
mechanisms such as platelet binding, platelet activation,
acceleration of coagulation, decrease of fibrinolysis, absorption
of fluid or physical barrier effects. This invention envisions
synthetic copolypeptides that are hemostatic in nature, as well as
those that have combined antimicrobial and hemostatic activities
(FIGS. 27-32, FIG. 39 (Table 7)).
Experimental
[0106] General. Dry tetrahydrofuran (THF) was prepared by passing
it through a column packed with alumina under nitrogen prior to
use. Molecular weights (Mn) and polydispersities (PDIs) were
obtained by tandem gel permeation chromatography/light scattering
(GPC/LS). performed at 60.degree. C. on a SSI pump equipped with a
Wyatt DAWN EOS light scattering detector and Wyatt Optilab DSP with
10.sup.5, 10.sup.4, and 10.sup.3 A Phenomenex 5 .mu.m columns using
0.1 M LiBr in DMF as eluent and polypeptide concentration of
approximately 5 mg/mL. Fourier transform infrared spectra (FTIR)
were recorded on a Perkin Elmer RX1 FTIR Spectrophotometer
calibrated using polystyrene film. .sup.1H NMR spectra were
recorded on a Bruker AVANCE 400 MHz spectrometer. Deionized (DI)
water was purified using a Purelab Option 560 reverse osmosis
purifier. Millipore water was obtained from a Millipore Milli-Q
Biocel A10 purification unit.
[0107] Block Copolypeptide Synthesis--General. The .alpha.-amino
acid-N-carboxyanhydride NCA monomers were synthesized using
previously published literature protocols. All of the block
copolypeptides were polymerized using the (PMe.sub.3).sub.4Co
initiator. The resulting polypeptides were characterized using GPC,
.sup.1H NMR and IR spectroscopy. The compositions of the copolymers
were determined by analysis of the integration values of the
.sup.1FI NMR spectra recorded in d-TFA. All compositions were found
to be within 5% of predicted values. Polymer chain length
distributions ranged (Mw/Mn) from 1.1 to 1.3.
[0108]
Poly(N.sub..epsilon.--CBZ-L-lysine).sub.55-b-poly(rac-leucine).sub.-
20, Z-K.sub.55(rac-L).sub.20. In the drybox,
N.sub..epsilon.-CBZ-L-lysine, Z-K NCA (11.34 g, 37 mmol) was placed
in a 500 mL flat bottom flask with a stir bar. Dry THF (227 mL) was
added and then sealed with a plastic stopper. An aliquot of
(PMe.sub.3).sub.4Co (18.9 mL of a 40 mg/mL in dry THF, 2.1 mmol)
was then added via syringe and the flask sealed and stirred for 45
minutes. An aliquot (50 .mu.L) was removed from the polymerization
for GPC analysis (Mn=14.7.times.10.sup.3 g/mol, Mw/Mn=1.12). The
stock poly(N.sub..epsilon.--CBZ-L-lysine).sub.55 was then divided
equally among 8 fractions (0.26 mmol (PMe.sub.3).sub.4Co initiator
in each) and placed in 125 mL flat bottomed flasks. To each
fraction, a different amount of hydrophobic D,L NCA was added as
needed. For example, to synthesize Z-K.sub.55(rac-L).sub.20 an
aliquot of D,L leucine (L) NCA (5.3 mL of a 50 mg/mL in THF, 1.7
mmol) was added and allowed to polymerize overnight.
[0109] A similar procedure was used to produce the following block
copolymers: Z-K.sub.55(rac-L).sub.5, D,L leucine NCA (1.3 mL of a
50 mg/mL in THF, 0.42 mmol); Z--K.sub.55(rac-L).sub.10, D,L leucine
NCA (2.7 mL of a 50 mg/mL in THF, 0.84 mmol);
Z--K.sub.55(rac-L).sub.30, D,L leucine NCA (7.9 mL of a 50 mg/mL in
THF, 2.5 mmol); Z--K.sub.55(rac-I).sub.20, D,L isoleucine (I) NCA
(5.3 mL of a 50 mg/mL in THF, 1.7 mmol);
Z-K.sub.55(rac-L/F).sub.20, D,L leucine NCA (2.6 mL of a 50 mg/mL
in THF, 0.84 mmol) and D,L phenylalanine (F) NCA (3.2 mL of a 50
mg/mL in THF, 0.84 mmol); Z--K.sub.55(rac-A).sub.20, D,L alanine
(A) NCA (3.9 mL of a 50 mg/mL in THF, 1.7 mmol); and
Z--K.sub.55(rac-V).sub.20, D,L valine (V) NCA (5.3 mL of a 50 mg/mL
in THF, 1.7 mmol).
[0110] Poly(L-LysineHCl).sub.55-b-poly-(rac-Leucine).sub.20,
K.sub.55(rac-L).sub.20. The
poly(N.sub..epsilon.--CBZ-L-lysine).sub.55-b-poly(rac-leucine).sub.20
was removed from the drybox. The THF was removed under reduced
pressure then dissolved in trifluoroacetic acid (TFA) (50 mL).
Next, the flask was placed in an ice bath followed by the addition
of HBr (33% in acetic acid, 6.0 mL, 19.7 mmol) and stirred for two
hrs. The deprotected polymer was isolated by addition of diethyl
ether to the reaction mixture (50 mL), followed by centrifugation
(three min at 3,000 rpm). The precipitated polymer was then washed
and centrifuged two more times with diethyl ether. The isolated
polymer was then dissolved in Millipore water and dialyzed (2,000
MWCO membrane) against tetrasodium EDTA (3 mmol, four days), 0.1 M
HCl (two days), DI water (one day), 0.1 M NaCl (two days),
Millipore water (two days), changing each solution two times/day.
The dialyzed polymer was isolated by freeze-drying to give the
product as a dry white powder (0.80 g, 84%).
[0111] A similar procedure was used to produce the following block
copolymers: K.sub.55(rac-L).sub.5 (0.51 g, 62%),
K.sub.55(rac-L).sub.10(0.70 g, 81%), K.sub.55(rac-L).sub.30 (0.77
g, 74%), K.sub.55(rac-I)).sub.20 (0.78 g, 81%),
K.sub.55(rac-L/F).sub.20 (0.74 g, 79%), K.sub.55(rac-A).sub.20(0.82
g, 92%), and K.sub.55(rac-V).sub.20 (0.82 g, 88%).
[0112] Poly(ethylene glycol).sub.205-b-poly(rac-leucine).sub.20,
PEG.sub.204(rac-L).sub.20. Prior to use, 0.50 g of .omega.-amino
terminated poly(ethylene glycol) monomethyl ether,
PEG.sub.205-NH.sub.2, (Mn=9,000 g/mol, PDI=1.08) was dried by
dissolving in dry benzene followed by removal of the solvent by
distillation to yield a dry solid. In a drybox,
PEG.sub.205-NH.sub.2 (0.50 g, 5.6.times.10.sup.-5 moles) was
dissolved in 4.0 mL of dry DMF. Next, L-Leucine NCA (83 mg, 0.53
mmol) and D-Leucine NCA (83 mg, 0.53 mmol) were dissolved in dry
DMF (2.5 mL) and then added to the polymerization mixture. The
solution stirred for three days at room temperature until fully
polymerized. It was then removed from the drybox and 5 mL of
Millipore water was added and then transferred to a dialysis
membrane (2,000 MWCO membrane) and dialyzed against Millipore water
(three days), changing each solution two times/day. The dialyzed
polymer was isolated by freeze-drying to give the product as a dry
white powder (0.51 g, 82%). .sup.1H-NMR
[0113] Poly(L-glutamate-Na).sub.54-b-poly(rac-leucine).sub.20,
E.sub.64(rac-L).sub.20. In the drybox, .gamma.-benzyl-L-glutamate,
Bzl-Glu NCA (5.00 g, 19 mmol) was placed in a 250 mL flat bottom
flask with a stir bar. Dry THF (100 mL) was added and then sealed
with a plastic stopper. An aliquot of (PMe.sub.3).sub.4Co (11.5 mL
of a 40 mg/mL in dry THF, 1.27 mmol) was then added via syringe and
the flask sealed and stirred for 1 hour. An aliquot (50 .mu.L) was
removed from the polymerization for GPC analysis
(Mn=13.9.times.10.sup.3 g/mol, Mw/Mn=1.27). Next, an aliquot of D,L
leucine (L) NCA (18.7 mL of a 50 mg/mL in THF, 6.0 mmol) was added
and allowed to polymerize overnight. Next, the THF was removed
under reduced pressure and then dissolved in dry CH.sub.2Cl.sub.2
(100 ml). To remove the benzyl protecting groups,
iodotrimethylsilane was added via syringe (10.8 mL, 76 mmol). A
reflux condenser was attached to the flask and refluxed overnight
at 40.degree. C. Next, the solvent was removed under reduced
pressure and 1 M NaOH was added and stirred overnight, then
filtered to remove precipitate and dialyzed (6-8,000 MWCO membrane)
against 5 mM sodium bisulfite and 0.1 M NaOH (three days), then
Millipore water (four days), changing each solution two times/day.
The clear solution was then freeze dried to afford a white fluffy
solid (1.26 g, 36%).
[0114] Poly(L-homoarginineHCl).sub.55-b-poly(rac-Leucine).sub.20,
R.sup.H.sub.55(rac-L).sub.20. To a 500 mL round bottom flask
containing a stir bar, K.sub.55(rac-L).sub.20 (1.00 g, 0.09 mmol)
was added and then dispersed in 1 M NaOH (137 mL). Next,
3,5-dimethyl-1-pyrazole formamidinium nitrate was added (3.93 g,
19.6 mmol). The pH was adjusted to pH=10 using HCl and then placed
into a 40.degree. C. oil bath and stirred for 48 hours. To quench
the reaction, the solution was acidified with 0.1 M HCl to a pH=3
then placed in a dialysis bag (2,000 MWCO) and dialyzed against
Millipore water (five days), changing each solution two times/day.
The dialyzed polymer was isolated by freeze-drying to give the
product as a white powder (0.95 g, 78%).
[0115] Poly(L-LysineHCl).sub.80-co-poly(L-Lysine-).sub.80,
K.sub.80(rac-L).sub.20. To a 50 mL polypropylene centrifuge tube
containing a stir bar, Poly(L-LysineHCl).sub.80, K.sub.80 (75 mg,
5.7 .mu.mol) was added and then dissolved in 50 mM
2-(N-morpholino)ethanesuffonic acid (MES) buffer (15 mL). Next,
tetrahydrofuran (THF) was added (14.3 mL). To this solution,
N-hydroxy succinimide (530 .mu.L of a 10 mg/mL solution in
THF/water, 46 .mu.mol), octanoic acid (660 .mu.L of a 10 mg/mL
solution in THF, 46 .mu.mol), and 1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide (2.6 mL of a 50 mg/mL solution in THF/water, 0.68
mmol) were added. The solution was allowed to stir overnight. The
next day, the solution was placed into a dialysis bag (2,000 MWCO)
and dialyzed against Millipore water (three days), 0.01 M HCl (two
days), 0.01 M NaOH (one day), 0.01 M HCl (one day), Millipore water
(two days), changing each solution two times/day. The dialyzed
polymer was isolated by freeze-drying to give the product as a
white powder (68 mg, 85%).
[0116] Critical Aggregation Concentration (CAC) via Pyrene
Fluorescence. Polypeptide solutions (2 mL) were dispersed in water
at a range of concentrations (2.0.times.10.sup.-3 to
2.0.times.10.sup.-12 M). A stock pyrene solution was made by
dissolving pyrene in acetone (6.0.times.10.sup.-2 M). Next, an
appropriate amount of the pyrene stock solution was added to give a
final concentration of 12.times.10.sup.-7 M in water and the
acetone was evaporated off. To each polypeptide solution, 2.0 mL of
the aqueous stock pyrene solution was added to afford a final
concentration of 6.0.times.10.sup.7 M. Then, each solution was
allowed to equilibrate overnight prior to measurements. To record
fluorescence spectra, 3.0 mL of each polypeptide solution was added
to a 4.0 mL polystyrene covet. The excitation spectra were recorded
within a range of 300-360 nm at an emission wavelength of 390 nm.
All spectra were run with an integration time of 1 sec/0.5 nm. The
ratio of the intensities of two peaks I338/I333 was plotted as a
function of polypeptide concentration (M) for each sample. The CACs
were determined as the intersection of the extrapolated linear fits
of the plot.
[0117] Emulsion Preparation. In a typical formulation, 800 .mu.L of
a 1 w/v % polypeptide solution was added to a 1.5 mL sterile
centrifuge tube. Next, 200 .mu.L of oil phase, typically
polydimethylsiloxane (PDMS) with a viscosity of 10 cSt (sterilized
by filtered through a 0.2 .mu.m sterile filter), was added to give
a final volume fraction, .PHI.=0.2. The solution was emulsified for
one minute using a hand-held ultrasonic homogenizer (Cole-Parmer
4710 Series Model ASI at an output of 35-40%) to form nanoscale
droplets (.about.400-500 nm in diameter based on dynamic light
scattering DLS measurements).
[0118] The following claims are thus to be understood to include
what is specifically illustrated and described above, what is
conceptually equivalent, what can be obviously substituted and also
what essentially incorporates the essential idea of the invention.
Those skilled in the art will appreciate that various adaptations
and modifications of the just-described preferred embodiment can be
configured without departing from the scope of the invention. The
illustrated embodiment has been set forth only for the purposes of
example and that should not be taken as limiting the invention.
Therefore, it is to be understood that, within the scope of the
appended claims, the invention may be practiced other than as
specifically described herein.
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