WO1999043357A1 - Indolicidin and cationic peptides conjugated with polymers - Google Patents

Abstract

Compositions and methods for modifying therapeutics with activated polyoxyalkylenes are provided. Therapeutics that may be modified contain or are derivatized to contain an amino or nucleophilic group. Therapeutics include peptides, proteins, antibiotics, and the like. Activation of polyoxyalkylenes is light-induced or chemical-induced.

Description

INDOLICIDIN AND CATIONIC PEPTIDES CONJUGATED WITH POLYMERS

TECHNICAL FIELD The present invention relates generally to polymer-modified therapeutics, such as polypeptides and antibiotics, and their uses.

BACKGROUND OF THE INVENTION

Candidate therapeutics may have sufficient efficacy but unacceptable toxicity, half life, or immunogenicity.

Approaches to reducing toxicity have included development of a derivative or delivery system that masks structural elements involved in the toxic response or that improves the efficacy at lower doses. Other approaches under evaluation include liposomes and micellular systems to improve the clinical effects of peptides, proteins, and hydrophobic drugs, and cyclodextrins to sequester hydrophobic surfaces during administration in aqueous media. For example, attachment of polyethylene glycol (PEG) polymers, most often by modification of amino groups, improves the medicinal value of some proteins such as asparaginase and adenosine deaminase, and increases circulatory half-lives of peptides and small proteins such as interleukins.

In the particular case of cationic peptides, none of these approaches are shown to improve administration. For example, methods for the stepwise synthesis of polysorbate derivatives that can modify peptides by acylation reactions have been developed, but acylation alters the charge of a modified cationic peptide and frequently reduces or eliminates the antimicrobial activity of the compound. Thus, for delivery of cationic peptides, as well as other peptides and proteins, there is a need for a system combining the properties of increased circulatory half-lives with the ability to form a micellular structure.

The present invention discloses methods and compositions for modifying peptides, proteins, antibiotics and the like to reduce toxicity, as well as providing other advantages. SUMMARY OF THE INVENTION

The present invention generally provides methods and compositions for modifying therapeutic compounds. In one aspect, the invention provides a compound that contains a polypeptide or antibiotic linked to a conjugate of a polyoxyalkylene and a lipophilic moiety, wherein the polypeptide or antibiotic is linked to the conjugate by a secondary amine. In preferred embodiments, the polyoxyalkylene is polyoxyethylene and/or the lipophilic moiety is a C₁₂-C₂₂ hydrocarbon group, where the hydrocarbon group may be derived from, for example, a fatty acid. The conjugate may further include sorbitan linking the polyoxyethylene and fatty acid, and/or may include polysorbate. The polypeptide is preferably a cationic peptide, where suitable cationic peptides include indolicidin or an indolicidin analogue. The secondary amine group may have the structure -NH-CH-, but does not have the -NH-C(O)- structure.

In other aspects, the invention provides a method of making a compound as described above. The method includes activating a conjugate of a polyoxyalkylene and a lipophilic moiety; freezing a mixture comprising the activated conjugate and a polypeptide or antibiotic; and lyophilizing the frozen mixture to form the compound. The mixture being frozen may include other components, including acetate buffer. In another method, the invention provides for making a compound modified with a conjugate of an activated polyoxyalkylene and a lipophilic moiety, the method including the steps of mixing the conjugate of an activated polyoxyalkylene and lipophilic moiety with the compound; the compound selected from polypeptides and antibiotics having a free amino group, for a time sufficient to form modified compounds, wherein the mixture is in a carbonate buffer having a pH greater than 8.5.

In the above methods, the conjugate or polyoxyalkylene may be activated by irradiation with UN light or by treatment with ammonium persulfate. Also in the above methods, the compound may be isolated by reverse-phase HPLC and/or by precipitation of the compound from an organic solvent. In the methods, the polypeptide is preferably a cationic polypeptide.

In other aspects, the invention provides a pharmaceutical composition that includes at least one compound as described above and a physiologically acceptable buffer. The pharmaceutical composition may further include one or more of an antibiotic agent, an antiviral agent, an antiparasitic agent and an antifungal agent. Also according to the present invention, the above-described compounds and pharmaceutical compositions may be used in treating an infection, where the treatment includes the step of administering to a patient a therapeutically effective amount of at least one of the above-described compounds and compositions. The infection may be due to, for example, a microorganism such as a bacterium, fungus, parasite and virus.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures, polypeptides, and/or compositions, and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C present RP-HPLC traces analyzing samples for APO- peptide formation after treatment of activated polysorbate with a reducing agent. APO- MBI-11CN peptides are formed via lyophilization in 200 mM acetic acid-NaOH, pH 4.6, 1 mg/ml MBI 11CN, and 0.5% activated polysorbate 80. The stock solution of activated 2.0% polysorbate is treated with (a) no reducing agent, (b) 150 mM 2- mercaptoethanol, or (c) 150 mM sodium borohydride for 1 hour immediately before use.

Figures 2A and 2B present RP-HPLC traces monitoring the formation of APO-MBI l lCN over time in aqueous solution. The reaction occurs in 200 mM sodium carbonate buffer pH 10.0, 1 mg/ml MBI 11CN, 0.5% activated polysorbate 80. Aliquots are removed from the reaction vessel at the indicated time points and immediately analyzed by RP-HPLC.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that are used herein. The amino acid designations herein are set forth as either the standard one-or three- letter code. A capital letter indicates an L-form amino acid; a small letter indicates a D-form amino acid. As used herein, an "antibiotic agent" refers to a molecule that tends to prevent, inhibit, or destroy life. The term "antimicrobial agent" refers to an antibiotic agent specifically directed to a microorganism.

"Polypeptide" as utilized herein refers to a series of amino acids linked by a peptidic bond. Polypeptides include dipeptides, peptides (e.g., a molecule composed of amino acids and having a molecule weight of less than about 10 kDa), proteins (e.g., a molecule composed of amino acids and having a molecular weight of greater than about lOkDa). Polypeptides may be either linear or branched, and may be produced, for example, by isolating naturally occurring polypeptides, synthetically, or, by recombinant means.

I. POLYMER MODIFICATION OF THERAPEUTICS

As noted herein, the present invention provides methods and compositions for modifying a compound with a free amine group. The amine group may be part of the native structure of the compound or added by a chemical method. Thus, peptides, proteins, certain antibiotics, nucleic acids and the like can be modified with an activated polyoxyalkylene and derivatives. When the compounds are peptides or proteins, the modified or derivatized forms .are referred to herein as "APO-modified peptides" or "APO-modified proteins". Similarly, modified forms of antibiotics are referred to as "APO-modified antibiotics." APO-modified compounds (e.g., APO- cationic peptides) generally exhibit improved pharmacological properties.

A. Characteristics of an activated polyoxyalkylene reagent

As discussed herein, a suitable reagent for formation of APO-modified compounds (e.g., peptides and proteins) comprises a hydrophobic region and a hydrophilic region, and optionally a linker. The hydrophobic region is a lipophilic compound with a suitable functional group for conjugation to the hydrophilic region or linker. The hydrophilic region is a polyoxyalkylene. As used herein, "polyoxyalkylene" refers to polymers (where the term polymers includes oligomers) having 2 or 3 carbon atom chains between oxygen atoms, i.e., having repeating units selected from (-0-C-C-) and (-0-C-C-C-). The carbon atoms of the repeating units may be substituted with, for example, alkyl or aryl groups. The polymer chain typically contains 2-100 repeating units, however may contain more than 100 repeating units. Polyoxyalkylenes having 2 carbon atom chains include polyoxyethylene and its derivatives, polyethylene glycol (PEG) of various molecular weights, and its derivatives such as polysorbate. Polyoxyalkylenes having 3 carbon atom chains include polyoxypropylene and its derivatives, and polypropylene glycol and its derivatives. Derivatives include alkyl- and aryl- polyoxyethylene compounds. The polyoxyalkylene preferably contains a free hydroxyl group.

The hydrophobic region is a lipophilic moiety, generally a fatty acid, but may be a fatty alcohol, fatty thiol, hydrocarbons (such as 4-(l,l,3,3-tetramethylbutyl)- cyclohexyl), aryl compounds (such as 4-(l,l,3,3-tetramethylbutyl)-phenyl) and the like, which are also lipophilic compounds. The fatty acid may be saturated or unsaturated

Chain lengths of Cj ₂-₂₂ ^are preferred.

The hydrophilic region is a polyoxyalkylene, such as polyethylene glycol monoether (for example Triton XI 14) and polysorbate. For polysorbate, the ether function is formed by the linkage between the polyoxyethylene chain, preferably having a chain length of from 2 to 100 monomeric units, and the sorbitan group.

Polymethylene glycol is unsuitable for administration in animals due to formation of formaldehydes, and glycols with a chain length of > 4 may be insoluble. Mixed polyoxyethylene-polyoxypropylene chains are also suitable. Suitable compounds include polyoxyethylenesorbitans and polyoxyethylene alkyl ethers. These and other suitable compounds may be synthesized by standard chemical methods, or may be obtained from commercial sources such as Aldrich (Milwaukee, WI) and J.B. Baker (New Jersey).

B. Activation of reagent

A polyoxyalkylene-containing compound, also referred to herein the reagent, is activated by exposure of the reagent to UV light with free exchange of air or by chemical treatment with ammonium persulfate, or a combination of these methods.

Exposure of the reagent to UV light, also referred to as photoactivation, may be achieved using a lamp that irradiates at 254 nm or 302 nm. Preferably, the output is centered at 254 nm. The use of longer wavelengths may require longer activation time. While some evidence exists that fluorescent room light can activate the polysorbates, experiments have shown that use of UV light at 254 nm yields maximal activation before room light yields a detectable level of activation.

Air plays an important role in the activation of the polyoxyalkylene reagent. Access to air doubles the rate of activation relative to activations performed in sealed containers. A shallow reaction chamber with a large surface area may be used to facilitate oxygen exchange. It is not yet known which gas is responsible; an oxygen derivative is likely, although peroxides are not involved. UV exposure of compounds with ether linkages is known to generate peroxides, which can be detected and quantified using peroxide test strips. In a reaction, hydrogen peroxide at 1 to 10 fold higher level than found in UV-activated material was added to a polysorbate solution in the absence of light. No activation was obtained.

The reagent is placed in a suitable vessel for irradiation. Studies with 2% polysorbate 80 indicate that 254 nm light at 1800 μW/cm2 is completely absorbed by the solution at a depth of 3-4 cm. Thus, the activation rate can be maximized by irradiating a relatively thin layer.

As such, a consideration for the vessel is the ability to achieve uniform irradiation. As noted above, a large shallow reaction chamber is desirable, however, it may be difficult to achieve on a large scale. To compensate, simple stirring that facilitates the replenishment of air in the solution achieves an equivalent result. Thus, if the pathlength is long or the reaction chamber is not shallow, the reagent may be mixed or agitated. The reagent can be activated in any aqueous solution and buffering is not required.

An exemplary activation takes place in a cuvette with a 1 cm liquid thickness. The reagent is irradiated at a distance of less than 9 cm at 1500 μW/cm² (initial source output) for approximately 24 hours. After treatment under these conditions, the activated reagent converts a minimum of 85% of the peptide to APO- peptide.

As noted above, the polyoxyalkylenes can be activated via chemical oxidation with ammonium persulfate. The activation is rapid and the extent of activation increases with the concentration of ammonium persulfate. Ammonium persulfate can be used in a range from about 0.01 % -0.5%, and most preferably from 0.025 to 0.1%), where percentage values are based on weight. If the levels of ammonium persulfate are too high, the peroxide byproducts can have an adverse effect on the compounds being modified. This adverse effect can be diminished by treatment of activated polyoxyalkylenes with mercaptoethanol, or another mild reducing agent, which does not inhibit the formation of APO-therapeutics. Peroxides generated from UV treatment can also be reduced by treatment with mercaptoethanol. Furthermore, as noted above, the UV procedure can be performed in conjunction with chemical activation.

C. Modification of peptides or proteins with activated reagent The therapeutics are reacted with the APO reagent in either a liquid or solid phase and become modified by the attachment of the APO derivative. The methods described herein for attachment offer the advantage of maintaining the charge on the therapeutic, such as a peptide or protein. When the charge of the peptide is critical to its function, such as the antibiotic activity of cationic peptides described herein, these attachment methods offer additional advantages. Methods that attach groups via acylation result in the loss of positive charge via conversion of amino to amido groups. In addition, no bulky or potentially antigenic linker, such as a triazine group, is known to be introduced by the methods described herein.

As noted above, APO-therapeutic formation occurs in solid phase or in aqueous solution. By way of example, briefly, in the solid phase method, a peptide or other therapeutic is suspended in a suitable buffer, such as an acetate buffer. Other suitable buffers that support APO-therapeutic formation may also be used. The acetate buffer may be sodium, potassium, lithium, and the like. Other acetate solutions, such as HAc or HAc-NaOH, are also suitable. A preferred pH range for the buffer is from 2 to 8.3, although a wider range may be used. When the starting pH of the acetic acid- NaOH buffer is varied, subsequent lyophilization from 200 mM acetic acid buffer yields only the Type I modified peptide. The presence of an alkaline buffer component results in the formation of Type II modified peptides. A typical peptide concentration is 1 mg/ml, which results in 85-95%> modified peptide, however other concentrations are suitable. The major consideration for determining concentration appears to be economic. The activated polymer (APO) is added in molar excess to the therapeutic. Generally, a starting ratio of approximately 2.5:1 (APO:therapeutic) to 5:1 (APO: therapeutic) generates APO-modified therapeutic in good yield.

The reaction mix is then frozen (e.g., -80°C) and lyophilized. Sodium acetate disproportionates into acetic acid and NaOH during lyophilization; removal of the volatile acetic acid by the vacuum leaves NaOH dispersed throughout the result solid matrix. This loss of acetic acid is confirmed by a pH increase detected upon dissolution of the lyophilizate. No APO-modified therapeutic is formed in acetate buffer if the samples are only frozen then thawed.

The modification reaction can also take place in aqueous solution. However, APO modifications do not occur at ambient temperature in any acetate buffer system tested regardless of pH. APO modifications also are not formed in phosphate buffers as high as pH 11.5. APO modification does occur in a sodium carbonate buffer at a pH greater than about 8.5. Other buffers may also be used if they support derivitization. A pH range of 9-11 is also suitable, and pH 10 is most commonly used. The reaction occurs in two phases: Type I peptides form first, followed by formation of

Type II peptides.

In the present invention, linkage occurs at an amino or a nucleophilic group. The amino group may be a primary amine, a secondary amine, or an aryl amine.

Nucleophilic groups that may be APO-modified include, but are not limited to, hydrazine derivatives and hydroxylamine derivatives. Preferably, the modification occurs at an amino group, more preferably at a primary or secondary amino group, and most preferably at a primary amino group. Examples of compounds that have been modified by the solid phase method are listed in Table 1 below.

Table 1

Compound Action Modification

.Amoxicillin penicillin antibiotic Yes

Amphotericin B anti-fungal No

Ampicillin penicillin antibiotic Yes

Bacitracin peptide antibiotic Yes

Cephalosporin C antibiotic No

Ciprofloxacin quinolone antibiotic Uncertain*

4,4'-Diaminodiphenyl Sulfone anti-leprotic Yes

Gentamicin aminoglycoside antibiotic Yes

Gramicidin S peptide antibiotic Yes Sulfadiazine sulfonamide antibiotic No

Vancomycin glycopeptide antibiotic Yes

*Ciprofloxacin was partially destroyed by the process.

For a peptide, linkage to the APO can occur at the α-NH₂ of the N- terminal amino acid or ε-NH₂ group of lysine. Other primary and secondary amines may also be modified. Complete blocking of all amino groups by acylation (MBI 11CN-Y1) inhibits APO-peptide formation. Thus, modification of arginine or tryptophan residues does not occur. If the only amino group available is the α-amino group (e.g., MBI 11B9CN and MBI 11G14CN), the Type I form is observed. The inclusion of a single lysine (e.g., MBI 11B1CN, MBI 11B7CN, MBI 11B8CN), providing an ε-amino group, results in Type II forms as well. The amount of Type II formed increases for peptides with more lysine residues.

Many antibiotics have free amine groups. Such antibiotics include but are not limited to ampicillin, amoxicillin, amikacin, ciprofloxacin, gentamicin, teicoplanin, tobramycin, and vancomycin. A number of these have been successfully modified by this protocol as summarized in Table 1.

Other types of compounds may be modified by introducing an APO group as described herein. For example, viruses lacking an envelope are candidates. Modification may reduce the antigenicity to viral vectors, such as adenoviruses, which are used in gene delivery protocols. Using the methods described herein, various peptides, including indolicidin, indolicidin analogues, gramicidin and bacitracin have been polymer modified.

D. Purification and physical properties of APO-modified therapeutics

The APO-modified therapeutics may be purified. In circumstances in which the free therapeutic, such as a peptide, is toxic, purification may be necessary to remove unmodified therapeutic and/or unreacted polyoxyalkylenes. .Any of a variety of purification methods may be used. Such methods include reverse phase HPLC, precipitation by organic solvent to remove polysorbate, size exclusion chromatography, 10

ion exchange chromatography, filtration and the like. RP-HPLC is preferred. Procedures for these separation methods are well known.

APO-therapeutic formation can result in the generation of products that are more hydrophobic than the parent compound. This property can be exploited to effect separation of the conjugate from free compound by RP-HPLC. As shown herein, peptide-conjugates are resolved into two populations based on their hydrophobicity as determined by RP-HPLC; the Type I population elutes slightly earlier than the Type II population.

The MBI 11 series of peptides have molecular weights between 1600 and 2500. When run on a Superose 12 column, a size exclusion column, these peptides adsorb to the resin, giving long retention times. In contrast, the APO-modified peptides do not adsorb and elute at 50 kDa (MBI 11CN-Tw80) and at 69 kDa (MBI 11A3CN-

Tw80), thus demonstrating a large increase in apparent molecular mass (Stokes radius).

An increase in apparent molecular mass could enhance the pharmacokinetics of peptides in particular because increased molecular mass reduces the rate at which peptides and proteins are removed from blood. Micelle formation may offer additional benefits by delivering "packets" of peptide molecules to microorganisms rather than relying on the multiple binding of single peptide molecules. In addition, APO-modified peptides are soluble in methylene chloride or chloroform (e.g., to at least 10 mg/mL), whereas the parent peptide is essentially insoluble. This increased organic solubility may significantly enhance the ability to penetrate tissue barriers and may be exploited for a simplified purification of the .APO-peptide. The increased solubility in organic media may also allow the formulation of peptides in oil or lipid based delivery systems which target specific sites, such as solid tumors. In addition, by circular dichroism (CD) studies, APO-modified peptides are observed to have an altered 3 -dimensional conformation. As shown in the Examples, MBI 11CN and MBI 11B7CN have unordered structures in phosphate buffer or 40%) aqueous trifluoroethanol (TFE) and form a β-turn conformation only upon insertion into liposomes. In contrast, CD spectra for .APO-modified MBI 11CN and APO-modified MBI 11B7CN indicate β-turn structure in phosphate buffer.

Cationic peptides appear to maintain their original charge after modification with an APO, thereby preventing loss of activity sometimes caused by 11

acylation reactions. Moreover, the present methods are not known to introduce antigenic linkers.

E. Biological properties of APO-modified therapeutics

The biological properties of APO-modified therapeutics appear to be improved compared to unmodified therapeutics. For example, modified and unmodified peptides have been compared. Because the product consists a peptide of known composition coupled to one or more polyoxyalkylene components derived from a polymeric mixture, defining an exact molecular weight for concentration calculations is not readily achieved. It is possible, however, to determine the concentration by spectrophotometric assay. Such a measurement is used to normalize APO-peptide concentrations for biological assays. For example, a lmg/mL MBI 11CN-Tw80 solution contains the same amount of cationic peptide as a 1 mg/mL solution of the parent peptide, thus allowing direct comparison of toxicity and efficacy data. The modified peptides have an equivalent MIC to unmodified peptides. In vivo, however, the modified peptides demonstrate a lower LC50 than the unmodified peptides against a panel of tumor cell lines. Thus, formation of .APO-peptides increases the potency of cationic peptides against cancer cells in culture.

In general, the efficacy of a modified therapeutic is determined by in vitro and in vivo assays used for the unmodified therapeutic. Thus, the assays employed depend upon the therapeutic. Assays for the therapeutics disclosed herein are well known. Such assays are available to those skilled in the art.

II. THERAPEUTICS

The present invention provides polymer-modified therapeutics. These therapeutics include any medically relevant compound and need only have a group suitable for modification. As noted herein, many therapeutics have such a group (e.g., amino group). Others can be derivatized to contain a suitable group.

A. Polypeptides

Within the context of the present invention, any polypeptide that has an amino group available for modification may be used. Generally, peptides and proteins have an NH₂-terminus. In some cases, the N-terminus may be blocked. In such cases, 12

modification can still occur at an ε-amino group of lysine, other nucleophilic group, or the protein (or peptide) can be reacted with a suitable reagent, such as Traut's reagent if a cysteine residue is present, to provide a primary amine for modification.

As used herein, a "peptide" is at least 5 amino acids in length. Unless otherwise indicated, a named amino acid refers to the L-form. Also included within the scope of peptides and proteins are variants that contain amino acid derivatives that have been altered by chemical means, such as methylation (e.g., a methylvaline), amidation, especially of the C-terminal amino acid by an alkylamine (e.g., ethylamine, ethanolamine, and ethylene diamine) and alteration of an amino acid side chain, such as acylation of the ε-amino group of lysine. Other amino acids that may be incorporated include any of the D-amino acids corresponding to the 20 L-amino acids commonly found in proteins, imino amino acids, rare amino acids, such as hydroxylysine, or non- protein amino acids, such as homoserine and ornithine. A peptide or protein may have none or one or more of these derivatives, and D-amino acids. In addition, a peptide may also be synthesized as a retro-, inverto- or retro-inverto-peptide.

Representative examples of polypeptides suitable for use within the present invention include cationic peptides such as indolicidin analogues (see, e.g., PCT Publication No. WO 98/07745). Other representative polypeptides are described in U.S Patent Nos. 4,822,608; 4,962,277; 4,980,163; 5,028,530; 5,096,886; 5,166,321 5,179,078 5,202,420 5,212,073 5,242,902 5,254,537 5,278,287 5,300,629 5,304,540 5,324,716 5,344,765 5,422,424 5,424,395 5,446,127 5,459,235 5,464,823 5,466,671 5,512,269 5,516,682 5,519,115 5,519,116 5,547,939 5,556,782 5,610,139 5,645,966 5,567,681 5,585,353 5,589,568 5,594,103 5,610,139 5,631,144 5,635,479 5,656,456 5,707,855 5,731,149 5,714,467 5,726,155 5,747,449; 5,756,462; PCT Publication Nos. WO 89/00199; WO 90/11766 WO 90/11771; WO 91/00869; WO 91/12815; WO 91/17760; WO 94/05251; WO 94/05156; WO 94/07528; WO 95/21601; WO 97/00694; WO 97/11713; WO 97/18826; WO 97/02287; WO 98/03192; WO 98/07833; WO 98/06425 European Application Nos. EP 17785; 349451; 607080; 665239; and Japanese Patent/Patent Application Nos. 4341179; 435883; 7196408; 798381; and 8143596.

Peptides may be synthesized by standard chemical methods, including synthesis by automated procedure. In general, peptides are synthesized based on the 13

standard solid-phase Fmoc protection strategy with HATU as the coupling agent. The peptide is cleaved from the solid-phase resin with trifluoroacetic acid containing appropriate scavengers, which also deprotects side chain functional groups. Crude peptide is further purified using preparative reversed-phase chromatography. Other purification methods, such as partition chromatography, gel filtration, gel electrophoresis, or ion-exchange chromatography may be used. Other synthesis techniques, known in the art, such as the tBoc protection strategy, or use of different coupling reagents or the like can be employed to produce equivalent peptides.

Peptides may be synthesized as a linear molecule or as branched molecules. Branched peptides typically contain a core peptide that provides a number of attachment points for additional peptides. Lysine is most commonly used for the core peptide because it has one carboxyl functional group and two (alpha and epsilon) amine functional groups. Other diamino acids can also be used. Preferably, either two or three levels of geometrically branched lysines are used; these cores form a tetrameric and octameric core structure, respectively (Tarn, Proc. Natl. Acad. Sci. USA 55:5409, 1988). To synthesize these multimeric peptides, the solid phase resin is derivatized with the core matrix, and subsequent synthesis and cleavage from the resin follows standard procedures. The multimeric peptide is typically then purified by dialysis against 4 M guanidine hydrochloride then water, using a membrane with a pore size to retain only multimers. The multimeric peptides may be used within the context of this invention as for any of the linear peptides.

Peptides may alternatively be synthesized by recombinant production. Recombinant production is preferred for proteins. A variety of host systems are suitable for production, including bacteria (e.g., E. colϊ), yeast (e.g., Saccharomyces cerevisiae), insect (e.g., Sf9), and mammalian cells (e.g., CHO, COS-7). Many expression vectors have been developed and are available for each of these hosts. Generally, bacteria cells and vectors that are functional in bacteria are used in this invention. However, at times, it may be preferable to have vectors that are functional in other hosts. Vectors and procedures for cloning and expression in E. coli and other organisms are discussed herein and, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1987) and in Ausubel et al. (Current Protocols in Molecular Biology, Greene Publishing Co., 1995). 14

Peptides and proteins are isolated by standard techniques, such as affinity, size exclusion, or ionic exchange chromatography, HPLC and the like. An isolated peptide or protein should preferably show a major band by Coomassie blue stain of SDS-PAGE that is at least 90% of the material.

B. Antibiotic Agents

An antibiotic agent includes any molecule that tends to prevent, inhibit or destroy life. As such, agents include anti-bacterial agents, anti-fungicides, anti-viral agents, and anti-parasitic agents. These agents may be isolated from an organism that produces the agent or procured from a commercial source (e.g., pharmaceutical company, such as Eli Lilly, Indianapolis, IN; Sigma, St. Louis, MO). Many of these agents have an amino or nucleophilic group for modification with the disclosed polymers. If no acceptable group is available, the agent may be derivatized by standard chemical methods to incorporated an amino group.

Anti-bacterial antibiotic agents include, but are not limited to, penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, sulfonamides, and fluoroquinolones (see Table 2 below).

15

Table 2

Class of Antibiotic Antibiotic Mode of Action

PENICILLINS Blocks the formation of new cell walls in bacteπa

Natural Penicillin G, Benzylpenicillm Penicillin V, Phenoxymethylpenicillm

Pemcillmase resistant Methicillm, Nafcillm, Oxacillm Cloxacillm, Dicloxacilhn

Acylammo-penicillms Ampicillm, Amoxicillm

Carboxy-penicilhns Ticarcillm, Carbenicillm

Ureido-penicillms Mezlocillin, Azlocillm, Piperacillm

CARBAPENEMS Imipenem, Meropenem Blocks the formation of new cell walls in bacteria

MONOBACTAMS Blocks the formation of new cell walls in bacteria

Aztreonam

CEPHALOSPORINS Prevents formation of new cell walls in bacteπa

1 st Generation Cephalothm, Cefazolm 2nd Generation Cefaclor, Cefamandole

Cefuroxime, Cefonicid,

Cefmetazole, Cefotetan, Cefprozil

3rd Generation Cefetamet, Cefoperazone

Cefotaxime, Ceftizoxime

Ceftπaxone, Ceftazidime

Cefixιme,Cefpodoxιme, Cefsulodm 4th Generation Cefepime

CARBACEPHEMS Loracarbef Prevents formation of new cell walls m bacteria CEPHAMYCINS Cefoxitm Prevents formation of new cell walls in bacteria

QUINOLONES Fleroxacm, Nahdixic Acid Inhibits bacterial DNA Norfloxacm, Ciprofloxacm synthesis

Ofloxacin, Enoxacm Lomefloxacm, Cmoxacm

16

Class of Antibiotic Antibiotic Mode of Action

TETRACYCLINES Doxycyclme, Mmocyclme, Inhibits bacterial protein Tetracyclme synthesis, binds to 30S πbosome subunit.

AMINOGLYCOSIDES Amikacm, Gentamicm, Kanamycm, Inhibits bacterial protein

Netilmicm, Tobramycm, synthesis, binds to 30S

Streptomycin πbosome subunit.

MACROLIDES Azithromycm, Clarithromycm, Inhibits bacterial protein Erythromycm synthesis, binds to 50S πbosome subunit

Derivatives of Erythromycm estolate, Erythromycm Erythromycm stearate

Erythromycm ethylsuccmate

Erythromycm gluceptate

Erythromycm lactobionate

GLYCOPEPTIDES Vancomycin, Teicoplanm Inhibits cell wall synthesis, prevents peptidoglycan elongation.

MISCELLANEOUS Chloramphemcol Inhibits bacterial protein synthesis, binds to 50S πbosome subunit. Chndamycm Inhibits bacterial protein synthesis, binds to 50S πbosome subunit.

Tπmethopπm Inhibits the enzyme dihydrofolate reductase, which activates folic acid. Sulfamethoxazole Acts as antimetabohte of P.ABA & inhibits synthesis of fohc acid

Nitrofurantom Action unknown, but is concentrated m urme where it can act on urinary tract bacteria

Rifamp Inhibits bacterial RNA polymerase Mupirocm Inhibits bacterial protein

synthesis 17

Anti-fungal agents include, but are not limited to, terbinafine hydrochloride, nystatin, amphotericin B, griseofulvin, ketoconazole, miconazole nitrate, flucytosine, fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic acid, and selenium sulfide. Anti- viral agents include, but are not limited to, amantadine hydrochloride, rimantadin, acyclovir, famciclovir, foscarnet, ganciclovir sodium, idoxuridine, ribavirin, sorivudine, trifluridine, valacyclovir, vidarabin, didanosine, stavudine, zalcitabine, zidovudine, interferon alpha, and edoxudine.

.Anti-parasitic agents include, but are not limited to, pirethrins/piperonyl butoxide, permethrin, iodoquinol, metronidazole, diethylcarbamazine citrate, piperazine, pyrantel pamoate, mebendazole, thiabendazole, praziquantel, albendazole, proguanil, quinidine gluconate injection, quinine sulfate, chloroquine phosphate, mefioquine hydrochloride, primaquine phosphate, atovaquone, co-trimoxazole (sulfamethoxazole/trimethoprim), and pentamidine isethionate.

C. Other Therapeutics

Other therapeutics are also useful within the context of this invention. Such therapeutics include any medically relevant compound that has a suitable group available for modification or can be derivatized to contain a suitable group. Examples of therapeutics include, but are not limited to, analgesics, antidiabetic agents, chemotherapeutics, viruses, antiarthritic compounds, anti-inflammatory compounds, antineoplastic agents, hormones, cardioprotective agents, contraceptives, migraine preparations, psychotherapeutic agents, respiratory drugs, and the like. Derivatization, if necess.ary, to provide an amino or nucleophilic group is performed by standard chemical methods known to those skilled in the art.

III. FORMULATIONS AND ADMINISTRATION

As noted above, the present invention provides compositions for modifying therapeutics for treating and preventing diseases and syndromes by administering to a patient a therapeutically effective amount of an .APO-modified therapeutic. Patients suitable for such treatment may be identified by well-established hallmarks. 18

Infections that may be treated with APO-peptides or APO-antibiotics include those caused by or due to microorganisms. Examples of microorganisms include bacteria (e.g., Gram-positive, Gram-negative), fungi, (e.g., yeast and molds), parasites (e.g., protozoans, nematodes, cestodes and trematodes), viruses, and prions. Specific organisms in these classes are well known (see for example, Davis et al., Microbiology, 3^rd edition, Harper & Row, 1980). Infections include, but -are not limited to, toxic shock syndrome, diphtheria, cholera, typhus, meningitis, whooping cough, botulism, tetanus, pyogenic infections, dysentery, gastroenteritis, anthrax, Lyme disease, syphilis, rubella, septicemia and plague. Effective treatment of infection may be examined in several different ways. The patient may exhibit reduced fever, reduced number of organisms, lower level of inflammatory molecules (e.g., IFN-γ, IL-12, IL-1, TNF), and the like.

The modified therapeutics of the present invention are preferably administered as a pharmaceutical composition. Briefly, pharmaceutical compositions of the present invention may comprise one or more of the APO-therapeutics described herein, in combination with one or more physiologically acceptable carriers, diluents, or excipients. As noted herein, the formulation buffer used may affect the efficacy or activity of the peptide analogue. A suitable formulation buffer contains buffer and solubilizer. The formulation buffer may comprise buffers such as sodium acetate, sodium citrate, neutral buffered saline, phosphate-buffered saline, and the like or salts, such as NaCl. Sodium acetate is preferred. In general, an acetate buffer from 5 to 500mM is used, and preferably from 100 to 200 mM. The pH of the final formulation may range from 3 to 10, and is preferably approximately neutral (about pH 7-8). Solubilizers, such as polyoxyethylenesorbitans (e.g., Tween 80, Tween 20) and polyoxyethylene ethers (e.g., Brij 56) may also be added if the compound is not already APO-modified. Although the formulation buffer is exemplified herein with peptide of the present invention, this buffer is generally useful and desirable for delivery of other therapeutics.

Additional compounds may be included in the compositions. These include, for example, carbohydrates such as glucose, mannose, sucrose or dextrose, mannitol, other proteins, polypeptides or amino acids, chelating agents such as EDTA or glutathione, adjuvants and preservatives. As noted herein, pharmaceutical 19

compositions of the present invention may also contain one or more additional active ingredients, such as an antibiotic or cytokine.

The compositions may be administered in a delivery vehicle. For example, the composition can be encapsulated in a liposome (see, e.g., WO 96/10585; WO 95/35094), complexed with lipids, encapsulated in slow-release or sustained release vehicles, such as poly-lactide, and the like. Within other embodiments, compositions may be prepared as a lyophilizate, utilizing appropriate excipients to provide stability.

Pharmaceutical compositions of the present invention may be administered in various manners, by intravenous injection, intraperitoneal injection or implantation, subcutaneous injection or implantation, intradermal injection, lavage, inhalation, implantation, intramuscular injection or implantation, intrathecal injection, bladder wash-out, suppositories, pessaries, topical (e.g., creams, ointments, skin patches, eye drops, ear drops, shampoos) application, enteric, oral, or nasal route. The modified therapeutic may be applied locally as an injection, drops, spray, tablets, cream, ointment, gel, and the like. The therapeutic may be administered as a bolus or as multiple doses over a period of time.

The level of therapeutic in serum and other tissues after administration can be monitored by various well-established techniques such as bacterial, chromatographic or antibody based, such as ELISA, and the like.

Pharmaceutical compositions of the present invention are administered in a manner appropriate to the infection or disease to be treated. The amount, route, and frequency of administration will be determined by factors such as the condition of the patient, the cause of the infection, and the severity of the infection. Appropriate dosages may be determined by clinical trials.

The modified compounds, especially the labeled ones, may be used in image analysis and diagnostic assays or for targeting sites in eukaryotic multicellular and single cell cellular organisms and in prokaryotes. As a targeting system, the modified compounds may be coupled with other peptides, proteins, nucleic acids, antibodies and the like. 20

The following examples are offered by way of illustration, and not by way of limitation.

21

EXAMPLES

EXAMPLE 1

ACTIVATION OF POLYSORBATE 80 BY ULTRAVIOLET LIGHT

A solution of 2% (w/w) polysorbate 80 is prepared in water and 200ml are placed in a 250mL crystallizing dish or over suitable container. Containers must have a clear light path. Cover the vessel with a piece of UV transparent plastic wrap or other UV transparent material. In addition, the material should allow the exchange of air but minimize evaporation. The solution is irradiated with ultraviolet light using a lamp emitting at

254 nm. Irradiation can also be performed using a lamp emitting at 302 nm. The solution should be stirred continuously to maximize the rate of activation. The activation is complete within 72 hours using a lamp with a output of 1800μW/cm². The reaction is monitored by a reversed-phased HPLC assay, which measures the formation of APO-MBI 11CN-Tw80 when the light-activated polysorbate is reacted with MBI 11CN.

Some properties of activated polysorbate are determined. Because peroxides are a known by-product of exposing ethers to UV light, peroxide formation is examined through the effect of reducing agents on the activated polysorbate. As seen in Figure 1, graph a, activated polysorbate readily reacts with MBI 11CN. Pre-treatment with 2-mercaptoethanol (Figure 1, graph b), a mild reducing agent, eliminates detectable peroxides, but does not cause a loss of conjugate forming ability. Treatment with sodium borohydride (Figure 1, graph c), eliminates peroxides and eventually eliminates the ability of activated polysorbate to modify peptides. Hydrolysis of the borohydride in water raises the pH and produces borate as a hydrolysis product. However, neither a pH change nor borate are responsible.

These data indicate that peroxides are not involved in the modification of peptides by activated polysorbate. Sodium borohydride should not affect epoxides or esters in aqueous media, suggesting that the reactive group is an aldehyde or ketone. The presence of aldehydes in the activated polysorbate is confirmed by using a formaldehyde test, which is specific for aldehydes including aldehydes other than formaldehyde. 22

Furthermore, activated polysorbate is treated with 2,4- dinitrophenylhydr.azine (DNPH) in an attempt to capture the reactive species. Three DNPH-tagged components are purified and analyzed by mass spectroscopy. These components are polysorbate-derived with molecular weights between 1000 and 1400. This indicates that low molecular weight aldehydes, such as formaldehyde or acetaldehyde, are involved.

EXAMPLE 2 ACTIVATION OF POLYSORBATE 80 BY AMMONIUM PERSULFATE

A 200 mL solution of 2% (w/w) polysorbate 80 is prepared in water. To this solution, 200 mg of ammonium persulfate is added while stirring. The reaction is stirred for 1-2 hours with protection from ambient light. If a solution of less than 0.1% (w/w) ammonium persulfate is used, then exposure to ultraviolet light at 254 nm during this period is used to help complete the reaction. The peroxide level in the reaction is determined using a test kit. Peroxides are reduced by titration with 2-mercaptoethanol.

EXAMPLE 3

FORMATION OF APO-MODIFIED PEPTIDES

APO-modified peptides are prepared either in solid phase or liquid phase. For solid phase preparation, 0.25 ml of 4 mg/ml of MBI 11CN is added to 0.5 ml of 0.4 M Acetic acid-NaOH pH 4.6 followed by addition of 0.25ml of UV-activated polysorbate. The reaction mix is frozen by placing it in a -80°C freezer. After freezing, the reaction mix is lyophilized overnight.

For preparing the conjugates in an aqueous phase, a sample of UV activated polysorbate 80 is first adjusted to a pH of 7.5 by the addition of 0.1M NaOH. This pH adjusted solution (0.5 ml) is added to 1.0 ml of 100 mM sodium carbonate, pH 10.0, followed immediately by the addition of 0.5 ml of 4 mg/ml of MBI 11CN. The reaction mixture is incubated at ambient temperature for 22 hours. The progress of the reaction is monitored by analysis at various time points using RP-HPLC (Figure 2). In 23

Figure 2, peak 2 is unreacted peptide, peak 3 is APO-modified peptide. Type 1 is the left-most of peak 3 and Type 2 is the right-most of peak 3.

Polysorbate 80 (TWEEN 80) at 2% (w/w) in water is activated. Ammonium persulfate (AP) is present at 0.05%> in the .AP and the .AP + UV samples. The UV and AP + UV samples are exposed to ultraviolet light at 254 nm using a lamp with an output of 1750-2000 μW/cm² during the time period. The reaction is stirred continuously with a magnetic stirrer at 100-200 rpm. Aliquots are removed and stored in the dark at -80°C until assayed. Aliquots are reacted with MBI11CN using the lyophilization method and the generation of MBI11CN-Tw80 was measured by RP- HPLC (Figure 2B).

Table 3 below summarizes data from several experiments. Unless otherwise noted in the table, the .APO-modified peptides are prepared via the lyophilization method in 200mM acetic acid-NaOH buffer, pH 4.6.

TABLE 3

COMPLEX

SEQUENCE NAME TYPE 1 TYPE 2

ILKKWPWWPWRRKamide 11CN

Solid phase, pH 2.0 Yes Low

Solid phase, pH 4.6 Yes Yes

Solid phase, pH 5.0 Yes Yes

Solid phase, pH 6.0 Yes Yes

Solid phase, pH 8.3 Yes Yes

Solution, pH 2.0 Trace Trace

Solution, pH 10.0 Yes Yes-Slow

(Ac)₄-ILKKWPWWPWRRKamide 11CN-Y1 No No

ILRRWPWWPWRRKamide 11B1CN Yes Lowered

ILRWPWWPWRRKamide 11B7CN Yes Lowered

ILWPWWPWRRKamide 11B8CN Yes Lowered

ILRRWPWWPWRRRamide 11B9CN Yes Trace

ILKKWPWWPWKKKamide 11B10CN Yes Yes iLKKWPWWPWRRkamide 11E3CN Yes Yes

ILKKWNWWPWRRKamide 11F3CN Yes Yes

ILKKWPWWPWKamide 11G13CN Yes Yes

ILKKWPWWPWRamide 11G14CN Yes Trace 24

The modification of amino groups is further analyzed by determining the number of primary amino groups lost during attachment. The unmodified and modified peptides are treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) (R.L. Lundblad in Techniques in Protein Modification and Analysis pp. 151-154, 1995). Briefly, a stock solution of MBI 11 CN at 4 mg/ml and an equimolar solution of APO-modified MBI 11CN are prepared. A 0.225 ml aliquot of MBI 11CN or APO-modified MBI 11CN is mixed with 0.225 ml of 200 mM sodium phosphate buffer, pH 8.8. A 0.450 ml aliquot of 1% TNBS is added to each sample, and the reaction is incubated at 37°C for 30 minutes. The absorbance at 367 nm is measured, and the number of modified primary amino groups per molecule is calculated using an extinction coefficient of 10,500 M^"1 cm^"1 for the trinitrophenyl (TNP) derivatives.

The primary amino group content of the parent peptide is then compared to the corresponding APO-modified peptide. As shown below in table 4, the loss of a single primary amino group occurs during formation of modified peptide. Peptides possessing a 3,4 lysine pair consistently give results that are 1 residue lower than expected, which may reflect steric hindrance after titration of one member of the doublet.

TABLE 4

TΝP/APO-

PEPTIDE SEQUENCE TΝP/PEPTIDE modified CHANGE peptide

ILKKWPWWPWRRKamide 2.71 1.64 1.07

ILRRWPWWPWRRKamide 1.82 0.72 1.10

IlKKWPWWPWRRkamide 2.69 1.61 1.08

ILKKWNWWPWRRKamide 2.62 1.56 1.06

Stability of APO-modified peptide analogues

.APO-modified peptides demonstrate a high degree of stability under conditions that promote the dissociation of ionic or hydrophobic complexes. APO- modified peptide in formulation D is prepared as 800 μg/ml solutions in water, 0.9%> saline, 8M urea, 8M guanidine-HCl, 67% 1-propanol, 1M HC1 and 1M NaOH and 25

incubated for 1 hour at room temperature. Samples are analyzed for the presence of free peptide using reversed phase HPLC and the following chromatographic conditions:

Solvent A: 0.1 % trifiuoroacetic acid (TFA) in water Solvent B: 0.1% TFA / 95% acetonitrile in water Media: POROS R2-20 (polystyrene divinylbenzene)

Elution: 0%> B for 5 column volumes

0-25%) B in 3 column volumes

25%o B for 10 column volumes

25-95%o B in 3 column volumes 95% B for 10 column volumes

Under these conditions, free peptide elutes exclusively during the 25 % B step and formulation-peptide complex during the 95%> B step. None of the dissociating conditions mentioned above, with the exception of 1M NaOH in which some degradation is observed, are successful in liberating free peptide from APO-modified peptide. Additional studies are carried out with incubation at 55°C or 85°C for one hour. APO-modified peptide is equally stable at 55°C and is only slightly less stable at

85°C. Some acid hydrolysis, indicated by the presence of novel peaks in the HPLC chromatogram, is observed with the 1M HC1 sample incubated at 85°C for one hour.

EXAMPLE 4

PURIFICATION OF APO-MODIFIED MBI 1 ICN

A large scale preparation of APO-modified MBI 11CN is purified. Approximately 400 mg of MBI 1 ICN is APO-modified and dissolved in 20ml of water. Unreacted MBI 11CN is removed by RP-HPLC. The solvent is then evaporated from the APO-modified MBI 11CN pool, and the residue is dissolved in 10 ml methylene chloride. The modified peptide is then precipitated with 10 ml diethyl ether. After 5 min at ambient temperature, the precipitate is collected by centrifugation at 5000xg for 10 minutes. The pellet is washed with 5 ml of diethyl ether and again collected by centrifugation at 5000xg for 10 minutes. The supernatants are pooled for analysis of unreacted polysorbate by-products. The precipitate is dissolved in 6 ml of water and 26

then flushed with nitrogen by bubbling for 30 minutes to remove residual ether. The total yield from the starting MBI 1 ICN was 43%.

The crude APO-MBI29-Tw80 prepared from 200 mg of MBI 29 is suspended in 40mL of methylene chloride and sonicated for 5 minutes to disperse large particles. The suspension is centrifuged in appropriate containers (Corning glass) at

3000-4000 x g for 15 minutes at 10°C to sediment insoluble material. The supernatant is decanted and saved.

The sediment is extracted twice more by adding 40 mL portions methylene chloride to the sediment and repeating the sonication/centrifugation step. The supematants from the three extractions are pooled and concentrated 8-10 fold using a rotary evaporator. The solution is transferred to centrifuge tubes and 3 volumes of diethyl ether are added. The mixture is incubated for 15 minutes, then centrifuged at 3000-4000 x g for 15 minutes at 10°C to sediment the product. The supernatant is decanted and discarded. The residual ether may be removed with a stream of nitrogen.

EXAMPLE 5 IN VITRO ASSAYS TO MEASURE APO-CATIONIC PEPTIDE ACTIVITY

A. Agarose Dilution Assay

The agarose dilution assay measures antimicrobial activity of peptides and peptide analogues, which is expressed as the minimum inhibitory concentration (MIC) of the peptides.

In order to mimic in vivo conditions, calcium and magnesium supplemented Mueller Hinton broth is used in combination with a low EEO agarose as the bacterial growth medium. The more commonly used agar is replaced with agarose as the charged groups in agar prevent peptide diffusion through the media. The media is autoclaved and then cooled to 50 - 55° C in a water bath before aseptic addition of antimicrobial solutions. The same volume of different concentrations of peptide solution are added to the cooled molten agarose that is then poured to a depth of 3 - 4 mm. The bacterial inoculum is adjusted to a 0.5 McFarland turbidity standard

(PML Microbiological) and then diluted 1:10 before application on to the agarose plate. 27

The final inoculum applied to the agarose is approximately 10⁴ CFU in a 5 - 8 mm diameter spot. The agarose plates are incubated at 35 - 37°C for 16 to 20 hours.

The MIC is recorded as the lowest concentration of peptide that completely inhibits growth of the organixm as determined by visual inspection. Representative MICs for various modified and unmodified cationic peptides are shown in Table 5 below.

28

TABLE 5

Corrected MIC(μg/mL)

Organism Organism # APO-Peptide APO-Peptide Peptide

A. calcoaceticus AC002 MBI11CN-Tw80 4 4

A. calcoaceticus AC002 MBI11B1CN-Tw80 4 2

A. calcoaceticus AC002 MBI11B7CN-Tw80 4 2

A. calcoaceticus AC002 MBI11B7CN-Txl l4r 2 2

A. calcoaceticus AC002 MBI11B7CN-F12-Txl l4r 1 1

A. calcoaceticus AC002 MBI11E3CN-Tw80 2 1

A. calcoaceticus AC002 MBI11F3CN-Tw80 8 2

A. calcoaceticus AC002 MBI11F4CN-Tw80 4 4

A. calcoaceticus AC002 MBI29-Tw80 4 1

E. cloacae ECL007 MBI11CN-Tw80 >128 >128

E. cloacae ECL007 MBI11B1CN-Tw80 128 >128

E. cloacae ECL007 MBI11B7CN-Tw80 >128 128

E. cloacae ECL007 MBI11B7CN-Txl l4r 128 128

E. cloacae ECL007 MBI11B7CN-F12-Txl l4r >128 >128

E. cloacae ECL007 MBI11E3CN-Tw80 128 >128

E. cloacae ECL007 MBI11F3CN-Tw80 128 >128

E. cloacae ECL007 MBI11F4CN-Tw80 64 32

E. cloacae ECL007 MBI29-Tw80 32 >64

E.coli ECO005 MBI11CN-Tw80 16 8

E.coli ECO005 MBI11B1CN-Tw80 8 8

E.coli ECO005 MBI11B7CN-Tw80 16 4

E.coli ECO005 MBI11B7CN-Txl l4r 16 4

E.coli ECO005 MBIl 1B7CN-F12-Txl 14r 32 16

E.coli ECO005 MBI11E3CN-Tw80 8 4

E. coli ECO005 MBI11F3CN-Tw80 128 16

E. coli ECO005 MBI11F4CN-Tw80 8 8

E.coli ECO005 MBI29-Tw80 16 4

E. faecalis EFS001 MBI11CN-Tw80 8 32

E. faecalis EFS001 MBI11B1CN-Tw80 4 32

E. faecalis EFS001 MBI11B7CN-Tw80 8 8

E. faecalis EFS001 MBI11B7CN-Txl l4r 0.5 0.5

E. faecalis EFS001 MBIl 1B7CN-F12-Txl 14r 0.5 0.5

E. faecalis EFS001 MBI11E3CN-Tw80 4 8

E. faecalis EFS001 MBI11F3CN-Tw80 8 32

E. faecalis EFS001 MBI29-Tw80 0.5 0.5

E. faecalis EFS004 MBI11CN-Tw80 4 8

E. faecalis EFS004 MBI11B1CN-Tw80 4 8

E. faecalis EFS004 MBI11B7CN-Tw80 8 8

E. faecalis EFS004 MBI11E3CN-Tw80 4 2

E. faecalis EFS004 MBI11F3CN-Tw80 4 16

E. faecalis EFS008 MBI11CN-Tw80 1 16

E. faecalis EFS008 MBI11B1CN-Tw80 1 2

E. faecalis EFS008 MBI11B7CN-Tw80 1 2

E. faecalis EFS008 MBI11B7CN-Txl l4r 2 4

E. faecalis EFS008 MBIl 1B7CN-F12-Txl 14r 2 2

E. faecalis EFS008 MBI11E3CN-Tw80 1 2

E. faecalis EFS008 MBI11F3CN-Tw80 4 16

E. faecalis EFS008 MBI11F4CN-Tw80 2 2

E. faecalis EFS008 MBI29-Tw80 2 0.5

K. pneumoniae KP001 MBI11CN-Tw80 8 16 29

Corrected MIC(μg/mL)

Organism Organism # APO-Peptide APO-Peptide Peptide

K. pneumoniae KP001 MBI11B1CN-Tw80 8 8

K. pneumoniae KP001 MBI11B7CN-Tw80 8 4

K. pneumoniae KP001 MBI11B7CN-Txl l4r 8 8

K. pneumoniae KP001 MBI11B7CN-F12-Txl l4r 32 16

K. pneumoniae KP001 MBI11E3CN-Tw80 4 8

K. pneumoniae POOl MBI11F3CN-Tw80 128 64

K. pneumoniae KP001 MBIl 1F4CN-Tw80 8 4

K. pneumoniae KPOOl MBI29-Tw80 16 2

P. aeruginosa PA004 MBI11CN-Tw80 >128 128

P. aeruginosa PA004 MBI11B1CN-Tw80 128 64

P. aeruginosa PA004 MBI11B7CN-Tw80 128 128

P. aeruginosa PA004 MBI11B7CN-Txl l4r 128 128

P. aeruginosa PA004 MBI11B7CN-F12-Txl l4r >128 >128

P. aeruginosa PA004 MBI11E3CN-Tw80 64 32

P. aeruginosa PA004 MBI11F3CN-Tw80 128 128

P. aeruginosa PA004 MBI11F4CN-Tw80 128 32

P. aeruginosa PA004 MBI29-Tw80 >64 16

S. aureus SAO 10 MBI11B1CN 4 1

S. aureus SAO 10 MBI11B7CN 4 1

S. aureus SA010 MBIl ICN 4 2

S. aureus SAO 10 MBI11E3CN 2 1

S. aureus SAO 10 MBI11F3CN 4 2

S. aureus SAO 11 MBI11CN-Tw80 16 8

S. aureus SA011 MBI11B1CN-Tw80 16 4

S. aureus SA011 MBI11B7CN-Tw80 16 4

S. aureus SA011 MBI11E3CN-Tw80 16 4

S. aureus SA011 MBI11F3CN-Tw80 16 8

S. aureus SA014 MBI11CN-Tw80 2 1

S. aureus SA014 MBI11B1CN-Tw80 2 1

S. aureus SA014 MBI11B7CN-Tw80 1 2

S. aureus SA014 MBI11B7CN-Txl l4r 2 1

S. aureus SA014 MBI11B7CN-F12-Txl l4r 2 2

S. aureus SA014 MBI11E3CN-Tw80 1 1

S. aureus SA014 MBI11F3CN-Tw80 8 8

S. aureus SA014 MBI11F4CN-Tw80 2 2

S. aureus SA014 MBI29-Tw80 2 1

S. aureus SAO 18 MBI11CN-Tw80 64 64

S. aureus SAO 18 MBI11B1CN-Tw80 32 16

S. aureus SAO 18 MBI11B7CN-Tw80 32 16

S. aureus SA018 MBI11E3CN-Tw80 32 16

S. aureus SAO 18 MBI11F3CN-Tw80 64 16

S. aureus SA025 MBI11CN-Tw80 2 4

S. aureus SA025 MBI11B1CN-Tw80 4 1

S. aureus SA025 MBI11B7CN-Tw80 2 1

S. aureus SA025 MBI11E3CN-Tw80 2 1

S. aureus SA025 MBI11F3CN-Tw80 4 2

S. aureus SA093 MBI11CN-Tw80 2 2

S. aureus SA093 MBI11B1CN-Tw80 2 1

S. aureus SA093 MBI11B7CN-Tw80 2 1

S. aureus SA093 MBI11B7CN-Txl l4r 1 1

S. aureus SA093 MBIl 1B7CN-F12-Txl 14r 1 1

S. aureus SA093 MBI11E3CN-Tw80 2 1 30

Corrected M3C(μg/mL)

Organism Organism # APO-Peptide APO-Peptide Peptide

S. aureus SA093 MBI11F3CN-Tw80 2 1

S. aureus SA093 MBI29-Tw80 1 0.5

S. epidermidis SE010 MBI11B7CN-Txl l4r 4 2

S. epidermidis SE010 MBI11B7CN-F12-Txl l4r 4 8

S. epidermidis SE010 MBI29-Tw80 >64 4

S. maltophilia SMA002 MBI11CN-Tw80 32 >128

S. maltophilia SMA002 MBI11B1CN-Tw80 32 32

S. maltophilia SMA002 MBI11B7CN-Tw80 64 16

S. maltophilia SMA002 MBI11B7CN-Txl l4r 32 16

S. maltophilia SMA002 MBIl 1B7CN-F12-Txl 14r 64 64

S. maltophilia SMA002 MBI11E3CN-Tw80 128 64

S. maltophilia SMA002 MBI11F3CN-Tw80 128 64

S. maltophilia SMA002 MBI11F4CN-Tw80 32 16

S. maltophilia SMA002 MBI29-Tw80 8 2

S. marcescens SMS003 MBI11CN-Tw80 >128 >128

S. marcescens SMS003 MBI11B1CN-Tw80 >128 >128

S. marcescens SMS003 MBI11B7CN-Tw80 >128 >128

S. marcescens SMS003 MBI11B7CN-Txl l4r >128 >128

S. marcescens SMS003 MBI11B7CN-F12-Txl l4r >128 >128

S. marcescens SMS003 MBI11E3CN-Tw80 128 >128

S. marcescens SMS003 MBI11F3CN-Tw80 128 >128

S. marcescens SMS003 MBI11F4CN-Tw80 >128 >128

S. marcescens SMS003 MBI29-Tw80 >64 >128

B. Broth Dilution Assay

This assay also uses calcium and magnesium supplemented Mueller Hinton broth as the growth medium. Typically 100 μl of broth is dispensed into each well of a 96-well microtitre plate and 100 μl volumes of two-fold serial dilutions of the peptide analogue are made across the plate. One row of wells receives no peptide and is used as a growth control. Each well is inoculated with approximately 5 x 10⁵ CFU of bacteria and the plate is incubated at 35 - 37°C for 16-20 hours. The MIC is again recorded at the lowest concentration of peptide that completely inhibits growth of the organism as determined by visual inspection.

C. Time Kill Assay

Time kill curves are used to determine the antimicrobial activity of cationic peptides over a time interval. Briefly, in this assay, a suspension of microorganisms equivalent to a 0.5 McFarland Standard is prepared in 0.9%> saline. This suspension is then diluted such that when added to a total volume of 9 ml of cation-adjusted Mueller Hinton broth, the inoculum size is 1 x 10⁶ CFU/ml. An aliquot 31

of 0.1 ml is removed from each tube at pre-determined intervals up to 24 hours, diluted in 0.9%o saline and plated in triplicate to determine viable colony counts. The number of bacteria remaining in each sample is plotted over time to determine the rate of cationic peptide killing. Generally a three or more log₁₀ reduction in bacterial counts in the antimicrobial suspension compared to the growth controls indicate an adequate bactericidal response.

As shown in Figure 3, all peptides demonstrated a three or more log₁₀ reduction in bacterial counts in the antimicrobial suspension compared to the growth controls indicating that these peptides have met the criteria for a bactericidal response.

D. Synergy Assay

Treatment with a combination of peptide analogues and conventional antibiotics can have a synergistic effect. Synergy is assayed using the agarose dilution technique, where an array of plates, each containing a combination of peptide and antibiotic in a unique concentration mix, is inoculated with the bacterial isolates. Synergy is investigated for peptide analogues in combination with a number of conventional antibiotics including, but not limited to, penicillins, cephalosporins, carbapenems, monobactams, aminoglycosides, macrolides, fluoroquinolones.

Synergy is expressed as a Fractional Inhibitory Concentration (FIC), which is calculated according to the equation below. An FIC of less than or equal to 0.5 is evidence of synergy, although combinations with higher values may be therapeutically useful.

FIC = MIC (peptide in combination) + MIC (^'antibiotic in combination) MIC (peptide alone) MIC (antibiotic alone)

E. In vitro Assays to Measure APO-Antibiotic Activity

Representative MICs for various antibiotics and modified forms thereof .are provided below in tables 6 to 8.

TABLE 6

MIC COMPARISON OF VANCOMYCIN AND MODIFIED- VANCOMYCIN

Organism | Number | Vancomycin | Vancomycin-Tw80 32

Organism Number Vancomycin Vancomycin-Tw80

A. calcoaceticus AC002 4 32

E. cloacae ECL007 >128 >128

E. coli ECO005 >128 >128

K. pneumoniae KPOOl >128 >128

P. aeruginosa PA004 >128 >128

S. maltophilia SMA002 128 >128

S. marcescens SMS003 >128 >128

C. jeikeium CJK005 ≤0.25 2

E. faecalis EFS001 1 4

S. aureus SA014 1 4

S. epidermidis SE010 1 4

S. mitis SMT014 0.5 2

S. pnuemoniae SPN002 <0.25 <0.25

S. pyogenes SPY001 <0.25 2

C albicans CA002 >128 >128

C. neoformans CNE001 >128 >128

TABLE 7

MIC COMPARISON OF VANCOMYCIN, AMOXICILLIN, AND APO-MODIFIED VERSIONS

Organism Number Vancomycin Vancomycin- .Amoxicillin .Amoxicillin Tw80 -Tw80

A. calcoaceticus AC002 4 32 16 >64

E. cloacae ECL007 >128 >128 >128 >64

E. coli ECO005 >128 >128 >128 >64

K. pneumoniae KPOOl >128 >128 >128 >64

P. aeruginosa PA004 >128 >128 >128 >64

S. maltophilia SMA002 128 >128 >128 >64

S. marcescens SMS003 >128 >128 >128 >64

C. jeikeium CJK005 <0.25 2 >128 >64

E. faecalis EFS001 1 4 32 >64

S. aureus SA014 1 4 64 >64

S. epidermidis SE010 1 4 4 >64

S. mitis SMT014 0.5 2 4 32

S. pnuemoniae SPN002 <0.25 <0.25 0.5 2

S. pyogenes SPY001 <0.25 2 <0.25 2

C. albicans CA002 >128 >128 >128 >64

C neoformans CNE001 >128 >128 32 2 33

TABLE 8

EFFECT OF MODIFICATION WITH POLYOXYETHYLENE ETHERS ON THE MIC OF 11B7CN

Organism Number 11B7CN 11B7CN- 11B7CN- 11B7CN- EG5C8 EG10C18Δ9 EG20C16

A. calcoaceticus AC002 4 4 8 8

E. cloacae ECL007 >64 >64 >64 >64

E. coli ECO005 8 >64 32 32

K. pneumoniae KPOOl 64 >64 32 32

P. aeruginosa PA004 128 >64 >64 >64

S. maltophilia SMA002 16 >64 32 32

S. marcescens SMS003 >128 >64 >64 >64

C. jeikeium CJK005 1 1 2 2

EXAMPLE 6 BIOLOGICAL ASSAYS USING APO-MODIFIED PEPTIDE

All biological assays that compare APO-modified peptides with unmodified peptides are performed on an equimolar ratio. The concentration of .APO- modified peptides can be determined by spectrophotometric measurement, which is used to normalize concentrations for biological assays. For example, a 1 mg/ml APO- modified MBI 11CN solution contains the same amount of peptide as a 1 mg/ml MBI 1 ICN solution, thus allowing direct comparison of toxicity and efficacy data.

APO-modified peptides are at least as potent as the parent peptides in in vitro assays performed as described herein. MIC values against gram positive bacteria are presented for several .APO-modified peptides and compared with the values obtained using the parent peptides (Table 9). The results indicate that the modified peptides are at least as potent in vitro as the parent peptides and may be more potent than the parent peptides against E. faecalis strains. Toxicities of APO-modified peptides and unmodified peptides are examined in Swiss CD-I mice. Groups of up to 6 mice are injected intravenously (0.1 ml to 0.25 ml volumne) with single doses of peptide in 0.9% saline. The dose levels used are between 0 and 128 mg/kg. Mice are monitored at 1, 3, and 6 hrs post-injection for the first day, then twice daily for 7 days. From the survival data, the maximum tolerated dose (MTD) of test article is determined. The results of the MTD 34

determination of selected peptides and their APO modified peptides is described in Table 6. The data show that APO-modified peptides are significantly less toxic than the parent peptides.

TABLE 9 MTD VALUES OF APO MODIFIED PEPTIDES AND THEIR PARENT PEPTIDES

IV. Test Peptide MTD (mg/kg)

MBI-11CN-TFA 5

APO-MBI- 1 ICN >13

MBI-11A3CN-TFA 4

APO-MBI-11A3CN 48

MBI-11B7CN-TFA 8

APO-MBI- 11B7CN 16

MBI-11B7CN-EG20C16 20

MBI-11B7CN-EG20C18:9 10

MBI-11B7CN-EG9C12 16

MBI-11J02CN-TFA 20

APO-MBI- 11J02CN 48

In addition, APO-peptides and parent peptides are tested against a panel of cancer cell lines. Cell death is measured using the Cytotox (Promega) assay kit which measures the release of lactate dehydrogenase. As shown below in table 10, the modified peptides had increased activity over the parent peptides.

TABLE 10

CELL LINE, LC₅₀, μg/mL±J 5.E.

Peptide PBL HUVEC H460 K562 DoHH-2 P388 P388ADR MCF-7 MCF-7ADR

1 1CN 57 >190 200 30 25 1 1.8+9 17±1

1 1CN-Tw80 6±6 16+4 16±4 1.9+5 3.5+2 11

1 1A3CN >500 >500 >500 >500 >500 >300 >300

1 1A3CN-Tw80 12.7+15 17±9 15+4 6 3.3+0.05 5.6±2 6.6±3 28 13

11B7CN 24+10 90±23 26+25 34+25 16.5+3 13.8 >700

1 1B7CN-Tw80 3.8+1 12.8±8 >100 4.7+3 3.3+1 5.1 12

11E3CN 22+11 117±7 18 9 3.6 13.9±3 7.9±3 5.6±2 5.3+1

11E3CN-Tw80 4.5+2 12.8±2 8.2+4 4.9±3 3.5±0.7 5.9+3 8.4+1 8.1+5 7.6±2

21A11 30±15 184+100 48 56±33 9.8+0.3

21A1 1-Tw80 4.5±4 17+9.9 21 4.3±2 4.7±0.6 8.1+3.4 9 18

29 12+10 10 12.6±10 1 2.1±0.5 1.4+0.5 2+0.2 4±2 3.2+1

29-Tw80 8.7±6 9.3+2 1.7 2.1±0.5 4±0.5 7.6+2.4 7.6+2 15.5+6 9.1+5

PBL, peripheral blood lymphocytes; HUVEC, human umbilical vein endothelial cells; H460, non-small lung tumor; K562, chronic myelogenous leukemia; DoHH-2, B-cell 35

cell lymphoma; P388, lymphocytic leukemia; P388.ADR, lymphocytic leukemia, multidrug resistant; MCF-7, breast carcinoma; MCF-7ADR, breast carcinoma, multidrug resistant.

EXAMPLE 7

STRUCTURAL ANALYSIS OF APO-INDOLICIDIN VARIANTS USING CIRCULAR DICHROISM SPECTROSCOPY Circular dichroism (CD) is a spectroscopic technique that measures secondary structures of peptides and proteins in solution, see for example, R.W. Woody, (Methods in Enzymology, 246: 34, 1995). The CD spectra of α-helical peptides is most readily interpretable due to the characteristic double minima at 208 and 222 nm. For peptides with other secondary structures however, interpretation of CD spectra is more complicated and less reliable. The CD data for peptides is used to relate solution structure to in vitro activity.

CD measurements of indolicidin analogues are performed in three different aqueous environments, (1) 10 mM sodium phosphate buffer, pH 7.2, (2) phosphate buffer and 40%> (v/v) trifluoroethanol (TFE) and (3) phosphate buffer and large (100 nm diameter) unilamellar phospholipid vesicles (liposomes) (Table 9). The organic solvent TFE and the liposomes provide a hydrophobic environment intended to mimic the bacterial membrane where the peptides are presumed to adopt an active conformation.

The results indicate that the peptides in Table 9 are unordered in phosphate buffer (a negative minima at around 200 nm) with the exception of MBI 11F4CN, which displays an additional minima at 220 nm (see below). In the presence of liposomes, peptides MBI 11CN and MBI 11B7CN, which are unordered in TFE, display β-turn structure (a negative minima at around 230 nm) (Figure 4). Hence, liposomes appear to induce more ordered secondary structure than TFE. A β-turn is the predominant secondary structure that appears in a hydrophobic environment, suggesting that it is the primary conformation in the active, membrane-associated form

CD spectra are recorded for APO-modified parent peptides (Table 11). The results show that these compounds have significant β-turn secondary structure in 36

phosphate buffer, which is only slightly altered in TFE. This suggests that the peptide moiety in APO-peptides adopts a conformation similar to the conformation adopted by the parent peptide when it interacts with a membrane.

TABLE 11

Peptide Phosphate buffer Conformation TFE Conformation min λ 1 Max λ in buffer min λ max λ in TFE polymer-modified peptides:

MBI l ICN 202, 229 220 β-tum 203 223 β-tum

MBI 11ACN 200, 229 - β-tum 202 222 β-tum

MBI 11B7CN 202, 230 223 β-tum 199 230 β-tum

MBI 11E3CN 202, 229 220 β-tum 199 - β-tum

MBI 11F3CN 205 - PpII helix 203 230 ppll helix

It will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

37CLAIMS We claim:

1. A compound comprising a polypeptide or antibiotic linked to a conjugate of a polyoxyalkylene and a lipophilic moiety, wherein the polypeptide or antibiotic is linked to the conjugate by a secondary amine.

2. The compound of claim 1, wherein the polyoxyalkylene is polyoxyethylene.

3. The compound of claim 1, wherein the lipophilic moiety is a C╬╣₂-C₂₂ hydrocarbon.

4. The compound of claim 1, wherein the polyoxyalkylene is polyoxyethylene and the lipophilic moiety is a fatty acid.

5. The compound of claim 4, wherein the conjugate further comprises sorbitan linking the polyoxyethylene and fatty acid.

6. The compound of claim 4, wherein the conjugate comprises polysorbate.

7. The compound of claim 1, wherein the polypeptide is a cationic peptide.

8. The compound of claim 8, wherein the polypeptide is indolicidin or an indolicidin analogue.

9. The compound of claim 1 wherein the polypeptide or antibiotic is linked to the conjugate by a -NH-CH- group.

10. A method of making a compound according to claim 1, comprising: (a) activating a conjugate of a polyoxyalkylene and a lipophilic moiety; 38

(b) freezing a mixture comprising the activated conjugate of step (a) and a polypeptide or antibiotic; and

(c) lyophilizing the frozen mixture to form the compound.

11. The method of claim 10 wherein the mixture of step (b) further comprises acetate buffer.

12. A method of making a compound modified with a conjugate of an activated polyoxyalkylene and a lipophilic moiety, comprising mixing the conjugate of an activated polyoxyalkylene and lipophilic moiety with the compound; the compound selected from polypeptides and antibiotics having a free amino group, for a time sufficient to form modified compounds, wherein the mixture is in a carbonate buffer having a pH greater than 8.5.

13. The method of claims 10 or 12, wherein the conjugate or polyoxyalkylene is activated by irradiation with UV light.

14. The method of claims 10 or 12, wherein the conjugate or polyoxyalkylene is activated by treatment with ammonium persulfate.

15. The method of claims 10 or 12, further comprising isolating the compound by reverse-phase HPLC and/or by precipitation of the compound from an organic solvent.

16. The method of claims 10 or 12 wherein the polypeptide is a cationic polypeptide.

17. A pharmaceutical composition comprising at least one compound according to any of claims 1-9 and a physiologically acceptable buffer.

18. The pharmaceutical composition according to claim 17, comprising at least one of an antibiotic agent, an antiviral agent, an antiparasitic agent and an antifungal agent.

19. A method of treating an infection, comprising administering to a patient a therapeutically effective amount of a compound according to claim 1 or a pharmaceutical composition according to claim 17.

20. The method of claim 19, wherein the infection is due to a microorganism selected from the group consisting of bacterium, fungus, parasite and virus.