USOO6503881B2 (12) United States Patent (10) Patent No.: US 6,503,881 B2 Krieger et al. (45) Date of Patent: *Jan. 7, 2003

(54) COMPOSITIONS AND METHODS FOR WO WO 97/08199 3/1997 TREATING INFECTIONS USING CATIONC WO WO 97/31942 9/1997 PEPTIDES ALONE OR IN COMBINATION W W SS 6.C. WITH ANTIBIOTICS WO WO 99/43357 9/1999 (75) Inventors: Timothy J. Krieger, Richmond (CA); OTHER PUBLICATIONS Robert Taylor, Surrey (CA); Douglas Erfle, Vancouver (CA); Janet R. Uchida et al., Pept. Chem. Volume date 1995, 33rd pp. Fraser, Vancouver (CA); Michael H. P. 229-232, 1996.* West, Vancouver (CA); Patricia J. Bundgaard (Ed) “Design of Prodrugs” (Elsevier Publishing) McNichol, Coquitlam (CA) pp. 1-24, 1983.* Chalk et al., “Purification of An Insect Defensin from the 73) Assignee:9. Micrologix Biotech Inc., Vancouver Mosquito, Aedes aegypti, ' Insect Biochem. Molec. Biol. (CA) 24(4):403-410, 1994. Chalk et al., “Full Sequence and Characterization of Two * ) Notice: Subject to anyy disclaimer, the term of this Insect Defensins: Immune Peptides from the Mosquito patent is extended or adjusted under 35 Aedes Aegypti,” Proc. R. Soc. Land. B. 261 (1361):217-221, U.S.C. 154(b) by 0 days. 1995. Darveau et al., “B-Lactam Antibiotics Potentiate Magainin This patent is Subject to a terminal dis 2 Antimicrobial Activity In Vitro and In Vivo, Antimicro claimer. bial Agents & Chemotherapy 35(6):1153–1159, 1991. Engström et al., “The Antibacterial Effect of Attacins from (21) Appl. No.: 09/030,619 the Silk Moth Hyalophoracecropia is Directed Against the Outer Membrane of Escherichia coli,” EMBO J. (22) Filed: Feb. 25, 1998 3(13):3347–3351, 1984. Hancock, “The Role of Fundamental Research and Biotech (65) Prior Publication Data nology in Finding Solutions to the Global Problem of US 2002/0035061 A1 Mar. 21, 2002 Antibiotic Resistance,” Clin. Infectious Diseases 24(Supp 1)S148–S150, 1997. Related U.S. Application Data Piers et al., “Improvement of Outer Membrane-Permeabi lizing and Lipopolysaccharide-Binding Activities of an (63) Continuation-in-part of application No. 08/915,314, filed on Antimicrobial Cationic Peptide by C-Terminal Modifica Aug. 20, 1997, now Pat. No. 6,180,604 (60) Provisional application No. 60/060,099, filed on Sep. 26, tion,” Antimicrobial Agents & Chemotherapy 1997, provisional application No. 60/040,649, filed on Mar. 38(10):2311-2316, 1994. 10, 1997, provisional application No. 60/034.949, filed on Vaara, “The Outer Membrane as the Penetration Barrier Jan. 13, 1997, and provisional application No. 60/024.754, Against Mupirocin in Gram-Negative Enteric Bacteria, J. filed on Aug. 21, 1996. Antimicrob. Chemother, 29(2):221-222, 1992. Vaara, “Agents That Increase the Permeability of the Outer (51) Int. Cl...... A61K 38/03; CO7K 7/00 Membrane,” Microbiological Reviews 56(3):395-411, 1992. (52) U.S. Cl...... 514/2; 514/17; 514/18; Vaara and Porro, “Group of Peptides That Act Synergisti 514/19; 514/20, 530/324; 530/325; 530/326; cally with Hydrophobic Antibiotics Against Gram-Negative 530/327; 530/328; 530/329; 530/330 Enteric Bacteria, Antimicrobial Agents & Chemotherapy (58) Field of Search ...... 530/324-330; 40(8): 1801–1805, 1996. 514/2, 17–20 Vaara and Vaara, “Polycations Sensitive Enteric Bacteria to Antibiotics,” Antimicrobial Agents & Chemotherapy (56) References Cited 24(1):107–113, 1983. Vaara and Vaara, “Sensitization of Gram-Negative Bacteria U.S. PATENT DOCUMENTS to Antibiotics and Complement by a Nontoxic Oligopep 4,510,132 A 4/1985 Vaara ...... 514/11 tide,” Nature 303:526–528, 1983. 5,324,716 A 6/1994 Selsted et al...... 514/14 5,359,030 A 10/1994 Ekwuribe ...... 530/303 (List continued on next page.)

5,438,040 A 8/1995 Ekwuribe ...... 514/3 5,523.288 A 6/1996 Cohen et al...... 514/12 Primary Examiner Bennett Celsa 5,547,939 A 8/1996 Selsted ...... 514/14 (74) Attorney, Agent, or Firm-Seed Intellectual Property 5,578,572 A 11/1996 Horwitz et al...... 514/12 Law Group PLLC 5,593.866 A 1/1997 Hancock et al...... 435/69.7 6,191,254 B1 2/2001 Falla et al...... 530/300 (57) ABSTRACT FOREIGN PATENT DOCUMENTS Compositions and methods for treating infections, especially bacterial infections, are provided. Indolicidin peptide ana EP 590 O70 B1 2/1998 logues containing at least two basic amino acids are pre WO WO 91/12815 9/1991 WO WO 92/22308 12/1992 pared. The analogues are administered as modified peptides, WO WO95/22338 8/1995 preferably containing photo-oxidized Solubilizer. WO WO 96/38473 12/1996 WO WO 97/04796 2/1997 62 Claims, 22 Drawing Sheets US 6,503,881 B2 Page 2

OTHER PUBLICATIONS Subbalakshmi and Sitaram, “Mechanism of Antimicrobial Vaara and Vaara, “Ability of Cecropin B to Penetrate the Action of Indolicidin,” FEMS Microbiology Letters Enterobacterial Outer Membrane,” Antimicrobial Agents & 160:91-96, 1998. Chemotherapy 38(10):2498-2501, 1994. Subbalakshmi et al., “Interaction of Indolicidin, a 13-Resi Falla et al., “Mode of Action of the Antimicrobial Peptide due Peptide Rich in Tryptophan and Proline and its Ana Indolicidin,” The Journal of Biological Chemistry 271 (32): logues with Model Membranes,” J. Biosci. 23(1):9-13, 19298–19303, Aug. 9, 1996. 1998. Falla and Hancock, “Improved Activity of a Synthetic Subbalakshmi et al., “Requirements for antibacterial and Indolicidin Analog,” Antimicrobial Agents and Chemo hemolytic activities in the bovine neutrophil derived therapy 41(4): 771–775, 1997. 13-residue peptide indolicidin,” FEBS Letters 395(1): Ladokhin et al., “CD Spectra of Indolicidin Antimicrobial 48-52, 1996. Peptides Suggest Turns, Not Polyproline Helix,” Biochem Tanchak et al., “Tryptophanins: Isolation and Molecular istry 38:12313–12319, 1999. Characterization of Oat cDNA Clones Encoding Proteins Lawyer et al., “Antimicrobial Activity of a 13 Amino Acid Structurally Related to Puroindoline and Wheat Grain Soft Tryptophan-Rich Peptide Derived From a Putative Porcine ness Proteins,” Plant Science 137:173–184, 1998. Precursor Protein of a Novel Family of Antibacterial Pep Uchida et al., “Stucture-Activity of Antibacterial Peptide tides.” FEBS Letters 390:95–98, 1996. Indolicidin and Analogs.” Peptide Science, pp. 221-224, Robinson, Jr. et al., “Anti-HIV-1 Activity of Indolicidin, an 1998. Antimicrobial Peptide from Neutrophils,” Journal of Leu Van Abel et al., “Synthesis and Characterization of Indoli kocyte Biology 63:94-100, 1998. cidin, a Tryptophan-Rich Antimicrobial Peptide from Selsted et al., “Indolicidin, a Novel Bactericidal Tridecapep Bovine Neutrophils,” Int. J. Peptide Protein Res. tide Amide from Neutrophils.” The Journal Of Biological 45:401–409, 1995. Chemistry 267(7):4292–4295, Mar. 5, 1992. Wakabayashi et al., “N-Acylated and D Encantiomer Selsted et al., “Purification, Characterization, Synthesis and Derivatives of a Nonamer Core Peptide of Lactoferricin B cDNA Cloning of Indolicidin: A Tryptophan-Rich Micro Showing Improved Antimicrobial Activity,” Antimicrobial bicidal Tridecapeptide from Neutrophils,” Proceedings of the 12" American Peptide Symposium, Jun. 16–21, 1991, Agents And Chemotherapy 43(5):1267-1269, May 1999. Cambridge, MA, pp. 905–907. * cited by examiner U.S. Patent Jan. 7, 2003 Sheet 1 of 22 US 6,503,881 B2

U.S. Patent Jan. 7, 2003 Sheet 2 of 22 US 6,503,881 B2 Time Kill Assay:

w MB 11 CN (64 ug/ml) vs. S. aureus SAO6 s >< 14 -- CONTROL 1 A 12 --0-- CONTROL 2 2 10 -A-TEST 1 S. 8 --o-- TEST 2 c 6 SP 4. 2 2. C O S O 10 20 30 60 120 180 240 24h Fig. 3A TIME (MIN) Time Kill Assay: MB 11 F4CN (8 ug/ml) vs. S. aureus SAO6

se X -- CONTROL 1 2 --0-- CONTROL 2 2 -A-TEST 1 9 --0-- TEST 2 s 5 2. - F. S O 10 20 30 60 120 180 240 24h Fig. 3B TIME (MIN) Time Kill Assay: MB 11 B70N (16 ug/ml) vs. S. aureus SA06

s > 2. -- CONTROL 1 2 16 --0-- CONTROL 2 2. 1: -A-TEST 1 Se 10 - - - - - TEST 2 8 S2 6 4 2 2 C O S O 1 O 20 30 60 120 180 240 24h Fig. 3C TIME (MIN) U.S. Patent Jan. 7, 2003 Sheet 3 of 22 US 6,503,881 B2

MB 11 F4CN at its MIC against S. aureus SAO6

-O-CONTROL 3 Logo reduction O-TEST

O 10 20 30 60 120 180 240 24h Time (min) Fig. 3D MB 26 & Vancomycin in Combination Against E. faecium EFMO17

--GROWTH CONTROL -O-MB 26 (1/2MIC) -O-Vancomycin (1/8 MIC) -HMB 26 + Wanc,

O 15 30 45 60 120 180 240 24h Time (min) Fig. 3E U.S. Patent Jan. 7, 2003 Sheet 4 of 22 US 6,503,881 B2

L. 120 g r c 100 Y f W 2 80 - 2 60 1. gt 40 - 20 O 200 400 600 800 PEPTIDE SOLUTION CONCENTRATION (ug/ml) Fig. 4

MB 1 OB in Formulations C1 and D

FREE PEPTIDE O.O7 0.07 100 100 0.06 0.06 0.05 0.05 0.04 0.04 i. 0.03 50 SS 0.05 50 ge 0.02 0.02 FREE PEPTIDE 0.01 0.01 0.00 0.00 O.O 2.5 5.0 7.5 O.O 2.5 5.0 7.5 MN. MN. Fig. 5A Fig. 5B U.S. Patent Jan. 7, 2003 Sheet 5 of 22 US 6,503,881 B2

CD Spectra of MBI 1CN / 5

O

m 5

-10 -0- buffer -- POPC liposomes - 15

-20 195 205 215 225 235 WAVELENGTH (nm)

CD Spectra of MB 11 B7CN

-0- Free -- Free +PC/PG

195 205 215 225 235 WAVELENGTH (nm) Fig. 6 U.S. Patent Jan. 7, 2003 Sheet 6 of 22 US 6,503,881 B2

ANTS/DPX Dye Release of Egg PC / Liposomes by MBI 11 CN

2 g s SS

O 100 200 300 400 500 TIME (SEC)

ANTS/DPX Dye Release of Egg PC/Cholesterol (55:45) Liposomes by MBI 11 CN

t

s SS

O 100 200 300 400 500 TIME (SEC) Fig. 7 U.S. Patent Jan. 7, 2003 Sheet 7 of 22 US 6,503,881 B2 MB 11B7CN grown in TB / 7

O 100 200 300 400 500 TIME (MIN)

MB 11B7CN grown in LB

f

O 100 200 300 400 500 TIME (MIN) Fig. 8 U.S. Patent US 6,503,881 B2

BW||(NIN)

90700 U.S. Patent Jan. 7, 2003 Sheet 9 of 22 US 6,503,881 B2

MB 11 B7CN Activity in the presence of NaCl or Mg 2+

xouA

W al S. C C CC) S. S.C XXX C SSX s C NCONTROL CONTROL NOC NaCl Mg2+ Mg2+ CONDITION

ig. 10

3000000 2500000 2OOOOOO

1500000

500000

20 40 60 80 100 120 140 160 18O TIME (MINUTES) Fig. 11 U.S. Patent Jan. 7, 2003 Sheet 10 of 22 US 6,503,881 B2

Stability of MB-11B7CN-Cl in Heat-inactivated Rabbit Serum 35000000 30000000 25000000 20000000 15000000 10000000 5000000 O O 1 2 3 4. 5 6 Time (hours) Time Area O 31027612 2 12767092 Fig. 12 6 28.04593 U.S. Patent Jan. 7, 2003 Sheet 11 of 22 US 6,503,881 B2

8% 8%

OI 09 00T 09

609LOQ*Tr?O“OG– 608*80qOG–Id() L U?IN

0||G IG00 GTº IºO ?????????????????GO* GT* I•O ?GO* ~~00^ U.S. Patent Jan. 7, 2003 Sheet 12 of 22 US 6,503,881 B2

O. O1 OO O. O1 OO

O. O.O 75 OOO 75

O. OO50 O. OO 50

0.0025 O. OO25

sooooo

10 U.S. Patent Jan. 7, 2003 Sheet 13 of 22 US 6,503,881 B2

50 S. 40 -0- MB 11 CN s 30 G 20 ? S 10 O ances O 5 10 15 20 TIME (MINUTES)

Fig. 15

is O 20 40 60 80 100 120 140 TIME (MINUTES) Fig. 16 U.S. Patent Jan. 7, 2003 Sheet 14 of 22 US 6,503,881 B2

------H -- H - - - - o Dose (mg/kg): . .2 ------Ampicillin cy -0- 38 M 5 S -CC s d 2. S S S S S S S E o d o Cs d o O d

- Cen c.co- C-, ch, Cen, (b> co- cbC o Cs o s O C d d C o O o O O C O TIME POST-INFECTION Fig. 17

10 ------d Dose (mg/kg): is 8 So ------Ampicilin9 ?h 6 o E 4 a. v- 2 O is O S S S S S S S E d o d o C c O c

> ( Co- C-, >n., Co-, bCa C-N- cb> d o o o o d o o O -o o O O O C o TIME POST-INFECTION Fig. 18 U.S. Patent Jan. 7, 2003 Sheet 15 of 22 US 6,503,881 B2

TIME POST-INFECTION Fig. 19

CTTfºr)CN

TIME POST-INFECTION Fig. 20 U.S. Patent Jan. 7, 2003 Sheet 16 of 22 US 6,503,881 B2

-X-X- X-X-X-X-X-X-X-X- Xe X

-0- F3 24 mg/kg -o- F3 18 mg/kg “T©ºroCNoLr><+oo?cc->oro -H F3 12mg/kg -A- Formulation Control ——————— -%- Ampicillin

TIME POST-INFECTION Fig. 21

TIME POST-INFECTION Fig. 22 U.S. Patent Jan. 7, 2003 Sheet 17 of 22 US 6,503,881 B2

TIME POST-INFECTION Fig. 23

Yé-x-x-x-x-x-x-x-x -0- B1 24 mg/kg -O- B1 18 mg/kg C)(TTºroCNLr><+I,coobco -H B1 12mg/kg -A- Formulation Control -X- Ampicillin

TIME POST-INFECTION Fig. 24 U.S. Patent Jan. 7, 2003 Sheet 18 of 22 US 6,503,881 B2

Ye-x-x-x-x-x-x-x -0- B7 24 mg/kg -O- B7 18 mg/kg -H B7 12mg/kg -A- Formulation Control -- Ampicillin

TIME POST-INFECTION Fig. 25

-H B8 12mg/kg -A- Formulation Control ->{- Ampicillin

TIME POST-INFECTION Fig. 26 U.S. Patent Jan. 7, 2003 Sheet 19 of 22 US 6,503,881 B2

-0- G4 24mg/kg TTC^^)c)*uro<+coorocot -o- G4 18 mg/kg -H G4 12mg/kg -A- Formulation Control ->{- Ampicillin

TIME POST-INFECTION Fig. 27

DAY OF OBSERVATION Fig. 29 U.S. Patent Jan. 7, 2003 Sheet 20 of 22 US 6,503,881 B2

a) peptide injection 15 minutes post-infection

------Dose (mg/kg):

TIME POST-INFECTION

b) peptide injection 60 minutes post-infection

- H ------Dose (mg/kg): ------

TIME POST-INFECTION

Fig. 28 U.S. Patent US 6,503,881 B2

5 Fig. 304

O MINUTES

10 MINUTES Fig. 300 U.S. Patent Jan. 7, 2003 Sheet 22 of 22 US 6,503,881 B2

/ 0.100

0.075

0.050

i 0.025

0.000

0.100

0.075

0.050

s i. 0.025

0.000

0.100

0.075

0.050 US 6,503,881 B2 1 2 COMPOSITIONS AND METHODS FOR mechanism against microorganisms. When isolated, these TREATING INFECTIONS USING CATIONC peptides are toxic to a wide variety of microorganisms, PEPTIDES ALONE OR IN COMBINATION including bacteria, fungi, and certain enveloped viruses. One WITH ANTIBIOTICS cationic peptide found in neutrophils is indolicidin. While indolicidin acts against many pathogens, notable exceptions CROSS-RELATED APPLICATIONS and varying degrees of toxicity exist. Although cationic peptides show efficacy in vitro against The present application claims priority from U.S. Provi a variety of pathogenic cells including gram-positive sional Application No. 60/040,649, filed Mar. 10, 1997, and bacteria, gram-negative bacteria, and fungi, these peptides U.S. Provisional Application No. 60/060,099, filed Sep. 26, are generally toxic to mammals when injected, and thera 1997, and is a continuation-in-part of U.S. application Ser. peutic indices are usually quite Small. Approaches to reduc No. 08/915,314 now U.S. Pat. No. 6,180,604, filed Aug. 20, ing toxicity have included development of a derivative or 1997, which claims priority from U.S. Provisional Applica delivery System that masks Structural elements involved in tion No. 60/024,754, filed Aug. 21, 1996, U.S. Provisional the toxic response or that improves the efficacy at lower Application No. 60/034,949, filed Jan. 13, 1997. 15 doses. Other approaches under evaluation include liposomes and micellular Systems to improve the clinical effects of TECHNICAL FIELD peptides, proteins, and hydrophobic drugs, and cyclodex The present invention relates generally to methods of trins to Sequester hydrophobic Surfaces during administra treating microorganism-caused infections using cationic tion in aqueous media. For example, attachment of polyeth peptides or a combination of cationic peptides and antibiotic ylene glycol (PEG) polymers, most often by modification of agents, and more particularly to using these peptides and amino groups, improves the medicinal value of Some pro antibiotic agents to overcome acquired resistance, tolerance, teins Such as asparaginase and adenosine deaminase, and and inherent resistance of an infective organism to the increases circulatory half-lives of peptides Such as interleu antibiotic agent. kins. 25 None of these approaches are shown to improve admin BACKGROUND OF THE INVENTION istration of cationic peptides. For example, methods for the For most healthy individuals, infections are irritating, but Stepwise Synthesis of polySorbate derivatives that can not generally life-threatening. Many infections are Success modify peptides by acylation reactions have been fully combated by the immune system of the individual. developed, but acylation alters the charge of a modified Treatment is an adjunct and is generally readily available in cationic peptide and frequently reduces or eliminates the developed countries. However, infectious diseases are a antimicrobial activity of the compound. Thus, for delivery of Serious concern in developing countries and in immunocom cationic peptides, as well as other peptides and proteins, promised individuals. there is a need for a system combining the properties of In developing countries, the lack of adequate Sanitation increased circulatory half-lives with the ability to form a and consequent poor hygiene provide an environment that 35 micellular structure. fosters bacterial, parasitic, fungal and viral infections. Poor The present invention discloses analogues of indolicidin, hygiene and nutritional deficiencies may diminish the effec designed to broaden its range and effectiveness, and further tiveness of natural barriers, Such as Skin and mucous provide other related advantages. The present invention also membranes, to invasion by infectious agents or the ability of provides methods and compositions for modifying peptides, the immune System to clear the agents. AS well, a constant 40 proteins, antibiotics and the like to reduce toxicity, as well onslaught of pathogens may streSS the immune System as providing other advantages. defenses of antibody production and phagocytic cells (e.g., In addition neither antibiotic therapy alone of cationic polymorphic neutrophils) to Subnormal levels. A breakdown peptide therapy alone can effectively combat all infections. of host defenses can also occur due to conditions Such as By expanding the categories of microorganisms that respond circulatory disturbances, mechanical obstruction, fatigue, 45 to therapy, or by overcoming the resistance of a microor Smoking, excessive drinking, genetic defects, AIDS, bone ganism to antibiotic agents, health and welfare will be marrow transplant, cancer, and diabetes. An increasingly improved. Additionally quality of life will be improved, due prevalent problem in the World is opportunistic infections in to, for example, decreased duration of therapy, reduced individuals who are HIV positive. 50 hospital stay including high-care facilities, with the con Although Vaccines may be available to protect against comitant reduced risk of Serious nosocomial (hospital Some of these organisms, vaccinations are not always acquired) infections. feasible, due to factorS Such as inadequate delivery mecha The present invention discloses cationic peptides, includ nisms and economic poverty, or effective, due to factorS Such ing analogues of indolicidin, cecropin/melittin fusion as delivery too late in the infection, inability of the patient 55 peptides, in combination with antibiotics Such that the to mount an immune response to the vaccine, or evolution of combination either Synergistic, able to overcome microor the pathogen. For other pathogenic agents, no vaccines are ganismal tolerance, able to overcome resistance to antibiotic available. When protection against infection is not possible, treatment, or further provides other related advantages. treatment of infection is generally pursued. The major SUMMARY OF THE INVENTION weapon in the arsenal of treatments is antibiotics. While 60 antibiotics have proved effective against many bacteria and The present invention generally provides the thus Saved countleSS lives, they are not a panacea. The co-administration of cationic peptides with an antibiotic overuse of antibiotics in certain situations has promoted the agent and also provides indolicidin analogues. Spread of resistant bacterial Strains. And of great importance, In related aspects, an indolicidin analogue is provided, antibacterials are useleSS against Viral infections. 65 comprising up to 25 amino acids and containing the formula: A variety of organisms make cationic (positively charged) RXZXXZXB(SEQ ID NO: 1); BXZXXZXB(SEQ ID NO: peptides, molecules used as part of a non-Specific defense 2) wherein at least one Z is valine; BBBXZXXZXB(SEQ ID US 6,503,881 B2 3 4 NO:3); BXZXXZXBBB (AA), MILBBAGS(SEQ ID NO: The invention also provides a method of making a com 5-8); BXZXXZXBB(AA), M(SEQ ID NO: 9–10); pound modified with a conjugate of an activated polyoxy LBBXZXXZXRK(SEQ ID NO: 18–18); alkylene and a lipophilic moiety, comprising: (a) freezing a LKXZXXZXRRK(SEQ ID NO: 19–20); mixture of the conjugate of an activated polyoxyalkylene BBXZXXZXBBB(SEQ ID NO: 21), wherein at least two X and lipophilic moiety with the compound; and (b) lyophiliz residues are phenylalanine; BBXZXXZXBBB(SEQ ID NO: ing the frozen mixture; wherein the compound has a free 22), wherein at least two X residues are tyrosine; and amino group. In preferred embodiments, the compound is a wherein Z is proline or valine, X is a hydrophobic residue, peptide or antibiotic. In other preferred embodiments, the B is a basic amino acid, AA is any amino acid, and n is 0 or mixture in Step (a) is in an acetate buffer. In a related aspect, 1. In preferred embodiments, Z is proline, X is tryptophan the method comprises mixing the conjugate of an activated and B is arginine or lysine. In other aspects, indolicidin polyoxyalkylene and lipophilic moiety with the compound; analogues having Specific Sequences are provided. In certain for a time Sufficient to form modified compounds, wherein embodiments, the indolicidin analogues are coupled to form the mixture is in a carbonate buffer having a pH greater than a branched peptide. In other embodiments, the analogue has 8.5 and the compound has a free amino group. The modified one or more amino acids altered to a corresponding D-amino 15 compound may be isolated by reversed-phase HPLC and/or acid, and in certain preferred embodiments, the N-terminal precipitation from an organic Solvent. and/or the C-terminal amino acid is a D-amino acid. Other The invention also provides a pharmaceutical composi preferred modifications include analogues that are acety tion comprising at least one modified compound and a lated at the N-terminal amino acid, amidated at the physiologically acceptable buffer, and in certain C-terminal amino acid, esterified at the C-terminal amino embodiments, further comprises an antibiotic agent, antivi acid, modified by incorporation of homoserine/homoserine ral agent, an antiparasitic agent, and/or antifimgal agent. The lactone at the C-terminal amino acid, and conjugated with composition may be used to treat an infection, Such as those polyethylene glycol or derivatives thereof. caused by a microorganism (e.g., bacterium, fungus, parasite In other aspects, the invention provides an isolated and virus). nucleic acid molecule whose Sequence comprises one or 25 This invention also generally provides methods for treat more coding Sequences of the indolicidin analogues, expres ing infections caused by a microorganism using a combi Sion vectors, and host cells transfected or transformed with nation of cationic peptides and antibiotic agents. In one the expression vector. aspect, the method comprises administering to a patient a Other aspects provide a pharmaceutical composition com therapeutically effective dose of a combination of an anti prising at least one indolicidin analogue and a physiologi biotic agent and a cationic peptide, wherein administration cally acceptable buffer, optionally comprising an antibiotic of an antibiotic agent alone is ineffective. Preferred peptides agent. are provided. In other embodiments, the pharmaceutical composition In another aspect, a method of enhancing the activity of further comprises an antiviral agent, an antiparasitic agent; an antibiotic agent against an infection in a patient caused by and an antifungal agent. In yet other embodiments, the 35 a microorganism is provided, comprising administering to composition is incorporated in a liposome or a slow-release the patient a therapeutically effective dose of the antibiotic vehicle. agent and a cationic peptide. In yet another aspect, a method In yet another aspect, the invention provides a method of is provided for enhancing the antibiotic activity of lysozyme treating an infection, comprising administering to a patient or nisin, comprising administering lysozyme or nisin with a a therapeutically effective amount of a pharmaceutical com 40 cationic peptide. position. The infection may be caused by, for example, a In other aspects, methods of treating an infection in a microorganism, Such as a bacterium (e.g., Gram-negative or patient caused by a bacteria that is tolerant to an antibiotic Gram-positive bacterium or anaerobe, parasite or virus. agent, caused by a microorganism that is inherently resistant In other aspects, a composition is provided, comprising an 45 to an antibiotic agent, or caused by a microorganism that has indolicidin analogue and an antibiotic. In addition, a device, acquired resistance to an antibiotic agent, comprises admin which may be a medical device, is provided that is coated istering to the patient a therapeutically effective dose of the with the indolicidin analogue and may further comprise an antibiotic agent and a cationic peptide, thereby overcoming antibiotic agent. tolerance, inherent or acquired resistance to the antibiotic In other aspects, antibodies that react specifically with any 50 agent. one of the analogues described herein are provided. The In yet other related aspects, methods are provided for antibody is preferably a monoclonal antibody or Single chain killing a microorganism that is tolerant, inherently resistant, antibody. or has acquired resistance to an antibiotic agent, comprising In a preferred aspect, the invention provides a composi contacting the microorganism with the antibiotic agent and tion comprising a compound modified by derivatization of 55 a cationic peptide, thereby overcoming tolerance, inherent an amino group with a conjugate comprising activated resistance or acquired resistance to the antibiotic agent. polyoxyalkylene and a lipophilic moiety. In preferred These and other aspects of the present invention will embodiments, the conjugate comprises Sorbitan linking become evident upon reference to the following detailed polyoxyalkylene glycol and fatty acid, and more preferably description and attached drawings. In addition, various is polySorbate. In preferred embodiments, the fatty acid is 60 references are set forth below which describe in more detail from 12-18 carbons, and the polyoxyalkylene glycol is certain procedures or compositions (e.g., plasmids, etc.), and polyoxyethylene, Such as with a chain length of from 2 to are therefore incorporated by reference in their entirety. 100. In certain embodiments, the compound is a peptide or protein, Such as a cationic peptide (e.g., indolicidin or an BRIEF DESCRIPTION OF THE DRAWINGS indolicidin analogue). In preferred embodiments, the poly 65 FIG. 1 is an SDS-PAGE showing the extraction profile of oxyalkylene glycol is activated by irradiation with ultravio inclusion bodies (ib) from whole cells containing MBI-11 let light or by treatment with ammonium perSulfate. fusion protein. The fusion protein band is indicated by the US 6,503,881 B2 S 6 arrow head. Lane 1, protein Standards, lane 2, total lysate of FIG. 21 is a graph showing the results of in Vivo testing XL1 Blue without plasmid; lane 3, total lysate of XL1 Blue of: MBI-11F3CN against S. aureus (Smith). Formulated (pR2h-11, p.GP1-2), cultivated at 30° C.; lane 4, total lysate peptide at various concentrations is administered by ip of XL1 Blue (pR2h-11, pGP1-2), induced at 42 C.; lane 5, injection one hour after infection with S. aureus (Smith) by insoluble fraction of inclusion bodies after Triton X100 ip injection. wash; lane 6, organic extract of MBI-11 fusion protein; lane FIG. 22 is a graph showing the results of in Vivo testing 7, concentrated material not Soluble in organic extraction of MBI-11G2CN against S. aureus (Smith). Formulated Solvent. peptide at various concentrations is administered by ip FIG. 2 is an SDS-PAGE showing the expression profile of injection one hour after infection with S. aureus (Smith) by the MBI-11 fusion protein using plasmid pPDR2h-11. Lane 1O ip injection. 1, protein Standards, lane 2, organic Solvent extracted MBI FIG. 23 is a graph showing the results of in Vivo testing 11; lane 3, total lysate of XL1 Blue (pPDR2h-11, pGP1-2), of MBI-11CN against S. aureus (Smith). Formulated peptide cultured at 30° C.; lane 4, total lysate of XL1 Blue (pPDR2h at various concentrations is administered by ip injection one 11, pGP 1-2), induced at 42 C. hour after infection with S. aureus (Smith) by ip injection. FIGS. 3A-E presents time kill assay results for MBI FIG. 24 is a graph showing the results of in Vivo testing 11CN, MBI 11F4CN, MBI 11B7CN, MBI 11 F4CN, and 15 of MBI-11B1CN against S. aureus (Smith). Formulated MBI 26 plus Vancomycin. The number of colony forming peptide at various concentrations is administered by ip unitsx10" is plotted versus time. injection one hour after infection with S. aureus (Smith) by FIG. 4 is a graph presenting the extent of solubility of ip injection. MBI 11CN peptide in various buffers. FIG. 25 is a graph showing the results of in vivo testing FIG. 5 is a reversed phase HPLC profile of MBI 11CN in of MBI-11B7CN against S. aureus (Smith). Formulated formulation C1 (left graph panel) and formulation D (right peptide at various concentrations is administered by ip graph panel). injection one hour after infection with S. aureus (Smith) by FIG. 6 presents CD spectra of MBI 11CN and MBI ip injection. 11B7CN. 25 FIG. 26 is a graph showing the results of in Vivo testing FIG. 7 presents results of ANTS/DPX dye release of egg of MBI-11B8CN against S. aureus (Smith). Formulated PC liposomes at various ratioS of lipid to protein. peptide at various concentrations is administered by ip FIG. 8 presents graphs showing the activity of MBI injection one hour after infection with S. aureus (Smith) by 11B7CN against mid-log cells grown in terrific broth (TB) ip injection. or Luria-Bretani broth (LB). FIG. 27 is a graph showing the results of in vivo testing FIG. 9 shows results of treatment of bacteria with MBI of MBI-11G4CN against S. aureus (Smith). Formulated 10CN, MBI 11 CN, or a control peptide alone or in com peptide at various concentrations is administered by ip bination with Vancomycin. injection one hour after infection with S. aureus (Smith) by FIG. 10 is a graph showing treatment of bacteria with ip injection. MBI 11 B7CN in the presence of NaCl or Mg". 35 FIGS. 28A and 28B display a graph showing the number FIG. 11 is a graph presenting the in vitro amount of free of animals Surviving an S. epidermidis infection after intra MBI 11CN in plasma over time. Data is shown for peptide venous injection of MBI 10CN, gentamicin, or vehicle. in formulation C1 and formulation D. Panel A, i.v. injection 15 min post-infection; panel B, i.V. FIG. 12 is a graph showing the stability of MBI-11B7CN injection 60 min post-infection. cl in heat-inactivated rabbit Serum. 40 FIG. 29 is a graph showing the number of animals FIG. 13 presents HPLC tracings showing the effects of Surviving an MRSA infection mice after intravenous injec amastatin and bestatin on peptide degradation. tion of MBI 11 CN, gentamicin, or vehicle. FIG. 14 is a chromatogram showing extraction of peptides FIGS. 30A-30C present RP-HPLC traces analyzing in rabbit plasma. samples for APS-peptide formation after treatment of acti 45 vated polysorbate with a reducing agent. APS-MBI-11CN FIG. 15 is a graph presenting change in in vivo MBI 11 peptides are formed via lyophilization in 200 mM acetic CN levels in blood at various times after intravenous injec acid-NaOH, pH 4.6, 1 mg/ml MBI 11CN, and 0.5% acti tion. vated polysorbate 80. The stock solution of activated 2.0% FIG. 16 is a graph presenting change in in vivo MBI polySorbate is treated with (a) no reducing agent, (b) 150 11CN levels in plasma at various times after intraperitoneal 50 mM 2-mercaptoethanol, or (c) 150 mM sodium borohydride injection. for 1 hour immediately before use. FIG. 17 is a graph showing the number of animals FIG. 31 presents RP-HPLC traces monitoring the forma surviving an MSSA infection after intraperitoneal injection tion of APS-MBI 11CN over time in aqueous solution. The of MBI 10CN, amplicillin, or vehicle. reaction occurs in 200 mM sodium carbonate buffer pH 10.0, FIG. 18 is a graph showing the number of animals 55 1 mg/ml MBI 11CN, 0.5% activated polysorbate 80. Ali surviving an MSSA infection after intraperitoneal injection quots are removed from the reaction vessel at the indicated of MBI 11 CN, amplicillin, or vehicle. time points and immediately analyzed by RP-HPLC. FIG. 19 is a graph showing the results of in vivo testing DETAILED DESCRIPTION OF THE of MBI-11A1CN against S. aureus (Smith). Formulated INVENTION peptide at various concentrations is administered by ip 60 injection one hour after infection with S. aureus (Smith) by Prior to setting forth the invention, it may be helpful to an ip injection. understanding thereof to Set forth definitions of certain terms FIG. 20 is a graph showing the results of in vivo testing that are used herein. of MBI-11E3CN against S. aureus (Smith). Formulated The amino acid designations herein are Set forth as either peptide at various concentrations is administered by ip 65 the Standard one- or three-letter code. A capital letter indi injection one hour after infection with S. aureus (Smith) by cates an L-form amino acid; a Small letter indicates a D-form ip injection. amino acid. US 6,503,881 B2 7 8 AS used herein, an “antibiotic agent” refers to a molecule AS used herein “inherent resistance' of a microorganism that tends to prevent, inhibit, or destroy life. The term to an antibiotic agent refers to a natural resistance to the “antimicrobial agent” refers to an antibiotic agent Specifi action of the agent even in the absence of prior exposure to cally directed to a microorganism. the agent. (R. C. Moellering Jr., Principles of Anti-infective As used herein, “cationic peptide” refers to a peptide that 5 Therapy, In. Principles and Practice of Infectious Diseases, has a net positive charge within the pH range of 4-10. A 4" Edition, Eds.; G. L. Mandell, J. E. Bennett, R. Dolin. cationic peptide is at least 5 amino acids in length and has Churchill Livingstone, New York USA, 1995, page 200). at least one basic amino acid (e.g., arginine, lysine, AS used herein, “acquired resistance' of a microorganism histidine). Preferably, the peptide has measurable anti- to an antibiotic agent refers to a resistance that is not microbial activity when administered alone. 10 inhibited by the normal achievable serum concentrations of AS used herein, “indolicidin” refers to an antimicrobial a recommended antibiotic agent based on the recommended cationic peptide. Indolicidins may be isolated from a variety dosage. (NCCLS guidelines). of organisms. One indolicidin is isolated from bovine neu- AS used herein, “tolerance' of a microorganism to an trophils and is a 13 amino acid peptide amidated at the antibiotic agent refers to when there is microStatic, rather carboxy-terminus in its native form (Selsted et al., J Biol. than microcidal effect of the agent. Tolerance is measured by Chem. 1992). An amino acid sequence of indolicidin is an MBC:MIC ratio greater than or equal to 32. (Textbook of presented in SEQ ID NO: 1. Diagnostic Microbiology, Eds., C. R. Mahon and G. AS used herein, a "peptide analogue”, “analogue', or Manuselis, W. B. Saunders Co., Toronto Canada, 1995, page “variant' of a cationic peptide, Such as indolicidin, is at least 2O 92). 5 amino acids in length, has at least one basic amino acid As noted above, this invention provides methods of (e.g., arginine and lysine) and has anti-microbial activity. treating infections caused by a microorganism, methods of Unless otherwise indicated, a named amino acid refers to the killing a microorganism, and methods of enhancing the L-form. Basic amino acids include arginine, lysine, histidine activity of an antibiotic agent. In particular, these methods and derivatives. Hydrophobic residues include tryptophan, as are especially applicable when a microorganism is resistant phenylalanine, isoleucine, leucine, Valine, and derivatives. to an antibiotic agent, by a mechanism, Such as tolerance, Also included within the Scope of the present invention inherent resistance, or acquired resistance. In this invention, are amino acid derivatives that have been altered by chemi- infections are treated by administering a therapeutically cal means, Such as methylation (e.g., C. methylvaline), effective dose of a cationic peptide alone or in combination amidation, especially of the C-terminal amino acid by an With an antibiotic agent to a patient With an infection. alkylamine (e.g., ethylamine, ethanolamine, and ethylene Similarly, the combination can be contacted with a micro diamine) and alteration of an amino acid side chain, Such as organism to effect killing. st of the e-amino group of lysine. Other amino acids I. CATIONIC PEPTIDES at may be incorporated in the analogue include any of the D-amino acids corresponding to the 20 L-amino acids 35 AS noted above, a cationic peptide is a peptide that has a commonly found in proteins, imino amino acids, rare amino net positive charge within the pH range 4-10. A peptide is acids, Such as hydroxylysine, or non-protein amino acids, at least 5 amino acids long and preferably not more than 25, Such as homoserine and omithine. A peptide analogue may 27, 30, 35, or 40 amino acids. Peptides from 12 to 30 have none or one or more of these derivatives, and D-amino residues are preferred. Examples of native cationic peptides acids. In addition, a peptide may also be Synthesized as a include, but are not limited to, representative peptides pre retro-, inverto- or retro-inverto-peptide. sented in the following table.

TABLE 1. Cationic Peptides

Accession Group Name Peptide Origin Sequence Number Reference Abaecins Abaecin Honey bee YVPLPNVPOPGRRPFPTFPGQGPFNPKIK P15450 Casteels P. et al., (1990) (Apis mellifera) WPOGY (SEQ ID NO: 156) Andropins Andropin Fruit fly VFIDILDKVENAIHNAAOVGIGFAKPFEK P21663 Samakovlis, C. et al., (Drosophilia LINPK (SEQ ID NO: 157) (1991) melanogaster) Apidaecins Apidaecin IA Lymph fluid of honey GNNRPVYIPQPRPPHPRI (SEQ ID NO: 158) P11525 Casteels, P. et al., (1989) bee (Apis mellifera) Apidaecin IB Lymph fluid of honey GNNRPVYIPQPRPPHPRL (SEQ ID P11526 Casteels, P. et al., (1989) bee (Apis mellifera) NO: 159) Apidaecin II Lymph fluid of honey GNNRPIYIPQPRPPHPRL (SEQ ID NO: 160) P11527 Casteels P. et al., (1989) bee (Apis mellifera) AS AS-48 Streptococcus faecalis 7.4 kDa Galvez, A., el al., (1989) subsp. Liquefacines S-48 Bactenecins Bactenecin Cytoplasmic granules of RLCRIVVIRVCR (SEQ ID NO: 161) A33799 Romeo, D et al., (1988) bovine neutrophils Bac Bac5 Cytoplasmic granules of RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPP B36589 Frank, R. W. et al., (1990) bovine neutrophils FRPPLRFP (SEQ ID NO: 162) Bacf Cytoplasmic granules of RRIRPRPPRLPRPRPRPLPFPRPGPRPIPRPL A36589 Frank, R. W. et al., (1990) bovine neutrophils PFPRPGPRPIPRPLPFPRPGPRPIPRP (SEQ ID NO: 163) US 6,503,881 B2 10

TABLE 1-continued Cationic Peptides Accession Group Name Peptide Origin Sequence Number Reference Bactericidins Bactericidin B2 Tobacco hornworm WNPFKELERAGORVRDAVISAAPAVATV P14662 Dickinson, L. et al., (1988) larvae hemolymph GQAAAIARG* (SEQ ID NO: 164) (Manduca Sexta) Bactericidin B-3 Tobacco hornworm WNPFKELERAGORVRDAIISAGPAVATV P14663 Dickinson, L. et al., (1988) larvae hemolymph GQAAAIARG (SEQ ID NO: 165) (Manduca Sexta) Bactericidin B-4 Tobacco hornworm WNPFKELERAGORVRDAIISAAPAVATV P14664 Dickinson, L. et al., (1988) larvae hemolymph GQAAAIARG* (SEQ ID NO: 166) (Manduca Sexta) Bactericidin B Tobacco hornworm WNPFKELERAGORVRDAVISAAAVATV P14665 Dickinson, L., et al., larvae hemolymph GQAAAIARGG* (SEQ ID NO: 167) (1988) (Manduca Sexta) Bacteriocins Bacteriocin Streptococcus mutants 4.8 kDa Takada, K., et al., (1984) C3603 Bacteriocin Staphylococcus aureus 5 kDa Nakamura, T, et al., 1Y 52 (1983) Bombinins Bomb inin Yellow-bellied toad GIGALSAKGALKGLAKGLAZHEAN PO1505 Csordas, A., and Michl, H. (Bombina variegata) (SEQ ID NO: 168) (1970) BLP-1 Asian Toad GIGASILSAGKSALKGLAKGLAEHFAN M76483 Gibson, B. W. et al., (1991) (Bombina Orientalis) (SEQ ID NO: 169) BLP-2 Asian Toad GIGSALSAGKSALKGLAKGLAEHFAN B41575 Gibson, B. W. et al., (1991) (Bombina Orientalis) (SEQ ID NO: 170) Bombolitins Bomb olitin BI Bumblebee venom IKITTMLAKLGKVLAHV* (SEQ ID P10521 Argiolas, A. and Pisano, (Megabombus NO: 171) J. J. (1985) pennsylvanicus) Bomb olitin BII Bumblebee venom SKITDILAKLGKVLAHV* (SEQ ID PO7493 Argiolas, A. and Pisano, (Megabombus NO: 172) J. J. (1985) pennsylvanicus) BPTI Bovin Bovine Pancreas RPDFCLEPPYTGPCKARRYFYNAKAGL POO974 Creighton, T. and Charles, Pancreatic COTFWYGGCRAKRNNFKSAEDCMRTCG I. G. (1987) Trypsin GA (SEQ ID NO: 173) Inhibitor (BPTI) Brewinins Brewinin-1E European frog FLPLLAGLAANFLPKIFCKITRKC (SEQ ID S33729 Simmaco, M. et al., (1993) (Rana esculenta) NO: 174) Brewinin-2E GIMDTLKNLAKTAGKGALOSLLNKASCK S33730 Simmaco, M. et al., (1993) LSGQC (SEQ ID NO: 175) Cecropins Cecro pin. A Silk moth KWKLFKKIEKVGONIRDGIIKAGPAVAV M63845 Gudmundsson, G. H. et al., (Hyalophora cecropia) VGQATOIAK (SEQ ID NO: 176) (1991) Cecro bin B Silk moth KWKVFKKEKMGRNRNGVKAGPAAV ZO74.04 Xanthopoulos, G. et al. (Hyalophora cecropia) LGEAKAL (SEQ ID NO: 177) (1988) Cecro pin C Fruit fly GWLKKLGKRIERIGOEITRDATIOGLGIAO Z11167 Tryselius, Y. et al. (1992) (Drosophila QAANVAATARG (SEQ ID NO: 178) melanogaster) Cecro bin D Silk moth pupae WNPFKELEKVGORVRDAVISAGPAVATV PO1510 Hultmark, D. et al., (1982) (Hyalophora cecropia) AQATALAK (SEQ ID NO: 179) Cecro pin P. Pig small intestine SWLSKTAKKLENSAKKRISEGIAIAIOGGP P14661 Lee, J. -Y. et al., (1989) (Sus scrofa) R (SEQ ID NO: 180) Charybdtoxins Chary bdtoxin Scorpion venom (Leiurus ZFTNVSCTTSKECWSVCORLHINTSRGKC P13487 Schweitz, H. et al., (1989) quin-questriatus MNKKCRCYS (SEQ ID NO: 181) hebraeus) Coleoptericins Coleo ptericin Beetle 8.1 kDa A41711 Bulet, P. et al., (1991) (Zophobas atratus) Crabolins Craboli European hornet venom FLPLILRKIVTAL* (SEQ ID NO: 182) AO1781 Argiolas, A. and Pisano, (Vespa crabo) J. J. (1984) Defensins Cryptdin 1 Mouse intestine LRDLVCYCRSRGCKGRERMNGTCRKGH A43279 Selsted, M. E. et al., (1992) alpha (Mus musculus) LLYTLCCR (SEQ ID NO: 183) Cryptdin 2 Mouse intestine LRDLVCYCRTRGCKRRERMNGTCRKGH C43279 Selsted, M. E. et al., (1992) (Mus musculus) LMYTLCCR (SEQ ID NO:184) MCP1 Rabbit alveolar VVCACRRALCLPRERRAGFCRRGRHPL M28883 Selsted, M. et al., (1983) macrophages CCRR (SEQ ID NO:185) (Oryctolagus cuniculus) MCP2 Rabbit alveolar VVCACRRALCLPLERRAGFCRIRGRHPL M28073 Ganz, T. et al., (1989) macrophages CCRR (SEQ ID NO: 186) (Oryctolagus cuniculus) GNCP-1 Guinea pig RRCICTTRTCRFPYRRLGTCIFQNRVYTF S21169 Yamashita, T. and Saito, (Cavia cutteri) CC (SEQ ID NO: 187) K., (1989) GNCP-2 Guinea pig RRCICTTRTCRFPYRRLGTCLFONRVYTF X63676 Yamashita, T. and Saito, (Cavia cutteri) CC (SEQ ID NO: 188) K., (1989) HNP-1 AZurophil granules of ACYCRIPACIAGERRYGTCIYOGRLWAFC P11479 Lehrer, R. et al., (1991) human neutrophils C (SEQ ID NO: 189) HNP-2 AZurophil granules of CYCRIPACIAGERRYGTCIYOGRLWAFCC P11479 Lehrer, R. et al., (1991) human neutrophils (SEQ ID NO:190) NP-1 Rabbit neutrophils VVCACRRALCLPRERRAGFCRRGRHPL PO1376 Ganz, T. et al., (1989) (Oryctolagus cuniculus) CCRR (SEQ ID NO: 191) US 6,503,881 B2 11 12

TABLE 1-continued Cationic Peptides Accession Group Name Peptide Origin Sequence Number Reference NP-2 Rabbit neutrophils VVCACRRALCLPLERRAGFCRIRGRHPL PO1377 Ganz, T. et al., (1989) (Oryctolagus cuniculus) CCRR (SEQ ID NO: 192) RatNP-1 Rat neutrophils VTCYCRRTRCGFRERLSGACGYRGRIYR Eisenhauer, P. B. et al., (Rattus norvegicus) LCCR (SEQ ID NO: 193) (1989) RatNP-2 Rat neutrophils VTCYCRSTRCGFRERLSGACGYRGRYR Eisenhauer, P. B. et al, (Rattus norvegicus) LCCR (SEQ ID NO: 194) (1989) Defensins BNBD-1 Bovine neutrophils DFASCHTNGGICLPNRCPGHMIOIGICFRP 127951 Selsted, M. E. et al., (1993) beta RVKCCRSW (SEQ ID NO: 195) BNBD-2 Bovine neutrophils VRNHVTCRINRGFCVPIRCPGRTROIGTC 127952 Selsted, M. E., et al., FGPRIKCCRSW (SEQ ID NO: 196) (1993) TAP Bovine tracheal mucosa NPVSCVRNKGICVPIRCPGSMKOIGTCVG P25068 Diamond, G. et al., (1991) (Bos taurus) RAVKCCRKK (SEQ ID NO: 197) Defensins Sapecin Flesh fly ATCDLLSGTGINHSACAAHCLLRGNRGG JO4053 Hanzawa, H. et al., (1990) insect (Sacrophaga peregrina) YCNGKAVCVCRN (SEQ ID NO: 198) Insect defensin Dragonfly larvae GFGCPLDOMOCHRHCOTITGRSGGYCSG Bulet, P. et al., (1992) (Aeschna cyanea) PLKLTCTCYR (SEQ ID NO: 199) Defensins Scorpion Scorpion GFGCPLNOGACHRHCRSIRRRGGYCAGF Cociancich, S. et al., scorpion defensin (Leiurus quinquestriatus) FKQTCTCYRN (SEQ ID NO: 200) (1993) Dermaseptins Dermaseptin South American arboreal ALWKTMLKKLGTMALHAGKAALGAAD Mor, A., et al., (1991) frog TISQTO (SEQ ID NO: 201) (Phyllomedusa Sauvagii) Diptericins Diptericin Nesting-suckling blowfly 9 kDa X15851 Reichhardt, J. M. et al., (Phormia terranovae) (1989) Drosocins Drosocin Fruit fly GKPRPYSPRPTSHPRPIRV (SEQ ID S35984 Bulet, P. et al., (1993) (Drosophila NO: 202) melanogaster) Esculentins Esculentin European frog GIFSKLGRKKIKNLLISGLKNVGKEVGMD S33731 Simmaco, M. et al., (1993) (Rana esculenta) VVRTGIDIAGCKIKGEC (SEQ ID NO: 203) Indolicidins Indolicidin Bovine neutrophils ILPWKWPWWPWRR (SEQ ID NO: 204) A42387 Selsted, M. et al., (1992) Lactoferricins Lactoferricin B N terminal region of FKCRRWOWRMKKLGAPSITCVRRAF M63502 Bellamy, W. et al., bovine lactoferrin (SEQ ID NO: 205) (1992b) Lantibiotics Nisin Lactococcus lactis ITSISLCTPGCKTGALMGCNMKTATCHCS P13068 Hurst, A. (1981) subsp. Lactis (bacterium) IHVSK (SEQ ID NO: 206) Pep 5 Staphylococcus TAGPAIRASVKOCOKTLKATRLFTVSCK P19578 Keletta, C. et al., (1989) epidermidis GKNGCK (SEQ ID NO: 207) Subtilin Bacilius Subtilis MSKFDDFDLDVVKVSKODSKITPOWKSE P10946 Banerjee, S. and Hansen, (bacterium) SLCTPGCVTGALOTCFLOTL TCNCKISK J. N. (SEQ ID NO: 208) (1988) Leukocins Leukocin LeuconoStoc geidun KYYGNGVHCTKSGCSVNWGEAFSAGVH S65611 Hastings, J. W. et al., A-val 187 UAL 187 RLANGGNGFW (SEQ ID NO: 209) (1991) (bacterium) Magainins Magainin I Amphibian skin GIGKFLHSAGKFGKAFVGEIMKS (SEQ A29771 Zasloff, M. (1987) (Xenopus laevis) ID NO: 210) Magainin II Amphibian skin GIGKFLHSAKKFGKAFVGEIMNS (SEQ A29771 Zasloff, M. (1987) (Xenopus laevis) ID NO: 211) PGLa Amphibian skin GMASKAGAIAGKIAKVALKAL* (SEQ ID X13388 Kuchler, K. et al., (1989) (Xenopus laevis) NO: 212) PGO Amphibian stomach GVLSNVIGYLKKLGTGALNAVLKQ (SEQ Moore, K. S. et al., (1989) (Xenopus laevis) ID NO: 213) XPF Amphibian skin GWASKIGOTLGKLAKVGLKELIOPK PO7198 Sures, I. And Crippa, M. (Xenopus laevis) (SEQ ID NO: 214) (1984) Mastoparans Mastoparan Wasp venom INLKALAALAKKIL* (SEQ ID NO: 215) PO1514 Bernheimer, A. and Rudy, (Vespula lewisii) B. (1986) Melittins Melittin Bee venom GIGAVLKVLTTGLPALISWIKRKROQ PO1504 Tosteson, M. T. and (Apis mellifera) (SEQ ID NO: 216) Tosteson, D. C. (1984) Phormicins Phormicin. A Nestling-suckling ATCDLLSGTGINHSACAAHCLLRGNRGG P10891 Lambert, J. et al., (1989) blowfly YCNGKGVCVCRN (SEQ ID NO: 217) (Phormia terranovae) Phormicin B Nestling-suckling ATCDLLSGTGINHSACAAHCLLRGNRGG P10891 Lambert, J. et al., (1989) blowfly YCNRKGVCVRN (SEQ ID NO: 218) (Phormia terranovae) Polyphemusins Polyphemusin I Atlantic horseshoe crab RRWCFRVCYRGFCYRKCR* (SEQ ID P14215 Miyata, T. et al., (1989) (Limulus polyphemus) NO: 219) Polyphemusin II Atlantic horseshoe crab RRWCFRVCYKGFCYRKCR* (SEQ ID P14216 Miyata, T. et al., (1989) (Limulus polyphemus) NO: 220) Protegrins Protegrin I Porcine leukocytes RGGRLCYCRRRFCVCVGR (SEQ ID S34585 Kokryakov, V. N. et al., (Sus scrofa) NO: 221) (1993) Protegrin II Porcine leukocytes RGGRLCYCRRRFCICV (SEQ ID NO: 222) 53.4586 Kikryakov, V. N. et al., (Sus scrofa) (1993) Protegrin III Porcine leukocytes RGGGLCYCRRRFCVCVGR (SEQ ID 534587 Kokryakov, V. N. et al., (Sus scrofa) NO: 223) (1993) Royalisins Royalisin Royal Jelly VTCDLLSFKGOVNDSACAANCLSLGKAG P17722 Fujiwara, S. et al., (1990) (Apis mellifera) GHCEKGVCICRKTSFKDLWDKYF (SEQ ID NO: 224) US 6,503,881 B2 13 14

TABLE 1-continued Cationic Peptides Accession Group Name Peptide Origin Sequence Number Reference Sarcotoxins Sarcotoxin IA Flesh fly GWLKKIGKKIERVGOHTRDATIOGLGIA P08375 Okada, M. and Natori S., (Sacrophaga peregrina) QQAANVAATAR* (SEQ ID NO: 225) (1985b) Sarcotoxin IB Flesh fly GWLKKIGKKIERVGOHTRDATIOVIGVA P08376 Okada, M. and Natori S., (Sacrophaga peregrina) QQAANVAATAR* (SEQ ID NO: 226) (1985b) Seminal Seminalplasmin Bovine seminal plasma SDEKASPDKHHRFSLSRYAKLANRLANP SO8184. Reddy, E. S. P. and plasmins (Bos taurus) KLLETFLSKWIGDRGNRSV (SEQ ID Bhargava, P. M. (1979) NO: 227) Tachyplesins Tachyplesin I Horseshoe crab KWCFRVCYRGICYRRCR* (SEQ ID P23684 Nakamura, T. et al., (1988) (Tachypleus tridentatus) NO: 228) Tachyplesin II Horseshoe crab RWCFRVCYRGICYRKCR* (SEQ ID P14214 Muta, T. et al., (1990) (Tachypleus tridentatus) NO: 229) Thionins Thionin Barley leaf KSCCKDTLARNCYNTCRFAGGSRPVCAG SOO825 Bohlmann, H. et al., BTH6 (Hordeum vulgare) ACRCKIISGPKCPSDYPK (SEQ ID (1988) NO: 230) Toxins Toxin 1 Waglers pit viper venom GGKPDLRPCIIPPCHYIPRPKPR (SEQ ID P24335 Schmidt, J. J. et al., (1992) (Trimeresurus wagleri) NO: 231) Toxin 2 Sahara scorpion WKDGYIVDDVNCTYFCGRNAYCNEECT PO1484 Bontems, F, et al., (1991) (Androctonus australis KLKGESGYCOWASPYGNACYCKLPDHV Hector) RTKGPGRCH (SEQ ID NO. 232) Arigiolas and Pisano, (1984). JBC 259, 101.06; Argiolas and Pisano, (1985). JBC 260, 1437; Banerjee and Hansen, (1988). JBC 263, 9508; Bellamy et al., (1992). J. Appl. Bacter. 73, 472; Bernheimer and Rudy, (1986). BBA 864, 123; Bohlmann et al., (1988). EMBO J. 7, 1559; Bontems et al., (1991). Science 254, 1521: Bulet et al., (1991). JBC 266, 24520; Bulet et al. (1992). Eur. J. Biochem. 209, 977; Bulet et al., (1993). JBC 268, 14893; Casteels et al., (1989). EMBO J. 8, 2387: Casteels et al., (1990). Eur J. Biochem. 187, 381; Cociancich et al., (1993). BBRC 194, 17: Creighton and Charles, (1987). J. Mol. Biol. 194, 11; Csordas and Michl, (1970). Monatsh Chemistry 101, 182; Diamond et al., (1991). PNAS 88, 3952; Dickinson et al., (1988). JBC 263, 19424; Eisenhauer et al., (1989). Infect. and Imm. 57, 2021; Frank et al., (1990). JBC 265, 18871; Fujiwara et al., (1990). JBC 265, 11333; Galvez et al., (1989). Antimicrobial Agents and Chemotherapy 33, 437; Ganz et al., (1989). J. Immunol. 143, 1358; Gibson et al., (1991). JBC 266, 23103; Gudmundsson et al., (1991). JBC 266, 11510; Hanzawa et al., (1990). FEBS Letters 269, 413; Hastings et al., (1991). J. of Bacteriology 173, 7491; Hultmark et al., (1982). Eur. J. Biochem. 127, 207; Hurst, A. (1981). Adv. Appl. Micro. 27, 85; Kaletta et al., (1989). Archives of Microbiol ogy 152, 16; Kokryakov et al., (1993). FEBS Letters 327, 231: Kuchler et al., (1989) Eur. J. Biochem. 179, 281; Lambert et al., (1989). PNAS 86, 262, Lee et al., (1989). PNAS 86, 9159; Lehrer et al., (1991). Cell 64, 229; Miyata et al., (1989). J. of Biochem. 106, 663; Moore et al., (1991). JBC 266, 19851; Mor et al., (1991). Biochemistry 30, 8824; Muta et al., (1990). J. Biochem. 108, 261; Nakamura et al., (1988). JBC 263, 16709; Nakamura et al., (1983). Infection and Immunity 39, 609; Okada and Natori (1985). Biochem. J. 229, 453; Reddy and Bhargava, (1979). Nature 279,725; Reichhart et al., (1989). Eur. J. Biochem. 182, 423; Romeo et al., (1988). JBC 263,9573; Samakovlis et al., (1991). EMBO J. 10, 163; Schmidt et al., (1992). Toxi con 30, 1027; Schweitz et al., (1989). Biochem. 28,9708; Selsted et al., (1983). JBC 258, 14485; Selsted et al., (1992). JBC 267, 4292; Simmaco et al., (1993). FEBS Letters 324, 159; Sures and Crippa (1984). PNAS 81,380; Takada et al., (1984). Infect. and Immun. 44, 370; Tosteson and Tosteson, (1984). Biophysical J. 45, 112: Tryselius et al., (1992). Eur. J. Biochem. 204, 395; Xanthopoulos et al., (1988). Eur. J. Biochem. 172, 371; Yamashita and Saito, (1989). Infect. and Imm. 57, 2405; Zasloff, M. (1987). PNAS 84,5449.

In addition to the peptides listed above, chimeras and 40 ation of an amino acid Side chain, Such as acylation of the analogues of these peptides are useful within the context of e-amino group of lysine. Other amino acids that may be the present invention. For this invention, analogues of native incorporated include any of the D-amino acids correspond cationic peptides must retain a net positive charge, but may ing to the 20 L-amino acids commonly found in proteins, contain D-amino acids, amino acid derivatives, insertions, rare amino acids, Such as hydroxylysine, or non-protein deletions, and the like, Some of which are discussed below. amino acids, Such as homoserine and omithine. A peptide Chimeras include fusions of cationic peptide, such as the may have none or one or more of these derivatives, and may peptides of fragments thereof listed above, and fusions of contain D-amino acids (specified as a lower case letter when cationic peptides with non-cationic peptides. using the 1-letter code). Furthermore, modification of the N AS described herein, modification of any of the residues or C-terminus is within the Scope of the invention. A including the N- or C-terminus is within the scope of the preferred modification of the C-terminus is amidation. Other invention. A preferred modification of the C-terminus is 50 modifications of the C-terminus include ester additions. amidation. Other modifications of the C-terminus include esterification and lactone formation. N-terminal modifica N-terminal modifications include acetylation, myristlyation, tions include acetylation, acylation, alkylation, PEGylation, and the like. myristylation, and the like. Additionally, the peptide may be A. Indolicidin and Analogues modified to forman polymer-modified peptide as described 55 AS noted above, the present invention provides cationic below. The peptides may also be labeled, such as with a peptides, including indolicidin and indolicidin analogues. radioactive label, a fluorescent label, a mass spectrometry Analogues include peptides that have one or more tag, biotin and the like. insertions, deletions, modified amino acids, D-amino acids Unless otherwise indicated, a named amino acid refers to and the like. These analogues may be Synthesized by chemi the L-form. Basic amino acids include arginine, lysine, 60 cal methods, especially using an automated peptide histidine, and derivatives. Hydrophobic residues include Synthesizer, or produced by recombinant methods. The tryptophan, phenylalanine, isoleucine, leucine, Valine, and choice of an amino acid Sequence is guided by a general derivatives. The peptide may contain derivatives of amino formula presented herein. acids that have been altered by chemical means, Such as The indolicidin analogues of the present invention are at methylation (e.g., C.-methylvaline), amidation, especially of 65 least 5 or 7 amino acids in length and preferably not more the C-terminal amino acid by an alkylamine (e.g., than 15, 20, 25, 27, 30, or 35 amino acids. Analogues from ethylamine, ethanolamine, and ethylene diamine) and alter- 9 to 14 residues are preferred. General formulas for peptide US 6,503,881 B2 15 16 analogues in the Scope of the present invention may be set K K W W RR X (SEQ ID NOS: 134–144) forth as: K K W W K X (SEQ ID NO: 145-155) RXZXXZXB (SEQ ID NO: 1) (1) wherein R is a hydrophobic amino acid residue, R is a hydrophilic amino acid residue, and X is from about 14 to BXZXXZXB (SEQ ID NO: 2) (2) 24 amino acid residues. BBBXZXXZXB (SEQ ID NO:3) (3) E. Drosocin and Analogues BXZXXZXBBB (AA), MILBBAGS (SEQ ID NOS: AS noted herein, cationic peptides include droSocin and 5-8) (4) droSocin analogues. DroSocins are isolated from Drosophila BXZXXZXBB(AA),M (SEQ ID NOS:9-20) (5) melanogaster. An exemplary droSocin is a 19 amino acid LBBXZXXZXRK (SEQ ID NOS: 11–18)(6) peptide having the sequence: GKPRPYSPRPTSHPRPIRV (SEQ ID NO. 202; GenBank Accession No. S35984). Ana LKXZXXZXRRK (SEQ ID NOS: 19–20)(7) logues of droSocin include peptides that have insertions, BBXZXXZXBBB (SEQ ID NO: 21) (8) deletions, modified amino acids, D-amino acids and the like. BBXZXXZXBBB (SEQ ID NO: 22) (9) F. Peptide Synthesis BXXBZBXBXZB (SEQ ID NO: 4) (10) Peptides may be synthesized by standard chemical wherein Standard Single letter amino abbreviations are 15 methods, including Synthesis by automated procedure. In used and; Z is proline, glycine or a hydrophobic residue, and general, peptide analogues are Synthesized based on the preferably Z is proline or valine, X is a hydrophobic residue, standard Solid-phase Fmoc protection strategy with HATU Such as tryptophan, phenylalanine, isoleucine, leucine and as the coupling agent. The peptide is cleaved from the Valine, and preferably tryptophan; B is a basic amino acid, Solid-phase resin with trifluoroacetic acid containing appro preferably arginine or lysine, AA is any amino acid, and n is priate Scavengers, which also deprotects Side chain func 0 or 1. In formula (2), at least one Z is valine; in formula (8), tional groups. Crude peptide is further purified using pre at least two XS are phenylalanine; and in formula (9), at least parative reversed-phase chromatography. Other purification two XS are tyrosine. Additional residues may be present at methods, Such as partition chromatography, gel filtration, gel the N-terminus, C-terminus, or both. 25 electrophoresis, or ion-exchange chromatography may be B. Cecropin Peptides used. Cecropins are cationic peptides that have antimicrobial Other Synthesis techniques, known in the art, Such as the activity against both Gram-positive and Gram-negative bac tBoc protection Strategy, or use of different coupling teria. Cecropins have been isolated from both invertebrates reagents or the like can be employed to produce equivalent (e.g., insect hemolymph) as well as vertebrates (e.g. pig peptides. intestines). Generally, these peptides are 35 to 39 residues. Peptides may be Synthesized as a linear molecule or as An exemplary cecropin has the sequence KWKLFK branched molecules. Branched peptides typically contain a KIEKVGQNIRDGIIKAGPAVAVVGQATOIAK (SEQ ID core peptide that provides a number of attachment points for NO.176). Some additional cecropin sequences are presented additional peptides. Lysine is most commonly used for the in Table 1. Within the context of this invention, cecropins core peptide because it has one carboxyl functional group include analogues that have one or more insertions, 35 and two (alpha and epsilon) amine functional groups. Other deletions, modified amino acids, D-amino acids and the like. diamino acids can also be used. Preferably, either two or C. Melittin Peptides three levels of geometrically branched lysines are used; Melittin is a cationic peptide found in bee venom. An these cores form a tetrameric and octameric core Structure, amino acid Sequence of an exemplary melittin peptide is respectively (Tam, Proc. Natl. Acad Sci. USA 85:5409, GIGAVLKVLTTGLPALISWIKRKKRQQ (SEQ ID NO. 40 1988). Schematically, examples of these cores are repre 216). Like the cecropins, melittin exhibits antimicrobial activity sented as shown (SEQ ID NO:29): against both Gram-positive and Gram-negative bacteria. Within the context of this invention, melittin includes ana D Lys n logues that have one or more insertions, deletions, modified 45 N Lys amino acids, D-amino acids and the like. 1. ty D Lys1 N D. Cecropin-melittin Chimeric Peptides AS noted herein, cationic peptides include fusion peptides D Lys season N1. Lys n Lys yo of native cationic peptides and analogues of fusion peptides. N L 1 In particular, fusions of cecropin and melittin are provided. 50 1. y An exemplary fusion has the Sequence: cecropin A (residues 1-8)/melittin (residues 1-18). Other fusion peptides useful within the context of this invention are described by the The attachment points for the peptides are typically at general formulas below. their carboxyl functional group to either the alpha or epsilon 55 amine groups of the lysines. To Synthesize these multimeric KW K R. R. R. R. R. R. R. R. R. R. R. R V LTT peptides, the Solid phase resin is derivatized with the core G LP A LIS (SEQ ID NO: 128) matrix, and Subsequent Synthesis and cleavage from the KW K R. R. R. R. R. R. R. R. R. R. R. R. V V TT resin follows Standard procedures. The multimeric peptides A K P L I SS (SEQ ID NO: 129) may be used within the context of this invention as for any KWKR. R. R. R. R. R. R. R. R. R. R. I LTTG 60 of the linear peptides and are preferred for use in generating L PAL I S (SEQ ID NO:130) antibodies to the peptides. KW K R. R. R. R. R. R. R. R. R. R. R G G LL G. Recombinant Production of Peptides S N IV T S L (SEQ ID NO: 131) Peptides may alternatively be synthesized by recombinant KW K R. R. R. R. R. R. R. R. R. GPI LA production (see e.g., U.S. Pat. No. 5,593.866). A variety of N L V S I V (SEQ ID NO: 132) 65 host Systems are Suitable for production of the peptide KKWW RR R. R. R. R. R. R. R. GPALS NV (SEQ analogues, including bacteria (e.g., E. coli), yeast (e.g., ID NO: 133) Saccharomyces cerevisiae), insect (e.g., Sf9), and mamma US 6,503,881 B2 17 18 lian cells (e.g., CHO, COS-7). Many expression vectors At minimum, the expression vector should contain a have been developed and are available for each of these promoter Sequence. However, other regulatory Sequences hosts. Generally, bacteria cells and Vectors that are func may also be included. Such Sequences include an enhancer, tional in bacteria are used in this invention. However, at ribosome binding Site, transcription termination Signal times, it may be preferable to have vectors that are func Sequence, Secretion signal Sequence, origin of replication, tional in other hosts. Vectors and procedures for cloning and Selectable marker, and the like. The regulatory Sequences are operationally associated with one another to allow transcrip expression in E. coli are discussed herein and, for example, tion and Subsequent translation. In preferred aspects, the in Sambrook et al. (Molecular Cloning. A Laboratory plasmids used herein for expression include a promoter Manual, Cold Spring Harbor Laboratory Press, Cold Spring designed for expression of the proteins in bacteria. Suitable Harbor, N.Y., 1987) and in Ausubel et al. (Current Protocols 1O promoters, including both constitutive and inducible in Molecular Biology, Greene Publishing Co., 1995). promoters, are widely available and are well known in the A DNA sequence encoding a cationic peptide is intro art. Commonly used promoters for expression in bacteria duced into an expression vector appropriate for the host. In include promoters from T7, T3, T5, and SP6 phages, and the preferred embodiments, the gene is cloned into a vector to trp, lpp, and lac operons. Hybrid promoters (see, U.S. Pat. create a fusion protein. The fusion partner is chosen to 15 No. 4,551,433), such as tac and trc, may also be used. contain an anionic region, Such that a bacterial host is Within a preferred embodiment, the vector is capable of protected from the toxic effect of the peptide. This protective replication in bacterial cells. Thus, the vector may contain a region effectively neutralizes the antimicrobial effects of the bacterial origin of replication. Preferred bacterial origins of peptide and also may prevent peptide degradation by host replication include f1-ori and col E1 ori, especially the ori proteases. The fusion partner (carrier protein) of the inven derived from pUC plasmids. Low copy number vectors (e.g., tion may further function to transport the fusion peptide to pPD100) may also be used, especially when the product is inclusion bodies, the periplasm, the Outer membrane, or the deleterious to the host. extracellular environment. Carrier proteins Suitable in the The plasmids also preferably include at least one Select context of this invention Specifically include, but are not able marker that is functional in the host. A selectable limited to, glutathione-S-transferase (GST), protein A from 25 marker gene conferS a phenotype on the host that allows StaphylococcuS aureus, two synthetic IgG-binding domains transformed cells to be identified and/or Selectively grown. (ZZ) of protein A, outer membrane protein F, Suitable Selectable marker genes for bacterial hosts include f-galactosidase (lacZ), and various products of bacterioph the chloroamphenicol resistance gene (Cm'), amplicillin age 2 and bacteriophage T7. From the teachings provided resistance gene (Amp), tetracycline resistance gene (Tc) herein, it is apparent that other proteins may be used as kanamycin resistance gene (Kan), and others known in the carriers. Furthermore, the entire carrier protein need not be art. To function in Selection, Some markers may require a used, as long as the protective anionic region is present. To complementary deficiency in the host. facilitate isolation of the peptide Sequence, amino acids In Some aspects, the Sequence of nucleotides encoding the Susceptible to chemical cleavage (e.g., CNBr) or enzymatic peptide also encodes a Secretion Signal, Such that the result cleavage (e.g., V8 protease, trypsin) are used to bridge the 35 ing peptide is Synthesized as a precursor protein, which is peptide and fusion partner. For expression in E. coli, the Subsequently processed and Secreted. The resulting Secreted fusion partner is preferably a normal intracellular protein protein may be recovered from the periplasmic space or the that directs expression toward inclusion body formation. In fermentation medium. Sequences of Secretion signals Suit Such a case, following cleavage to release the final product, able for use are widely available and are well known (von there is no requirement for renaturation of the peptide. In the 40 Heijne, J. Mol. Biol. 184:99-105, 1985). present invention, the DNA cassette, comprising fusion The vector may also contain a gene coding for a repressor partner and peptide gene, may be inserted into an expression protein, which is capable of repressing the transcription of a vector, which can be a plasmid, Virus or other vehicle known promoter that contains a repressor binding site. Altering the in the art. Preferably, the expression vector is a plasmid that physiological conditions of the cell can depress the pro contains an inducible or constitutive promoter to facilitate 45 moter. For example, a molecule may be added that com the efficient transcription of the inserted DNA sequence in petitively binds the repressor, or the temperature of the the host. Transformation of the host cell with the recombi growth media may be altered. Repressor proteins include, nant DNA may be carried out by Ca"-mediated techniques, but are not limited to the E. coli lacI repressor (responsive by electroporation, or other methods well known to those to induction by IPTG), the temperature sensitive cI857 skilled in the art. 50 repressor, and the like. Briefly, a DNA fragment encoding a peptide is derived Examples of plasmids for expression in bacteria include from an existing cDNA or genomic clone or Synthesized. A the pT expression vectors pFT3a, pFT 11a, pFT 12a-c, and convenient method is amplification of the gene from a pET 15b (see U.S. Pat. No. 4,952,496; available from Single-Stranded template. The template is generally the prod Novagen, Madison, Wis.). Low copy number vectors (e.g., uct of an automated oligonucleotide Synthesis. Amplification 55 pPD100) can be used for efficient overproduction of pep primers are derived from the 5' and 3' ends of the template tides deleterious to the E. coli host (Dersch et al., FEMS and typically incorporate restriction sites chosen with regard Microbiol. Lett. 123:19, 1994). to the cloning site of the vector. If necessary, translational Bacterial hosts for the T7 expression vectors may contain initiation and termination codons can be engineered into the chromosomal copies of DNA encoding T7 RNA polymerase primer Sequences. The Sequence encoding the protein may 60 operably linked to an inducible promoter (e.g., lacUV pro be codon-optimized for expression in the particular host. moter; see, U.S. Pat. No. 4,952,496), such as found in the E. Thus, for example, if the analogue fusion protein is coli strains HMS 174(DE3)pLysS, BL21 (DE3)pLyss, expressed in bacteria, codons are optimized for bacterial HMS174(DE3) and BL21 (DE3). T7 RNA polymerase can usage. Codon optimization is accomplished by automated also be present on plasmids compatible with the T7 expres Synthesis of the entire gene or gene region, ligation of 65 Sion vector. The polymerase may be under control of a multiple oligonucleotides, mutagenesis of the native lambda promoter and repressor (e.g., p.GP1-2, Tabor and Sequence, or other techniques known to those in the art. Richardson, Proc. Natl. Acad. Sci. USA 82: 1074, 1985). US 6,503,881 B2 19 20 The peptide product is isolated by Standard techniques, administered as an emulsion in an adjuvant Such as Freund's Such as affinity, Size exclusion, or ionic exchange complete or incomplete adjuvant in order to increase the chromatography, HPLC and the like. An isolated peptide immune response. The animal is generally boosted at least should preferably show a major band by Coomassie blue once prior to harvest of Spleen and/or lymph nodes and Stain of SDS-PAGE that is at least 90% of the material. immortalization of those cells. Various immortalization H. Generation of Analogues by Amplification-based Semi techniques, Such as mediated by Epstein-Barr virus or fusion random Mutagenesis to produce a hybridoma, may be used. In a preferred Cationic peptide analogues can be generated using an embodiment, immortalization occurs by fusion with a Suit able myeloma cell line to create a hybridoma that Secretes amplification (e.g., PCR)-based procedure in which primers monoclonal antibody. Suitable myeloma lines include, for are designed to target Sequences at the 5' and 3' ends of an example, NS-1 (ATCC No. TIB 18), and P3X63-Ag 8.653 encoded parent peptide, for example indolicidin. Amplifi (ATCC No. CRL 1580). The preferred fusion partners do not cation conditions are chosen to facilitate misincorporation of express endogenous antibody genes. After about Seven days, nucleotides by the thermostable polymerase during Synthe the hybridomas may be Screened for the presence of anti Sis. Thus, random mutations are introduced in the original bodies that are reactive against a telomerase protein. A wide Sequence, Some of which result in amino acid alteration(s). 15 variety of assays may be utilized (see Antibodies: A Labo Amplification products may be cloned into a coat protein of ratory Manual, Harlow and Lane (eds.), Cold Spring Harbor a phage Vector, Such as a phagemid Vector, packaged and Laboratory Press, 1988). amplified in an acceptable host to produce a display library. Other techniques may also be utilized to construct mono These libraries can then be assayed for antibiotic activity clonal antibodies (see Huse et al., Science 246:1275-1281, of the peptides. Briefly, bacteria infected with the library are 1989; Sastry et al., Proc. Natl. Acad. Sci. USA plated, grown, and overlaid with agarose containing a bac 86:5728-5732, 1989; Alting-Mees et al., Strategies in terial Strain that the phage are unable to infect. Zones of Molecular Biology 3: 1-9, 1990; describing recombinant growth inhibition in the agarose overlay are observed in the techniques). These techniques include cloning heavy and area of phage expressing an analogue with anti-bacterial light chain immunoglobulin cDNA in Suitable vectors, Such activity. These inhibiting phage are isolated and the cloned 25 as ImmunoZap(H) and ImmunoZap(L). These recombi peptide Sequence determined by DNA sequence analysis. nants may be Screened individually or co-expressed to form The peptide can then be independently Synthesized and its Fab fragments or antibodies (see Huse et al., Supra; Sastry et antibiotic activity further investigated. al., Supra). Positive plaques may Subsequently be converted 5. Antibodies to Cationic Peptides to a non-lytic plasmid that allows high level expression of Antibodies may be generated to a specific peptide ana monoclonal antibody fragments from E. coli. logue using multiple antigenic peptides (MAPs) that contain Similarly, portions or fragments, Such as Fab and FV approximately eight copies of the peptide linked to a Small fragments, of antibodies may also be constructed utilizing non-immunogenic peptidyl core to form an immunogen. conventional enzymatic digestion or recombinant DNA (See, in general, Harlow and Lane, Supra.) Alternatively, the techniques to yield isolated variable regions of an antibody. target peptide can be conjugated to bovine Serum albumin 35 Within one embodiment, the genes which encode the vari (BSA), ovalbumin or another suitable conjugate. The MAP able region from a hybridoma producing a monoclonal or peptide conjugate is injected Subcutaneously into rabbits antibody of interest are amplified using nucleotide primers or into mice or other rodents, where they may have Suffi for the variable region. In addition, techniques may be ciently long half-lives to facilitate antibody production. utilized to change a “murine' antibody to a “human” After twelve weeks blood Samples are taken, Serum is 40 antibody, without altering the binding Specificity of the Separated and tested in an ELISA assay against the original antibody. peptide, with a positive result indicating the presence of antibodies specific to the target peptide. This Serum can then II. TESTING be Stored and used in ELISA assays to specifically measure Cationic peptides of the present invention are assessed the amount of the Specific analogue. Alternatively, other 45 either alone or in combination with an antibiotic agent or Standard methods of antibody production may be employed, another analogue for their potential as antibiotic therapeutic for example generation of monoclonal antibodies. agents using a Series of assayS. Preferably, all peptides are Within the context of the present invention, antibodies are initially assessed in Vitro, the most promising candidates are understood to include monoclonal antibodies, polyclonal Selected for further assessment in Vivo, and then candidates antibodies, anti-idiotypic antibodies, antibody fragments 50 are Selected for pre-clinical Studies. The in Vitro assays (e.g., Fab, and F(ab'), F. Variable regions, or complemen include measurement of antibiotic activity, toxicity, tarity determining regions). Antibodies are generally Solubility, pharmacology, Secondary Structure, liposome per accepted as Specific against indolicidin analogues if they meabilization and the like. In Vivo assays include assess bind with a K of greater than or equal to 107M, preferably ment of efficacy in animal models, antigenicity, toxicity, and greater than of equal to 10 M. The affinity of a monoclonal 55 the like. In general, in Vitro assays are initially performed, antibody or binding partner can be readily determined by followed by in vivo assays. one of ordinary skill in the art (see Scatchard, Ann, N.YAcad Generally, cationic peptides are initially tested for (1) Sci. 51:660-672, 1949). Once suitable antibodies have been anti-microbial activity in vitro, (2) in vitro toxicity to normal obtained, they may be isolated or purified by many tech mammalian cells, and (3) in Vivo toxicity in an animal niques well known to those of ordinary skill in the art. 60 model. Peptides that have Some anti-microbial activity are Monoclonal antibodies may also be readily generated preferred, although Such activity may not be necessary for from hybridoma cell lines using conventional techniques enhancing the activity of an antibiotic agent. Also, for in (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4.543,439, and Vivo use, peptides should preferably demonstrate acceptable 4,411,993; see also Antibodies. A Laboratory Manual, Har toxicity profiles, as measured by Standard procedures. Lower low and Lane (eds.), Cold Spring Harbor Laboratory Press, 65 toxicity is preferred. Additional assays may be performed to 1988). Briefly, within one embodiment, a subject animal demonstrate that the peptide is not immunogenic and to Such as a rat or mouse is injected with peptide, generally examine antimicrobial activity in Vivo. US 6,503,881 B2 21 22 A. In Vitro Assays permeabilization, and bacterial inner membrane permeabi Cationic peptides, including indolicidin analogues, are lization. In general, it is desirable that analogues are Soluble assayed by, for example, an agarose dilution MIC assay, a and perform better than the parent peptide (e.g., indolicidin). broth dilution, time-kill assay, or equivalent methods. Anti B. In Vivo Assays biotic activity is measured as inhibition of growth or killing Peptides, including peptide analogues, Selected on the of a microorganism (e.g., bacteria, fungi). basis of the results from the in vitro assays can be tested in Briefly, a candidate peptide in Mueller Hinton broth vivo for efficacy, toxicity and the like. Supplemented with calcium and magnesium is mixed with The antibiotic activity of selected peptides may be assessed in vivo for their ability to ameliorate microbial molten agarose. Other broths and agarS may be used as long infections using animal models. A variety of methods and as the peptide can freely diffuse through the medium. The animal models are available. Within these assays, a peptide agarose is poured into petri dishes or Wells, allowed to is useful as a therapeutic if inhibition of microorganismal Solidify, and a test Strain is applied to the agarose plate. The growth compared to inhibition with vehicle alone is Statis test Strain is chosen, in part, on the intended application of tically significant. This measurement can be made directly the peptide. Thus, by way of example, if an indolicidin from cultures isolated from body fluids or sites, or indirectly, analogue with activity against S. aureuS is desired, an S. 15 by assessing Survival rates of infected animals. For assess aureuS Strain is used. It may be desirable to assay the ment of antibacterial activity Several animal models are analogue on Several Strains and/or on clinical isolates of the available, Such as acute infection models including those in test Species. Plates are incubated overnight and inspected which (a) normal mice receive a lethal dose of Visually for bacterial growth. A minimum inhibitory con microorganisms, (b) neutropenic mice receive a lethal dose centration (MIC) of a cationic peptide is the lowest concen of microorganisms or (c) rabbits receive an inoculum in the tration of peptide that completely inhibits growth of the heart, and chronic infection models. The model selected will organism. Peptides that exhibit good activity against the test depend in part on the intended clinical indication of the Strain, or group of Strains, typically having an MIC of leSS analogue. than or equal to 16 lig/ml are Selected for further testing. By way of example, in a normal mouse model, mice are Alternatively, time kill curves can be used to determine 25 inoculated ip or iv with a lethal dose of bacteria. Typically, the differences in colony counts over a set time period, the dose is such that 90–100% of animals die within 2 days. typically 24 hours. Briefly, a Suspension of organisms of The choice of a microorganismal Strain for this assay known concentration is prepared and a candidate peptide is depends, in part, upon the intended application of the added. Aliquots of the Suspension are removed at Set times, analogue, and in the accompanying examples, assays are diluted, plated on medium, incubated, and counted. MIC is carried out with three different StaphylococcuS Strains. measured as the lowest concentration of peptide that com Briefly, shortly before or after inoculation (generally within pletely inhibits growth of the organism. In general, lower 60 minutes), analogue in a Suitable formulation buffer is MIC values are preferred. injected. Multiple injections of analogue may be adminis Candidate cationic peptides may be further tested for their tered. Animals are observed for up to 8 days post-infection toxicity to normal mammalian cells. An exemplary assay is 35 and the Survival of animals is recorded. Successful treatment a red blood cell (RBC) (erythrocyte) hemolysis assay. either rescues animals from death or delays death to a Briefly, in this assay, red blood cells are isolated from whole Statistically significant level, as compared with non blood, typically by centrifugation, and washed free of treatment control animals. Analogues that show better effi plasma components. A 5% (v/v) Suspension of erythrocytes cacy than indolicidin itself are preferred. in isotonic Saline is incubated with different concentrations 40 In Vivo toxicity of a peptide is measured through admin of peptide analogue. Generally, the peptide will be in a istration of a range of doses to animals, typically mice, by a suitable formulation buffer. After incubation for approxi route defined in part by the intended clinical use. The mately 1 hour at 37 C., the cells are centrifuged, and the Survival of the animals is recorded and LDs, LDoooo, and absorbance of the Supernatant at 540 nm is determined. A maximum tolerated dose (MTD) can be calculated to enable relative measure of lysis is determined by comparison to 45 comparison of analogues. Indolicidin analogues leSS toxic absorbance after complete lysis of erythrocytes using NHCl than indolicidin are preferred. or equivalent (establishing a 100% value). A peptide with Furthermore, for in Vivo use, low immunogenicity is <10% lysis at 100 lug/ml is suitable. Preferably, there is <5% preferred. To measure immunogenicity, peptides are injected lysis at 100 tug/ml. Such peptides that are not lytic, or are into normal animals, generally rabbits. At various times after only moderately lytic, are desirable and suitable for further 50 a single or multiple injections, Serum is obtained and tested Screening. Other in vitro toxicity assays, for example mea for antibody reactivity to the peptide analogue. Antibodies to Surement of toxicity towards cultured mammalian cells, may peptides may be identified by ELISA, immunoprecipitation be used to assess in vitro toxicity. assays, Western blots, and other methods. (See, Harlow and Solubility of the peptide in formulation buffer is an Lane, Antibodies: A Laboratory Manual, Cold Spring Har additional parameter that may be examined. Several differ 55 bor Laboratory Press, Cold Spring Harbor, N.Y., 1988). No ent assays may be used, Such as appearance in buffer. Briefly, or minimal antibody reactivity is preferred. Additionally, peptide is Suspended in Solution, Such as broth or formula pharmacokinetics of the analogues in animals and histopa tion buffer. The appearance is evaluated according to a Scale thology of animals treated with analogues may be deter that ranges from (a) clear, no precipitate, (b) light, diffuse mined. precipitate, to (c) cloudy, heavy precipitate. Finer gradations 60 Selection of cationic peptides as potential therapeutics is may be used. In general, leSS precipitate is more desirable. based on in vitro and in Vivo assay results. In general, However, Some precipitate may be acceptable. peptides that exhibit low toxicity at high dose levels and Additional in Vitro assays may be carried out to assess the high efficacy at low dose levels are preferred candidates. potential of the peptide as a therapeutic. Such assays include peptide Solubility in formulations, pharmacology in blood or 65 III. ANTIBIOTICAGENTS plasma, Serum protein binding, analysis of Secondary An antibiotic agent includes any molecule that tends to Structure, for example by circular dichroism, liposome prevent, inhibit or destroy life and as Such, includes anti US 6,503,881 B2 23 24 bacterial agents, anti-fuingicides, anti-Viral agents, and anti No. 12650-69-0); Metronidazole (CAS Registry No.: 443 parasitic agents. These agents may be isolated from an 48-1); Cephalexin (CAS Registry No.: 15686-71-2); organism that produces the agent or procured from a com Roxithromycin (CAS Registry No.: 80214-83-1); mercial Source (e.g., pharmaceutical company, Such as Eli Co-amoxiclavuanate; combinations of Piperacillin and Lilly, Indianapolis, Ind., Sigma, St. Louis, Mo.). TaZobactam, and their various Salts, acids, bases, and other Anti-bacterial antibiotic agents include, but are not lim derivatives. ited to, penicillins, cephalosporins, carbacephems, cepha my cins, carb ape ne mS, monobact a mS, A table presenting categories of antibiotics, their mode of aminoglycosides, glycopeptides, quinolones, tetracyclines, action, and examples of antibioticS is shown below. macrollides, and fluoroquinolones (see Table below). Examples of antibiotic agents include, but are not limited to, TABLE 2 Penicillin G (CAS Registry No. 61-33-6); Methicillin (CAS Registry No. 61-32-5); Nafcillin (CAS Registry No.: 147 Class of Antibiotic Antibiotic Mode of Action 52-4); Oxacillin (CAS Registry No.: 66-79-5); Cloxacillin PENCILLINS (CAS Registry No. 61-72-3); Dicloxacillin (CAS Registry 15 Natural Penicillin G, Blocks the formation No. 3116-76-5); Ampicillin (CAS Registry No.: 69-53-4); Benzylpenicillin of new cell walls in Amoxicillin (CAS Registry No.: 26787-78-0); Ticarcillin Penicillin V, bacteria (CAS Registry No. 34787-01-4); Carbenicillin (CAS Reg Phenoxymethylpenicillin Penicillinase resistant Methicillin, Nafcillin, istry No.: 4697-36-3); Mezlocillin (CAS Registry No.: Oxacillin 51481-65-3); Azlocillin (CAS Registry No.: 37091-66-0); Cloxacillin, Dicloxacillin Piperacillin (CAS Registry No. 61477-96-1); Imipenem Acylamino-penicillins Ampicillin, Amoxicillin (CAS Registry No.: 74431-23-5); Aztreonam (CAS Registry Carboxy-penicillins Ticarcillin, Carbenicillin Ureido-penicillins Mezlocillin, Azlocillin, No. 78110-38-0); Cephalothin (CAS Registry No.: 153-61 Piperacillin 7); Cefazolin (CAS Registry No.: 25953-19-9); Cefaclor CARBAPENEMS Imipenem, Meropenem Blocks the formation (CAS Registry No. 70356-03-5); Cefamandole formate 25 of new cell walls in sodium (CAS Registry No.: 42540-40-9); Cefoxitin (CAS bacteria MONOBACTAMS Aztreonam Blocks the formation Registry No.: 35607-66-0); Cefuroxime (CAS Registry No.: of new cell walls in 55268-75-2); Cefonicid (CAS Registry No. 61270-58-4); bacteria Cefnmetazole (CAS Registry No. 56796-20-4); Cefotetan CEPHALOSPORINS (CAS Registry No.: 69712-56-7); Cefprozil (CAS Registry 1st Generation Cephalothin, Cefazolin Prevents formation of No. 92665-29-7); Loracarbef (CAS Registry No.: 121961 2nd Generation Cefaclor, Cefamandole new cell walls in 22-6); Cefetamet (CAS Registry No. 65052-63-3); Cefop Cefuroxime, Cefonicid, bacteria eraZone (CAS Registry No. 62893-19-0); Cefotaxime (CAS Cefnetazole, Cefotetan: Registry No.: 63527-52-6); Ceftizoxime (CAS Registry No.: Cefprozil 35 3rd Generation Cefetamet, Cefoperazone 68401-81-0); Ceftriaxone (CAS Registry No.: 73384-59-5); Cefotaxime, Ceftizoxime Ceftazidime (CAS Registry No. 72558-82-8); Cefepime Ceftriaxone, Ceftazidime (CAS Registry No.: 88040-23-7); Cefixime (CAS Registry Cefixime, Cefpodoxime, No. 79350-37-1); Ce?podoxime (CAS Registry No.: 80210 Cefsulodin 4th Generation Cefepime 62-4); Cefsulodin (CAS Registry No.: 62587-73-9); CARBACEPHEMS Loracarbef Prevents formation of Fleroxacin (CAS Registry No. 79660-72-3); Nalidixic acid 40 new cell walls in (CAS Registry No. 389-08-2); Norfloxacin (CAS Registry bacteria No. 70458-96-7); Ciprofloxacin (CAS Registry No. 85721 CEPHAMYCINS Cefoxitin Prevents formation of new cell walls in 33-1); Ofloxacin (CAS Registry No. 82419-36-1); Enoxa bacteria cin (CAS Registry No. 74011-58-8); Lomefloxacin (CAS OUINOLONES Fleroxacin, Nalidixic Inhibits bacterial DNA Registry No.: 98.079-51-7); Cinoxacin (CAS Registry No.: 45 Acid Norfloxacin, synthesis 28657-80-9); Doxycycline (CAS Registry No. 564-25-0); Ciprofloxacin, Ofloxacin, Minocycline (CAS Registry No.: 10118-90-8); Tetracycline Enoxacin Lomefloxacin, Cinoxacin (CAS Registry No. 60-54-8); Amikacin (CAS Registry No.: TETRACYCLINES Doxycycline, Mino Inhibits bacterial 37517-28-5); Gentamicin (CAS Registry No.: 1403-66-3); cycline, Tetracycline protein synthesis, Kanamycin (CAS Registry No. 8063-07-8); Netilmicin 50 binds to 30S (CAS Registry No. 56391-56-1); Tobramycin (CAS Reg ribosome subunit. AMINO Amikacin, Gentamicin, Inhibits bacterial istry No.: 32986-56-4); Streptomycin (CAS Registry No.: GLYCOSIDIES Kanamycin, Netilmicin, protein synthesis, 57-92-1); Azithromycin (CAS Registry No. 83905-01-5); Tobramycin, Strepto binds to 30S Clarithromycin (CAS Registry No. 81103-11-9); Erythro mycin ribosome subunit 55 MACROLIDES Azithromycin, Clarithro Inhibits bacterial mycin (CAS Registry No.: 114-07-8); Erythromycinestolate mycin, Erythromycin protein synthesis, (CAS Registry No. 3521-62-8); Erythromycin ethyl succi binds to 5OS nate (CAS Registry No.: 41342-53-4); Erythromycin glu ribosome subunit coheptonate (CAS Registry No.: 23067-13-2); Erythromy Derivatives of Erythromycin estolate, Erythromycin Erythromycin stearate cin lactobionate (CAS Registry No. 3847-29-8); Erythromycin ethyl Erythromycin stearate (CAS Registry No.: 643-22-1); Van 60 Succinate comycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Erythromycin gluceptate Registry No. 61036-64-4); Chloramphenicol (CAS Regis Erythromycin lacto try No. 56-75-7); Clindamycin (CAS Registry No.: 18323 biomate GLYCOPEPTIDES Vancomycin, Teico Inhibits cell wall 44-9); Trimethoprim (CAS Registry No.: 738-70-5); Sul planin synthesis, prevents fame thoxazole (CAS Registry No.: 723-46-6); 65 peptidoglycan Nitrofurantoin (CAS Registry No.: 67-20-9); Rifampin elongation. (CAS Registry No.: 13292-46-1); Mupirocin (CAS Registry US 6,503,881 B2 25 26 A. Enhancement of Antibiotic Agent or Cationic Peptide TABLE 2-continued Activity A Synergistic combination of cationic peptide and antibi Class of Antibiotic Antibiotic Mode of Action otic agent may permit a reduction in the dosage of one or MISCELLANEOUS Chloramphenicol Inhibits bacterial both agents in order to achieve a similar therapeutic effect. protein synthesis, This would allow Smaller doses to be used, thus, decreasing binds to 5OS the incidence of toxicity (e.g., from aminoglycosides) and ribosome subunit. lowering costs of expensive antibiotics (e.g., Vancomycin). Clindamycin Inhibits bacterial protein synthesis, Concurrent or Sequential administration of peptide and anti binds to 5OS biotic agent is expected to provide more effective treatment ribosome subunit. of infections caused by micro-organisms (bacteria, Viruses, Trimethoprim Inhibits the enzyme fungi, and parasites). In particular, this could be achieved by dihydrofolate using doses that the peptide or antibiotic agent alone would reductase, which activates folic acid. not achieve therapeutic Success. Alternatively, the antibiotic Sulfamethoxazole Acts as antimetabolite 15 agent and peptide can be administered at therapeutic doses of PABA & inhibits for each, but wherein the combination of the two agents synthesis of folic acid provides even more potent effects. Nitrofurantoin Action unknown, but is concentrated in As used herein, “synergy” refers to the in vitro effect of urine where it can act administration of a combination of a cationic peptide and on urinary tract antibiotic agent Such that (1) the fractional inhibitory con bacteria centration (FIC) is less than or equal to 0.5 in an FIC assay Rifampin Inhibits bacterial RNA polymerase described herein; or (2) there is at least a 100-fold (2logo) Mupirocin Inhibits bacterial increase in killing at 24 hours for the combination as protein synthesis compared with the antibiotic agent alone in a time kill curve assay as described herein. 25 Such Synergy is conveniently measured in an in vitro Anti-fungal agents include, but are not limited to, terbin assay, Such as kinetic kill Studies or a fractional inhibitory afine hydrochloride, nyStatin, amphotericin B, griseofulvin, concentration (FIC) assay as determined by agarose or broth ketoconazole, miconazole nitrate, flucytosine, fluconazole, dilution assay. The agarose dilution assay is preferred. itraconazole, clotrimazole, benzoic acid, Salicylic acid, and Briefly, in the dilution assay, a checkerboard array of Selenium Sulfide. cationic peptides and antibiotic agents titrated in doubling Anti-Viral agents include, but are not limited to, amanta dilutions are inoculated with a microbial (e.g., bacterial) dine hydrochloride, rimantadin, acyclovir, famciclovir, isolate. The FIC is determined by observing the impact of foscarnet, ganciclovir Sodium, idoxuridine, ribavirin, one antibiotic agent on the MIC (“minimal inhibitory Sorivudine, trifluridine, Valacyclovir, Vidarabin, didanosine, concentration”) of the cationic peptide and Vice versa. FIC Stavudine, Zalcitabine, Zidovudine, interferon alpha, and 35 is calculated by the following formula: edoxudine. MIC(peptide in combination) -- MIC(antibiotic in combination) Anti-parasitic agents include, but are not limited to, FC = pire thrins/piperonyl butoxide, permethrin, iodoquinol, MIC(peptide alone) MIC(antibiotic alone) metronidazole, diethylcarbamazine citrate, piperazine, pyr antel pamoate, mebendazole, thiabendazole, praziquantel, 40 albendazole, proguanil, quinidine gluconate injection, qui An FIC of s ().5 is evidence of synergy. An additive nine Sulfate, chloroquine phosphate, mefloquine response has an FIC alue of >0.5 and less than or equal to hydrochloride, primaquine phosphate, atovaquone, 1, while an indifferent response has an FIC value of >1 and co-trimoxazole (Sulfamethoxazole/trimethoprim), and pen s2. Although a Synergistic effect is preferred, an additive 45 effect may still indicate that the combination of antibiotic tamidine isethionate. agent and cationic peptide are therapeutically useful. IV. ENHANCED ACTIVITY OF B. Overcoming Tolerance COMBINATIONS OF CATIONIC PEPTIDES Tolerance is associated with a defect in bacterial cellular AND ANTIBIOTICAGENTS autolytic enzymes Such that an antibacterial agent demon 50 strates bacteriostatic rather than bactericidal activity (Mahon Enhanced activity occurs when a combination of peptide and Manuselis, Textbook of Diagnostic Microbiology, W. B. and antibiotic agent potentiates activity beyond the indi Saunders Co., Toronto, Canada, p. 92, 1995). For antibiotic vidual effects of the peptide or antibiotic agent alone or agents that have only bacterioStatic activity, the administra additive effects of peptide plus antibiotic agent. Enhanced tion of cationic peptides in combination with antibiotic activity is especially desirable in at least four Scenarios: (1) 55 agents can restore bactericidal activity. Alternatively, the the microorganism is Sensitive to the antibiotic agent, but the addition of a peptide to an antibiotic agent may increase the dosage has associated problems; (2) the microorganism is rate of a bactericidal effect of an antibiotic. tolerant to the antibiotic agent, and is inhibited from growing Bactericidal effects of antibiotics can be measured in vitro but is not killed; (3) the microorganism is inherently resis by a variety of assays. Typically, the assay is a measurement tant to the antibiotic agent; and (4) the microorganism has 60 of MBC (“minimal bactericidal concentration”), which is an acquired resistance to the antibiotic agent. Enhanced effi extension of the MIC determination. The agarose dilution cacy resulting from administration of the antibiotic agent in assay is adapted to provide both MBC and MIC for an combination with a cationic peptide in the above Scenarios: antimicrobial agent alone and the agent in combination with (1) allows for administration of lower dosages or antibiotic a cationic peptide. Alternatively, kinetic time-kill (or agent and cationic peptide; (2) restores a cytocidal effect; (3) 65 growth) curves can be used to determine MIC and MBC. overcomes inherent resistance; and (4) overcomes acquired Briefly, following determination of MIC, MBC is deter resistance. mined from the assay plates by Swabbing the inocula on US 6,503,881 B2 27 28 plates containing antibiotic agent in concentrations at and Lysozymes disrupt certain bacteria by cleaving the gly above the MIC, resuspending the Swab in Saline or medium, cosidic bond between N-acetylglucosamine and and plating an aliquot on agarose plates. If the number of N-acetylmuramic acid in the polysaccharide component of colonies on these agarose plates is less than 0.1% of the bacterial cell walls. However, lysozyme exhibits only weak initial inoculum (as determined by a plate count immediately antibacterial activity with a narrow spectrum of activity. The after inoculation of the MIC test plates), then 299.9% addition of cationic peptide may improve the effectiveness killing has occurred. The MBC end point is defined as the of this activity and broaden the Spectrum of activity. lowest concentration of the antimicrobial agent that kills 99.9% of the test bacteria. Nisins are 34-residue peptide lantibiotics with primarily Thus, tolerance of a microorganism to an antimicrobial anti-Gram-positive bacterial activity. Nisin is used in the agent is indicated when the number of colonies growing on food processing industry as a preservative, especially for subculture plates exceeds the 0.1% cutoff for several suc cheese, canned fruits and vegetables. Nisin forms transient cessive concentrations above the observed MIC. A combi potential-dependent pores in the bacterial cytoplasmic mem nation of antimicrobial agent and cationic peptide that branes but also exhibits weak antibacterial activity with a breaks tolerance results in a decrease in the MBC:MIC ratio narrow Spectrum of activity. The addition of cationic peptide to <32. 15 may improve the effectiveness of nisin and broaden the C. Overcoming Inherent Resistance Spectrum of activity. The combination of a cationic peptide with an antibiotic F. In Vivo Assays agent, for which a microorganism is inherently resistant (i.e., In vivo testing involves the use of animal models of the antibiotic has never been shown to be therapeutically infection. Typically, but not exclusively, mice are used. The effective against the organism in question), is used to test organism is chosen according to the intended combina overcome the resistance and confer Susceptibility of the tion of cationic peptide and antibiotic to be evaluated. microorganism to the agent. Overcoming inherent resistance Generally, the test organism is injected intraperitoneally (IP) is especially useful for infections where the causative organ or intravenously (IV) at 10 to 100 times the fifty percent ism is becoming or has become resistant to most, if not all, lethal dose (LDso). The LDso is calculated using a method of the currently prescribed antibiotics. Additionally, admin istering a combination therapy provides more options when 25 described by Reed and Muench (Reed LJ and Muench H. toxicity of an antibiotic agent and/or price are a consider The American Journal of Hygiene, 27:493-7). The antibi ation. otic agent and the cationic peptide are injected IP, IV, or Overcoming resistance can be conveniently measured in subcutaneously (SC) individually as well as in combination vitro. Resistance is overcome when the MIC for a particular to different groups of mice. The antimicrobial agents may be antibiotic agent against a particular microorganism is given in one or multiple doses. Animals are observed for 5 decreased from the resistant range to the Sensitive range to 7 days. Other models of infection may also be used (according to the National Committee for Clinical Labora according to the clinical indication for the combination of tory Standards (NCCLS)) (see also, Moellering, in Prin antibiotic agents. ciples and Practice of Infectious Diseases, 4th edition, The number of mice in each group that Survive the Mandell et al., eds. Churchill Livingstone, NY, 1995). 35 infectious insult is determined after 5 to 7 days. In addition, NCCLS standards are based on microbiological data in when bacteria are the test organisms, bacterial colony counts relation to pharmacokinetic data and clinical Studies. Resis from blood, peritoneal lavage fluid, fluid from other body tance is determined when the organism causing the infection Sites, and/or tissue from different body Sites taken at various is not inhibited by the normal achievable Serum concentra time intervals can be used to assess efficacy. Samples are tions of the antibiotic agent based on recommended dosage. 40 serially diluted in isotonic saline and incubated for 20-24 Susceptibility is determined when the organism responds to hours, at 37 C., on a suitable growth medium for the therapy with the antibiotic agent used at the recommended bacterium. dosage for the type of infection and microorganism. Synergy between the cationic peptide and the antibiotic D. Overcoming Acquired Resistance agent is assessed using a model of infection as described Acquired resistance in a microorganism that was previ 45 above. For a determination of Synergy, one or more of the ously Sensitive to an antibiotic agent is generally due to following should occur. The combination group should mutational events in chromosomal DNA, acquisition of a resistance factor carried via plasmids or phage, or transpo show greater Survival rates compared to the groups treated Sition of a resistance gene or genes from a plasmid or phage with only one agent; the combination group and the antibi to chromosomal DNA. otic agent group have equivalent Survival rates with the When a microorganism acquires resistance to an 50 combination group receiving a lower concentration of anti antibiotic, the combination of a peptide and antibiotic agent biotic agent; the combination group has equivalent or better can restore activity of the antibiotic agent by overcoming the Survival compared to an antibiotic agent group with a lower resistance mechanism of the organism. This is particularly microorganismal load at various time points. useful for organisms that are difficult to treat or where Overcoming tolerance can be demonstrated by lower current therapy is costly or toxic. The ability to use a leSS 55 bacterial colony counts at various time points in the com expensive or less toxic antibiotic agent, which had been bination group over the antibiotic agent group. This may effective in the past, is an improvement for certain current also result in better Survival rates for the combination group. therapies. The re-introduction of an antibiotic agent would Similar animal models to those described above can be enable previous clinical Studies and prescription data to be used to establish when inherent or acquired resistance is used in its evaluation. Activity is measured in vitro by MICs 60 overcome. The microorganism Strain used is, by definition, or kinetic kill curves and in Vivo using animal and human resistant to the antibiotic agent and So the Survival rate in the clinical trials. antibiotic agent group will be close, if not equal, to Zero E. Enhancement of Effect of Lysozyme and Nisin percent. Thus, overcoming the inherent resistance of the The combination of cationic peptides and lysozyme or microorganism to the antibiotic agent is demonstrated by nisin may improve their antibacterial effectiveness and allow 65 increased Survival of the combination group. Testing for use in Situations in which the Single agent is inactive or reversing acquired resistance may be performed in a similar inappropriate. C. US 6,503,881 B2 29 30 V. COMBINATIONS OF PEPTIDES AND ANTIBIOTICAGENTS TABLE 4

ANTIMICROBIAL AS discussed herein, cationic peptides are administered in BACTERIAL SPECIES AGENTS PEPTIDE Enterococcus species Ampicillin (Amino- MBI 21A10 combination with antibiotic agents. The combination penicillins) Piperacillin enhances the activity of the antibiotic agents. Such combi (Penicillins, anti nations may be used to effect a Synergistic result, overcome pseudomonal) Enterococcus species Gentamicin (Amino- MBI 29 tolerance, overcome inherent resistance, or overcome glycosides) acquired resistance of the microorganism to the antibiotic Enterococcus species Vancomycin, Teicoplanin MBI 26 agent. (glycopeptides) StreptococcuS pneumoniae Penicillins MBI 29A3 Salmonella typhi Chloramphenicol MBI 11A1CN To achieve a Synergistic effect, a combination of antibiotic Campylobacter jejuni Erythromycin (Macrollides) MBI 11B4CN agent and cationic peptide is administered to a patient or 15 administered in Such a manner as to contact the microor To overcome inherent resistance, a combination of anti ganism. Any combination of antibiotic agent and cationic biotic agent and cationic peptide is administered to a patient peptide may result in a Synergistic effect and, thus, is useful or administered in Such a manner as to contact the micro within the context of this invention. organism. Any combination of antibiotic agent and cationic peptide that overcomes resistance is usefull within the In particular, certain microorganisms are preferred targets. context of this invention. In particular, certain In conjunction with these microorganisms, certain com microorganisms, which exhibit inherent resistance to Spe monly used antibiotic agents are preferred to be enhanced. cific antibiotic agents are preferred targets. The table below The table below Sets out these microorganisms, antibiotic 25 Sets out these microorganisms, antibiotic agents, and cat agents, and cationic peptide combinations that are preferred. ionic peptide combinations that are preferred. TABLE 5 TABLE 3 ANTIMICROBIAL ANTIMICROBIAL BACTERIAL SPECIES AGENTS PEPTIDE BACTERIAL SPECIES: AGENTS PEPTIDE Methicillin-resistant S. aureus Amikacin MBI 29F1 A. baun annii Gentamicin MB21A2 S. maltophilia Gentamicin MBI 11D18CN B. cepacia Ceftriaxone MBI 11JO2CN S. maltophilia Gentamicin MBI 26 E. cloacae Ciprofloxacin MBI 29A2 S. maltophilia Tobramycin MBI 29A3 E. faecalis Amikacin MBI, 11B16CN 35 Methicillin-resistant S. aureus Tobramycin MBI 21A1 E. faecium Vancomycin MBI 29 E. coli Mupirocin MBI 21A1 P. aeruginosa Mupirocin MBI 28 S. maltophilia Amikacin MBI, 11B16CN P. aeruginosa Tobramycin MBI 11 G13CN S. maltophilia Amikacin MB 26 S marceScens Piperacillin MBI 11G7CN B. cepacia Amikacin MBI 29A3 S. aureus Piperacillin MBI 11CN Methicillin resistant S. aureus Gentamicin MBI 11D18CN S. maltophilia Tobramycin REWH 53ASCN 40 ANTIFUNGAL MYCOSES ANTIFUNGALAGENTS PEPTIDE MYCOSES AGENTS PEPTIDE

Candida species Fluconazole MBI 28 Aspergillosis Fluconazole MBI 11D18CN Cryptococcus Fluconazole MBI 29A3 Candida species Griseofulvin MBI 29 Aspergillis species Itraconazole MB 26 45 VIRUSES ANTIVIRAL AGENTS PEPTIDE To overcome acquired resistance, a combination of anti Herpes simplex virus Acyclovir MBI 11A2C N biotic agent and cationic peptide is administered to a patient Influenza A virus Amantadine- MBI 21A1 or administered in Such a manner as to contact the micro rimantadine organism. Any combination of antibiotic agent and cationic 50 ANTIPARASITIC peptide that overcomes resistance is useful within the con PARASITES AGENTS PEPTIDE text of this invention. In particular, certain microorganisms, which exhibit acquired resistance to specific antibiotic Trichomonas vaginalis Metronidazole MBI 29 agents are preferred targets. The table below Sets out these Plasmodium falciparum Chloroquine MBI 11D18CN microorganisms, antibiotic agents, and cationic peptide 55 combinations that are preferred. To overcome tolerance, a combination of antibiotic agent TABLE 6 and cationic peptide is administered to a patient or admin BACTERIA ANTIMICROBIAL AGENT PEPTIDE istered in Such a manner as to contact the microorganism. 60 Enterococcus spp. Vancomycin MB 26 Any combination of antibiotic agent and cationic peptide P. aeruginosa Ceftriaxone MB 26 that overcomes tolerance is useful within the context of this S. aureus Ciprofloxacin MBI 29A2 invention. In particular, certain microorganisms, which E. cloacae Piperacillin MBI 11F4CN P. aeruginosa Tobramycin MBI 21A1 exhibit tolerance to specific antibiotic agents are preferred P. aeruginosa Ciprofloxacin MBI 29A2 targets. The table below Sets out these microorganisms, 65 P. aeruginosa Gentamicin MBI, 11B16CN antibiotic agents, and cationic peptide combinations that are S. epidermidis Gentamicin MBI 11D18CN preferred. US 6,503,881 B2 32 The hydrophobic region is a lipophilic moiety, generally TABLE 6-continued a fatty acid, but may be a fatty alcohol, fatty thiol, hydro carbons (such as 4-(1,1,3,3-tetramethylbutyl)-cyclohexyl), Acinetobacter spp. Tobramycin MB 11F3CN aryl compounds (Such as 4-(1,1,3,3-tetramethylbutyl)- Enterococcus spp. Vancomycin MB 11A1CN phenyl) and the like, which are also lipophilic compounds. MYCOSES ANTIFUNGALAGENTS PEPTIDE The fatty acid may be Saturated or unsaturated. The chain length does not appear to be important, although typically Candida species Fluconazole MB 11CN commercially available fatty acids are used and have chain Cryptococcus Fluconazole MB 11A1CN lengths of Cs. The length may be limited however by VIRUSES ANTIVIRAL AGENTS PEPTIDE Solubility or Solidity of the compound, that is longer lengths Herpes simplex virus Acyclovir MBI 29 of fatty acids are Solid at room temperature. Fatty acids of Respiratory Syncytial Ribavirin MB 26 12 carbons (lauryl), 14 carbons, 16 carbons (palmitate), and Virus (RSV) 18 carbons (monostearate or oleate) are preferred chain Influenza A virus Amantadine-rimantadine MB 26 lengths. 15 The hydrophilic region is a polyoxyalkylene, Such as PARASITES ANTIPARASITICAGENTS PEPTIDE polyethylene, polypropylene glycol monoether (for example Trichomonas vaginalis Metronidazole MBI 29 Triton X114), and polysorbate. For polysorbate, the ether Pneumocystis carini Cotrimoxazole MBI 29A3 function is formed by the linkage between the polyoxyeth Plasmodium falciparum Chloroquine MB 26 ylene chain, preferably having a chain length of from 2 to 100 monomeric units, and the Sorbitan group. Polymethyl Additional preferred combinations for indolicidin ana ene glycol is unsuitable for administration in animals due to logues are listed below: formation of formaldehydes, and glycols with a chain length of 24 may be insoluble. Mixed polyoxyethylene polyoxypropylene chains are also Suitable. 25 A linker for bridging the hydrophilic and hydrophobic ANTIEIOTIC PEPTIDE regions is not required, but if used, should be able to bridge Ciprofloxacin MB 11A1CN both a polyoxyalkylene and the hydrophobic region. Suit Vancomycin MB 11A1CN able linkers include Sorbitan, Sugar alcohols, ethanolamine, Piperacillin MB 11B9CN ethanolthiol, 2-mercaptoethanol, 1,6-diaminohexane, an Gentamicin MB 11B16CN Piperacillin MB 11D18CN amino acid (e.g., glutamine, lysine), other reduced Sugars, Tobramycin MB 11D18CN and the like. For example, Sorbitan forms an ester linkage Vancomycin MB 11D18CN with the fatty acid in a polySorbate. Piperacillin MB 11E3CN Suitable compounds include polyoxyethyleneSorbitans, Tobramycin MB 11F3CN Piperacillin MB 11F4CN Such as the monolaurate, monooleate, monopalmitate, 35 monoStearate, trioleate, and tristearate esters. These and other Suitable compounds may be Synthesized by Standard VI. POLYMER MODIFICATION OF PEPTIDES chemical methods or obtained commercially (e.g., Sigma AND PROTEINS Chemical Co., Mo.; Aldrich Chemical Co., Wis.; J. B. Baker, AS noted herein, the present invention provides methods N.J.). and compositions for modifying a compound with a free 40 B. Activation of Reagent amine group. The amine group may be part of the native The reagent is activated by exposure to UV light with free Structure of the compound or added by a chemical method. eXchange of air or by chemical treatment with ammonium Thus, peptides, proteins, and antibiotics and the like can be perSulfate, or a combination of these methods. modified with an activated polyoxyalkylene and derivatives. Photoactivation is achieved using a lamp that irradiates at When the compounds are peptides or proteins, the modified 45 254 nm or 302 nm. Preferably, the output is centered at 254 or derivatized forms are referred to herein as “APO nm. Longer wave lengths may require longer activation modified peptides” or “APO-modified proteins”. Similarly, time. While some evidence exists that fluorescent room light modified forms of antibiotics are referred to as “APO can activate the polySorbates, experiments have shown that modified antibiotics.” APO-modified compounds (e.g., APO use of UV light at 254mm yields maximal activation before cationic peptides) generally exhibit improved pharmacologi 50 room light yields a detectable level of activation. cal properties. Air plays an important role in the activation of the A. Characteristics of an Activated Polyoxyalkylene Reagent polySorbates. Access to air doubles the rate of activation AS discussed herein, a Suitable reagent for formation of APO-modified compounds (e.g., peptides and proteins) relative to activations performed in Sealed containers. A comprises a hydrophobic region and a hydrophilic region, shallow reaction chamber with a large Surface area would and optionally a linker. The hydrophobic region is a lipo 55 facilitate oxygen exchange. It is not yet known which gas is philic compound with a Suitable functional group for con responsible; an oxygen derivative is likely, although perOX jugation to the hydrophilic region or linker. The hydrophilic ides are not involved. UV exposure of compounds with ether region is a polyoxyalkylene. AS used herein, "polyoxyalky linkages is known to generate peroxides, which can be lene' refers to 2 or 3 carbon polyoxyalkylene polymers. The detected and quantified using peroxide test Strips. In a polymer chain is of a length 2 units or greater. Two carbon 60 reaction, hydrogen peroxide at 1 to 10 fold higher level than polyoxyalkylenes include polyoxyethylene and its found in UV-activated material was added to a polysorbate derivatives, polyethylene glycol (PEG) of various molecular Solution in the absence of light. No activation was obtained. weights, and its derivatives, Such as polySorbate. Three The reagent is placed in a Suitable vessel for irradiation. carbon polyoxyalkylenes include polyoxypropylene and Studies with 2% polysorbate 80 indicate that 254 nm light at derivatives and polypropylene glycol and its derivatives. 65 1800 W/cm2 is completely absorbed by the solution at a Derivatives include alkyl- and aryl-polyoxyethylene com depth of 3-4 cm. Thus, the activation rate can be maximized pounds. by irradiating a relatively thin layer. US 6,503,881 B2 33 34 AS Such, a consideration for the vessel is the ability to The reaction mix is then frozen (e.g., -80 C.) and achieve uniform irradiation. AS noted above, a large shallow lyophilized. Sodium acetate disproportionates into acetic reaction chamber is desirable, however, it may be difficult to acid and NaOH during lyophilization; removal of the vola achieve on a large Scale. To compensate, Simple Stirring that tile acetic acid by the vacuum leaves NaOH dispersed facilitates the replenishment of air in the Solution achieves throughout the result Solid matrix. This loSS of acetic acid is an equivalent result. Thus, if the pathlength is long or the confirmed by a pH increase detected upon dissolution of the reaction chamber is not shallow, the reagent may be mixed lyophilizate. No APO-modified therapeutic is formed in or agitated. The reagent can be activated in any aqueous acetate buffer if the samples are only frozen then thawed. Solution and buffering is not required. The modification reaction can also take place in aqueous An exemplary activation takes place in a cuvette with a 1 Solution. However, APO modifications do not occur at cm liquid thickness. The reagent is irradiated at a distance of ambient temperature in any acetate buffer System tested less than 9 cm at 1500 uW/cm° (initial source output) for regardless of pH. APO modifications also are not formed in approximately 24 hours. Under these conditions, the acti phosphate buffers as high as pH 11.5. APO modification vated reagent converts a minimum of 85% of the peptide to does occur in a Sodium carbonate buffer at a pH greater than APO-peptide. about 8.5. Other buffers may also be used if they support AS noted above, the polyoxyalkylenes can be activated 15 derivatization. A pH range of 9-11 is also suitable, and pH via chemical oxidation with ammonium perSulfate. The 10 is most commonly used. The reaction occurs in two activation is rapid and the extent of activation increases with phases: Type I modified peptides form first, followed by the concentration of ammonium perSulfate. Ammonium formation of Type II modified peptides. persulfate can be used in a range from about 0.01%-0.5%, In the present invention, linkage occurs at an amino or a and most preferably from 0.025 to 0.1%. If the levels of nucleophilic group. The amino group may be a primary ammonium perSulfate are too high, the peroxide byproducts amine, a Secondary amine, or an aryl amine. Nucleophilic can have an adverse effect on the compounds being modi groups that may be APO-modified include, but are not fied. This adverse effect can be diminished by treatment of limited to, hydrazine derivatives, hydroxylamine activated polyoxyalkylenes with mercaptoethanol, or derivatives, and sulfhydryl compounds. Preferably, the another mild reducing agent, which does not inhibit the 25 modification occurs at an amino group, more preferably at a formation of APO-therapeutics. Peroxides generated from primary or Secondary amino group, and most preferably at UV treatment can also be reduced by treatment with mer a primary amino group. captoethanol. Furthermore, as noted above, the UV proce For a peptide, linkage can occur at the C-NH2 of the dure can be performed in conjunction with chemical acti N-terminal amino acid or e-NH group of lysine. Other Vation. primary and Secondary amines may also be modified. Com C. Modification of Peptides or Proteins with Activated plete blocking of all amino groups by acylation (MBI Reagent 11CNY1) inhibits APO-peptide formation. Thus, modifica The therapeutics are reacted with the APO reagent in tion of arginine or tryptophan residues does not occur. If the either a liquid or Solid phase and become modified by the only amino group available is the O-amino group (e.g., MBI attachment of the APO derivative. The methods described 35 11B9CN and MBI 11 G14CN), the Type I form is observed. herein for attachment offer the advantage of maintaining the The inclusion of a single lysine (e.g., MBI 11B1CN, MBI charge on the therapeutic, Such as a peptide or protein. When 11B7CN, MBI 11B8CN), providing an e-amino group, the charge of the peptide is critical to its function, Such as the results in Type II forms as well. The amount of Type II antibiotic activity of cationic peptides described herein, formed increases for peptides with more lysine residues. these attachment methods offer additional advantages. 40 Many antibiotics have free amine groupS. Such antibiotics Methods that attach groups via acylation result in the loSS of include but are not limited to amplicillin, amoxicillin, positive charge via conversion of amino to amido groups. In amikacin, ciprofloxacin, gentamicin, teicoplanin, addition, no bulky or potentially antigenic linker, Such as a tobramycin, and Vancomycin. Using the methods described triazine group, is known to be introduced by the methods herein, Several peptides, including indolicidin, indolicidin described herein. 45 analogues, gramicidin and bacitracin-2 have been polymer As noted above, APO-therapeutic formation occurs in modified. Solid phase or in aqueous Solution. By way of example, Examples of compounds that have modified by the solid briefly, in the Solid phase method, a peptide or other thera phase method are listed in the table below. peutic is Suspended in a Suitable buffer, Such as an acetate buffer. Other suitable buffers that support APO-therapeutic 50 TABLE 7 formation may also be used. The acetate buffer may be Compound Action Modification Sodium, potassium, lithium, and the like. Other acetate Amoxicillin penicillin antibiotic Yes Solutions, Such as HAc or HAc-NaOH, are also Suitable. A Amphotericin B anti-fungal No preferred pH range for the buffer is from 2 to 8.3, although Ampicillin penicillin antibiotic Yes a wider range may be used. When the starting pH of the 55 Bacitracin peptide antibiotic Yes acetic acid-NaOH buffer is varied, subsequentlyophilization Cephalosporin C aminoglycoside antibiotic No Ciprofloxacin quinolone antibiotic Uncertain from 200 mM acetic acid buffer yields only the Type I 4,4'-Diaminodiphenyl Sulfone anti-leprotic Yes modified peptide (see Example 14). The presence of an Gentamicin aminoglycoside antibiotic Yes alkaline buffer component results in the formation of Type II Gramicidin S peptide antibiotic Yes modified peptides. A typical peptide concentration is 1 60 Sulfadiazine sulfonamide antibiotic No mg/ml, which results in 85-95% modified peptide, however Vancomycin glycopeptide antibiotic Yes other concentrations are Suitable. The major consideration *Ciprofloxacin was partially destroyed by the process. for determining concentration appears to be economic. The activated polymer (APO) is added in molar excess to the D. Purification and Physical Properties of APO-modified therapeutic. Generally, a starting ratio of approximately 65 Therapeutics 2.5:1 (APO: therapeutic) to 5:1 (APO: therapeutic) generates The APO-modified therapeutics may be purified. In cir APO-modified therapeutic in good yield. cumstances in which the free therapeutic, Such as a peptide US 6,503,881 B2 35 36 is toxic, purification may be necessary to remove unmodi 1 mg/mL Solution of the parent peptide, thus allowing direct fied therapeutic and/or unreacted polyoxyalkylenes. Any of comparison of toxicity and efficacy data. The modified a variety of purification methods may be used. Such methods peptides have an equivalent MIC to unmodified peptides. In include reversed phase HPLC, precipitation by organic Vivo, however, the modified peptides demonstrate a lower Solvent to remove poly Sorbate, Size exclusion LCso than the unmodified peptides against a panel of tumor chromatography, ion exchange chromatography, filtration cell lines. Thus, formation of APO-peptides increases the and the like. RP-HPLC is preferred. Procedures for these potency of cationic peptides against cancer cells in culture. Separation methods are well known. In general, the efficacy of a modified therapeutic is APO-therapeutic formation can result in the generation of determined by in vitro and in vivo assays used for the products that are more hydrophobic than the parent com unmodified therapeutic. Thus, the assays employed depend pound. This property can be exploited to effect Separation of upon the therapeutic. ASSays for the therapeutics disclosed the conjugate from free compound by RP-HPLC. As shown herein are well known. ASSays include those for biological herein, peptide-conjugates are resolved into two populations activity, pharmacokinetics, toxicity, adverse reactions, based on their hydrophobicity as determined by RP-HPLC; immunogenicity, and the like. Such assays are available to the Type I population elutes slightly earlier than the Type II 15 those skilled in the art. population. VII. FORMULATIONS AND ADMINISTRATION The MBI 11 series of peptides have molecular weights between 1600 and 2500. When run on a Superose 12 AS noted above, the present invention provides methods column, a size exclusion column, these peptides adsorb to for treating and preventing infections by administering to a the resin, giving long retention times. In contrast, the APO patient a therapeutically effective amount of a peptide ana modified peptides do not adsorb and elute at 50 kDa logue of indolicidin as described herein. Patients suitable for (MBI11CN-Tw80) and at 69 kDa (MBI 11A3CN-Tw80), such treatment may be identified by well-established hall thus demonstrating a large increase in apparent molecular marks of an infection, Such as fever, pus, culture of mass (Stokes radius). organisms, and the like. Infections that may be treated with An increase in apparent molecular mass could enhance 25 peptide analogues include those caused by or due to micro the pharmacokinetics of peptides in particular because organisms. Examples of microorganisms include bacteria increased molecular mass reduces the rate at which peptides (e.g., Gram-positive, Gram-negative), fungi, (e.g., yeast and and proteins are removed from blood. Micelle formation molds), parasites (e.g., protozoans, nematodes, cestodes and may offer additional benefits by delivering “packets” of trematodes), viruses, and prions. Specific organisms in these peptide molecules to microorganisms rather than relying on classes are well known (see for example, Davis et al., the multiple binding of Single peptide molecules. In Microbiology, 3' edition, Harper & Row, 1980). Infections addition, APO-modified peptides are soluble in methylene include, but are not limited to, toxic shock Syndrome, chloride or chloroform (e.g., to at least 10 mg/mL), whereas diphtheria, cholera, typhus, meningitis, whooping cough, the parent peptide is essentially insoluble. This increased botulism, tetanus, pyogenic infections, dysentery, organic Solubility may significantly enhance the ability to 35 gastroenteritis, anthrax, Lyme disease, Syphilis, rubella, Sep penetrate tissue barriers and may be exploited for a simpli ticemia and plague. fied purification of the APO-peptide. The increased solubil More Specifically, clinical indications include, but are not ity in organic media may also allow the formulation of limited to: 1/infections following insertion of intravascular peptides in oil or lipid based delivery Systems which target devices or peritoneal dialysis catheters, 2/infection associ Specific Sites, Such as Solid tumors. 40 ated with medical devices or prostheses; 3/infection during In addition, by circular dichroism (CD) studies, APO hemodialysis; 4/S. aureus nasal and extra-nasal carriage; modified peptides are observed to have an altered 5/burn wound infections; 6/Surgical wounds, 7/acne, includ 3-dimensional conformation. AS shown in the Examples, ing Severe acne Vulgaris; 8/nosocomial pneumonia; MBI 11 CN and MBI 11B7CN have unordered structures in 9/meningitis; 10/cystic fibrosis; 11/infective endocarditis; phosphate buffer or 40% aqueous trifluoroethanol (TFE) and 45 12/osteomyelitis, and 13/Sepsis in an immunocompromised form a B-turn conformation only upon insertion into lipo host. somes. In contrast, CD spectra for APO-modified MBI 1/Infections following insertion of contaminated intravas 11CN and APO-modified MBI 11B7CN indicate B-turn cular devices, Such as central venous catheters, or peritoneal Structure in phosphate buffer. dialysis catheters. These catheters are cuffed or non-cuffed, Cationic peptides appear to maintain their original charge 50 although the infection rate is higher for non-cuffed catheters. after modification with an APO, thereby preventing loss of Both local and Systemic infection may result from contami activity Sometimes caused by acylation reactions. Moreover, nated intravascular devices, more than 25,000 patients the present methods are not known to introduce antigenic develop device related bacteremia in the United States each linkers. year. The main organisms responsible are coagulase E. Biological Properties of APO-modified Therapeutics 55 negative staphylococci (CoNS), StaphylococcuS aureus, The biological properties of APO-modified therapeutics Enterococcus spp., E. coli and Candida spp. appear to be improved compared to unmodified therapeutics. The peptide and/or antibiotic, preferably as an ointment or For example, modified and unmodified peptides are com cream, can be applied to the catheter Site prior to insertion pared. Because the product consists of a peptide of known of the catheter and then again at each dressing change. The composition coupled to one or more polyoxyalkylene com 60 peptide may be incorporated into the ointment or cream at a ponents derived from a polymeric mixture, defining an exact concentration preferably of about 0.5 to about 2% (w/v) molecular weight for concentration calculations is not 2/Infection associated with medical devices or prostheses, readily achieved. It is possible, however, to determine the e.g. catheter, grafts, prosthetic heart Valves, artificial joints, concentration by Spectrophotometric assay. Such a measure etc. One to five percent of indwelling prostheses become ment is used to normalize APO-peptide concentrations for 65 infected which usually requires removal or replacement of biological assays. For example, a 1 mg/mL MBI11CN-Tw80 the prostheses. The main organisms responsible for these Solution contains the same amount of cationic peptide as a infections are CoNS and S. aureus. US 6,503,881 B2 37 38 Preferably, the peptide and/or antibiotic can be coated, in the wound prior to and during closure of the wound. either covalently bonded or by any other means, onto the Following closure the peptide antibiotic could be applied at medical device either at manufacture of the device or after dressing changes. For wounds that are infected, the peptide manufacture but prior to insertion of the device. In Such an antibiotic could be applied topically and/or Systemically. application, the peptide antibiotic is preferably applied as a 7/Acne, including Severe acne Vulgaris. This condition is 0.5 to 2% solution. due to colonization and infection of hair follicles and 3/Infection during hemodialysis. Infection is the Second Sebaceous cysts by Propionibacterium acne. Most cases leading cause of death in patients on chronic hemodialysis. remain mild and do not lead to Scarring although a Subset of Approximately 23% of bacteremias are due to access site patients develop large inflammatory cysts and nodules, infections. The majority of graft infections are caused by which may drain and result in Significant Scarring. coagulate-positive (S. aureus) and coagulate-negative sta The peptide alone or with an antibiotic can be incorpo phylococci. To combat infection, the peptide alone or in rated into Soap or applied topically as a cream, lotion or gel combination with an antibiotic can be applied as an ointment to the affected areas either once a day or multiple times or cream to the dialysis site prior to each hemodialysis during the day. The length of treatment may be for as long procedure. 15 as the lesions are present or used to prevent recurrent 4/S. aureuS nasal and extra-nasal carriage. Infection by lesions. The peptide antibiotic could also be administered this organism may result in impetigenous lesions or infected orally or Systemically to treat or prevent acne lesions. wounds. It is also associated with increased infection rates 8/Nosocomial pneumonia. NoSocomial pneumonias following cardiac Surgery, hemodialysis, orthopedic Surgery account for nearly 20% of all nosocomial infections. and neutropenia, both disease induced and iatrogenic. Nasal Patients most at risk for developing nosocomial pneumonia and extra-nasal carriage of Staphylococci can result in hos are those in an intensive care units, patients with altered pital outbreaks of the same Staphylococci Strain that is levels of consciousness, elderly patients, patients with colonizing a patient's or hospital worker's nasal passage or chronic lung disease, ventilated patients, SmokerS and post extra-nasal site. Much attention has been paid to the eradi operative patients. In a Severely compromised patient, cation of nasal colonization, but the results of treatment have 25 multiantibiotic-resistant noSocomial pathogens are likely to been generally unsatisfactory. The use of topical antimicro be the cause of the pneumonia. bial Substances, Such as Bacitracin, Tetracycline, or The main organisms responsible are P aeruginosa, S. Chlorhexidine, results in the Suppression of nasal aureus, Klebsiella pneumoniae and Enterobacter spp. The colonization, as opposed to its eradication. peptide alone or in combination with other antibiotics could The peptide alone or in combination with an antibiotic are be administered orally or Systemically to treat pneumonia. preferably applied intra-nasally, formulated for nasal Administration could be once a day or multiple administra application, as a 0.5 to 2% ointment, cream or Solution. tions per day. Peptide antibiotics could be administered Application may occur once or multiple times until the directly into the lung via inhalation or via installation of an colonization of Staphylococci is reduced or eliminated. 35 endotracheal tube. 5/Burn wound infections. Although the occurrence of 9/Meningitis. Bacterial meningitis remains a common invasive burn wound infections has been Significantly disease worldwide. Approximately 25,000 cases occur reduced, infection remains the most common cause of annually, of which 70% occur in children under 5 years of morbidity and mortality in extensively burned patients. age. Despite an apparent recent decline in the incidence of Infection is the predominant determinant of wound healing, Severe neurologic Sequelae among children Surviving bac incidence of complications, and outcome of burn patients. 40 terial meningitis, the public health problems as a result of The main organisms responsible are Pseudomonas this disease are significant worldwide. The main responsible aeruginosa, S. aureus, Streptococcus pyogenes, and various organisms are H. influenzae, StreptococcuS pneumoniae and gram-negative organisms. Frequent debridements and estab Neisseria meningitidis. Community acquired drug resistant lishment of an epidermis, or a Surrogate Such as a graft or a 45 S. pneumoniae are emerging as a widespread problem in the skin Substitute, is essential for prevention of infection. United States. The peptide alone or in combination with The peptide alone or in combination with antibiotics can known antibiotics could be administered orally or Systemi be applied to burn wounds as an ointment or cream and/or cally to treat meningitis. The preferred route would be administered Systemically. Topical application may prevent intravenously either once a day or multiple administration Systemic infection following Superficial colonization or 50 per day. Treatment would preferably last for up to 14 dayS. eradicate a Superficial infection. The peptide is preferably 10/Cystic fibrosis. Cystic fibrosis (CF) is the most com administered as a 0.5 to 2% cream or ointment. Application mon genetic disorder of the Caucasian population. Pulmo to the skin could be done once a day or as often as dressings nary disease is the most common cause of premature death are changed. The Systemic administration could be by in cystic fibrosis patients. Optimum antimicrobial therapy intravenous, intramuscular or Subcutaneous injections or 55 for CF is not known, and it is generally believed that the infusions. Other routes of administration could also be used. introduction of better anti-pseudomonal antibiotics has been 6/Surgical wounds, especially those associated with for the major factor contributing to the increase in life expect eign material, e.g. Sutures. AS many as 71% of all noSoco ancy for CF patients. The most common organisms associ mial infections occur in Surgical patients, 40% of which are ated with lung disease in CF are S. aureus, P. aeruginosa infections at the operative site. Despite efforts to prevent 60 and H. influenzae. infection, it is estimated that between 500,000 and 920,000 The peptide alone or in combination with other antibiotics Surgical wound infections complicate the approximately 23 could be administrated orally or Systemically or via aeroSol million Surgical procedures performed annually in the United to treat cystic fibrosis. Preferably, treatment is effected for up States. The infecting organisms are varied but Staphylococci to 3 weeks during acute pulmonary disease and/or for up to are important organisms in these infections. 65 2 weeks every 2–6 months to prevent acute exacerbations. The peptide alone or with an antibiotic may be applied as 11/Infective endocarditis. Infective endocarditis results an ointment, cream or liquid to the wound Site or as a liquid from infection of the heart Valve cusps, although any part of US 6,503,881 B2 39 40 the endocardium or any prosthetic material inserted into the Briefly, pharmaceutical compositions of the present inven heart may be involved. It is usually fatal if untreated. Most tion may comprise one or more of the peptide analogues infections are noSocomial in origin, caused by pathogens described herein, in combination with one or more physi increasingly resistant to available drugs. The main organ ologically acceptable carriers, diluents, or excipients. AS isms responsible are Viridans Streptococci, EnterococcuS noted herein, the formulation buffer used may affect the spp., S. aureus and CoNS. efficacy or activity of the peptide analogue. A Suitable The peptide alone or in combination with other antibiotics formulation buffer contains buffer and solubilizer. The for could be administered orally or Systemically to treat mulation buffer may comprise bufferS Such as Sodium endocarditis, although Systemic administration would be acetate, Sodium citrate, neutral buffered Saline, phosphate preferred. Treatment is preferably for 2-6 weeks in duration buffered Saline, and the like or salts, Such as NaCl. Sodium and may be given as a continuous infusion or multiple 1O acetate is preferred. In general, an acetate buffer from 5 to administration during the day. 500 mM is used, and preferably from 100 to 200 mM. The 12/Osteomyelitis. In early acute disease the vascular pH of the final formulation may range from 3 to 10, and is Supply to the bone is compromised by infection extending preferably approximately neutral (about pH 7-8). into Surrounding tissue. Within this necrotic and ischemic Solubilizers, Such as polyoxyethyleneSorbitans (e.g., Tween tissue, the bacteria may be difficult to eradicate even after an 15 80, Tween 20) and polyoxyethylene ethers (e.g., Brij 56) intense host response, Surgery, and/or antibiotic therapy. The may also be added if the compound is not already polymer main organisms responsible are S. aureus, E. coli, and P modified. aeruginosa. Although the formulation buffer is exemplified herein The peptide antibiotic could be administered Systemically with peptide analogues of the present invention, this buffer alone or in combination with other antibiotics. Treatment is generally useful and desirable for delivery of other would be 2-6 weeks in duration. The peptide antibiotic peptides. Peptides that may be delivered in this formulation could be given as a continuous infusion or multiple admin buffer include indolicidin, other indolicidin analogues (see, istration during the day. Peptide antibiotic could be used as PCT WO95/22338), bacteriocins, gramicidin, bactenecin, an antibiotic-impregnated cement or as antibiotic coated droSocin, polyphemusins, defensins, cecropins, melittins, beads for joint replacement procedures. 25 cecropin/melittin hybrids, magainins, Sapecins, apidaecins, 13/Sepsis in immunocompromised host. Treatment of protegrins, tachyplesins, thionins, IL-1 through 15, infections in patients who are immunocompromised by corticotropin-releasing hormone, human growth hormone; Virtue of chemotherapy-induced granulocytopenia and insulin; erythropoietin; thrombopoietin; myelin basic pro immunosuppression related to organ or bone marrow trans tein peptides, various colony Stimulating factorS Such as plantation is always a big challenge. The neutropenic patient M-CSF, GM-CSF, kit ligand; and peptides and analogues of is especially Susceptible to bacterial infection, So antibiotic these and Similar proteins. therapy should be initiated promptly to cover likely Additional compounds may be included in the composi pathogens, if infection is Suspected. Organisms likely to tions. These include, for example, carbohydrates Such as cause infections in granulocytopenic patients are: S. 35 glucose, mannose, Sucrose or dextrose, mannitol, other epidermidis, S. aureus, S. viridans, EnterococcuS spp., E. proteins, polypeptides or amino acids, chelating agents Such coli, Klebsiella spp., P. aeruginosa and Candida spp. as EDTA or glutathione, adjuvants and preservatives. AS The peptide alone or with an antibiotic is preferably noted herein, pharmaceutical compositions of the present administered orally or Systemically for 2-6 weeks in dura invention may also contain one or more additional active tion. The peptide antibiotic could be given as a continuous 40 ingredients, Such as an antibiotic (see discussion herein on infusion or multiple administration during the day. Synergy) or cytokine. Effective treatment of infection may be examined in The compositions may be administered in a delivery several different ways. The patient may exhibit reduced vehicle. For example, the composition can be encapsulated fever, reduced number of organisms, lower level of inflam in a liposome (see, e.g., WO 96/10585; WO95/35094), matory molecules (e.g., IFN-y, IL-12, IL-1, TNF), and the 45 complexed with lipids, encapsulated in slow-release or SuS like. tained release vehicles, Such as poly-galactide, and the like. The in Vivo therapeutic efficacy from administering a Within other embodiments, compositions may be prepared cationic peptide and antibiotic agent in combination is based as a lyophilizate, utilizing appropriate excipients to provide on a Successful clinical outcome and does not require 100% stability. elimination of the organisms involved in the infection. 50 Pharmaceutical compositions of the present invention Achieving a level of antimicrobial activity at the Site of may be administered in various manners. For example, infection that allows the host to Survive or eradicate the cationic peptides with or without antibiotic agents may be microorganism is Sufficient. When host defenses are maxi administered by intravenous injection, intraperitoneal injec mally effective, Such as in an otherwise healthy individual, tion or implantation, Subcutaneous injection or implantation, only a minimal antimicrobial effect may Suffice. Thus, 55 intradermal injection, lavage, inhalation, implantation, intra reducing the organism load by even one log (a factor of 10) muscular injection or implantation, intrathecal injection, may permit the defenses of the host to control the infection. bladder wash-out, Suppositories, pessaries, topical (e.g., In addition, clinical therapeutic Success may depend more creams, ointments, Skin patches, eye drops, ear drops, on augmenting an early bactericidal effect than on the Shampoos) application, enteric, oral, or nasal route. The long-term effect. These early events are a significant and 60 combination is preferably administered intravenously. SyS critical part of therapeutic Success, because they allow time temic routes include intravenous, intramuscular or Subcuta for the host defense mechanisms to activate. This is espe neous injection (including a depot for long-term release), cially true for life-threatening infections (e.g. meningitis) intraocular or retrobulbar, intrathecal, intraperitoneal (e.g. and other serious chronic infections (e.g. infective by intraperitoneal lavage), transpulmonary using aero endocarditis). 65 Solized or nebulized drug or transdermal. Topical routes Peptides and antibiotic agents of the present invention are include administration in the form of Salves, ophthalmic preferably administered as a pharmaceutical composition. drops, ear drops, or irrigation fluids (for, e.g. irrigation of US 6,503,881 B2 41 42 wounds). The compositions may be applied locally as an The peptides, especially the labeled analogues, may be injection, drops, Spray, tablets, cream, ointment, gel, and the used in image analysis and diagnostic assays or for targeting like. They may be administered as a bolus or as multiple Sites in eukaryotic multicellular and Single cell cellular doses over a period of time. organisms and in prokaryotes. As a targeting System, the The level of peptide in serum and other tissues after analogues may be coupled with other peptides, proteins, administration can be monitored by various well-established techniques Such as bacterial, chromatographic or antibody nucleic acids, antibodies and the like. based, Such as ELISA, assayS. Pharmaceutical compositions of the present invention are The following examples are offered by way of illustration, administered in a manner appropriate to the infection or disease to be treated. The amount and frequency of admin and not by way of limitation. istration 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 EXAMPLES clinical trials, but will generally range from about 0.1 to 50 15 mg/kg. The general range of dosages for the antibiotic agents are presented below. Example 1 TABLE 8 ANTIMICROBAL AGENT DOSE RANGE Synthesis Purification and Characterization of Cationic Pep Ciprofloxacin 400-1500 mg/day tides and Analogues Gentamicin 3 mg/kg/day Tobramycin 3 mg/kg/day Imipenem 1500 mg/kg every 12 h 25 Peptide Synthesis is based on the Standard Solid-phase Piperacillin 24 g/day Fmoc protection Strategy. The instrument employed is a Vancomycin, Teicoplanin 6-30 mg/kg/day 9050 Plus PepSynthesiser (PerSeptive BioSystems Inc.). Streptomycin 500 mg-1 g/every 12 h Methicillin 100-300 mg/day Polyethylene glycol polystyrene (PEG-PS) graft resins are Ampicillin, Amoxicillin 250-500 mg/every 8 h employed as the Solid phase, derivatized with an Fmoc Penicillin 200,000 units/day protected amino acid linker for C-terminal amide Synthesis. Ceftriaxone 4 g/day Cefotaxime 12 g/day HATU (O-(7- a Zabenzo triazole-1-yl)-1,1,3,3- Metronidazole 4 g/day tetramethyluronium hexafluorophosphate) is used as the Tetracycline 500 mg/every 6 h coupling reagent. During Synthesis, coupling Steps are con Rifampin 600 mg/day Fluconazole 150-400 mg/day tinuously monitored to ensure that each amino acid is Acyclovir 200-400 mg/day 35 incorporated in high yield. The peptide is cleaved from the Ribavirin 20 mg/ml (aerosol) Solid-phase resin using trifluoroacetic acid and appropriate Amantadine-rimantadine 200 mg/day Metronidazole 2 g/day Scavengers and the crude peptide is purified using prepara Cotrimoxazole 15-20 mg/kg/day tive reversed-phase chromatography. Typically the peptide Chloroquine 800 mg/day is prepared as the trifluoroacetate Salt, but other Salts, Such 40 as acetate, chloride and Sulfate, can also be prepared by Salt In addition, the compositions of the present invention may eXchange. be used in the manner of common disinfectants or in any Situation in which microorganisms are undesirable. For All peptides are analyzed by mass Spectrometry to ensure example, these peptides may be used as Surface 45 that the product has the expected molecular mass. The disinfectants, coatings, including covalent bonding, for product should have a single peak accounting for >95% of medical devices, coatings for clothing, Such as to inhibit the total peak area when Subjected to analytical reversed growth of bacteria or repel mosquitoes, in filters for air phase high performance liquid chromatography (RP purification, Such as on an airplane, in water purification, HPLC), a separation method that depends on the hydropho constituents of Shampoos and Soaps, food preservatives, 50 bicity of the peptide. In addition, the peptide should show a cosmetic preservatives, media preservatives, herbicide or single band accounting for >90% of the total band intensity insecticides, constituents of building materials, Such as in when Subjected to acid-urea gel electrophoresis, a separation Silicone Sealant, and in animal product processing, Such as method based on the charge to mass ration of the peptide. curing of animal hides. AS used herein, "medical device' refers to any device for use in a patient, Such as an implant 55 Peptide content, the amount of the product that is peptide or prosthesis. Such devices include, Stents, tubing, probes, rather than retained water, Salt or Solvent, is measured by cannulas, catheters, Synthetic vascular grafts, blood moni quantitative amino acid analysis, free amine derivatization toring devices, artificial heart Valves, needles, and the like. or spectrophotometric quantitation. Amino acid analysis also For these purposes, typically the peptides alone or in provides information on the ratio of amino acids present in conjunction with an antibiotic are included in compositions 60 commonly employed or in a Suitable applicator, Such as for the peptide, which assists in confirming the authenticity of applying to clothing. They may be incorporated or impreg the peptide. nated into the material during manufacture, Such as for an air filter, or otherwise applied to devices. The peptides and Peptide analogues and their names are listed below. In this antibiotics need only be Suspended in a Solution appropriate 65 list, and elsewhere, the amino acids are denoted by the for the device or article. Polymers are one type of carrier that one-letter amino acid code and lower case letters represent can be used. the D-form of the amino acid.

US 6,503,881 B2 45 46

- continued 11H6CN (SEQ ID NO: 85) I L R W P A W P W R R K 11HTCN (SEQ ID NO: 86) I L R W P W A P W R R K 11H8CN (SEQ ID NO: 87) I L R W P W W A W R R K 11H9CN (SEQ ID NO: 88) I L R W P W W P A R R K 11H10CN (SEQ ID NO: 89) I L R W P W W P W A R K 11H11CN (SEQ ID NO: 90) I L R W P W W P W R A K 11H12CN (SEQ ID NO: 91) I L R W P W W P W R R A 11JO1CN (SEQ ID NO: 64) R. R. I W K P K W R L. P K R 11JO2CN (SEQ ID NO: 32) W R W W K P K W R W P K W 21A1 (SEQ ID NO: 115) K K W W R R W L S G L K T PA I Q S W L N K 21A2 (SEQ ID NO: 116) K K W W R R A L Q G L K T PA I Q S W L N K 21A10 (SEQ ID NO: 117) K K W W R R V L K G L S S A L S N W 22A1 (SEQ ID NO: 118) K K W W R R A L Q A L K N P A L I S 26 (SEQ ID NO: 119) K W K S F I K K L T S A. A W W T T A K P L S S 27 (SEQ ID NO: 120) K W K L F K K I G I G A V W L T T G L P A L I S 28 (SEQ ID NO: 121) K W K L F K K I G I G A V W L T T. G. L. P. A. L. K. L. T. K. 29 (SEQ ID NO: 122) K W K S F I K K L T T A V W L T T G L P A L IS 29A2 (SEQ ID NO: 123) K W K S F I K. N. L T K V L W W T T. A. L. P. A. L. I. S 29A3 (SEQ ID NO: 124) K W K S F I K K L T S A. A W L T T G L P A L I S 29F1 (SEQ ID NO: 125) K W K L F I K K L T P A V W L L T G L P A L I S 31 (SEQ ID NO: 126) G K P R P Y S P I P T S P I R Y REWH 53A5 (SEQ ID NO: 127) R L A R I V V I R V A. R. CN suffix = amidated C-terminus H suffix = homoserine at C-terminus M suffix = MAP branched Peptide R suffix = retro-synthesized Peptide

Example 2 exceSS methyl iodide. The reaction mixture is stirred over Synthesis of Modified Peptides night at room temperature, extracted with organic Solvent, Cationic peptides, Such as indolicidin analogues, are neutralized and purified as for an unmodified peptide. Using modified to alter the physical properties of the original this procedure, a peptide is not fully methylated; methyla peptide, either by use of modified amino acids in Synthesis tion of MBI 11CN yielded an average of 6 methyl groups. or by post-Synthetic modification. Such modifications Thus, the modified peptide is a mixture of methylated include: acetylation at the N-terminus, Fmoc-derivatized products. N-terminus, polymethylation, peracetylation, and branched Peracetylation. A purified peptide in DMF solution is derivatives. 35 Cl-N-terminal acetylation. Prior to cleaving the peptide treated with N-acetylimidazole for 1 hour at room tempera from the resin and deprotecting it, the fully protected peptide ture. The crude product is concentrated, dissolved in water, is treated with N-acetylimidazole in DMF for 1 hour at room lyophilized, re-dissolved in water and purified as for an temperature, which results in Selective reaction at the Cl-N- unmodified peptide. Complete acetylation of primary amine terminus. The peptide is then deprotected/cleaved and puri 40 groups is observed. fied as for an unmodified peptide. Four/eight branch derivatives. The branched peptides are Fmoc-derivatized Cl-N-terminus. If the final Fmoc depro Synthesized on a four or eight branched core bound to the tection Step is not carried out, the Cl-N-terminus Fmoc group resin. Synthesis and deprotection/cleavage proceed as for an remains on the peptide. The peptide is then Side-chain unmodified peptide. These peptides are purified by dialysis deprotected/cleaved and purified as for an unmodified pep 45 against 4 M guanidine hydrochloride then water, and ana tide. lyzed by mass spectrometry. Polymethylation. The purified peptide in a methanol solu Peptides modified using the above procedures are listed in tion is treated with excess sodium bicarbonate, followed by Table 9.

TABLE 9 Peptide Peptide Sequence Modification I Acetylated C-N-terminus I Acetylated C-N-terminus I Acetylated C-N-terminus I Fmoc-derivatized N-terminus I Polymethylated derivative I Peracetylated derivative I Four branch derivative I Eight branch derivative I Fmoc-derivatized N-terminus I Acetylated N-terminus I Acetylated N-terminus I Formylated Lys12 I Acetylated N-terminus W Eight branch derivative I Eight branch derivative US 6,503,881 B2 47 48

TABLE 9-continued Peptide Peptide modified name Sequence Modification

11G6CN 11 G6ACN I L. K. K. W. P. W. W. P. R. R. K. Acetylated C-N-terminus 11GCN 11 GTACN I L. K. K. W. P. W. W. P. W. R. R. Acetylated C-N-terminus

Example 3 called pPDR2h-11. FIG. 2 presents a gel electrophoresis Recombinant Production of Peptide Analogues analysis of the MBI-11 fusion protein expressed in this Peptide analogues are alternatively produced by recom vector. Expression level of MBI-11 fusion protein is com binant DNA technique in bacterial host cells. The peptide is parable with that obtained from plasmid pR2h-11. The CAT produced as a fusion protein, chosen to assist in transporting gene product is not apparent, presumably due to the low the fusion peptide to inclusion bodies, periplasm, outer 15 copy-number nature of this plasmid, CAT protein is not membrane or extracellular environment. expressed at high levels in pPDR2h-11. Construction of Plasmids Encoding MBI-11 Peptide Fusion Protein Example 4 Amplification by polymerase chain reaction is used to In Vitro Assays to Measure Cationic Peptide Activity synthesize double-stranded DNA encoding the MBI peptide Acationic peptide may be tested for antimicrobial activity genes from single-stranded templates. For MBI-11, 100 ul of alone before assessing its enhancing activity with antibiotic reaction mix is prepared containing 50 to 100 ng of template, agents. Preferably, the peptide has measurable antimicrobial 25 pmole of each primer, 1.5 mM MgCl2, 200 uM of each activity. dNTP, 2U of Taq polymerase in buffer supplied by the Agarose Dilution ASSay manufacturer. Amplification conditions are 25 cycles of 94 25 The agarose dilution assay measures antimicrobial activ C. for 30 sec., 55° C. for 30 sec., 74° C. for 30 sec., followed ity of peptides and peptide analogues, which is expressed as by 74 C. for 1 min. Amplified product is digested with the minimum inhibitory concentration (MIC) of the pep BamHI and HindIII and cloned into a plasmid expression tides. vector encoding the fusion partner and a Suitable Selection In order to mimic in Vivo conditions, calcium and mag marker. nesium supplemented Mueller Hinton broth is used in com Production of MBI-11Peptide Fusion in E. coli bination with a low EEO agarose as the bacterial growth The plasmid pR2h-11, employing a T7 promoter, high medium. Agarose, rather than agar, is used as the charged copy origin of replication, Apr marker and containing the groups in agar prevent peptide diffusion through the media. gene of the fusion protein, is co-electroporated with pCP1-2 The media is autoclaved and then cooled to 50-55 C. in a into E. coli strain XL1-Blue. Plasmid pGP1-2 contains a T7 35 water bath before aseptic addition of antimicrobial Solutions. RNA polymerase gene under control of a lambda promoter The same Volume of different concentrations of peptide and cl&57 repressor gene. Fusion protein expression is Solution are added to the cooled molten agarose that is then induced by a temperature shift from 30° C. to 42 C. poured to a depth of 3-4 mm. Inclusion bodies are washed with Solution containing Solu The bacterial inoculum is adjusted to a 0.5 McFarland bilizer and extracted with organic extraction Solvent. Pro 40 turbidity standard (PML Microbiological) and then diluted files of the samples are analyzed by SDS-PAGE. FIG. 1 1:10 before application on to the agarose plate. The final shows the SDS-PAGE analysis and an extraction profile of inoculum applied to the agarose is approximately 104 CFU inclusion body from whole cell. The major contaminant in in a 5-8 mm diameter Spot. The agarose plates are incubated the organic Solvent extracted material is P-lactamase (FIG. at 35–37° C. for 16 to 20 hours. 1). The expression level in these cells is presented in Table 45 The MIC is recorded as the lowest concentration of 10. peptide that completely inhibits growth of the organism as determined by visual inspection. Representative MICs for TABLE 10 various indolicidin analogues against bacteria are shown in % protein Table 11 and representative MICs against Candida are Fusion Mol. mass in whole % in inclusion % which is 50 shown in Table 12 below. protein (kDa) cell lysate body extract MBI-11 peptide MB-11 20.1 15 42 7.2 TABLE 11 Organism Organism # MIC (ug/ml) In addition, a low-copy-number vector, pPD100, which 55 has a chloramphenicol resistance gene, is used to express 1. MBI 10 MBI-11 in order to eliminate the need for using ampicillin, A. calcoaceticus ACOO1 128 thereby reducing the appearance of B-lactamase in extracted E. coli ECOOO2 128 material. This plasmid allows Selective gene expression and E. faecalis EFSOO4 8 K. pneumoniae KPOO1 128 high-level protein overproduction in E. coli using the bac 60 P. aeruginosa PAOO3 >128 teriophage T7 RNA polymerase/T7 promoter system S. aureus SAOO7 2 (Dersch et al., FEMS Microbiol. Lett. 123: 19–26, 1994). S. maltophilia SMAOO1 128 pPD100 contains a chloramphenicol resistance gene (CAT) S. narceScenS SMSOO3 >128 as a Selective marker, a multiple cloning site, and an ori 2 MB 1OA Sequence derived from the low-copy-number vector 65 E. faecalis EFSOO4 16 pSC101. There are only about 4 to 6 copies of these plasmids E. faecium EFMOO3 8 per host cell. The resulting construct containing MBI-11 is

US 6,503,881 B2 65 66

TABLE 11-continued TABLE 11-continued Organism Organism # MIC (ug/ml) Organism Organism # MIC (ug/ml) E. coli ECOOO5 8 S. epidermidis SEO1O 32 E. faecalis EFSOO1 2 S. maltophilia SMAOO2 >128 E. faecalis EFSOO8 8 S. narceScenS SMSOO3 >128 K. pneumoniae KPOO1 32 107. MBI 11JO2CN aeruginosa PAOO4 >128 S. aureus SAO14 4 A. calcoaceticus ACOO2 4 S. aureus SAO93 1. 1O E. cloacae ECLOO7 64 S. epidermidis SEO1O 4 E. coli ECOOO5 4 S. maltophilia SMAOO2 32 E. faecalis EFSOO1 4 S. PCéSCéS SMSOO3 >128 E. faecalis EFSOO8 16 1. O2. MBI 11HO9CN K. pneumoniae KPOO1 4 P. aeruginosa PAOO4 32 A. Caicoaceticus ACOO2 15 S. aureus SAO14 2 E. cloacae ECLOO7 >128 S. aureus SAO93 1. E. coli ECOOO5 32 S. epidermidis SEO1O 2 E. faecalis EFSOO1 S. maltophilia SMAOO2 8 E. faecalis EFSOO8 64 S. narceScenS SMSOO3 >128 K. pneumoniae KPOO1 64 P. aeruginosa PAOO4 >128 S. aureus SAO14 S. aureus SAO93 TABLE 12 S. epidermidis SEO1O 16 S. maltophilia SMAOO2 64 MBI 11CN MB 11B7CN S. narceScenS SMSOO3 >128 Organism MIC (ug/ml) MIC (ug/ml) 103. MBI 11H10CN 25 C. albicans CA001 28 64 A. Caicoaceticus ACOO2 C. albicans CA002 64 32 E. cloacae ECLOO7 >128 C. albicans CA003 28 64 E. coli ECOOO5 16 C. albicans CA004 64 32 E. faecalis EFSOO1 C. albicans CA005 28 32 E. faecalis EFSOO8 C. albicans CA006 28 64 K. pneumoniae KPOO1 16 C. albicans CA007 28 64 aeruginosa PAOO4 >128 C. albicans CA008 64 32 S. aureus SAO14 C. albicans CA009 64 32 S. aureus SAO93 C. albicans CA010 28 64 S. epiderinidis SEO10 C. albicans CAO11 64 64 S. maltophilia SMAOO2 64 C. albicans CA012 28 64 S. PCéSCéS SMSOO3 >128 35 C. albicans CA013 28 64 1. O4. MBI 11H11CN C. albicans CA014 64 32 C. albicans CAO15 28 64 A. Caicoaceticus ACOO2 C. albicans CAO16 28 64 E. cloacae ECLOO7 >128 C. albicans CA017 28 64 E. coli ECOOO5 16 C. albicans CA018 28 64 EFSOO1 E. faecalis 40 C. albicans CA019 28 64 E. faecalis EFSOO8 C. albicans CA020 28 32 K. pneumoniae KPOO1 16 C. albicans CA021 28 32 P. aeruginosa PAOO4 >128 C. albicans CA022 32 32 S. aureus SAO14 C. albicans CAO23 28 64 S. aureus SAO93 C. albicans CA024 16 8 S. epidermidis SEO1O C. glabrata CGLOO1 >128 128 S. maltophilia SMAOO2 64 45 C. glabrata CGL002 >128 128 S. narceScenS SMSOO3 >128 C. glabrata CGL003 >128 128 105. MBI 11H12CN C. glabrata CGL004 >128 128 C. glabrata CGL005 >128 128 A. Caicoaceticus ACOO2 C. glabrata CGL009 >128 128 E. cloacae ECLOO7 >128 C. glabrata CGLO10 >128 128 E. coli ECOOO5 16 50 C. krusei CKROO1 0.5 1. E. faecalis EFSOO1 C. tropicalis CTR001 4 4 E. faecalis EFSOO8 C. tropicalis CTR002 4 8 K. pneumoniae KPOO1 16 C. tropicalis CTR003 8 8 P. aeruginosa PAOO4 >128 C. tropicalis CTR004 4 8 S. aureus SAO14 C. tropicalis CTR005 4 4 S. aureus SAO93 55 C. tropicalis CTR006 16 8 S. epidermidis SEO1O C. tropicalis CTR007 16 8 S. maltophilia SMAOO2 64 C. tropicalis CTR008 8 4 S. narceScenS SMSOO3 >128 C. tropicalis CTR009 8 4 106. MBI 1101CN A. Caicoaceticus ACOO2 Broth Dilution Assay E. cloacae ECLOO7 >128 60 Typically 100 ul of calcium and magnesium Supple E. coli ECOOO5 64 E. faecalis EFSOO1 128 mented Mueller Hinton broth is dispensed into each well of E. faecalis EFSOO8 >128 a 96-well microtitre plate and 100 ul volumes of two-fold K. pneumoniae KPOO1 >128 Serial dilutions of the peptide are prepared acroSS the plate. P. aeruginosa PAOO4 >128 One row.of wells receives no peptide and is used as a growth S. aureus SAO14 16 65 control. Each well is inoculated with approximately 5x10 S. aureus SAO93 CFU of bacteria and the plate is incubated at 35–37 C. for 16–20 hours. The MIC is recorded at the lowest concentra US 6,503,881 B2 67 68 tion of peptide that completely inhibits growth of the organ determine viable colony counts. The number of bacteria ism as determined by Visual inspection. remaining in each Sample is plotted over time to determine For example, MIC values in tug/ml are established by the rate of cationic peptide killing. Generally a three or more broth dilution assay (Table 13) or by agarose dilution assay logo reduction in bacterial counts in the antimicrobial (Table 14) for a series of cationic peptides against various 5 Suspension compared to the growth controls indicate an bacterial Strains. adequate bactericidal response. As shown in FIGS. 3A-E, most of the peptides demon TABLE 13 Strate a three or more logo reduction in bacterial counts in the antimicrobial Suspension compared to the growth Organism MBI 11CN MB 11A1CN 10 controls, indicating that these peptides have met the criteria A. calcoaceticus 8.191 256 >256 for a bactericidal response. E. cloacae 13047 >128 >256 E. coli KL4 64 256 Example 5 E. coli DH1 64 128 ASSays to Measure Enhanced Activity of Antibiotic Agent E. co ECOOO3 64 >256 and Cationic Peptide Combinations E. coli 25922 128 512 15 - - - - p E. faecalis 29212 64 >256 Killing Curves K. pneumoniae 13883 >128 >256 Time kill curves resulting from combination of cationic K. pneumoniae B44 64 >256 peptide and antibiotic agent are compared to that resulting P. aeruginosa H650 256 >256 from agent alone P. aeruginosa H652 256 >256 9. v. P. aeruginosa 27853 >128 >256 2O The assay is performed as described above except that P. aeruginosa 95.03024 >256 >256 duplicate tubes are Set up for each concentration of the P. aeruginosa 8509041 256 >256 antibiotic agent alone and of the combination of antibiotic S.P. aeruginosaaureus 25923 93.08077 12832 >256512 agent and cationic peptide. Synergy is demonstrated by at S. aureus 27217 64 >256 least a 100-fold (2 logio) increase in killing at 24 hours by S. aureus 33593 64 >256 25 the antibiotic agent and cationic peptide combination com S. aureus 292.13 32 5. pared to the antibiotic agent alone. A time kill assay is shown t C . 5. in FIG. 3E for MBI 26 in combination with Vancomycin S. aureus 8402093 32 >256 against a bacterial Strain. The combination of peptide and S. maltophilia 13637 128 256 antibiotic agent gave greater killing than either peptide or S. epidermidis 14990 8 512 3O antibiotic agent alone. S. maltophilia H361 64 256 S. narceScens 13880 >128 >256 FIC Measurements S. narceScens B21 >256 >256 In this method, Synergy is determined using the agarose dilution technique. An array of plates or tubes, each con taining a combination of peptide and antibiotic in a unique

TABLE 1.4 Peptide MIC values in ug/ml

MBI MBI MBI MBI MBI MBI MBI Organism MB 1OCN MB 11CN 11A1CN 11A3CN 11B4CN 11B8CN 11D18CN 11F1CN 11 G13CN E. co ATCC 25922 16 16 28 >128 32 32 16 8 64 E. coli ESS ND ND 16 >128 8 ND 2 ND 32 E. co NCTC 10418 8 4 16 64 8 4 2 2 16 E. faecium ATCC 29212 4 8 28 >128 8 8 8 8 32 P. aeruginosa ATCC 27853 >128 >128 >128 >128 >128 >128 128 64 >128 P. aeruginosa NCTC 10662 >128 >128 >128 >128 >128 >128 128 64 >128 P. vulgaris ATCC 13315 ND ND >128 >128 >128 ND 32 ND >128 S. airets ATCC 292.13 ND ND 2 16 1. ND 0.5 ND 1. S. aureus MRSA13 4 16 >128 >128 32 32 8 16 64 S. aureus MRSA17 2 4 32 >128 8 4 2 2 16 S. aureus MRSA9 1. 2 8 28 4 2 2 2 8 S. aureus SA206 ND ND 28 >128 16 ND 4 ND 32 S. narceScens SM76 ND ND >128 >128 >128 ND >128 ND >128 S. narceScens SM82 >128 >128 >128 >128 >128 >128 >128 >128 >128 S. pneumoniae 406LE8 >128 >128 >128 >128 128 >128 64 128 128 S. pneumoniae 60120 64 >128 >128 >128 >128 >128 128 128 >128 S. pneumoniae ATCC 49619 32 64 >128 >128 64 128 64 64 128

ND = Not determined

Time Kill Assay concentration mix, is inoculated with bacterial isolates. Time kill curves are used to determine the antimicrobial When performing Solid phase assays, calcium and magne activity of cationic peptides over a time interval. Briefly, in 60 sium supplemented Mueller Hinton broth is used in combi this assay, a Suspension of microorganisms equivalent to a nation with a low EEO agarose as the bacterial growth 0.5 McFarland Standard is prepared in 0.9% saline. This medium. Broth dilution assays can also be used to determine Suspension is then diluted Such that when added to a total Synergy. Synergy is determined for cationic peptides in volume of 9 ml of cation-adjusted Mueller Hinton broth, the combination with a number of conventional antibiotic inoculum size is 1x10 CFU/ml. An aliquot of 0.1 ml is 65 agents, for example, penicillins, cephalosporins, removed from each tube at pre-determined intervals up to 24 carbapenems, monobactams, aminoglycosides, macrollides, hours, diluted in 0.9% saline and plated in triplicate to fluoroquinolones, nisin and lysozyme. US 6,503,881 B2 69 70 Synergy is expressed as a fractional inhibitory concentra tion (FIC), which is calculated according to the equation TABLE 15-continued below. An FICs 0.5 is evidence of synergy. An additive response has an FIC values 0.5 ands 1, while an indifferent response has an FIC valued 1 and S2. MMicroorganism StrainStrai AntibiotiOC FC Peptideptide

FC = MIC(peptide in combination) -- MIC(antibiotic in combination) S. epidermidis SE8416 Ampicillin O.52 MB 31 MIC(peptide alone) MIC(antibiotic alone) S. epidermidis SE8416 Clindamycin O.56 MB 26 S. epidermidis SE8505 Ampicillin 1.OO MB 26 Tables 15, 16 and 17 present combinations of cationic 1O S. epidermidis SE8565 Amplicillin- - 1.OO MB 26 peptides and antibiotic agents that display an FIC value of S. epidermidis SH8575 Amplicillin O.27 MB 31 less than or equal to 1. Although FIC is measured in Vitro S. haemolyticus SA7797 Ampicillin OSO MB 31 and Synergy defined as an FIC of less than or equal to 0.5, S. haemolitics SA7817 Ampicillin O.26 MB 31 an additive effect may be therapeutically useful. As shown is - - below, although all the microorganisms are Susceptible S. haemolyticus SA7818 Amplicillin O.52 MB 31 (NCCLS breakpoint definitions) to the tested antibiotic S. haemolyticus SA7834 Amplicillin O.52 MB 31 agents, the addition of the cationic peptide improves the S. haemolyticus SA7835 Ampicillin OSO MB 31 efficacy of the antibiotic agent. S. haemolyticus SH8459 Ampicillin O.52 MB 26 2O S. haemolyticus SH8472 Ampicillin O.56 MB 26 TABLE 1.5 y S. haemolyticus SH8563 Ampicillin O.75 MB 26 Microorganism Strain Antibiotic FIC Peptide S. haemolyticus SH8564 Ampicillin O.62 MB 26 S. aureus SAO14 Ciprofloxacin O.63 MB 26 S. haemolyticus SH8575 Ampicillin O.75 MB 26 S. aureus SAO14 Ciprofloxacin O.75 MB 28 - - S. aureus SAO14 Ciprofloxacin 1.OO MBI 25 S. h.aenolyticus ivt. SH8576 A. CII O.62 MB 26 11A2CN S. haemolyticus SH8578 Ampicillin 1.OO MB 26 S. aureus SAO93 Ciprofloxacin 0.75 MBI S. haemolyticus SH8597 Ampicillin 1.OO MB 31 11A2CN S. aureus SAF609 Clindamycin O.25 MB 26 S. aureus SAF609 Methicillin O.56 MB 26 S. aureus SA761O Clindamycin O.63 MB 26 3O TABLE 16 S. aureus SA761O Methicillin O.31 MB 26 S. aureus SA7795 Ampicillin O.52 MB 26 Teicoplanin (ug/ml) MBI 26 (Lig/ml) S. aureus SA7795 Clindamycin O.5.3 MB 26 S. aureus SA7796 Ampicillin 1.OO MB 26 Microorganism Strain Alone +MBI 26 Alone +Teicoplanin S. aureus SAf796 Clindamycin O.51 MB 26 S. aureus SAf817 Ampicillin O.50 MB 26 35 E. faecium 97001 VanB 0.5 0.25 64 4 S. aureus SAf818 Ampicillin 1.OO MB 26 E. faecium 97002 VanB 0.5 0.25 64 S. aureus SAf818 Erythromycin O.15 MB 26 E. faecium 97003 VanB 0.5 0.25 64 S. aureus SAf818 Erythromycin O.15 MB 26 E. faecium 97005 VanB 1. 0.25 64 S. aureus SA7821 Erythromycin O.50 MB 26 E. faecium 97006 VanB 0.5 0.5 64 4 S. aureus SA7821 Erythromycin O.50 MB 26 E. faecium 97007 VanB 0.5 0.25 64 S. aureus SAf822 Ampicillin O.25 MB 26 40 E. faecium 97008 VanB 0.5 0.25 64 4 S. aureus SAf823 Ampicillin O.25 MB 26 E. faecium 97009 VanB 0.5 0.25 32 S. aureus SA7824 Ampicillin 1.OO MB 26 E. faecium 97010 VanB 0.5 0.25 64 4 S. aureus SAf825 Ampicillin 1.OO MB 26 E. faecium 97011 VanB 0.5 0.25 64 4 S. aureus SAf825 Erythromycin 1.OO MB 26 E. faecium 97012 VanB 8 0.25 64 4 S. aureus SAf825 Erythromycin 1.OO MB 26 S. aureus SA7834 Ampicillin O.5.3 MB 26 E. faecium 97O13 VanBal 8 0.25 64 8 45 E. faecium 97014 VanB 8 0.25 32 4 S. aureus SA7834 Clindamycin O.56 MB 26 E 97O15 VanB 0.5 0.25 64 4 S. aureus SAf835 Ampicillin O.5.3 MB 26 faecium al 4 4 S. aureus SAf836 Ampicillin O.75 MB 26 E. ?aecium 97016 VanB 0.5 0.25 6 S. aureus SAF837 Ampicillin 1.OO MB 26 E. faecalis 97040 VanB 0.5 0.25 64 8 S. aureus SAATCC25293 Methicillin O.50 MB 26 E. faecalis 97.04 VanB 1 0.25 64 8 S. aureus SAATCC29213 Methicillin O.31 MB 26 E. faecalis 97042 VanB 1 0.25 64 8 S. aureus SAW1133 Methicillin O.75 MB 26 50 E. faecalis 97043 VanB 0.5 0.25 64 8 S. epidermidis SE8406 Clindamycin O.50 MB 26

TABLE 1.7 1. Amikacin Amikacin MIC (ug/ml) Peptide MIC (ug/ml) Peptide Organism FIC Alone + Peptide Alone + Amikacin

MBI 11B16CN A. battmannii ABIOO1 OSO 32 O.125 32 16 A. baunanni ABIOO3 O.53 16 0.5 16 8 P. aeruginosa PAO22 O.38 64 8 64 16 P. aeruginosa PAO37 O.25 16 2 >128 32 S. maltophilia SMA018 O31 128 8 32 8 S. maltophilia SMA022 O.09 >128 8 >128 16 E. faecalis EFS008 O.28 32 8 8 0.25

US 6,503,881 B2 81 82

TABLE 17-continued

... baunanni ABIOO1 O31 16 al 64 ... baunanni ABIOO2 O.53 8 al 16 maltophilia SMA027 O.19 32 al >128 maltophilia SMA029 O16 128 al 32 aureus SAO 18 MRSA O.56 64 al 32 haemolyticus SHAO01 O.53 4 O.125 ... baunanni ABIOO1 O.53 16 O.5 32 ... baunanni ABIOO2 1.OO 4 2 16 P. aeruginosa PAO32 OSO 16 al P. maltophilia SMAO29 O.28 28 32 28 S. maltophilia SMAO30 O.26 28 28 S. aureus SAO 14 MRSA O.51 >128 2 4 S. haemolyticus SHAO05 O.56 4 O.25 MBI 11 G13CN A. baunanni ABIOO1 OSO 16 al 28 P. aeruginosa PAO22 O.56 8 al S. maltophilia SMAO29 OSO 28 64 28 S. maltophilia SMAO30 OSO 28 64 P. aureus SAO25 MRSA OSO >128 O.125 MBI 21A1 B. cepacia BC001 O.25 28 32 P. aeruginosa PAO22 O.53 8 al P. aeruginosa PAO26 O.51 8 al 16 maltophilia SMA029 O.28 28 al 28 maltophilia SMA030 O16 28 al aureus SAO14 MRSA OSO >128 O.125 32 aureus SAO25 MRSA OSO >128 O.125 haemolyticus SHAO01 OSO 2 O.5 16 haemolyticus SHAO05 O.38 4 32 MB22A1 maltophilia SMA030 O.26 28 32 maltophilia SMA031 O.25 28 O.5 32 aureus SAO14 MRSA 0.27 >128 al epidermidis SE072 OSO >128 O.125 16 epidermidis SEO73 OSO >128 O.125 16 epidermidis SEO80 O.56 32 16 1. 2 5 MB 26 maltophilia SMA029 O.05 28 al maltophilia SMA030 O.05 28 al epidermidis SEO67 O.38 >128 64 epidermidis SEO68 0.27 >128 al MBI 27 coli ECOOO6 O.56 8 O.5 maltophilia SMA029 OSO 64 16 maltophilia SMA031 O.53 28 al 8 MBI 29 A. baunanni ABIOO1 O.53 16 8 O.125 E. coli ECO004 O.53 2 O.125 E. coli ECOOO6 O.53 8 al O.125 K. pneumoniae KP008 0.52 0.5 O.25 O.125 P. aeruginosa PAO30 0.52 16 8 O.125 S. maltophilia SMAO31 OSO >128 O.25 S. maltophilia SMAO32 O.53 128 al S. epidermidis SE072 O.53 >128 8 MB 293 P. aeruginosa PAO22 O.56 8 al 0.25 P. aeruginosa PAO28 OSO 32 16 3 O.125 P. aeruginosa PAO29 O.51 32 16 O.125 S. maltophilia SMAO29 O.28 128 al S. maltophilia SMAO30 O.28 128 al REWHS3ASCN S. maltophilia SMAO29 O.08 128 al 16 S. maltophilia SMA030 O.13 128 O.25 32 S. aureus SAO14 MRSA OSO >128 O.125 8. Vancomycin Vancomycin MIC (ug/ml) Peptide MIC (ug/ml)

Peptide Organism FIC Alone + Peptide Alone + Vancomycin

MB 11A1CN E. faecalis EFS001 O.53 1. O.5 O.125 E. faecalis EFS006 OSO 8 4 128 0.25 E. faecalis EFS007 OSO 4 2 128 0.5 E. faecalis EFS010 0.27 16 4 128 2 E. faecalis EFS012 O.25 >128 32 64 8 E. faecalis EFSO14 O.51 128 1. 2 E. faecium EFM004 OSO >128 O.5 32 16 E. faecium EFM007 O.28 128 4 64 16 E. faecium EFM009 O.25 32 4 64 8 US 6,503,881 B2 83 84

TABLE 17-continued MBI 11D18CN E. faecalis EFS001 O.38 1. O.125 8 2 E. faecalis EFS004 OSO 2 O.5 8 2 E. faecalis EFSO11 OSO 64 32 64 O.125 E. faecalis EFS012 O.38 >128 64 16 2 E. faecalis EFSO14 O16 28 4 4 0.5 E. faecium EFM004 OSO >128 64 8 2 E. faecium EFM009 0.52 64 32 8 O.125 E. faecium EFM010 O.28 >128 64 8 0.25 E. faecium EFM011 OSO >128 64 8 2 MBI 21A1 E. faecalis EFS007 O.56 2 1. 16 1. E. faecalis EFS012 O16 28 16 32 1. E. faecalis EFS013 O.28 28 32 32 1. E. faecium EFM010 O.56 64 32 32 2 MB 26 E. faecalis EFS005 O31 16 4 >128 16 E. faecalis EFS012 O.O7 >128 2 16 1. E. faecalis EFS013 O.O7 >128 2 16 1. E. faecium EFM010 O31 32 2 32 8 E. faecium EFM011 O31 32 2 32 8 E. faecium EFM012 O31 32 2 64 16 E. faecium EFM014 0.27 >128 4 32 8 E. faecium EFMO16 O.51 28 1. 8 4 MBI 29 E. faecalis EFS005 O.38 16 4 32 4 E. faecalis EFS010 O.38 64 16 2 0.25 E. faecalis EFS012 OSO >128 64 2 0.5 E. faecium EFM005 O.53 28 4 4 E. faecium EFMO16 O.51 28 1. 4 2 MBI 29A3 E. faecalis EFS003 O.56 4 2 32 2 E. faecalis EFS005 O.28 16 4 32 1. E. faecalis EFSO11 OSO 16 4 32 8 E. faecalis EFSO14 0.52 64 1. 1. 0.5 E. faecium EFMOO6 0.52 >128 4 4 2

Example 6 MBC end point is defined as the lowest concentration of the Overcoming Tolerance by Administering a Combination of antimicrobial agent that kills 99.9% of the test bacteria. Thus, tolerance of a microorganism to an antimicrobial Antibiotic Agent and Cationic Peptide agent occurs when the number of colonies growing on Tolerance to an antibiotic agent is associated with a defect subculture plates exceeds the 0.1% cutoff for several Suc in bacterial cellular autolytic enzymes Such that an antimi 35 cessive concentrations above the observed MIC. A combi crobial agent is bacterioStatic rather than bactericidal. Tol nation of antimicrobial agent and cationic peptide that erance is indicated when a ratio of minimum bactericidal breaks tolerance results in a decrease in the MBC:MIC ratio concentration (MBC) to minimum inhibitory concentration to <32. Table 18 shows that the combination of Vancomycin (MIC) (MBC:MIC) is 232. and MBI 26 overcomes the tolerance of the organisms listed.

TABLE 1.8 Vancomycin Vancomycin + MBI 26

MIC MBC MIC MEC Organism (ug/ml) (ug/ml) MBC/MIC (ug/ml) (ug/ml) MBC/MIC E. casselliflavus ECAO01 2 >128 >64 0.5 2 4 E. faecium EFMOO1 0.5 >128 >256 0.5 0.5 1. E. faecium EFMO20 1. >128 >128 0.5 4 8 E. faecalis EFS001 1. >128 >128 0.5 4 8 E. faecalis EFS004 1. >128 >128 1. 2 2 E. faecalis EFS007 4 128 32 2 2 1. E. faecalis EFS009 4 >128 >32 4 4 1. E. faecalis EFSO15 1. >128 >128 0.5 0.5 1.

55 The agarose dilution assay is adapted to provide both the Example 7 MBC and MIC for an antimicrobial agent alone and an agent Overcoming Inherent Resistance by Administering a Com bination of Antibiotic Agent and Cationic Peptide in combination with a peptide. Following determination of Peptides are tested for their ability to overcome the MIC, MBC is determined from the agarose dilution assay inherent antimicrobial resistance of microorganisms, includ plates by Swabbing the inocula on plates at and above the 60 ing those encountered in hospital Settings, to specific anti MIC and resuspending the Swab in 1.0 ml of saline. A 0.01 microbials. Overcoming resistance is demonstrated when the antibiotic agent alone exhibits minimal or no activity ml aliquot is plated on agarose medium (Subculture plates) against the microorganism, but when used in combination and the resulting colonies are counted. If the number of with a cationic peptide, results in Susceptibility of the colonies is less than 0.1% of the initial inoculum (as 65 microorganism. determined by a plate count immediately after inoculation of The agarose dilution assay described above is used to the MIC test plates), thens 99.9% killing has occurred. The determine the minimum inhibitory concentration (MIC) of US 6,503,881 B2 85 86 antimicrobial agents and cationic peptides, alone and in combination. Alternatively, the broth dilution assay or time TABLE 20-continued kill curves can be used to determine MICs. Tables 19, 20, 21 and 22 present MIC values for antibiotic agents alone and in Vancomycin combination with peptide at the concentration shown. In all MIC (ug/ml) MBI 26 MIC (ug/ml) cases, the microorganism is inherently resistant to its mode of action, thus, the antibiotic agent is not effective against Microorganism Alone +MBI 26 Alone +Vancomycin the test microorganism. In addition, the antibiotic agent is not clinically prescribed against the test microorganism. E. casselliflavus 97057 4 2 2 0.5 1O In the data presented below, the MIC values for the VanC antibiotic agents when administered in combination with E. casselliflavus 97058 2 1. 4 O.25 peptide are decreased, from equal to or above the resistant VanC breakpoint to below it. E. casselliflavus 97059 4 2 32 0.5 15 VanC TABLE 1.9 E. casselliflavus 97060 2 2 0.5 O.25 Erythromycin VanC MIC (lig/ml MBI 26 MIC (ugml Microorganism Alone +MBI 26 Alone +Erythro. 20 TABLE 21 A. calcoaceticus AC001 32 1. 16 8 Teicoplanin K. pneumoniae KP001 32 0.25 16 8 MIC (tigml MBI 26 MIC (ug/ml K. pneumoniae KP002 256 0.5 64 32 P. aeruginosa PAO41 128 4 64 32 Microorganism Alone +MBI 26 Alone +Vancomycin 25 E. gallinarum 97044 0.5 0.25 64 1. VanC TABLE 2.0 E. gallinarum 97046 1. 0.25 8 1. VanC Vancomycin E. gallinarum 97047 8 0.25 64 32 MIC ml MBI 26 MIC (leg?ml 3O VanC E. gallinarum 97048 0.5 0.25 8 1. Microorganism Alone +MBI 26 Alone +Vancomycin VanC E. gallinarum 97049 2 0.25 64 32 E. gallinarum 97044 8 2 8 0.5 VanC VanC E. cassellifiavus 97056 0.5 0.25 64 2 E. gallinarum 97046 32 1. 2 4 VanC VanC 35 E. casselliflavus 97057 0.5 0.25 64 0.5 E. gallinarum 97047 128 16 64 8 VanC VanC E. casselliflavus 97058 0.5 0.25 32 0.5 E. gallinarum 97048 32 4 2 2 VanC VanC E. casselliflavus 97059 0.5 0.25 64 1. E. gallinarum 97049 128 4 64 16 VanC VanC 40 E. casselliflavus 97060 0.5 0.25 64 1. E. casselliflavus 97056 8 2 8 1. VanC VanC

TABLE 22 1. Amikacin Amikacin MIC (ug/ml) Peptide MIC (ug/ml) Peptide Organism FIC Alone + Peptide Alone + Amikacin

A baunanni ABIOO1 0.25 32 4 32 4 maltophilia SMA018 O.31 28 8 32 8 maltophilia SMA022 O.14 >128 4 >128 32 aureus SAO14 MRSA 0.75 32 8 8 4 aureus SAO25 MRSA O.63 32 4 8 4 MB21A2 maltophilia SMA018 O.53 >128 8 16 8 maltophilia SMA060 O.31 >128 16 >128 64 aureus SAO25 MRSA O.56 32 2 2 1. MB 26 maltophilia SMA022 O.19 28 8 64 8 maltophilia SMA037 O.19 28 16 >128 16 MBI 27 ... baunanni ABIOO1 1.OO 32 16 8 4 cepacia BC005 O.SO 64 16 >128 64 maltophilia SMA036 O.56 >128 16 64 32 maltophilia SMA037 O.31 64 4 64 16 aureus SAO25 MRSA 0.75 32 16 2 0.5 MBI 29A3 cepacia BC003 O.63 32 16 >128 32 cepacia BC005 O.38 28 32 >128 32 S. maltophilia SMA036 O.53 >128 8 64 32 S. maltophilia SMA063 O.56 >128 16 8 4

US 6,503,881 B2 89 90

TABLE 22-continued MB 11D18CN S. aureus SAO 18 MRSA O.56 64 MB 11F3CN S. aureus SAO 14 MRSA O.51 >128 4 MBI 11 G13CN S. aureus SAO25 MRSA O.SO >128 O.125 4 : MBI 21A1 S. aureus SAO 14 MRSA O.SO >128 O.125 S. aureus SAO25 MRSA O.SO >128 O.125 2 1. MB22A1 S. aureus SAO 14 MRSA 0.27 >128

1O Example 8 Similarly teicoplanin resistant enterococci become Sus Overcoming Acquired Resistance by Administering a Com ceptible to teicoplanin when teicoplanin is used in combi bination of Antibiotic Agent and Cationic Peptide nation with cationic peptides such as MBI 26. An antibiotic agent can become ineffective against a AS described previously, the agarose dilution assay is used previously Susceptible microorganism if the microorganism 15 acquires resistance to the agent. However, acquired resis to determine the MIC for antibiotic agents administered tance can be overcome when the agent is administered in alone and in combination with cationic peptide. Alterna combination with a cationic peptide. For example Vanco tively the broth dilution assay or time kill curves can be mycin resistant enterococci (VRE) become Susceptible to employed. Tables 23 and 25 presents results showing that Vancomycin when it is used in combination with a cationic administration of a cationic peptide in combination with an peptide such as MBI 26. This combination is likely to be antibiotic agent overcomes acquired resistance. Table 24 effective against other organisms acquiring resistance to presents results showing administration of MBI 26 in com Vancomycin including but not limited to Strains of methi bination with teicoplanin against teicoplanin resistant cillin resistant S. aureus (MRSA). enterococci.

TABLE 23

MC MIC alone comb. Peptide Microorganism Strain Antibiotic agent (ug/ml) (ug/ml) Peptide MIC A. Caicoaceticus 002 Tobramycin 8 1. MBI 29 4 A. Caicoaceticus 003 Ceftazidime 32 2 MB 26 32 A. Caicoaceticus 003 Ceftazidime 32 2 MBI 29 8 A. Caicoaceticus 003 Ciprofloxacin 8 1. MBI 29 16 A. Caicoaceticus 004 Ciprofloxacin 8 4 MB 26 4 A. Caicoaceticus 010 Ceftazidime 32 2 MB 26 32 E. faecium ATCC 29212 Mupirocin 1OO O.1 MB 11CN 8 E. faecium ATCC 29212 Mupirocin 1OO O.1 MB 11 G13CN 32 P. aeruginosa PA41 Ciprofloxacin 4 O.125 MB 21A1 16 P. aeruginosa PA41 Ciprofloxacin 4 1. MB21A2 16 P. aeruginosa PA41 Ciprofloxacin 8 2 MB 28 8 P. aeruginosa OO1 Piperacillin 128 64 MBI 27 8 P. aeruginosa O23 Piperacillin 128 64 MBI 29 8 P. aeruginosa O24 Tobramycin 64 1. MBI 29 8 P. aeruginosa O25 Ceftazidime 64 16 MBI 29 8 P. aeruginosa O27 Imipenem 16 8 MBI 29 16 P. aeruginosa O28 Imipenem 16 8 MBI 29 16 S. haemolyticus SH8578 Erythromycin 8 O.5 MB 31 1. S. aureus SAF338 Ampicillin 2 O.25 MB 26 O.25 S. aureus SAF609 Erythromycin 32 O.5 MB 26 1. S. aureus SAF835 Erythromycin 8 O.125 MB 26 2 S. aureus SATT95 Erythromycin 32 1. MB 26 8 S. aureus SAf796 Erythromycin 32 1. MB 26 2 S. aureus SATT95 Erythromycin 32 4 MB3 O.125 S. aureus SAF818 Erythromycin 32 2 MB3 O.125 S. aureus SAf796 Erythromycin 32 2 MB3 O.125 S. aureus SA7834 Methicillin 32 8 MB 26 4 S. aureus SAF835 Methicillin 32 4 MB 26 16 S. aureus SAf796 Methicillin 16 2 MB3 16 S. aureus SATT97 Methicillin 16 2 MB3 16 S. aureus SAF823 Methicillin 16 2 MB3 0.5 S. aureus SA7834 Methicillin 64 1. MB3 32 S. aureus SAF835 Methicillin 64 2 MB3 16 S. aureus SAOO7 Piperacillin 128 64 MBI 27 0.5 S. aureus MRSA9 Mupirocin >100 O.1 MB 11D18CN 2 S. aureus MRSA9 Mupirocin >100 O.1 MB 11 G13CN 8 S. aureus MRSA9 Mupirocin >100 O.1 MB 21A1 16 S. aureus MRSA9 Mupirocin >100 O.3 MB 21A10 32 S. aureus MRSA9 Mupirocin >100 O.1 MB 21A2 32 S. aureus MRSA9 Mupirocin >100 O.1 MB 26 4 S. aureus MRSA9 Mupirocin >100 O.1 MB 27 2 S. aureus MRSA 13 Mupirocin 1OO 3 MB 1OCN 4 S. aureus MRSA 13 Mupirocin 1OO O.1 MB 11CN 16 S. aureus MRSA 13 Mupirocin 1OO 3 MB 11F1CN 8 US 6,503,881 B2 91 92

TABLE 23-continued

MIC MIC alone comb. Peptide Microorganism Strain Antibiotic agent (ug/ml) (ug/ml) Peptide MC S. aureus O14 Ciprofloxacin 8 O.125 MB 21A2 4 S. aureus MRSA 17 Mupirocin >1OO 1. MB 1OCN 1. O.3 2 S. aureus MRSA 17 Mupirocin >1OO 1. MB 11A1CN 32 S. aureus MRSA 17 Mupirocin >1OO 1. MBI 16 11 G13CN S. aureus MRSA 17 Mupirocin >1OO O.3 MB 27 2 S. aureus MRSA 17 Mupirocin >1OO O. MB29A3 4 S. aureus O93 Ciprofloxacin 32 O.125 MB 21A1 2 S. aureus O93 Ciprofloxacin 32 1. MB21A2 4 S. aureus SAf818 Methicillin 16 4 MB 26 2 S. epidermidis SE8497 Clindamycin 32 O.125 MB 26 2 S. epidermidis SE8403 Erythromycin 8 O.125 MB 26 2 S. epidermidis SE841O Erythromycin 32 O.5 MB 26 S. epidermidis SE8411 Erythromycin 32 O.5 MB 26 S. epidermidis SE8497 Erythromycin 32 O.125 MB 26 S. epidermidis SE8503 Erythromycin 32 O.5 MB 26 S. epidermidis SE8565 Erythromycin 32 O.5 MB 26 S. epidermidis SE8403 Erythromycin 8 O.125 MB 31 2 S. epidermidis SE841O Erythromycin 32 O.5 MB 31 S. epidermidis SE8411 Erythromycin 32 O.5 MB 31 S. epidermidis SE8497 Erythromycin 32 O.125 MB 31 S. epidermidis SE8503 Erythromycin 32 O.5 MB 31 S. epidermidis SE8565 Erythromycin 32 O.5 MB 31 S. haemolyticus SH8459 Ampicillin 0.5 O.25 MB 26 O.25 S. haemolyticus SH8472 Ampicillin 2 O.25 MB 26 16 S. haemolyticus SH8564 Ampicillin 64 O.25 MB 26 32 S. haemolyticus SH8575 Ampicillin 0.5 O.25 MB 26 8 S. haemolyticus SH8578 Ampicillin 0.5 O.25 MB 26 4 S. haemolyticus SH8597 Clindamycin 16 O.125 MB 26 1. S. haemolyticus SH8463 Erythromycin 8 O.5 MB 26 0.5 S. haemolyticus SH8472 Erythromycin 8 O.5 MB 26 0.5 S. haemolyticus SH8575 Erythromycin 32 2 MB 26 0.5 S. haemolyticus SH8578 Erythromycin 8 O.5 MB 26 O1 S. haemolyticus SH8597 Erythromycin 32 O.5 MB 26 0.5 S. haemolyticus SH8463 Erythromycin 8 O.5 MB 31 0.5 S. haemolyticus SH8472 Erythromycin 8 O.5 MB 31 0.5 S. haemolyticus SH8564 Erythromycin 32 2 MB 31 0.5 S. haemolyticus SH8575 Erythromycin 32 2 MB 31 0.5 S. haemolyticus SH8563 Methicillin 64 O.25 MB 26 2 S. maltophilia O34 Tobramycin 8 1. MBI 29 4 S. maltophilia O37 Tobramycin 32 4 MBI 29 16 S. maltophilia O39 Ciprofloxacin 4 2 MBI 29 16 S. maltophilia O41 Tobramycin 16 1. MBI 29 8 S. maltophilia O43 Imipenem >256 4 MBI 29 16 S. maltophilia O44 Piperacillin >512 16 MB 26 32

TABLE 24 50 TABLE 24-continued Teicoplanin (leg?ml) MBI 26 (ugml) Teicoplanin (ug/ml) MBI 26 (ug/ml Microorganism Strain Alone + MBI 26 Alone + Teicoplanin Microorganism Strain Alone + MBI 26 Alone + Teicoplanin f C. A. VI. 8, 2. E. ?aecium 903 VanA 32 0.25 64 32 E. faecium 97019 VanA 32 0.5 64 16 f C 2. y : E. faecium 97020 VanA 32 0.5 64 16 E. faecium 97034 VanA 32 0.25 64 8 E. faecium 97021 VanA 32 0.5 64 32 E. faecium 97022 VanA 32 0.5 64 4 E. faecium 97.035 VanA 32 0.25 64 0.5 E. faecium 97023 VanA 32 0.25 64 4 go E. ?aecium 97.036 VanA is 0.25 8 4 E. faecium 97.024 VanA 32 0.25 64 8 E. faecalis 97050 VanA 32 0.25 64 8 E. faecium 97025 VanA 32 0.5 16 4 E. faecalis 97051 VanA 32 0.25 64 8 E. faecium 97O26 VanA 32 0.5 64 16 E. faecalis 97.052 VanA 32 0.25 64 8 E. faecium 97027 VanA 32 8 64 8 E. faecalis 97053 VanA 32 0.25 64 8 E. faecium 97028 VanA 32 0.25 8 8 E. faecalis 97054 VanA 32 0.25 64 8 E. faecium 97029 VanA 32 0.25 64 8 65 E. faecalis 97055 VanA 32 0.25 64 8 E. faecium 97030 VanA 32 0.25 64 32

US 6,503,881 B2

TABLE 25-continued MBI 29A3 E. faecalis EFS005 O.19 16 32 4 E. faecalis EFSO11 O.SO 16 32 8 E. faecalis EFSO14 0.52 64 1. 0.5 E. faecium EFMOO6 0.52 >128 4 2

These data show that acquired resistance can be over come. For example, the 5 acquired resistance of S. aureus, 1O TABLE 26 a Gram-positive organism, to piperacillin and ciprofloxacin is overcome when these antibiotic agents are combined with Code Formulation Buffer peptides MBI 27, MBI 21A1 or MBI 21A2 respectively. A. PBS 200 mM, pH 7.1 B Sodium Citrate 100 mM, pH 5.2 Similar results are obtained for peptides MBI 26 and MBI 31 15 C Sodium Acetate 200 mM, pH 4.6 in combination with methicillin, amplicillin and C1 Sodium Acetate 200 mM/0.5% Polysorbate 80, pH 4.6 erythromycin, and for peptide MBI 26 in combination with D Sodium Acetate 100 mM/0.5% Activated Polysorbate Vancomycin or teicoplanin against resistant enterococci. 80, pH 7.5: Lyophilized/Reconstituted Example 9 Solubility in Broth Synergt of Cationic Peptides and Lysozyme or Nisin The Solubility of peptide analogues is assessed in calcium and magnesium supplemented Mueller Hinton broth by The effectiveness of the antibiotic activity of lysozyme or Visual inspection. The procedure employed is that used for nisin is improved when either agent is administered in the broth dilution assay except that bacteria are not added to combination with a cationic peptide. The improvement is 25 the Wells. The appearance of the Solution in each well is demonstrated by measurement of the MICs of lysozyme or evaluated according to the Scale: (a) clear, no precipitate, (b) nisin alone and in combination with the peptide, whereby the light diffuise precipitate and (c) cloudy, heavy precipitate. lysozyme or nisin MIC is lower in combination than alone. Results show that, for example, MBI 10CN is less soluble The MICS can be measured by the agarose dilution assay, the than MBI 11 CN under these conditions and that MBI broth dilution assay or by time kill curves. 11 BCN analogues are less soluble than MBI 11 ACN ana logues. Example 10 Reversed Phase HPLC Analysis of Peptide Analogue For mulations Biochemical Characterization of Peptide Analogues Reversed-phase HPLC, which provides an analytical Solubility in Formulation Buffier 35 method for peptide quantification, is used to examine pep The primary factor affecting Solubility of a peptide is its tides in two different formulations. A 400 lug/mL solution of amino acid Sequence. Polycationic peptides are preferably MBI 11CN prepared in formulations C1 and D is analyzed freely Soluble in aqueous Solutions, especially under low pH by using a stepwise gradient to resolve free peptide from conditions. However, in certain formulations, polycationic other species. Standard chromatographic conditions are used peptides may form an aggregate that is removed in a 40 as follows: filtration Step. AS peptide Solutions for in Vivo assays are Solvent A: 0.1% trifluoroacetic acid (TFA) in water filtered prior to administration, the accuracy and reproduc Solvent B: 0.1% TFA /95% acetonitrile in water ibility of dosing levels following filtration are examined. Media: POROS(R) R2-20 (polystyrene divinylbenzene) Peptides dissolved in formulations are filtered through a As shown in FIG. 5, MBI 11CN could be separated in two hydrophilic 0.2 um filter membrane and then analyzed for 45 forms, as free peptide in formulation C1, and as a principally total peptide content using reversed-phase HPLC. A 100% formulation-complex peptide in formulation This complex Soluble Standard for each concentration is prepared by Survives the Separation protocol in gradients containing dissolving the peptide in MilliO water. Total peak area for acetonitrile, which might be expected to disrupt the Stability of the complex. A peak corresponding to a Small amount each condition is measured and compared with the peak area 50 of the Standard in order to provide a relative recovery value (<10%) of free peptide is also observed in formulation D. If the shape of the elution gradient is changed, the associated for each concentration/formulation combination. peptide elutes as a broad low peak, indicating that com MBI 11CN was prepared in four different buffer systems plexes of peptide in the formulation are heterogeneous. (A, B, C, and C1) (Table 26, below) at 50, 100, 200 and 400 tug/ml peptide concentrations. With formulations A or B, 55 Example 11 both commonly used for Solvation of peptides and proteins, Structural Analysis of Indolicidin Variants. Using Circular peptide was lost through filtration in a concentration depen Dichroism SpectroScopy dent manner (FIG. 4). Recovery only reached a maximum of Circular dichroism (CD) is a spectroscopic technique that 70% at a concentration of 400 tug/ml. In contrast, peptides measures Secondary Structures of peptides and proteins in dissolved in formulations C and C1 were fully recovered. 60 solution, see for example, R. W. Woody, (Methods in Buffers containing polyanionic ions appear to encourage Enzymology, 246:34, 1995). The CD spectra of ccC.-helical aggregation, and it is likely that the aggregate takes the form peptides is most readily interpretable due to the character of a matrix which is trapped by the filter. Monoanionic istic double minima at 208 and 222 nm. For peptides with counterions are more Suitable for the maintenance of pep other Secondary Structures however, interpretation of CD tides in a non-aggregated, Soluble form, while the addition 65 Spectra is more complicated and leSS reliable. The CD data of other Solubilizing agents may further improve the formu for peptides is used to relate Solution Structure to in Vitro lation. activity. US 6,503,881 B2 103 104 CD measurements of indolicidin analogues are performed Additionally CD spectra are recorded for APO-modified in three different aqueous environments, (1) 10 mM sodium peptides (Table 28). The results show that these compounds phosphate buffer, pH 7.2, (2) phosphate buffer and 40% (v/v) have significant B-turn Secondary Structure in phosphate trifluoroethanol (TFE) and (3) phosphate buffer and large buffer, which is only slightly altered in TFE. (100 nm diameter) unilamellar phospholipid vesicles 5 Again, the CD results Suggest that a f-turn structure (i.e. (liposomes) (Table 27). The organic solvent TFE and the membrane-associated) is the preferred active conformation liposomes provide a hydrophobic environment intended to among the indolicidin analogues tested.

TABLE 27 Phosphate buffer Conformation TFE Conformation

Peptide min W max w in buffer min W maxw in TFE

MB 1OCN 2O1 Unordered 2O3 --219 Unordered MBI 199 Unordered 202, 227 22O B-turn MBI 11 ACN 199 Unordered 2O3 219 Unordered MBI 1CN 2OO Unordered 2OO Unordered MB 11CNY1 2OO Unordered 2OO Unordered MBI 1B1CNW1 2O1 Unordered 2O1 Unordered MB 11B4ACN 2OO Unordered 2OO Unordered MBI 1B7CN 2OO Unordered 204, -2.19 Unordered MBI 1139ACN 2OO Unordered 2OO Unordered MB 11B9CN 2OO Unordered 2OO Unordered MB 11D1CN 2OO Unordered 2O)4 Unordered MB 11E1CN 2O1 Unordered 2O1 Unordered MB 11E2CN 2OO Unordered 2O1 Unordered MB 11E3CN 2O2 226 ppII helix 2OO Unordered MB 11F3CN 199 228 ppII helix 2O2 Unordered MB 11F4CN 202, 220 Unordered 206, 222 slight C-helix MB 11G4CN 199, 221 Unordered 201, 226 215 B-turn MB 11G6ACN 2OO Unordered 199 Unordered MBI 11 GTACN 2OO Unordered 2O2 221 Unordered

TABLE 28 APO-modified Phosphate buffer Conformation TFE Conformation

peptide min W maxw in buffer min W maxw in TFE MBI 1CN 202, 229 22O B-turn 2O3 223 B-turn MB 11BCN 200, 229 B-turn 2O2 222 B-turn MB 11B7CN 202, 230 223 B-turn 199 230 B-turn MB 11E3CN 202, 229 22O B-turn 199 B-turn MB 11F3CN 205 ppII helix 2O3 230 ppII helix mimic the bacterial membrane where the peptides are pre- Example 12 Sumed to adopt an active conformation. Membrane Permeabilization Assays 45 Liposome Dye Release The results indicate that the peptides are primarily unor A method for measuring the ability of peptides to perme dered in phosphate buffer (a negative minima at around 200 abilize phospholipid bilayers is described (Parente et al., nm) with the exception of MBI 11F4CN, which displays an Biochemistry, 29, 8720, 1990) Briefly, liposomes of a additional minima at 220 nm (see below). The presence of defined phospholipid composition are prepared in the pres TFE induces B-turn structure in MBI 11 and MBI 11G4CN, 50 ence of a fluorescent dye molecule. In this example, a dye and increases 60-helicity in MBI 11 F4CN, although most of pair consisting of the fluore Scent molecule the peptides remain unordered. In the presence of lipoSomes, 8-aminonapthalene-1,3,6-trisulfonic acid (ANTS) and its quencher molecule p-xylene-bis-pyridinium bromide (DPX) peptides MBI 11CN and MBI 11B7CN, which are unordered are used. The mixture of free dye molecules, dye free in TFE, display f-turn structure (a negative minima at liposomes, and liposomes containing encapsulated ANTS around 230 nm) (FIG. 6). Hence, liposomes appear to induce DPX are separated by size exclusion chromatography. In the more ordered secondary structure than TFE. assay, the test peptide is incubated with the ANTS-DPX A B-turn is the predominant Secondary Structure that containing liposomes and the fluorescence due to ANTS appears in a hydrophobic environment, Suggesting that it is release to the outside of the liposome is measured over time. 60 Using this assay, peptide activity, measured by dye the primary conformation in the active, membrane release, is shown to be extremely Sensitive to the composi associated form. In contrast, MBI 11 F4CN displays tion of the liposomes at many liposome to peptide ratioS increased 60 -helical conformation in the presence of TFE. (L/P) (FIG. 7). Specifically, addition of cholesterol to lipo Peptide MBI 11 F4CN is also the most insoluble and Somes composed of egg phosphotidylcholine (PC) virtually hemolytic of the peptides tested, Suggesting that C-helical 65 abolishes membrane permeabilizing activity of MBI 11CN, Secondary Structure may introduce unwanted properties in even at very high lipid to peptide molar ratios (compare with these analogues. egg PC liposomes containing no cholesterol). This in vitro US 6,503,881 B2 105 106 selectivity may mimic that observed in vitro for bacterial incubation at 37 C. for 1 hour with constant agitation, the cells in the presence of mammalian cells. Solution is centrifuged and the Supernatant measured for In addition, there is a size limitation to the membrane absorbance at 540 nm to detect released hemoglobin. When disruption induced by MBI 11CN. ANTS/DPX can be compared with the absorbance for a 100% lysed standard, a replaced with fluorescein isothiocyanate-labeled dextran relative measure of the amount of hemoglobin that has been (FD-4), molecular weight 4,400, in the egg PC liposomes. released from inside the red blood cells is determined and No increase in FD-4 fluorescence is detected upon incuba hence the ability of the peptide/formulation to cause red tion with MBI 11CN. These results indicate that MBI blood cell lysis. 11CN-mediated membrane disruption allows the release of Three peptide analogues, MBI 10CN, MBI 11 and MBI the relatively smaller ANTS/DPX molecules (Q400 Da), but 11CN, in formulation C1 at 800 tug/ml cause substantial not the bulkier FD-4 molecules. lysis, which is due primarily to the pH of the buffer. In E. coli ML-35 Inner Membrane Assay contrast, formulation D has a more neutral pH and causes An alternative method for measuring peptide-membrane significantly less lysis. Under these conditions, MBI 10CN, interaction uses the E. coli strain ML-35 (Lehrer et al., J. MBI 11, and MBI 11CN are essentially non-lytic, resulting Clin. Invest., 84. 553, 1989), which contains a chromosomal in 3.9, 2.3, and 3.2% lysis, respectively. copy of the lacZ gene encoding B-galactosidase and is 15 Various cationic peptides are tested for the extent of permease deficient. This Strain is used to measure the effect erythrocyte lysis. As shown in the following table, very little of peptide on the inner membrane through release of toxicity is observed. B-galactosidase into the periplasm. Release of B-galactosidase is measured by Spectrophotometrically TABLE 29 monitoring the hydrolysis of its SubStrate Cl-nitrophenol f3-D-galactopyranoside (ONPG). The maximum rate of Peptide # % Lysis hydrolysis (V) is determined for aliquots of cells taken at Apidaecin IA O.3 various growth points. MB 1OCN 4.3 MB 11CN O.8 A preliminary experiment to determine the concentration 25 MB 11A1CN 0.5 of peptide required for maximal activity against mid-log MB 11A2CN O.1 cells, diluted to 4x107 C.FU/ml, yields a value of 50 tug/ml, MB 11A3CN O.O which is used in all Subsequent experiments. Cells are grown MB 11A4CN O.3 MB 11A5CN O.3 in two different growth media, Terrific broth (TB) and Luria MB 11A6CN O.7 broth (LB) and equivalent amounts of cells are assayed MB 11A7CN 0.5 during their growth cycles. The resulting activity profile of MB 11B1CN 3.1 MBI 11B7CN is shown in FIG. 8. For cells grown in the MB 11B2CN 3.2 MB 11B3CN 3.3 enriched TB media, maximum activity occurs at early mid MB 11B4CN 1.6 log (140 min), whereas for cells grown in LB media, the MB 11B5CN 1.7 MB 11B7CN 3.2 maximum occurs at late mid-log (230 min). Additionally, 35 only in LB, a dip in activity is observed at 140 min. This MB 11B8CN 1.1 MB 11B9CN 0.4 drop in activity may be related to a transition in metabolism, MB 11B10CN O.2 Such as a requirement for utilization of a new energy Source MB 11D3CN O.8 due to depletion of the original Source, which does not occur MB 11D4CN O.9 in the more enriched TB media. A consequence of a metabo MB 11D5CN O.7 40 MB 11D6CN 1.1 lism Switch would be changes in the membrane potential. MB 11D11H O.7 To test whether membrane potential has an effect on MB 11D13H 1.7 peptide activity, the effect of disrupting the electrochemical MB 11D14CN 1.1 gradient using the potassium ionophore Vancomycin is MB 11D15CN O.9 MB 11D18CN O.8 examined. Cells pre-incubated with Vancomycin are treated 45 MB 11E1CN O.8 with peptide and for MBI 10CN and MBI 11CN ONPG MBF11E2CN 0.5 hydrolysis diminished by approximately 50% compared to MB 11E3CN 1.3 no pre-incubation with Vancomycin (FIG. 9). Another cat MB 11F1CN 2.1 MB 11F2CN 1.4 ionic peptide that is not Sensitive to Vancomycin is used as MB 11G3CN 0.5 a positive control. 50 MB 11GSCN O.6 Further delineation of the factors influencing membrane MB 11G6CN O.6 permeabilizing activity are tested. In an exemplary test, MBI MB 11G-7CN 1.5 MBI 11 G13CN O.2 11B7CN is pre-incubated with isotonic HEPES/Sucrose MBI 11 G14CN 1.1 buffer containing either 150 mM sodium chloride (NaCl) or MB21A2 0.5 MB 26 O.6 5 mM magnesium ions (Mg) and assayed as described 55 earlier. In FIG. 10, a significant inhibition is observed with MBI 27 2.7 MB 28 4.7 either Solution, Suggesting involvement of electroStatic MBI 29 1.9 interactions in the permeabilizing action of peptides. MBI 29A3 2.O Example 13 MB 31 O.3 Erythrocyte Hemolysis by Cationic Peptides 60 Cationic peptides are tested for toxicity towards eukary A combination of cationic peptide and antibiotic agent is otic cells by measuring the extent of lysis of mammalian red tested for toxicity towards eukaryotic cells by measuring the blood cells (RBC). Briefly, in this assay, red blood cells are extent of lysis of mammalian red blood cells. Briefly, red Separated from whole blood by centrifugation and washed blood cells are separated from whole blood by free of plasma components. A 5% (v/v) washed red blood 65 centrifugation, washed free of plasma components, and cell Suspension is prepared in isotonic Saline. An aliquot of resuspended to a 5% (v/v) Suspension in isotonic Saline. The peptide in formulation is then added and mixed in. After peptide and antibiotic agent are pre-mixed in isotonic Saline, US 6,503,881 B2 107 108 or other acceptable Solution, and an aliquot of this Solution Example 15 is added to the red blood cell Suspension. Following incu Pharmacology of Cationic Peptides in Plasma and Blood bation with constant agitation at 37 C. for 1 hour, the The in vitro lifetime of free peptides in plasma and in Solution is centrifuged, and the absorbance of the Superna blood is determined by measuring the amount of peptide tant is measured at 540 nm, which detects released hemo present after Set incubation times. Blood is collected from globin. Comparison to the Aso for a 100% lysed Standard sheep, treated with an anticoagulant (not heparin) and, for provides a relative measure of hemoglobin release from red plasma preparation, centrifuged to remove cells. Formulated blood cells, indicating the lytic ability of the cationic peptide peptide is added to either the plasma fraction or to whole blood and incubated. Following incubation, peptide is iden and antibiotic agcnt combination. tified and quantified directly by reversed phase HPLC or an A red blood cell (RBC) lysis assay is used to group antibody-based assay. The antibiotic agent is quantified by a peptides according to their ability to lyse RBC under Stan Suitable assay, Selected on the basis of its Structure. Chro dardized conditions compared with MBI 11 CN and matographic conditions are as described above. Extraction is Gramicidin-S. Peptide samples and washed sheep RBC are not required as the free peptide peak does not overlie any prepared in isotonic Saline with the final pH adjusted to 15 peaks from blood or plasma. between 6 and 7. Peptide samples and RBC suspension are A 1 mg/mL Solution of MBI 11CN in formulations C1 and mixed together to yield solutions that are 1% (v/v) RBC and D is added to freshly prepared Sheep plasma at a final 5, 50 or 500 tug/ml peptide. The assay is performed as peptide concentration of 100 lug/mL and incubated at 37 C. described above. Each set of assays also includes MBI 11CN At various times, aliquots of plasma are removed and (500 lug/ml) and Gramicidin-S (5ug/ml) as “low lysis” and analyzed for free peptide by reversed phase HPLC. From “high lysis’ controls, respectively. each chromatogram, the area of the peak corresponding to MBI11B7CN, MBI11F3CN and MBI11 F4CN are tested free peptide is integrated and plotted against time of incu using this procedure and the results are presented in Table 30 bation. As shown in FIG. 11, peptide levels diminish over below. time. Moreover, when administered in formulation D, up to 25 50% of the peptide is immediately released from TABLE 30 formulation-peptide complex on addition to the blood. The % lysis at % lysis at % lysis at decay curve for free peptide yields an apparent half-life in Peptide 5 ug/ml 50 tug/ml 500 ug/ml blood of 90 minutes for both formulation C1 and D. These results indicate that in sheep's blood MBI 11CN is relatively MBI, 11B7CN 4 13 46 resistant to plasma peptidases and proteases. New peaks that MBI 11F3CN 1. 6 17 MBI 11F4CN 4 32 38 appeared during incubation may be breakdown products of MBI 11CN N/D N/D 9 the peptide. Gramicidin-S 30 N/D N/D A 1 mg/mL solution of MBI 11B7CN in isotonic saline is added to freshly prepared heat-inactivated rabbit Serum, to N/D = not done 35 give a final peptide concentration of 100 lug/mL and is Peptides that at 5 ug/ml lyse RBC to an equal or greater incubated at 32° C. The peptide levels detected are shown in extent than Gramicidin-S, the “high lysis” control, are FIG. 12. considered to be highly lytic. Peptides that at 500 tug/ml lyse A Series of peptide Stability Studies are performed to RBC to an equal to or lesser extent than MBI 11CN, the “low investigate the action of protease inhibitors on peptide lysis” control, are considered to be non-lytic. The three 40 degradation. Peptide is added to rabbit Serum or plasma, analogues tested are all “moderately lytic' as they cause either with or without protease inhibitors, then incubated at more lysis than MBI 11CN and less than Gramicidin S. In 22 C. for 3 hrs. Protease inhibitors tested include amastatin, addition one of the analogues, MBI-11F3CN, is significantly bestatin, COMPLETE protease inhibitor cocktail, leupeptin, less lytic than the other two variants at all three concentra pepstatin A and EDTA. Amastatin and bestatin at 100 uM tions tested. 45 prevent the degradation of MBI 11B7CN in plasma over 3 hrs (FIG. 13). For this experiments 10 mM stock solutions of amastatin and bestatin are prepared in dimethylsulfoxide. Example 14 These solutions are diluted 1:100 in heat-inactivated rabbit Production of Antibodies to Peptide Analogues serum and incubated at 22 C. for 15 mins prior to addition Multiple antigenic peptides (MAPs), which contain four 50 of peptide. MBI 11B7CN is added to the serum at a final or eight copies of the target peptide linked to a Small concentration of 100 tug/mL and incubated for 3 hrs at 22 non-immunogenic peptidyl core, are prepared as immuno C. After the incubation period, the Serum Samples are gens. Alternatively, the target peptide is conjugated to analyzed on an analytical Cs column (Waters Nova Pak Cs bovine serum albumin (BSA) or ovalbumin. For example, 3.9x170 mm) with detection at 280 nm. In FIG. 13, MBI MBI 11B7 conjugated to ovalbumin is used as an immuno 55 11B7CN elutes at 25 min. gen. The immunogens are injected Subcutaneously into Peptide is extracted from plasma using CS Sep Pak rabbits to raise IgG antibodies using Standard protocols (see, cartridges at peptide concentrations between 0 and 50 Harlow and Lane, Antibodies. A Laboratory Manual, Cold lug/mL. Each extraction also contains MBI 11CN at 10 Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., tug/mL as an internal Standard. Immediately after addition of 1988). After repeated boosters (usually monthly), serum 60 the peptides to fresh rabbit plasma, the Samples are mixed from a blood Sample is tested in an ELISA against the target then diluted 1:10 with a 1% acqueous trifluoroacetic acid peptide. A positive result indicates the presence of antibod (TFA) solution, to give a final TFA concentration of 0.1%. ies and further tests determine the specificity of the antibody Five hundred pL of this solution is immediately loaded onto binding to the target peptide. Purified antibodies can then be a C Sep Pak cartridge and eluted with 0.1% TFA in 40% isolated from this serum and used in ELISAS to selectively 65 acetonitrile/60% HO. Twenty IL of this eluant is loaded identify and measure the amount of the target peptide in onto a 4.6x45mm analytical Cs column and is eluted with research and clinical Samples. an acetonitrile gradient of 25% to 65% over 8 column US 6,503,881 B2 109 110 Volumes. The peptides are detected at 280 nm. A chromato Intraperitoneal route. Groups of 6 mice are injected with gram showing the extraction MBI 11B7CN with MBI 11CN peptide doses of between 80 and 5 mg/kg in 500 ul dose as an internal Standard is shown in FIG. 14. MBI 11B7CN Volumes. After peptide administration, the mice are and MBI 11CN elute at 5 and 3 min respectively. MBI observed for a period of 5 days, at which time the dose 11B7CN is detected over background at concentrations of 5 causing 50% mortality (LDs), the dose causing 90–100% tug/mL and above. mortality (LDoo-oo) and maximum tolerated dose (MTD) Peptide levels in plasma in vivo are measured after iv or levels are determined. The LDso values are calculated using ip administration of 80–100% of the maximum tolerated the method of Reed and Muench (J. of Amer. Hyg 27: dose of peptide analogue in either formulation C1 or D. MBI 493–497, 1938). The results presented in Table 31 show that 11CN in formulation C1 is injected intravenously into the the LDs values for MBI 11CN and analogues range from 21 tail vein of CD11CRBR Strain mice. At various times to 52 mg/kg. post-injection, mice are anesthetized and blood is drawn by cardiac puncture. Blood from individual mice is centrifuiged TABLE 31 to Separate plasma from cells. Plasma is then analyzed by reversed phase HPLC column. The resulting elution profiles Peptide LDso LDoo-100 MTD 15 are analyzed for free peptide content by UV absorbance at MB 11CN 34 mg/kg 40 mg/kg 20 mg/kg 280 nm, and these data are converted to concentrations in MB 11B7CN 52 mg/kg >80 mg/kg 30 mg/kg blood based upon a calibrated Standard. Each data point MB 11E3CN 21 mg/kg 40 mg/kg <20 mg/kg represents the average blood level from two mice. In this MB 11F3CN 52 mg/kg 80 mg/kg 20 mg/kg assay, the detection limit is approximately 1 u/ml, less than 3% of the dose administered. The Single dose toxicity of a cationic peptide and antibi The earliest time point at which peptide can be measured otic agent combination is examined in outbred ICR mice. is three minutes following injection, thus, the maximum Intraperitoneal injection of the combination in isotonic observed concentration (in lug/ml) is extrapolated back to Saline is carried out at increasing dose levels. The Survival time zero (FIG. 15). The projected initial concentration of the animals is monitored for 7 days. The number of corresponds well to the expected concentration of between 25 35 and 45 lug/ml. Decay is rapid, however, and when the animals Surviving at each dose level is used to determine the curve is fitted to the equation for exponential decay, free maximum tolerated dose (MTD). In addition, the MTD can circulating peptide is calculated to have a half life of 2.1 be determined after administration of the peptide and agent minutes. Free circulating peptide was not detectable in the by different routes, at different time points, and in different blood of mice that were injected with MBI 11CN in formu formulations. lation D, Suggesting that peptide is not released as quickly from the complex as in vitro. TABLE 32 In addition, MBI 11CN is also administered to CD1 Peptide # MTD/mg/kg ICRBR Strain mice by a single ip injection at an efficacious Intraperitoneal injection dose level of 40 mg/kg. Peptide is administered in both 35 formulations C1 and D to determine if peptide complexation MB 1OCN >29 has any effect on blood levels. At various times post MB 11CN >40 injection, mice are anesthetized and blood is drawn by MB 26 >37 MBI 29 24 cardiac puncture. Blood is collected and analyzed as for the Intravenous injection iv injection. 40 MBI 11CN administered by this route demonstrated a MB 1OCN 5.6 quite different pharmacologic profile (FIG. 16). In formu MB 11CN 6.1 lation C1, peptide entered the blood Stream quickly, with a MB 26 >18 peak concentration of nearly 5 lug/ml after 15 minutes, which declined to non-detectable levels after 60 minutes. In The single dose toxicity of MBI 10CN and MBI 11CN is contrast, peptide in formulation D is present at a level above 45 examined in outbred ICR mice (Table 32). Intraperitoneal 2 tug/ml for approximately two hours. Therefore, formula injection (groups of 2 mice) of MBI 10CN in formulation D tion affects entry into, and maintenance of levels of peptide showed no toxicity up to 29 mg/kg and under the same in the blood. conditions MBI 11CN showed no toxicity up to 40 mg/kg. The in vivo lifetime of the cationic peptide and antibiotic Intravenous route. Groups of 6 mice are injected with agent combination is determined by administration, typi 50 peptide doses of 20, 16, 12, 8, 4 and 0 mg/kg in 100 ul cally by intravenous or intraperitoneal injection, of Volumes (4 ml/kg). After administration, the mice are 80-100% of the maximum tolerable dose of the combination observed for a period of 5 days, at which time the LDso, in a Suitable animal model, typically a mouse. At Set times LDoo and MTD levels are determined. The results from post-injection, each group of animals are anesthetized, blood the IV toxicity testing of MBI 11CN and three analogues are is drawn, and plasma obtained by centrifugation. The 55 shown in Table 33. The LDs, LDoo and MTD values amount of peptide or agent in the plasma Supernatant is range from 5.8 to 15 mg/kg, 8 to 20 mg/kg and <4 to 12 analyzed as for the in vitro determination. mg/kg respectively. Example 16 TABLE 33 Toxicity of Cationic Peptides iv Vivo 60 The acute, Single dose toxicity of various indolicidin Peptide LDso LDoo-100 MTD analogues is tested in Swiss CD1 mice using various routes MB 11CN 5.8 mg/kg 8.0 mg/kg <4 mg/kg of administration. In order to determine the inherent toxici MB 11B7CN 7.5 mg/kg 16 mg/kg 4 mg/kg ties of the peptide analogues in the absence of any MB 11F3CN 10 mg/kg 12 mg/kg 8 mg/kg formulation/delivery vehicle effects, the peptides are all 65 MB 11F4CN 15 mg/kg 20 mg/kg 12 mg/kg administered in isotonic saline with the final pH between 6 and 7. US 6,503,881 B2 111 112 Intravenous injection (groups of 10 mice) of MBI 10CN toms are reversible, disappearing in all mice by the Second in formulation D showed an MTD of 5.6 mg/kg. Injection of day of observations. 11 mg/kg gave 40% toxicity and 22 mg/kg gave 100% The single dose toxicity of MBI 10CN and MBI 11CN in toxicity. Intravenous injection of MBI 11CN in formulation different formulations is also examined in outbred ICR mice C (lyophilized) showed a MTD of 3.0 mg/kg. Injection at 6.1 5 (Table 36). Intraperitoneal injection (groups of 2 mice) of mg/kg gave 10% toxicity and at 12 mg/kg 100% toxicity. MBI 10CN in formulation D show no toxicity up to 29 mg/kg and under the same conditions MBI 11CN show no TABLE 34 toxicity up to 40 mg/kg. Intravenous injection (groups of 10 mice) of MBI 10CN MTD 1O in formulation D show a maximum tolerated dose (MTD) of Peptide Route # Animals Formulation (mg/kg) 5.6 mg/kg (Table 36). Injection of 11 mg/kg gave 40% MB 1OCN ip 2 formulation D 29 toxicity and 22 mg/kg result in 100% toxicity. Intravenous MB 11CN ip 2 formulation D 40 MB 1OCN iv. 1O formulation D 5.6 injection of MBI 11CN in formulation C (lyophilized) show MB 11CN iv. 1O formulation C 3.0 5 a MTD of 3.0 mg/kg. Injection at 6.1 mg/kg result in 10% (lyophilized) 1. toxicity and at 12 mg/kg 100% toxicity.

These results are obtained using peptide/buffer Solutions TABLE 36 that were lyophilized after preparation and reconstituted MTD with water. If the peptide solution is not lyophilized before Peptide Route # Animals Formulation (mg/kg) injection, but used immediately after preparation, an MB 1OCN ip 2 formulation D >29 increase in toxicity is Seen, and the maximum tolerated dose MBI 11CN ip 2 formulation D >40 can decrease by up to four-fold. For example, an intravenous MB 1OCN iv. 1O formulation D 5.6 injection of MBI 11CN as a non-lyophilized solution, for MBI 11CN iv. 1O formulation C 3.0 mulation C1, at 1.5 mg/kg gives 20% toxicity and at 3.0 (lyophilized) mg/kg gives 100% toxicity. HPLC analyses of the non 25 lyophilized and lyophilized formulations indicate that the These results are obtained using peptide/buffer Solutions MBI 11CN forms a complex with Tween 80, and this that are lyophilized after preparation and reconstituted with complexation of the peptide reduces its toxicity in mice. water. If the peptide solution is not lyophilized before In addition, mice are multiply injected by an intravenous injection, but used immediately after preparation, an route with MBI 11CN (Table 35). In one representative increase in toxicity is Seen, and the maximum tolerated dose experiment, peptide administered in 10 injections of 0.84 can decrease by up to four-fold. For example, an intravenous mg/kg at 5 minute intervals is not toxic. However, two injection of MBI 11CN as a non-lyophilized solution, for injections of peptide at 4.1 mg/kg administered with a 10 mulation C1, at 1.5 mg/kg results in 20% toxicity and at 3.0 minute interval results in 60% toxicity. mg/kg gave 100% toxicity. HPLC analyses of the non

TABLE 35

Dose Level # Time Peptide Route Formulation (mg/kg) Injections Interval Result MBI 11CN iv. formulation D O.84 1O 5 min no toxicity MBI 11CN iv. formulation D 4.1 2 10 min 66% toxicity

45 Subcutaneous route. The toxicity of MBI 11CN is also lyophilized and lyophilized formulations indicate that the determined after subcutaneous (SC) administration. For SC MBI 11CN forms a complex with polysorbate, and this toxicity testing, groups of 6 mice are injected with peptide doses of 128,96, 64, 32 and 0 mg/kg in 300 till dose volumes complexation of the peptide reduces its toxicity in mice. (12 mL/kg). After administration, the mice are observed for a period of 5 days. None of the animals died at any of the 50 In addition, mice are multiply injected by an intravenous dose levels within the 5 day observation period. Therefore, route with MBI 11CN (Table 37). In one representative the LDso, LDoo-oo and MTD are all taken to be greater than experiment, peptide administered in 10 injections of 0.84 128 mg/kg. Mice receiving higher dose levels showed mg/kg at 5 minute intervals is not lethal. However, two Symptoms similar to those Seen after IV injection Suggesting injections of peptide at 4.1 mg/kg administered with a 10 that peptide entered the Systemic circulation. These Symp minute interval results in 60% mortality.

TABLE 37

Dose # Time Peptide Route Formulation Level Injections Interval Result MB 11CN iv formulation D 0.84 1O 5 min no mortality MB 11CN iv formulation D 4.1 2 10 min 66% mortality *(mg/kg) US 6,503,881 B2 113 114 To assess the impact of dosing mice with peptide Example 17 analogue, a Series of histopathology investigations can be In vivo Efficacy of Cationic Peptides carried out. Groups of mice are administered analogue at Cationic peptides are tested for their ability to rescue mice dose levels that are either at, or below the MTD, or above the from lethal bacterial infections. The animal model used is an MTD, a lethal dose. Multiple injections may be used to mimic possible treatment regimes. Groups of control mice intraperitoneal (ip) inoculation of mice with 10°-10 Gram are not injected or injected with buffer only. positive organisms with Subsequent administration of pep Following injection, mice are Sacrificed at Specified times tide. The three pathogens investigated, methicillin-Sensitive and their organs immediately placed in a 10% balanced S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), formalin Solution. Mice that die as a result of the toxic or S. epidermidis are injected ip into mice. For untreated effects of the analogue also have their organs preserved mice, death occurs within 12-18 hours with MSSA and S. immediately. Tissue Samples are taken and prepared as epidermis and within 6-10 hours with MRSA. Stained micro-Sections on slides which are then examined Peptide is administered by two routes, intraperitoneally, at microscopically. Damage to tissues is assessed and this one hour post-infection, or intravenously, with Single or information can be used to develop improved analogues, multiple doses given at various times pre- and post improved methods of administration or improved dosing 15 infection. regimes. MSSA infection. In a typical protocol, groups of 10 mice To assess the impact of dosing mice with peptide are infected intraperitoneally with a LDoo-oo dose (5.2x10 analogue, a Series of histopathology investigations are car CFU/mouse) of MSSA (Smith, ATCC #19640) injected in ried out. Groups of two mice are administered MBI 11CN in brain-heart infuision containing 5% mucin. This strain of S. formulation D by ip and iv injection. The dose levels are aureuS is not resistant to any common antibiotics. At 60 either at or below the MTD or a lethal dose above MTD. minutes post-infection, MBI 10CN or MBI 11CN, in for Groups of control mice are uninjected or injected with buffer mulation D, is injected intraperitoneally at the Stated dose only. At 0, 70 and 150 minutes after injection, the major levels. An injection of formulation alone Serves as a negative organs of moribund or Sacrificed mice are examined histo control and administration of amplicillin Serves as a positive logically for evidence of toxicity. 25 control. The Survival of the mice is monitored at 1, 2, 3 and 4 hrs post-infection and twice daily thereafter for a total of 8 days. As shown in FIG. 17, MBI 10CN is maximally active Mice given an iv injection of MBI 11CN are identified as follows: against MSSA (70–80% survival) at doses of 14.5 to 38.0 Control Mouse A: No dose mg/kg, although 100% survival is not achieved. Below 14.5 Control Mouse B: Buffer Dose only (no peptide) mg/kg, there is clear dose-dependent Survival. At these M7OA, B: MBI 11CN, 4 mg/kg, 70 minute observation lower dose levels, there appears to be an animal-dependent M150A, B: MBI 11CN, 4 mg/kg, 150 minute observation threshold, as the mice either die by day 2 or survive for the MXA, B: MBI 11CN, 12 mg/kg (lethal dose) full eight day period. As seen in FIG. 18, MBI 11CN, on the Mice given an ip injection of MBI 11CN are identified as follows: 35 other hand, rescued 100% of the mice from MSSA infection Control Mouse A: No dose at a dose level of 35.7 mg/kg, and was therefore as effective Control Mouse B: Buffer Dose only (no peptide) as amplicillin. There was little or no activity at any of the M7OA, B: MBI 11CN, 40 mg/kg, 70 minute observation M150A, B: MBI 11CN, 40 mg/kg, 150 minute observation lower dose levels, which indicates that a minimum blood MXA, B: MBI 11CN, 80 mg/kg (lethal dose) Stream peptide level must be achieved during the time that 40 bacteria are a danger to the host. Following injection, the mice are Sacrificed at the times As shown above, blood levels of MBI 11CN can be indicated above and their organs immediately placed in a Sustained at a level of greater than 2 tug/ml for a two hour 10% balanced formalin solution. The tissue samples are period inferring that this is higher than the minimum level. prepared as Stained micro-Sections on Slides and then exam 45 Additionally, eight variants based on the sequence of MBI ined microscopically. 11CN are tested against MSSA using the experimental Mice given a non-lethal dose were always lethargic, with system described above. Peptides prepared in formulation D raised fur and evidence of edema and hypertension, but are administered at dose levels ranging from 12 to 24 mg/kg recovered to normal within two hours. Tissues from these and the Survival of the infected mice is monitored for eight animals indicate that there was Some damage to blood 50 days (FIGS. 19-27). The percentage survival at the end of vessels, particularly within the liver and lung at both the the observation period for each variant is Summarized in observation times, but other initial abnormalities returned to Table 38. As shown in the table, several of the variants normal within the 150 minute observation time. It is likely showed efficacy greater than or equal to MBI 11CN under that blood vessel damage is a consequence of continuous these conditions. exposure to high circulating peptide levels. 55 In contrast, mice given a lethal dose had completely TABLE 38 normal tissues and organs, except for the liver and heart of % the ip and iv. dosed mice, respectively. In general, this Survival 24 mg/kg 18 mg/kg 12 mg/kg damage is identified as disruption of the cells lining the 1OO 60 blood vessels. It appears as though the rapid death of mice 90 11B1CN, is due to this damage, and that the peptide did not penetrate 11F3CN beyond that point. Extensive damage to the hepatic portal 8O 70 11E3CN veins in the liver and to the coronary arterioles in the heart 60 11B7CN was observed. 50 11CN Further evidence points to a cumulative toxic effect, 65 40 11G2CN where the maximum dose iv is lethal when repeated after 10 3O 11B1CN minutes, but not when repeated after one hour. US 6,503,881 B2 116 the negative control, indicates that injections 1 hour apart, TABLE 38-continued even at a higher level, are not effective against MRSA. % Survival 24 mg/kg 18 mg/kg 12 mg/kg TABLE 39 2O 11G4CN Time of Observation Percentage of Animals Surviving 1O 11CN, 11B7CN, 11G2CN 11B8CN, 11F3CN (Hours post-infection) No Treatment Treatment O 11A1CN 11A1CN, 11G2CN, 11CN, 11A1CN, 11B1CN, 6 50% 70% 8 O 40% 1O O 30% 12 O 20% S. epidermidis infection. Peptide analogues generally have lower MIC values against S. epidermidis in vitro, therefore, lower blood peptide levels might be more effec Example 18 tive against infection. 15 Activation of Polysorbate 80 by Ultraviolet Light In a typical protocol, groups of 10 mice are injected A solution of 2% (w/w) polysorbate 80 is prepared in intraperitoneally with an LDoo-oo dose (2.0x10 CFU/ water and 200 ml are placed in a 250mL crystallizing dish mouse) of S. epidermidis (ATCC #12228) in brain-heart or over Suitable container. Containers must have a clear light infusion broth containing 5% mucin. This strain of S. path. Cover the vessel with a piece of UV transparent plastic epidermidis is 90% lethal after 5 days. At 15 mins and 60 wrap or other UV transparent material. In addition, the mins post-infection, various doses of MBI 11CN in formu material should allow the exchange of air but minimize lation D are injected intravenously via the tail vein. An evaporation. injection of formulation only Serves as the negitive control The Solution is irradiated with ultraViolet light using a and injection of gentamicin Serves as the positive control; lamp emitting at 254 nm. Irradiation can also be performed both are injected at 60 minutes post-infection. The Survival 25 using a lamp emitting at 302 nm. The Solution should be of the mice is monitored at 1, 2, 3, 4, 6 and 8 hrs post Stirred continuously to maximize the rate of activation. The activation is complete within 72 hours using a lamp with a infection and twice daily thereafter for a total of 8 days. output of 1800 uW/cm. The reaction is monitored by a As shown in FIGS. 28A and 28B, MBI 11CN prolongs the reversed-phased HPLC assay, which measures the formation survival of the mice. Efficacy is observed at all three dose of APO-MBI 11CN-Tw80when the light-activated polysor levels with treatment 15 minutes post-infection, there is leSS bate is reacted with MBI 11CN. activity at 30 minutes post-infection and no significant effect Some properties of activated polySorbate are determined. at 60 minutes post-infection. Time of administration appears Because peroxides are a known by-product of exposing to be important in this model System, with a Single injection ethers to UV light, peroxide formation is examined through of 6.1 mg/kg 15 minutes post-infection giving the best 35 the effect of reducing agents on the activated polySorbate. AS Survival rate. seen in FIG. 30A, activated polysorbate readily reacts with MRSA infection. MRSA infection, while lethal in a short MBI 11CN. Pre-treatment with 2-mercaptoethanol (FIG. period of time, requires a much higher bacterial load than 30B), a mild reducing agent, eliminates detectable MSSA. In a typical protocol, groups of 10 mice are inject peroxides, but does not cause a loSS of conjugate forming eded intraperitoneally with a LDoo-oo dose (4.2x10 CFU/ 40 ability. Treatment with sodium borohydride (FIG. 30C), mouse) of MRSA (ATCC #33591) in brain-heart infusion eliminates peroxides and eventually eliminates the ability of containing 5% mucin. The treatment protocols are as activated polysorbate to modify peptides. Hydrolysis of the follows, with the treatment times relative to the time of borohydride in water raises the pH and produces borate as a infection: hydrolysis product. However, neither a pH change nor 45 borate are responsible. These data indicate that peroxides are not involved in the 0 mg/kg Formulation D alone (negative control), injected at 0 mins modification of peptides by activated polySorbate. Sodium 5 mg/kg Three 5.5 mg/kg injections at -5, +55, and +115 mins borohydride should not affect epoxides or esters in aqueous 1 mg/kg (2 Five 1.1 mg/kg injections at -5, +55, +115, +175 and +235 media, Suggesting that the reactive group is an aldehyde or hr) mins 50 1 mg/kg (20 Five 1.1 mg/kg injections at -10, -5, 0, +5, and +10 mins ketone. The presence of aldehydes in the activated polySor min) bate is confirmed by using a formaldehyde test, which is Vancomycin (positive control) injected at 0 mins Specific for aldehydes including aldehydes other than form aldehyde. Furthermore, activated polysorbate is treated with 2,4- MBI 11CN is injected intravenously in the tail vein in 55 dinitrophenylhydrazine (DNPH) in an attempt to capture the formulation D. Survival of mice is recorded at 1, 2, 3, 4, 6, reactive Species. Three DNPH-tagged components are puri 8, 10, 12, 20, 24 and 30 hrs post-infection and twice daily fied and analyzed by mass spectroscopy. These components thereafter for a total of 8 days. There was no change in the are polySorbate-derived with molecular weights between number of surviving mice after 24 hrs (FIG. 29). 1000 and 1400. This indicates that low molecular weight The 1 mg/kg (20 min) treatment protocol, with injections 60 aldehydes, Such as formaldehyde or acetaldehyde, are 5 minutes apart centered on the infection time, delayed the involved. death of the mice to a significant extent with one Survivor remaining at the end of the Study. The results presented in Example 19 Table 39 suggest that a sufficiently high level of MBI 11CN Activation of Polysorbate 80 by Ammonium Persulfate maintained over a longer time period would increase the 65 A 200 mL solution of 2% (w/w) polysorbate 80 is number of mice Surviving. The 5 mg/kg and 1 mg/kg (2 hr) prepared in water. To this Solution, 200 mg of ammonium results, where there is no improvement in Survivability over perSulfate is added while Stirring. The reaction is Stirred for US 6,503,881 B2 117 118 1-2 hours with protection from ambient light. If a solution molecule is calculated using an extinction coefficient of of less than 0.1% (w/w) ammonium persulfate is used, then 10,500 M' cm for the trinitrophenyl (TNP) derivatives. exposure to ultraViolet light at 254 nm during this period is The primary amino group content of the parent peptide is used to help complete the reaction. The peroxide level in the then compared to the corresponding APO-modified peptide. reaction is determined using a test kit. Peroxides are reduced AS shown below, the loSS of a single primary amino group by titration with 2-mercaptoethanol. occurs during formation of modified peptide. Peptides pos Example 20 Sessing a 3.4 lysine pair consistently give results that are 1 Formation of APO-Modified Peptides residue lower than expected, which may reflect Steric hin APO-modified peptides are prepared either in Solid phase drance after titration of one member of the doublet. or liquid phase. For Solid phase preparation, 0.25 ml of 4 1O mg/ml of MBI 11CN is added to 0.5 ml of 0.4 M Acetic TABLE 41 acid-NaOH pH 4.6 followed by addition of 0.25 ml of TNP/APO UV-activated polysorbate. The reaction mix is frozen by modified placing it in a -80 C. freezer. After freezing, the reaction PEPTIDE SEQUENCE TNP/PEPTIDE peptide CHANGE mix is lyophilized overnight. 15 ILKKWPWWPWRRKande 2.71 1.64 1.07 For preparing the conjugates in an aqueous phase, a ILRRWPWWPWRRKande 1.82 0.72 1.10 sample of UV activated polysorbate 80 is first adjusted to a IIKKWPWWPWRRKande 2.69 1.61 1.08 pH of 7.5 by the addition of 0.1M NaOH. This pH adjusted ILKKWVWWPWRRKande 2.62 1.56 1.06 solution (0.5 ml) is added to 1.0 ml of 100 mM sodium carbonate, pH 10.0, followed immediately by the addition of Stability of APO-modified Peptide Analogues 0.5 ml of 4 mg/ml of MBI 11CN. The reaction mixture is APO-modified peptides demonstrate a high degree of incubated at ambient temperature for 22 hours. The progreSS Stability under conditions that promote the dissociation of of the reaction is monitored by analysis at various time ionic or hydrophobic complexes. APO-modified peptide in points using RP-HPLC (FIG. 31). In FIG. 31, peak 2 is formulation D is prepared as 800 ug/ml Solutions in water, unreacted peptide, peak 3 is APO-modified peptide. Type 1 25 0.9% saline, 8M urea, 8M guanidine-HC1, 67% 1-propanol, is the left-most of peak 3 and Type 2 is the right-most of peak 1M HCI and 1M NaOH and incubated for 1 hour at room 3. temperature. Samples are analyzed for the presence of free The table below summarizes data from several experi peptide using reversed phase HPLC and the following ments. Unless otherwise noted in the table, the APO chromatographic conditions: modified peptides are prepared via the lyophilization method Solvent A: 0.1% trifluoroacetic acid (TFA) in water in 200 mM acetic acid-NaOH buffer, pH 4.6. Solvent B: 0.1% TFA /95% acetonitrile in water Media POROS R2-20 (polystyrene divinylbenzene) TABLE 40 Elution: 0% B for 5 column volumes 0–25% B in 3 COMPLEX column volumes 25% B for 10 column volumes 35 25-95% B in 3 column volumes 95% B for 10 column SEOUENCE NAME TYPE 1. TYPE 2 Volumes ILKKWPWWPWRRKande 11CN Under these conditions, free peptide elutes exclusively Solid phase, pH 2.0 Yes Low during the 25% B Step and formulation-peptide complex Solid phase, pH 4.6 Yes Yes during the 95% B step. None of the dissociating conditions Solid phase, pH 5.0 Yes Yes 40 mentioned above, with the exception of 1M NaOH in which Solid phase, pH 6.0 Yes Yes Solid phase, pH 8.3 Yes Yes Some degradation is observed, are Successful in liberating Solution, pH 2.0 Trace Trace free peptide from APO-modified peptide. Additional studies Solution, pH 10.0 Yes Yes-Slow are carried out with incubation at 55 C. or 85 C. for one (Ac)-ILKKWPWWPWRRKamide 11CN-Y1 No No hour. APO-modified peptide is equally stable at 55 C. and ILRRWPWWPWRRKande 11B1CN Yes Lowered 45 ILRWPWWPWRRKande 11B7CN Yes Lowered is only slightly less stable at 85 C. Some acid hydrolysis, ILWPWWPWRRKande 11B8CN Yes Lowered indicated by the presence of novel peaks in the HPLC ILRRWPWWPWRRRanide 11B9CN Yes Trace chromatogram, is observed with the 1M HCI sample incu ILKKWPWWPWKKKande 11B10CN Yes Yes bated at 85° C. for one hour. LKKWPWWPWRRKande 11E3CN Yes Yes ILKKWVWWPWRRKande 11F3CN Yes Yes 50 Example 21 ILKKWPWWPWKande 11 G13CN Yes Yes Purification of APO-Modified MBI 11 CN ILKKWPWWPWRande 11 G14CN Yes Trace A large scale preparation of APO-modified MBI 11CN is purified. Approximately 400 mg of MBI 11CN is APO The modification of amino groups is further analyzed by modified and dissolved in 20 ml of water. Unreacted MBI determining the number of primary amino groups lost during 55 11CN is removed by RP-HPLC. The solvent is then evapo attachment. The unmodified and modified peptides are rated from the APO-modified MBI 11CN pool, and the treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) (R. residue is dissolved in 10 ml methylene chloride. The L. Lundblad in Techniques in Protein Modification and modified peptide is then precipitated with 10 ml diethyl Analysis pp. 151-154, 1995). ether. After 5 min at ambient temperature, the precipitate is Briefly, a stock solution of MBI 11CN at 4 mg/ml and an 60 collected by centrifugation at 5000xg for 10 minutes. The equimolar solution of APO-modified MBI 11CN are pre pellet is washed with 5 ml of diethyl ether and again pared. A 0.225 ml aliquot of MBI 11CN or APO-modified collected by centrifugation at 5000xg for 10 minutes. The MBI 11 CN is mixed with 0.225 ml of 200 mM Sodium Supernatants are pooled for analysis of unreacted polySor phosphate buffer, pH 8.8. A 0.450 ml aliquot of 1% TNBS bate by-products. The precipitate is dissolved in 6 ml of is added to each Sample, and the reaction is incubated at 37 65 water and then flushed with nitrogen by bubbling for 30 C. for 30 minutes. The absorbance at 367 nm is measured, minutes to remove residual ether. The total yield from the and the number of modified primary amino groups per starting MBI 11CN was 43%. US 6,503,881 B2 119 120 The crude APO-MB129-Tw80 prepared from 200 mg of lar ratio. The concentration of APO-modified peptides can MBI 29 is suspended in 40 mL of methylene chloride and be determined by Spectrophotometric measurement, which Sonicated for 5 minutes to disperse large particles. The is used to normalize concentrations for biological assayS. Suspension is centrifuged in appropriate containers (Coming For example, a 1 mg/ml APO-modified MBI 11CN solution glass) at 3000-4000xg for 15 minutes at 10° C. to sediment 5 contains the same amount of peptide as a 1 mg/ml MBI insoluble material. The Supernatant is decanted and Saved. 11CN Solution, thus allowing direct comparison of toxicity The sediment is extracted twice more by adding 40 mL and efficacy data. portions methylene chloride to the Sediment and repeating the Sonication/centrifugation Step. The Supernatants from the APO-modified peptides are at least as potent as the parent three extractions are pooled and concentrated 8-10 fold 10 peptides in in vitro assays performed as described herein. using a rotary evaporator. The Solution is transferred to MIC values against gram positive bacteria are presented for centrifuge tubes and 3 volumes of diethyl ether are added. several APO-modified peptides and compared with the val The mixture is incubated for 15 minutes, then centrifuged at ues obtained using the parent peptides (Table 5). The results 3000–4000 xg for 15 minutes at 10° C. to sediment the indicate that the modified peptides are at least as potent in product. The Supernatant is decanted and discarded. The 15 Vitro as the parent peptides and may be more potent than the residual ether may be removed with a stream of nitrogen. parent peptides against E. faecalis Strains. The agarose dilution assay measures antimicrobial activ Example 22 ity of peptides and peptide analogues, which is expressed as Biological Assays to Measure APO-Cationic Peptide Activ the minimum inhibitory concentration (MIC) of the pep ity 2O tides. This assay is performed as described above. Repre All biological assays that compare APO-modified pep sentative MICs for various modified and unmodified cat tides with unmodified peptides are performed on an equimo ionic peptides are shown in the Table below.

Organism calcoaceticiaS calcoaceticiaS calcoaceticiaS calcoaceticiaS calcoaceticiaS calcoaceticus calcoaceticiaS calcoaceticiaS calcoaceticiaS cloacae cloacae cloacae cloacae cloacae cloacae cloacae cloacae cloacae coli coli coli coli coli coli coli coli coli . faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis

US 6,503,881 B2

TABLE 42-continued MIC(ug/mL Organism Organism # APO-Peptide APO-Peptide Peptide S. narceScenS SMSOO3 MB11F3CN-Tw8O 128 >128 S. narceScenS SMSOO3 MB11F4CN-Tw8O >128 >128 S. narceScenS SMSOO3 MB29-Tw8O >64 >128

Toxicities of APO-modified MBI 11 CN and unmodified MBI 11CN are examined in Swiss CD-1 mice. Groups of 6 TABLE 44 mice are injected iv with Single doses of 0.1 ml peptide in 9% saline. The dose levels used are 0, 3, 5, 8, 10, and 13 15 Test Peptide LDso LDoo 100 MTD mg/kg. Mice are monitored at 1. 3, and 6 hrs post-injection MB 11CN 7 mg/kg 8 mg/kg 5 mg/kg for the first day, then twice daily for 4 days. The Survival APO-MB-11CN >13 mg/kg 213 mg/kg >13 mg/kg data for MBI 11CN mice are presented in Table 43. For APO-modified MBI 11CN, 100% of the mice Survived at all could not be calculated with available data. doses, including the maximal dose of 13 mg/kg.

TABLE 43

Peptide administered Cumulative No. Cumulative No. (mg/kg) No. Dead/Total Dead Surviving Dead/Total %. Dead

13 6/6 18 O 18/18 1OO 1O 6/6 12 O 12/12 1OO 8 6/6 6 O 6/6 1OO 5 Of6 O 6 Of6 O 3 Of6 O 12 O/12 O O Of6 O 18 Of 18 O

As Summarized below, the LDs for MBI 11CN is 7 In addition, APO-peptides and parent peptides are tested mg/kg (Table 7), with all Subjects dying at a dose of 8 against a panel of cancer cell lines. Cell death is measured mg/ml. The highest dose of MBI 11CN giving 100% sur 40 using the Cytotox (Promega) assay kit which measures the vival was 5 mg/kg. The data show that APO-modified release of lactate dehydrogenase. AS Shown below the modi peptides are significantly leSS toxic than the parent peptides. fied peptides had increased activity over the parent peptides.

TABLE 45 CELL LINE, LCso kg/mL it S.E.

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

11CN 57 >190 2OO 3O 25 11.89 17 - 1 11CN-Tw8O 6 6 16 - 4 16 - 4 1.9 - 5 3.5 - 2 11 11A3CN >500 >5OO >500 >5OO >500 >3OO >300 11A3CN-Tw8O 12.7 - 15 179 15 - 4 6 3.3 - 0.05 5.6 - 2 6.63 28 13 11B7CN 24 10 90 - 23 26 - 25 34 - 25 16.5 - 3 13.8 >7OO 11B7CN-Tw8O 3.8 - 1 12.88 -100 4.73 3.3 1 5.1 12 11E3CN 22 11 1177 18 9 3.6 13.93 7.93 5.6 - 2 5.3 + 1 11E3CN-Tw8O 4.5 - 2 12.8 2 8.2 4 4.93 3.5 O.7 5.93 8.4 + 1 8.15 6 - 2 21A11 30 - 15 184 100 48 5633 9.8 + 0.3 - 21A11-Tw8O 4.5 - 4 179.9 21 4.3 2 4.7 O.6 8.1 3.4 9 18 29 12 10 1O 12.6 10 1. 2.1 - 0.5 1.4 - 0.5 2 + 0.2 4 + 2 3.2 - 1 29-Tw8O 8.7 6 9.3 - 2 1.7 2.1 - 0.5 4 - 0.5 7.6 24 7.6 2 15.5 - 6 9.1 - 5 US 6,503,881 B2 125 126 PBL, peripheral blood lymphocytes; HUVEC, human It will be appreciated that, although Specific embodiments umbilical vein endothelial cells; H460, non-small lung of the invention have been described herein for purposes of tumor; K562, chronic myelogenous leukemia; DoHH-2, B-cell cell lymphoma; P388, lymphocytic leukemia; illustration, various modifications may be made without P388ADR, lymphocytic leukemia, multidrug resistant; departing from the Spirit and Scope of the invention. MCF-7, breast carcinoma, MCF-7ADR, breast carcinoma, Accordingly, the invention is not limited except as by the multidrug resistant. appended claims.

SEQUENCE LISTING

&160 NUMBER OF SEQ ID NOS: 232 SEQ ID NO 1 LENGTH 8 TYPE PRT ORGAN SM: Artificial Sequence FEATURE OTHE R INF O RMAT ON: Indolicidin Analogue NAME AKEY WAR ANT LOCATION (2) . . . (2) R IN RMAT ON: Xala Hydrophobic Residue AKEY WAR ANT LOCATION 3 ) . . . (3) R IN : RMAT ON: Xala Proline or Waline AKEY WA R ANT LOCATION (4 ) . . . (5) R IN RMAT ON: Xala Hydrophobic Residue AKEY WAR ANT LOCATION (6 ) . . . (6) R IN RMAT ON: Xala Proline or Waline AKEY WAR ANT LOCATION (7) . . . (7) R IN RMAT ON: Xala Hydrophobic Residue AKEY WAR ANT LOCATION (8) . . . (8) R IN RMAT ON: Xala Basic Amino Acid

<400 SEQU ENCE: 1 Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa 5

SEQ ID NO 2 LENGTH 8 TYPE PRT ORGAN SM: Artificial Sequence FEATURE OTHE R INF RMAT ON: Indolicidin Analogue NAME AKEY WAR ANT LOCATION (1) . . . (1) R IN RMAT ON: Xala Basic Amino Acid AKEY WAR ANT LOCATION (2) . . . (2) R IN : RMAT ON: Xala Hydrophobic Residue AKEY AR ANT LOCATION 3 ) . . . (3) R IN RMAT ON: Xala Proline or Waline AKEY AR ANT LOCATION (4) . . . (5) R IN RMAT ON: Xala Hydrophobic Residue AKEY WAR ANT LOCATION (6) . . . (6) R IN RMAT ON: Xala Proline or Waline AKEY WARA. ANT LOCATION ( 7 ) . . . (7) ER IN RMAT ON: Xala Hydrophobic Residue AKEY WAR ANT LOCATION (8) . . . (8) R IN RMAT ON: Xala Basic Amino Acid O R IN RMAT ON: At least one residue at positions 3 and 6 is

<400 SEQU ENCE: 2

Xaa Xala Xala Xala Xala Xala Xala Xala US 6,503,881 B2 127 128

-continued

5

SEQ ID NO 3 LENGTH 10 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFO RMATION: Indolicidin Analogue NAME/KEY: WARIANT LOCATION: (1) . . . (3) OTHER INFORMATION Xaa Basic Amino Acid NAME/KEY: WARIANT LOCATION: (4) . . . (4) OTHER INFORMATION Xaa Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (5) . . . (5) OTHER INFORMATION Xaa Proline or Waline NAME/KEY: WARIANT LOCATION: (6) . . . (7) OTHER INFORMATION Xaa Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (8) . . . (8) OTHER INFORMATION Xaa Proline or Waline NAME/KEY: WARIANT LOCATION: (9) . . . (9) OTHER INFORMATION Xaa Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (10 ) . . . (10) OTHER INFORMATION Xaa Basic Amino Acid

<400 SEQUENCE: 3

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala 1 5 10

SEQ ID NO 4 LENGTH 11 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Indolicidin Analogue NAME/KEY: WARIANT LOCATION: (1) . . . (1) OTHER INFO RMATION: Xaa- Basic Amino Acid NAME/KEY: WARIANT LOCATION: (2) . . . (3) OTHER INFORMATION Xaa Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (4) . . . (4) OTHER INFORMATION Xaa Basic Amino Acid NAME/KEY: WARIANT LOCATION: (5) . . . (5) OTHER INFORMATION Xaa Proline or Waline NAME/KEY: WARIANT LOCATION: (6) . . . (6) OTHER INFORMATION Xaa Basic Amino Acid NAME/KEY: WARIANT LOCATION: (7) . . . (7) OTHER INFORMATION Xaa Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (8) . . . (8) OTHER INFORMATION Xaa Basic Amino Acid NAME/KEY: WARIANT LOCATION: (9) . . . (9) OTHER INFORMATION Xaa Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (10 ) . . . (10) OTHER INFORMATION Xaa Proline or Waline NAME/KEY: WARIANT LOCATION: (11) . . . (11) OTHER INFORMATION Xaa Basic Amino Acid

<400 SEQUENCE: 4

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 1 5 10 US 6,503,881 B2 129 130

-continued

SEQ ID NO 5 LENGTH 17 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMA CION: Indolicidin Analogue NAME/KEY: WAR ANT LOCATION: (1) . . . (1) OTHER INFORMA CION: Xaa = Basic Amino Acid NAME/KEY: WAR ANT LOCATION: (2) . . . (2) OTHER INFORMA CION: Xaa = Hydrophobic Residue NAME/KEY: WAR ANT LOCATION: (3) . . . (3) OTHER INFORMA CION: Xaa = Proline or Waline NAME/KEY: WAR ANT LOCATION: (4) . . . (5) OTHER INFORMA CION: Xaa = Hydrophobic Residue NAME/KEY: WAR ANT LOCATION: (6) . . . (6) OTHER INFORMA ON: Xaa = Proline or Waline NAME/KEY: WAR ANT LOCATION: (7) . . . (7) OTHER INFORMA ON: Xaa = Hydrophobic Residue NAME/KEY: WAR ANT LOCATION: (8) . . . (9) OTHER INFORMA ON: Xaa = Basic Amino Acid NAME/KEY: WAR ANT LOCATION: (13) . . . (14) OTHER INFORMA ON: Xaa = Basic Amino Acid

<400 SEQUENCE: 5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Ile Leu Xaa Xaa Ala Gly 1 5 10 15

Ser

SEQ ID NO 6 LENGTH 18 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMA ON: Indolicidin Analogue NAME/KEY: WAR ANT LOCATION: (1) . . . (1) OTHER INFORMA ON: Xaa = Basic Amino Acid NAME/KEY: WAR ANT LOCATION: (2) . . . (2) OTHER INFORMA ON: Xaa = Hydrophobic Residue NAME/KEY: WAR ANT LOCATION: (3) . . . (3) OTHER INFORMA ON: Xaa = Proline or Waline NAME/KEY: WAR ANT LOCATION: (4) . . . (5) OTHER INFORMA ON: Xaa = Hydrophobic Residue NAME/KEY: WAR ANT LOCATION: (6) . . . (6) OTHER INFORMA ON: Xaa = Proline or Waline NAME/KEY: WAR ANT LOCATION: (7) . . . (7) OTHER INFORMA ON: Xaa = Hydrophobic Residue NAME/KEY: WAR ANT LOCATION: (8) . . . (10) OTHER INFORMA ON: Xaa = Basic Amino Acid NAME/KEY: WAR ANT LOCATION: (14) . . . (15) OTHER INFORMA ON: Xaa = Basic Amino Acid

<400 SEQUENCE: 6

Xaa Xala Xala Xala Xaa Xaa Xaa Xala Xala Xala Met Ile Leu Xaa Xaa Ala 1 5 10 15 Gly Ser US 6,503,881 B2 131 132

-continued SEQ ID NO 7 LENGTH 18 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Indolicidin Analogue NAME/KEY: WARIANT LOCATION: (1) . . . (1) OTHER INFORMATION Xaa = Basic Amino Acid NAME/KEY: WARIANT LOCATION: (2) . . . (2) OTHER INFORMATION Xaa = Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (3) . . . (3) OTHER INFORMATION Xaa = Proline or Waline NAME/KEY: WARIANT LOCATION: (4) . . . (5) OTHER INFORMATION Xaa = Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (6) . . . (6) OTHER INFORMATION Xaa = Proline or Waline NAME/KEY: WARIANT LOCATION: (7) . . . (7) OTHER INFORMATION Xaa = Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (8) ... (9) OTHER INFORMATION Xaa = Basic Amino Acid NAME/KEY: WARIANT LOCATION: (10 ) . . . (10) OTHER INFORMATION: Xaa-. Any Amino Acid NAME/KEY: WARIANT LOCATION: (14) . . . (15) OTHER INFORMATION Xaa = Basic Amino Acid

<400 SEQUENCE: 7

Xaa Xala Xala Xala Xaa Xaa Xaa Xala Xala Xala Met Ile Leu Xaa Xaa Ala 1. 5 10 15 Gly Ser

SEQ ID NO 8 LENGT H 19 TYPE PRT ORGAN SM: Artificial Sequence FEAT RE: OTHER N FORMATION: Indolicidin Analogue NAME/ KEY WARIANT ON: (1) . . . (1) NFORMATION Xaa Basic Amino Acid KEY WARIANT ON: (2) . . . (2) NFORMATION Xaa Hydrophobic Residue KEY WARIANT ON: (3) . . . (3) NFORMATION Xaa Proline or Waline KEY WARIANT ON: (4) . . . (5) NFORMATION Xaa Hydrophobic Residue KEY WARIANT ON: (6) . . . (6) NFORMATION Xaa Proline or Waline KEY WARIANT ON: (7) . . . (7) NFORMATION Xaa Hydrophobic Residue KEY WARIANT ON: (8) . . . (10) NFORMATION Xaa Basic Amino Acid KEY WARIANT ON: (11) . . . (11) NFORMATION Xaa Any Amino Acid KEY WARIANT ON: (15) . . . (16) NFORMATION Xaa Basic Amino Acid

<400 SEQUENCE: 8

Xaa Xala Xala Xala Xaa Xaa Xaa Xala Xala Xala Xaa Met Ile Leu Xaa Xala

US 6,503,881 B2 135 136

-continued ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Indolicidin Analogue NAME/KEY: WARIANT LOCATION: (2) . . . (2) OTHER INFORMATION Xaa Basic Amino Acid NAME/KEY: WARIANT LOCATION: (3) . . . (6) OTHER INFORMATION Xaa Hydrophobic Residue <400 SEQUENCE: 11 Leu Xaa Xala Xala Xala Xaa Arg Lys 1 5

SEQ ID NO 12 LENGTH 9 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Indolicidin Analogue NAME/KEY: WARIANT LOCATION: (2) . . . (3) OTHER INFORMATION Xaa Basic Amino Acid NAME/KEY: WARIANT LOCATION: (4) . . . (7) OTHER INFORMATION Xaa Hydrophobic Residue <400 SEQUENCE: 12 Leu Xaa Xala Xala Xala Xala Xala Arg Lys 1 5

SEQ ID NO 13 LENGTH 10 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Indolicidin Analogue NAME/KEY: WARIANT LOCATION: (2) . . . (3) OTHER INFORMATION: Xaa = Basic Amino Acid NAME/KEY: WARIANT LOCATION: (4) . . . (4) OTHER INFORMATION: Xaa = Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (5) . . . (5) OTHER INFORMATION Xaa Proline or Waline NAME/KEY: WARIANT LOCATION: (6) . . . (8) OTHER INFORMATION: Xaa = Hydrophobic Residue <400 SEQUENCE: 13 Leu Xaa Xala Xala Xala Xala Xala Xala Arg Lys 1 5 10

SEQ ID NO 14 LENGTH 11 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Indolicidin Analogue NAME/KEY: WARIANT LOCATION: (2) . . . (3) <223> Xaa = Basic Amino Acid NAME/KEY: WARIANT LOCATION: (4) . . . (4) OTHER INFORMATION Xaa Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (5) . . . (5) OTHER INFORMATION Xaa Proline or Waline NAME/KEY: WARIANT LOCATION: (6) . . . (7) OTHER INFORMATION Xaa Hydrophobic Residue NAME/KEY: WARIANT LOCATION: (8) . . . (8) US 6,503,881 B2 137 138

-continued

&223> OTHER INFORMATION Xaa Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (9) . . . (9) &223> OTHER INFORMATION Xaa Hydrophobic Residue <400 SEQUENCE: 14 Leu Xaa Xala Xala Xala Xala Xala Xala Xala Arg Lys 1 5 10

<210 SEQ ID NO 15 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <221 NAME/KEY: WARIANT <222> LOCATION: (2) . . . (2) <223> OTHER INFORMATION: Xaa = Basic Amino Acid <221 NAME/KEY: WARIANT <222> LOCATION: (3) . . . (5) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (6) . . . (6) &223> OTHER INFORMATION Xaa Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (7) . . . (7) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <400 SEQUENCE: 15 Leu Xaa Xala Xala Xala Xala Xala Arg Lys 1 5

<210> SEQ ID NO 16 &211's LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <221 NAME/KEY: WARIANT <222> LOCATION: (2) . . . (2) <223> OTHER INFORMATION: Xaa = Basic Amino Acid <221 NAME/KEY: WARIANT <222> LOCATION: (3) . . . (3) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (4) . . . (4) <223> OTHER INFORMATION:: Xaa = Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (5) . . . (6) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY WARIANT <222> LOCATION (7) . . . (7) &223> OTHER IN O RMATION: Xaa = Proline or Waline <221 NAME/KEY WARIANT <222> LOCATION (8) . . . (8) &223> OTHER IN ORMATION: Xaa = Hydrophobic Residue <400 SEQUENCE: 16 Leu Xaa Xala Xala Xala Xala Xala Xala Arg Lys 1 5 10

<210 SEQ ID NO 17 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <221 NAME/KEY: WARIANT <222> LOCATION: (2) . . . (3) &223> OTHER INFORMATION Xaa Basic Amino Acid <221 NAME/KEY: WARIANT <222> LOCATION: (4) . . . (6) &223> OTHER INFORMATION Xaa Hydrophobic Residue US 6,503,881 B2 139 140

-continued <221 NAME/KEY: WARIANT <222> LOCATION: (7) . . . (7) &223> OTHER INFORMATION Xaa Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (8) . . . (8) &223> OTHER INFORMATION Xaa Hydrophobic Residue <400 SEQUENCE: 17 Leu Xaa Xala Xala Xala Xala Xala Xala Arg Lys 1 5 10

<210> SEQ ID NO 18 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <221 NAME/KEY: WARIANT <222> LOCATION: (2) . . . (2) <223> OTHER INFORMATION: Xaa = Basic Amino Acid <221 NAME/KEY: WARIANT <222> LOCATION: (3) . . . (3) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (4) . . . (4) &223> OTHER INFORMATION Xaa Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (5) . . . (7) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <400 SEQUENCE: 18 Leu Xaa Xala Xala Xala Xala Xala Arg Lys 1 5

<210 SEQ ID NO 19 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <221 NAME/KEY: WARIANT <222> LOCATION: (2) . . . (2) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (3) . . . (3) <223> OTHER INFORMATION: Xaa = Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (4) . . . (5) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (6) . . . (6) <223> OTHER INFORMATION: Xaa = Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (7) . . . (7) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <400 SEQUENCE: 19 Leu Xaa Xala Xala Xala Xala Xala Arg Arg Lys 1 5 10

<210> SEQ ID NO 20 <211& LENGTH: 11 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <221 NAME/KEY: WARIANT <222> LOCATION: (3) . . . (3) &223> OTHER INFORMATION Xaa Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (4) . . . (4) &223> OTHER INFORMATION Xaa Proline or Waline <221 NAME/KEY: WARIANT US 6,503,881 B2 141 142

-continued <222> LOCATION: (5) . . . (6) &223> OTHER INFORMATION Xaa Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (7) . . . (7) &223> OTHER INFORMATION Xaa Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (8) . . . (8) &223> OTHER INFORMATION Xaa Hydrophobic Residue <400 SEQUENCE: 20 Leu Lys Xaa Xala Xala Xala Xala Xala Arg Arg Lys 1 5 10

<210> SEQ ID NO 21 <211& LENGTH: 11 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE &223> OTHER IN O RMATION: Indolicidin Analogue <221 NAME/KEY WARIANT <222> O CA. O N (1) . . . (2) &223> OTHER IN :ORMATION: Xaa = Basic Amino Acid <221 NAME/KEY: WARIANT <222> LOCATION: (3) . . . (3) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY WARIANT <222> O CA. O N (4) . . . (4) &223> OTHER IN O RMATION: Xaa = Proline or Waline <221 NAME/KEY WARIANT <222> LOCATION (5) . . . (6) &223> OTHER IN ORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (7) . . . (7) <223> OTHER INFORMATION: Xaa = Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (8) . . . (8) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (9) . . . (11) <223> OTHER INFORMATION: Xaa = Basic Amino Acid <223> OTHER INFORMATION: At leat two residues at positions 3, 5, 6 and 8 are Phenylalanine

<400 SEQUENCE: 21

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 1 5 10

<210> SEQ ID NO 22 <211& LENGTH: 11 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <221 NAME/KEY: WARIANT <222> LOCATION: (1) . . . (2) <223> OTHER INFORMATION: Xaa = Basic Amino Acid <221 NAME/KEY: WARIANT <222> LOCATION: (3) . . . (3) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (4) . . . (4) <223> OTHER INFORMATION: Xaa = Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (5) . . . (6) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (7) . . . (7) <223> OTHER INFORMATION: Xaa = Proline or Waline <221 NAME/KEY: WARIANT <222> LOCATION: (8) . . . (8) <223> OTHER INFORMATION: Xaa = Hydrophobic Residue <221 NAME/KEY: WARIANT <222> LOCATION: (9) . . . (11) <223> OTHER INFORMATION: Xaa = Basic Amino Acid <223> OTHER INFORMATION: At least two residues at positions 3, 5, 6 and US 6,503,881 B2 143 144

-continued 8 are Tyrosine <400 SEQUENCE: 22

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 1 5 10

<210> SEQ ID NO 23 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 23 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10

<210> SEQ ID NO 24 &2 11s LENGTH 2.0 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 24 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ile Leu Lys 1 5 10 15 Lys Ala Gly Ser 2O

<210> SEQ ID NO 25 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 25 Lys Arg Arg Trp Pro Trp Trp Pro Trp Llys Lys Lieu. Ile 1 5 10

<210> SEQ ID NO 26 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 26 Trp Arg Ile Trp Llys Pro Lys Trp Arg Lieu Pro Llys Trp 1 5 10

<210 SEQ ID NO 27 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 27 Ile Leu Arg Trp Val Trp Trp Val Trp Arg Arg Lys 1 5 10

<210> SEQ ID NO 28 &2 11s LENGTH 13 &212> TYPE PRT US 6,503,881 B2 145 146

-continued ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 28 Ile Leu Arg Arg Trp Val Trp Trp Val Trp Arg Arg Lys 1 5 10

SEQ ID NO 29 LENGTH 10 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 29 Leu Arg Trp Trp Trp Pro Trp Arg Arg Lys 1 5 10

SEQ ID NO 30 LENGTH 12 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 30 Ala Lieu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10

SEQ ID NO 31 LENGTH 12 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 31 Ile Leu Arg Trp Ala Trp Trp Pro Trp Arg Arg Lys 1 5 10

SEQ ID NO 32 LENGTH 13 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 32 Trp Arg Trp Trp Llys Pro Lys Trp Arg Trp Pro Llys Trp 1 5 10

SEQ ID NO 33 LENGTH 13 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 33 Ile Leu Lys Lys Ile Pro Ile Ile Pro Ile Arg Arg Lys 1 5 10

SEQ ID NO 34 LENGTH 13 TYPE PRT ORGANISM: Artificial Sequence US 6,503,881 B2 147 148

-continued

&220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 34 Ile Leu Lys Lys Tyr Pro Tyr Tyr Pro Tyr Arg Arg Lys 1 5 10

<210 SEQ ID NO 35 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 35 Ile Leu Lys Lys Tyr Pro Trp Tyr Pro Trp Arg Arg Lys 1 5 10

<210 SEQ ID NO 36 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 36 Ile Leu Lys Llys Phe Pro Trp Phe Pro Trp Arg Arg Lys 1 5 10

<210 SEQ ID NO 37 &2 11s LENGTH 13 212s. TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 37 Ile Leu Lys Llys Phe Pro Phe Trp Pro Trp Arg Arg Lys 1 5 10

<210 SEQ ID NO 38 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 38 Ile Leu Arg Tyr Val Tyr Tyr Val Tyr Arg Arg Lys 1 5 10

<210 SEQ ID NO 39 &2 11s LENGTH 15 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 39 Ile Leu Arg Trp Pro Trp Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10

<210> SEQ ID NO 40 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE US 6,503,881 B2 149 150

-continued <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 40 Trp Trp Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10

<210> SEQ ID NO 41 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 41 Ile Leu Arg Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10

<210> SEQ ID NO 42 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 42 Ile Leu Arg Arg Trp Pro Trp Trp Pro Trp Arg Lys 1 5 10

<210> SEQ ID NO 43 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 43 Ile Leu Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10

<210> SEQ ID NO 44 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 44 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Lys 1 5 10

<210> SEQ ID NO 45 <211& LENGTH: 11 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 45 Ile Leu Lys Trp Pro Trp Trp Pro Trp Arg Lys 1 5 10

<210> SEQ ID NO 46 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue US 6,503,881 B2 151 152

-continued

<400 SEQUENCE: 46 Lys Arg Arg Trp Pro Trp Trp Pro Trp Arg Lieu. Ile 1 5 10

<210> SEQ ID NO 47 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 47 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Ile Met Ile Leu 1 5 10 15 Lys Lys Ala Gly Ser 2O

<210> SEQ ID NO 48 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 48 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Asp Met Ile Leu 1 5 10 15 Lys Lys Ala Gly Ser 2O

<210 SEQ ID NO 49 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 49 Ile Leu Arg Trp Pro Trp Arg Arg Trp Pro Trp Arg Arg Lys 1 5 10

<210 SEQ ID NO 50 &2 11s LENGTH 2.8 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 50 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ile Leu Arg 1 5 10 15 Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ala Ala 2O 25

<210 SEQ ID NO 51 &2 11s LENGTH 2.0 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Indolicidin Analogue <400 SEQUENCE: 51 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Met Ile Leu Lys 1 5 10 15