Peptides with antibacterial activity

Abstract

The present disclosure provides compounds of formula I: S1-[block-1] m -x-[Block-2] n -y-[Block-3] O -z-[Block-4] p Also included are pharmaceutical compositions containing the peptides and methods of treating microbial infections using the peptides.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to novel arginine-containing peptides (ACPs) as therapeutic agents for fungal infections. The novel peptides contain 2-4 "blocks", each block containing 2-7 L-arginine and / or D-arginine and / or homoarginine amino acids linked by a linker containing a single amino acid or amino acid between any two "blocks" of arginine and / or homoarginine. These peptides may also contain modifications at the N-terminus and / or C-terminus. [Background technology]

[0002] 2. Background of the Invention Invasive candidiasis is a significant cause of morbidity and mortality in the United States and is associated with high mortality despite adequate antifungal therapy (Pfaller MA et al., Clin Microbiol Rev 2007,20(1):p.133-63). Candida albicans is the most common cause, followed by C. glabrata; together, these two pathogens account for approximately 70% of all candidemia cases. C. parapsilosis and C. tropicalis are responsible for the majority of the remaining cases, with various other species accounting for 3% or less of infections (Lockhart SR et al., J Clin Microbiol 2012, 50(11): p. 3435-42; Diagn Microbiol Infect Dis 2012, 74(4): p. 323-31; CDC, "Antibiotic Resistance Threats in the United States." CDC: Atlanta, GA. 2019, p. 1-138). There are three classes of antifungal drugs available for the treatment of invasive candidiasis: polyenes (e.g., amphotericin B), azoles, and echinocandins. Each of these classes has limitations, including a narrow therapeutic window with some amphotericin B products, drug-drug interactions and large variability in pharmacokinetics (PK) with the azoles, poor brain and urinary concentrations with the echinocandins, and high and / or increasing rates of antifungal resistance in various Candida species (Pappas PG et al., Clin Infect Dis. 2016, 62(4): p. e1-50; Ashley ESD et al. Pharmacology of Systemic Antifungal Agents, Clinical Infectious Diseases 43(Suppl 1) 2006: p. S28-S39; Wiederhold NP, Infect Drug Resist 10(doi) 2017: p. 249-259. PMC5587015; Bidaud AL et al., J Mycol Med 2018, 28(3): p. 568-573).

[0003] Approximately 7% of candidemias are resistant to at least one class of antifungal drugs (CDC, "Antibiotic Resistance Threats in the United States" CDC: Atlanta, GA. 2019, p. 1-138). C. glabrata is of particular concern due to the severity of the disease and the rising rate of echinocandin resistance on a background of high azole resistance (Vallabhaneni S et al., Open Forum Infect Dis 2015, 2(4): p. ofv163. PMC4677623). Although the overall prevalence is low, C. krusei also accounts for a large proportion of these resistant candidemias, as this species is primarily resistant to fluconazole (Lockhart SR et al., J Clin Microbiol 2012, 50(11): p. 3435-42). C. auris is another rare but concerning pathogen that has spread rapidly worldwide since it first emerged in 2009 with high resistance rates (90% resistant to one class, 30% resistant to two classes, and somewhat resistant to all available antifungal drugs) (Forsberg KK et al., Med Mycol. 2019, 57(1):p.1-12).

[0004] Antimicrobial peptides (AMPs) rich in cationic amino acids have attracted attention due to their positive properties of rapid bactericidal activity (interacting with negatively charged microbial membranes resulting in frequent destruction), low tendency to develop resistance, and low possibility of off-target effects or drug-drug interactions (Hancock RE et al., Nat Biotechnol. 2006, 24(12): p. 1551-7. doi: 10.1038 / nbt1267; Gordon YJ et al., Curr Eye Res. 2005, 30(7): p. 505-15; Lau JL et al., Bioorg Med Chem 2018, 26(10): p. 2700-2707; Lewies A et al., Probiotics Antimicrob Proteins 2019, 11(2): p. 370-381). Due to their structural differences and unique mechanisms, they are less likely to suffer from cross-resistance with conventional antimicrobial agents. Successful clinical development of AMPs has been significantly limited by toxicity due to host cell membrane disruption (e.g., hemolysis and cytotoxicity), reduced or lost activity under physiological conditions, and / or rapid enzymatic degradation in vivo (Koo HB et al., Peptide Science 2019, 111(5): p.e24122; Mahlapuu M et al., Frontiers in cellular and infection microbiology 2016, 6: p.194-194).

[0005] The need for new antibacterial therapeutics, especially new antifungal therapeutics, that are safe, effective, and can overcome fungal resistance remains a major unmet medical need. The compounds of the present invention are intended to meet this unmet medical need, especially the need for new therapeutics for treating fungal infections. The compounds of the present invention have strong antifungal activity, tolerability, selectivity, and stability. Summary of the Invention

[0006] The present invention relates to arginine-containing peptides (ACPs) for use in the treatment of microbial infections, particularly fungal infections.

[0007] In one aspect, the present invention provides a compound of formula I: S1-[Block-1] m -x-[Block-2] n -y-[Block-3] O -z-[Block-4] p -S2 Formula I SEQ ID NO:1 or a pharma- ceutical acceptable salt thereof, wherein m, n, o and p are independently 0 or 1, where 0 represents absence and 1 represents presence, and where at least two of m, n, o and p are 1; block-1, block-2, block-3, and block-4 independently comprise 2 to 7 amino acids each independently selected from L-arginine (R), D-arginine (r), and homoarginine (Har); S1 and S2 are each independently an amino acid or amino acid other than R, r, or Har, and are independently present or absent; x, y, and z are each a linker, each linker being independently present or absent, and each linker is selected from the group consisting of proline (P), glycine (G), 3-aminopropionic acid (β-alanine, Apr), 4-aminobutyric acid (Aba), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), 7-aminoheptanoic acid (Ahp), 8-aminooctanoic acid (Aoa), 9-aminononanoic acid (Ana), 10-amino Decanoic acid (Ada), 11-aminoundecanoic acid (Aun), 12-aminododecanoic acid (Ado), 13-aminotridecanoic acid (Atr), 14-aminotetradecanoic acid (Ata), 15-aminopentadecanoic acid (Apn), 16-aminohexadecanoic acid (Ahd), N-(3-aminopropyl)glycine (Apg), (S)-indoline-2-carboxylic acid (Ica), L-α-methylleucine (Leu( Me), and L-2-indanylglycine (Igl), 5-amino-3-oxapentanoic acid (Aea), N-(2-aminoethyl)glycine (Aeg or Aeg2), isonipecotic acid (Inp), 2-cyclohexylglycine, N-butylglycine (ButylGly), N-(4-piperidinyl)glycine (PipGly), 2-amino-3-guanidinopropionic acid (Agp), (4'-pyridyl ) alanine (4-PyrAla), (S)-N-(1-phenylethyl)glycine (Feg), N-benzylglycine (Bng), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (Tiq), and 4-guanidinophenylalanine (Phe(4-Ngu)); with the proviso that if m is 1 and n is 0, or m is 0 and n is 1, then x is absent; if n is 1 and o is 0, or n is 0 and o is 1, then y is absent; if o is 1 and p is 0, or o is 0 and p is 1, then z is absent; Optionally, the peptide may have a modified N-terminal amino acid in which the N-terminal -NH2 is replaced with -N(X1)(X2), where (X1) and (X2) are independently selected from H, R1, R2C(O), R3SO2, and R4R5NC(O), where R1, R2, and R3 are independently alkyl or alkaryl groups, and R4 and R5 are independently H, alkyl, or alkaryl groups, which alkyl and alkaryl groups may be further independently substituted with halogen, alkyl, amino, and / or oxygen moieties; Optionally, the peptide may have a modified C-terminal amino acid (carboxamide) in which the C-terminal -COOH is replaced with -CONH2. A peptide or a pharma- ceutically acceptable salt thereof is provided.

[0008] In some embodiments, the peptide of formula I is selected from the peptides of Table 1 (SEQ ID NOs: 2-98).

[0009] [Table 1] TIFF2024534845000003.tif236159TIFF2024534845000004.tif68159

[0010] [Table 2]

[0011] In another aspect, the invention provides a peptide conjugate comprising a peptide of Formula I or Table 1 and a group linked to the C-terminus or N-terminus or peptide, the group being selected from a polyethylene glycol (PEG) group, a glycosyl group, a lipid group, a cholesterol or sterol group, a peptide or protein group, and an oligonucleotide group.

[0012] In another aspect, the invention provides a pharmaceutical composition comprising a peptide of Formula I or Table 1, or a peptide conjugate comprising a peptide of Formula I or Table 1, and one or more pharma- ceutically acceptable carriers, binders, diluents, and / or excipients.

[0013] In another aspect, the present invention provides a method of treating a microbial infection in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising a peptide of Formula I or Table 1, or a peptide conjugate comprising a peptide of Formula I or Table 1.

[0014] In some embodiments, the microbial infection is a fungal infection. In some embodiments, the infection is a fungal infection, such as Absidia spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Arthroglasphis spp., Aspergillus spp., Basidiobolus spp., Beauveria spp., Blastomyces spp., Blastoschizomyces spp., Candida spp., Chrysosporium spp., Cladophialophora spp., Coccidioides spp., Conidiobolus spp., Cryptococcus spp., Cussudamaccabia spp., Emmonsia spp., Epidermophyton spp., Exophiala spp., Fonsecaea spp., Fusarium spp., Geotrichum spp., Graphium spp., Histoplasma spp., Lacazia spp., Leptosphaeria spp., Lomentospora spp., ra), Malassezia spp., Microsporum spp., Mucor spp., Neotestudina spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Paracoccidioides spp., Phialophora spp., Fama spp., Piedraia spp., Pneumocystis spp., Pseudoalescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsis spp., Sporobolomyces spp., Sporothrix spp., Syncephalastorum spp., Chinea spp., Trichoderma spp., Trichophyton spp., Trichosporon spp., Urocladium spp., Ustilago spp., Verticillium spp., and Wangiella spp. spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Arthrographis spp., Aspergillus spp., Basidiobolus spp., Beauveria spp., Blastomyces spp., Blastoschizomyces spp., Candida spp., Chrysosporium spp., Cladophialophora spp., Coccidioides spp., Conidiobolus spp., Cryptococcus spp., Cunninghamella spp., Emmonsia spp., Epidermophyton spp., Exophiala spp., Fonsecaea spp., Fusarium spp., Geotrichum spp., Graphium spp., Histoplasma spp., Lacazia spp., Leptosphaeria spp., Lomentaspora spp., Malassezia spp., Microsporum spp., Mucor spp., Neotestudina spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Paracoccidiomyces spp., Phialophora spp., Phoma spp., Piedraia spp., Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsis spp., Sporobolomyces spp., Sporotrix spp., Syncephalastrum spp., Tinea spp., Trichoderma spp., Trichophyton spp., Trichosporon spp., Ulocladium spp., Ustilago spp., Verticillium spp., and Wangiella spp.). In some embodiments, the method further comprises administering to the subject another antifungal agent.

[0015] In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the infection is caused by a gram-positive bacterium, a gram-negative bacterium, or a mycobacterium. For example, the bacterium can be Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella senftenberg, Shigella sonnei, Mycobacterium spp.

[0016] The details of one or more embodiments are set forth in the accompanying drawings and the following specification. Other features, objects, and advantages of the embodiments will become apparent from the description and drawings, and from the claims. All publications cited herein are incorporated by reference. [Brief description of the drawings]

[0017] [Figure 1] Figure 1 is a set of graphs showing the time-kill kinetics of SEQ ID NO: 7 and SEQ ID NO: 8 in Candida albicans ATCC 90028 (A) and Cryptococcus neoformans ATCC MYA-4564 (B). CFU is colony forming unit and MIC is minimum inhibitory concentration. [Diagram 2] FIG. 2 is a graph showing the mean plasma concentrations of SEQ ID NO:7 following single intravenous (IV) and intraperitoneal (IP) administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Abbreviations and Definitions As used herein, the term "amino acid" is understood to mean an organic compound that contains both a basic amino group and an acidic carboxyl group. The term includes conventional alpha amino acids (e.g., L-amino acids), isomers of alpha amino acids (e.g., D-amino acids), and known amino acids. Alpha amino acids include alanine [Ala (three-letter abbreviation); A (one-letter abbreviation)], arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; P ... e;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V); homoarginine (Har), homoleucine (hLeu), S-indoline-2-carboxylic acid (Ica), L-α-methylleucine (Leu(Me)), and L-2-indanylglycine (Igl), and L-2-cyclohexylglycine.

[0019] As used herein, the term "amine acid" refers to 3-aminopropionic acid (β-alanine, Apr), 4-aminobutyric acid (Aba), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), 7-aminoheptanoic acid (Ahp), 8-aminooctanoic acid (Aoa), 9-aminononanoic acid (Ana), 10-aminodecanoic acid (Ada), 11-aminoundecanoic acid (Aun), 12-aminododecanoic acid (Ado), 13-aminotridecanoic acid (Atr), 14-aminotetradecanoic acid (Ata), 15-aminopentadecanoic acid (Apn), 16-aminohexadecanoic acid (Ahd), N-(3-aminopropyl)glycine (Apg), (S)-indoline-2-carboxylic acid (Ica), L-α-methylleucine (Leu(Me)), and L- These include 2-indanylglycine (Igl), 5-amino-3-oxapentanoic acid (Aea), N-(2-aminoethyl)glycine (Aeg or Aeg2), isonipecotic acid (Inp), 2-cyclohexylglycine, N-butylglycine (ButylGly), N-(4-piperidinyl)glycine (PipGly), 2-amino-3-guanidinopropionic acid (Agp), (4'-pyridyl)alanine (4-PyrAla), (S)-N-(1-phenylethyl)glycine (Feg), N-benzylglycine (Bng), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (Tiq), and 4-guanidinophenylalanine (Phe(4-Ngu)).

[0020] The term "linker" or "linkage" refers to a single amino acid or amino acid between any two arginine and / or homoarginine blocks as defined above (under Formula 1).

[0021] As used herein, the term "peptide" refers in general terms to a number of amino acid residues and / or amino acids linked together by peptide bonds. It is used interchangeably and is meant to be the same as polypeptide and protein. The term includes peptides containing modified C- or N-termini.

[0022] The term "arginine-containing peptide" (ACP) refers to a peptide that contains 6-30 amino acid residues, primarily arginine and / or homoarginine, in "blocks" and further includes a "linker" for connecting the blocks. ACPs may also be conjugated at the C-terminus or N-terminus to polyethylene glycol (PEG), glycosyl groups, lipid groups, cholesterol or sterol groups, peptide or protein groups, and / or oligonucleotide groups. In general, the ACPs of the present invention are considered to be linear peptides.

[0023] The ACPs of the present invention are particularly useful as antimicrobial peptides against, for example, bacteria, fungi, yeasts, parasites, protozoa and viruses. The term "antimicrobial peptide" can be used herein to define any peptide having bactericidal and / or microbistatic activity, non-exclusively including any peptide described as having antibacterial, anti-fungal, anti-mycotic, antiparasitic, antiprotozoal, antiviral, anti-infectious, anti-infective and / or germicidal, algicidal, amebicidal, microbicidal, bactericidal, fungicidal, parasiticidal and protozoal properties.

[0024] The term "mycosis" refers to infectious diseases caused by pathogenic fungi in humans and animals. Mycoses are common, and a variety of environmental and physiological conditions can contribute to the development of fungal diseases.

[0025] The term "candidiasis" refers to a fungal infection caused by yeasts (a type of fungus) of the Candida family. Several types of Candida can cause infection in people, the most common being Candida albicans. Candida normally lives on the skin and inside the body in places such as the mouth, throat, intestines, vagina, and nails without causing any problems. However, it is an opportunistic pathogen and can cause infections if it overgrows or if it invades the bloodstream or certain organs such as the brain, lungs, kidneys, or heart.

[0026] The term "minimum inhibitory concentration" (MIC) refers to the lowest concentration of a therapeutic agent that prevents visible growth of a microorganism, particularly in the case of ACPs of formula I, a fungus or bacteria.

[0027] The terms "treating" or "treatment" refer to the administration of an effective amount of a therapeutic agent to a subject in need thereof for the purpose of curing, alleviating, ameliorating, ameliorating, or preventing a disease, its symptoms, or tendencies thereto. Such subjects may be identified by a medical professional based on the results of any appropriate diagnostic method.

[0028] As used herein, the terms "administer," "administering," "administration," and the like refer to methods that can be used to enable delivery of an agent or composition to a desired site of biological action.

[0029] The term "subject" refers to an animal, preferably a mammal, most preferably a human, that is the object of treatment, prevention, observation or experiment. Exemplary mammals include mice, rats, rodents, hamsters, gerbils, rabbits, guinea pigs, dogs, cats, sheep, goats, pigs, cows, horses, giraffes, platypuses, primates, such as monkeys, chimpanzees, apes, and humans. In addition, the subject may be a bird, including chickens and turkeys.

[0030] The phrase "pharmaceutical acceptable" is employed herein to refer to those agents, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings, or, as the case may be, animals, without excessive toxicity, irritation, allergic response, or other problem or complication, consistent with a reasonable benefit / risk ratio.

[0031] The term "alkyl" refers to an alkane group lacking one hydrogen. The general formula for the acyclic alkyl group to be attached is C n H 2n+1 The alkyl group includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, and hexadecyl groups.

[0032] The term "alkaryl" refers to an alkyl group terminated in an optionally substituted aryl or heteroaryl group, where the optionally substituted groups include halogen, alkyl, amino, and / or oxygen moieties.

[0033] Peptides of the Invention In one aspect, the present invention provides a compound of formula I: S1-[Block-1] m -x-[Block-2] n -y-[Block-3] O -z-[Block-4] p -S2 Formula I SEQ ID NO:1 (In the formula, m, n, o and p are independently 0 or 1, where 0 represents absence and 1 represents presence, and where at least two of m, n, o and p are 1; block-1, block-2, block-3, and block-4 independently comprise 2 to 7 (i.e., 1, 2, 3, 4, 5, 6, or 7) amino acids each independently selected from L-arginine (R), D-arginine (r), and homoarginine (Har); S1 and S2 are each independently an amino acid or amino acid other than R, r, or Har, and are independently present or absent; x, y, and z are each a linker, each of which may be independently present or absent, and may be selected from the group consisting of proline (P), glycine (G), 3-aminopropionic acid (β-alanine, Apr), 4-aminobutyric acid (Aba), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), 7-aminoheptanoic acid (Ahp), 8-aminooctanoic acid (Aoa), 9-aminononanoic acid (Ana), 10-amino Decanoic acid (Ada), 11-aminoundecanoic acid (Aun), 12-aminododecanoic acid (Ado), 13-aminotridecanoic acid (Atr), 14-aminotetradecanoic acid (Ata), 15-aminopentadecanoic acid (Apn), 16-aminohexadecanoic acid (Ahd), N-(3-aminopropyl)glycine (Apg), (S)-indoline-2-carboxylic acid (Ica), L-α-methylleucine (Leu( Me), and L-2-indanylglycine (Igl), 5-amino-3-oxapentanoic acid (Aea), N-(2-aminoethyl)glycine (Aeg or Aeg2), isonipecotic acid (Inp), 2-cyclohexylglycine, N-butylglycine (ButylGly), N-(4-piperidinyl)glycine (PipGly), 2-amino-3-guanidinopropionic acid (Agp), (4'-pyridyl ) alanine (4-PyrAla), (S)-N-(1-phenylethyl)glycine (Feg), N-benzylglycine (Bng), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (Tiq), and 4-guanidinophenylalanine (Phe(4-Ngu)); with the proviso that if m is 1 and n is 0, or m is 0 and n is 1, then x is absent; if n is 1 and o is 0, or n is 0 and o is 1, then y is absent; if o is 1 and p is 0, or o is 0 and p is 1, then z is absent; Optionally, the peptide has a modified N-terminal amino acid in which the N-terminal -NH2 is replaced by -N(X1)(X2), where (X1) and (X2) are independently selected from H, R1, RC(O), RSO2, and R4R5NC(O), where R1, R2, and R3 are independently alkyl or alkaryl groups, and R4 and R5 are independently H, alkyl, or alkaryl groups, which alkyl and alkaryl groups are optionally further substituted independently with halogen, alkyl, amino, and / or oxygen moieties; and Optionally, the peptide has a modified C-terminal amino acid (carboxamide) in which the C-terminal -COOH is replaced by -CONH2. or a pharma- ceutical acceptable salt thereof.

[0034] In some embodiments, the peptides disclosed herein may be provided as their pharma- ceutically acceptable salts. The term "pharma-ceutically acceptable salts" refers to salts of compounds that do not cause significant irritation or toxicity to the organism to which they are administered and do not inhibit the biological activity and properties of the peptide. In some embodiments, the salts are acid additions to the peptide. Pharmaceutical salts can be obtained by reacting the peptide with a mineral or organic acid, such as hydrochloric acid, hydrobromic acid, acetic acid, methanesulfonic acid, phosphoric acid, mesylic acid, oxalic acid, and the like.

[0035] Table 2 shows the chemical structures of representative amino acid linkers of the present invention.

[0036] [Table 3] TIFF2024534845000007.tif242159TIFF2024534845000008.tif238159

[0037] In another embodiment, the peptide of formula I is selected from the ACPs of Table 1.

[0038] biology In a preferred embodiment of the present invention, the microbial infection may be a fungal infection. Fungal infections include those caused by Candida spp. (e.g., Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida auris, Candida dubliniensis, Candida lusitaniae, Candida guilliermondii), Cryptococcus neoformans, Cryptococcus gatti, Fusarium spp., Lomentospora prolificans, and the like. prolificans), Coccidioides spp., Trichophyton spp., Microsporum spp., Epidermophyton spp., Aspergillus spp.ACP can be an infection with any of the following fungal species: Mucoromycetes, including Rhizopus arrhizus, Mucoromycetes, including Rhizopus arrhizus, and / or another fungal species. However, ACP can be an infection with any of the following fungal species: Exophiala spp., Chinea spp., Blastomyces spp., Blastoschizomyces spp., Cryptococcus spp., Histoplasma spp., Paracoccidioides spp., Sporothrix spp., Absidia spp., Cladophialophora spp., Fonsecaea spp., Phialophora spp., Lacazia spp., Arthrographis spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Emmonsia spp., Basidiobolus spp., Beauveria spp., Chrysosporium spp., Conidiobolus spp., Cusdamacca spp., Geotrichum spp., Graphium spp., Leptosphaeria spp., species, Malassezia species (e.g., Malassezia furfur), Mucosae species, Neotestudina species, Nocardia species, Nocardiopsis species, Paecilomyces species, Fama species, Piedraia species, Pneumocystis species, Pseudoalescheria species, Pyrenochaeta species, Rhizomucor species, Rhizopus species, Rhodotorula species, Saccharomyces species, Scopulariopsis species, Sporobolomyces species, Syncephalastrum species, Trichoderma species, Trichosporon species, Urocladium species, Ustilago species, Verticillium species, or Wangiella species spp., Tinea spp., Blastomyces spp., Blastoschizomyces spp., Cryptococcus spp., Histoplasma spp., Paracoccidiomyces spp., Sporotrix spp., Absidia spp., Cladophialophora spp., Fonsecaea spp., Phialophora spp., Lacazia spp., Arthrographis spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Emmonsia spp., Basidiobolus spp., Beauveria spp., Chrysosporium spp., Conidiobolus spp., Cunninghamella spp., Geotrichum spp., Graphium spp., Leptosphaeria spp., Malassezia spp. (eg Malassezia furfur), Mucor spp., Neotestudina spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Phoma spp., Piedraia spp., Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scopulariopsis spp., Sporobolomyces spp., Syncephalastrum spp., Trichoderma spp., Trichosporon spp., Ulocladium spp., Ustilago spp., It can also provide treatment for other fungi, such as Verticillium spp., or Wangiella spp.

[0039] Invasive candidiasis is an infection caused by a yeast (a type of fungus) called Candida. Unlike a Candida infection in the mouth and throat (oropharyngeal candidiasis, also called "thrush") or in the vagina (vulvovaginal candidiasis or "yeast infection"), invasive candidiasis is a serious infection that can affect the blood, heart, brain, kidneys, eyes, bones, and other parts of the body. Candidemia, a bloodstream infection with Candida, is a common infection in hospitalized patients.

[0040] Cryptococcus is an invasive fungus that causes cryptococcosis, an infection that is rare in healthy individuals but commonly associated with immunosuppressed individuals. The two species of cryptococcus commonly associated with infection in humans are Cryptococcus neoformans and Cryptococcus gattii. Cryptococcus can infect the meninges, resulting in cryptococcal meningitis.

[0041] Without being bound by any theory, it is hypothesized that the net negative charge of the cell wall and cell membrane of the microorganism may facilitate an interaction with the net positive charge of the ACP, causing lysis and killing of the cell wall and / or cell membrane, similar to the action of antimicrobial peptides.

[0042] The ACPs of the present invention are positively charged cationic peptides useful for treating infections or diseases caused by a wide variety of pathogenic yeasts, molds, bacteria and other microorganisms. The peptides of the present invention may also be useful in treating other conditions, including but not limited to, conditions associated with mucosal infections, such as cystic fibrosis, gastrointestinal, genitourinary, urinary (e.g., kidney infections or cystitis), vaginal or respiratory infections.

[0043] antifungal activity Table 3 shows the minimum inhibitory concentrations (MICs) for selected peptides listed in Table 1 that were tested against various Candida and Cryptococcus species (see Example 2). These peptides were found to have potent antifungal activity in Candida and Cryptococcus species, including strains resistant to current antifungal therapies, compared to the positive reference compounds fluconazole, caspofungin and amphotericin B.

[0044] [Table 4] TIFF2024534845000010.tif241159TIFF2024534845000011.tif27159

[0045] Table 4 shows the MIC values ​​of selected peptides from Table 1 tested against Coccidioides species (see Example 2). These peptides were found to have potent antifungal activity in Coccidioides compared to the positive reference compound fluconazole.

[0046] [Table 5]

[0047] Table 5 shows the MIC values ​​of selected peptides from Table 1 tested against filamentous fungi (see Example 2). These peptides were found to have potent antifungal activity in these species compared to the positive reference compounds fluconazole, voriconazole, posaconazole, caspofungin, and amphotericin B.

[0048] [Table 6] TIFF2024534845000014.tif21159

[0049] Table 6 shows the MIC values ​​for SEQ ID NO:5, SEQ ID NO:2, SEQ ID NO:34, and SEQ ID NO:32 tested against three dermatophyte species (see Example 2). These peptides were found to have potent antifungal activity in these species compared to the positive reference compounds fluconazole, caspofungin, and amphotericin B.

[0050] [Table 7]

[0051] ACPs exhibit rapid fungicidal activity (defined as a 3 log reduction in CFU / mL from time zero in a time-kill kinetic assay) as shown by SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:32 in C. albicans (Figure 1A) and C. neoformans (Figure 1B).

[0052] Antibacterial activity Table 7 shows the MIC values ​​of selected peptides from Table 1 tested against Gram-positive and Gram-negative bacterial species and mycobacterial species (see Example 4).

[0053] [Table 8]

[0054] Structure-activity relationships The role of the C-terminal cysteine ​​in the antifungal and antibacterial activity of arginine-containing peptides (ACPs) was investigated and found not to be required (Tables 3 and 7-8). SEQ ID NO:2, which contains a C-terminal cysteine, has similar activity against Candida species as SEQ ID NO:3, which has an identical sequence except that it lacks the C-terminal cysteine. In Table 7, only SEQ ID NO:5 contains a C-terminal cysteine ​​and its MIC value against bacteria is similar to the ACP structure lacking cysteine. The addition of a C-terminal cysteine ​​increases cytotoxicity (Table 9).

[0055] [Table 9]

[0056] [Table 10]

[0057] The role of arginine stereochemistry in antifungal activity in ACPs was investigated. Results showed that when all arginine amino acids in the ACP have L-stereochemistry, i.e., L-arginine, the ACP lacks antifungal activity or has significantly reduced antifungal activity (Table 10). The best antifungal activity is found in ACPs where all argins have D-stereochemistry, i.e., D-arginine, or ACPs with a mixture of L-arginine and D-arginine, or ACPs with homoarginine (Har) (Table 10).

[0058] [Table 11]

[0059] The effect of the total number of arginines in the ACP on the antifungal or antibacterial activity was investigated. The results showed that the optimal activity was found in ACPs with a total of 10 to 16 arginine amino acids, including a mixture of L-arginine and D-arginine. ACPs containing 14 arginine amino acids had the lowest MIC.50 or MIC 90 The antibacterial activity was observed in ACPs with fewer than 12 arginine amino acids in Candida species (Table 11) and Cryptococcus species and filamentous fungi (Table 12). ACPs with 14 arginines have antibacterial activity (Table 7), but antibacterial activity is lost in ACPs containing only 10 arginines (SEQ ID NO: 17, Table 7).

[0060] [Table 12]

[0061] [Table 13]

[0062] Enhanced antifungal activity is observed when arginines are constructed and arranged in 2-4 blocks with a linker placed between each of the two blocks of arginine and / or homoarginine (Table 13). The separation of these arginine and / or homoarginine containing blocks by a linker between any two blocks of arginine or homoarginine is essential for enhanced antifungal activity, and ACPs containing three linkers and four arginine and / or homoarginine blocks have the best activity, i.e., the lowest MIC. 90 The values ​​are shown (Table 13).

[0063] [Table 14]

[0064] Pharmaceutical Compositions The term "pharmaceutical composition" refers to a mixture of a therapeutic agent (i.e., ACP) disclosed herein with other chemical components, such as pharma- ceutical acceptable diluents, carriers, binders and / or excipients, that facilitate administration of the compound to a subject.

[0065] The present disclosure relates to pharmaceutical compositions comprising therapeutic physiologically acceptable carriers, binders, excipients and / or diluents that are well known in the pharmaceutical arts and described, for example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, Pa. (1990), which is incorporated herein by reference in its entirety.

[0066] The pharmaceutical compositions of the present invention can be manufactured in a manner that allows for various routes of administration. Delivery by parenteral administration, for example by bolus injection or continuous infusion, includes aqueous solutions or suspensions of the therapeutic agent, which may contain substances that increase the viscosity of the suspension, and in some cases, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions. Therapeutic arginine and / or homoarginine-containing peptides may be formulated as known in the art for direct topical application to the target area. For oral administration, the compounds can be readily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art to produce tablets, pills, dragees, capsules, sachets, liquids, gels, syrups, lozenges, slurries, suspensions, and the like, for oral ingestion by the patient to be treated. Additionally, these peptides may be formulated for administration to the airways as a nebulized liquid or dry powder, or as drops, such as eye drops or nose drops, or as a mouthwash.

[0067] Administration Suitable routes of administration may include, but are not limited to, oral, sublingual, transmucosal, inhalation, transdermal, topical, vaginal or rectal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary or intrathecal injection, as well as intranasal or intraocular injection. These compounds may also be administered in sustained or controlled release dosage forms, depot formulations, and continuous infusions via pumps or pulse administration at a predetermined rate.

[0068] Pharmaceutical compositions suitable for administration include compositions in which the active ingredient is contained in a therapeutically effective amount to prevent disease, alleviate or ameliorate symptoms, or prolong survival of the subject being treated. Determination of effective dosage levels, that is the dosage levels necessary to achieve the desired therapeutic result, can be performed by one of skill in the art using routine pharmacological methods. Administration of therapeutic agents according to the invention can be in a continuous or intermittent manner, in a single dose, in multiple doses, and both systemic and local administration are contemplated.

[0069] Treatment methods The present disclosure provides a method of treating a subject suffering from a fungal or other microbial infection by administering to the subject a compound of the present invention. Therapeutic treatment is initiated after diagnosis or onset of symptoms of a fungal or other microbial infection. One or more peptides listed in Table 1 or peptides of formula I of the present invention, or combinations thereof, can be used to treat or prevent fungal or microbial infections. Exemplary fungal infections include, but are not limited to, infections with Candida species, including, for example, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida auris, Candida dubliniensis, Cryptococcus neoformans, Cryptococcus gattii, Fusarium species, Scedosporium species, including Lomentospora prolificans, Coccidioides species, Trichophyton species, Microsporum species, Epidermophyton species, Aspergillus species, Mucoromycetes, including Rhizopus alhizus, and / or another fungal species.

[0070] Preventive or prophylactic antifungal therapy is routinely administered during the treatment of cancer patients and high-risk liver transplant recipients (Rex JH et al., Healthcare Epidemiology CID 2001:32, pp. 1191-1200). The primary fungi of concern in these patients or other transplant or immunocompromised patients are Candida species and various mold fungi, particularly Aspergillus species. In addition, health care providers may prescribe preventive or prophylactic antifungal therapy to patients at high risk of developing invasive candidiasis, such as critically ill patients in intensive care units, organ transplant patients, stem cell or bone marrow transplant patients with low white blood cell counts (neutropenia), and very low-weight (<2.2 lbs) infants and young children in nurseries who have a high incidence of invasive candidiasis.

[0071] The peptides of the present invention may also be administered prophylactically, for example, before a subject develops symptoms of a fungal infection, to prevent or delay the onset of a fungal infection. Treatment may be administered before, during, or after diagnosis of infection or onset of symptoms. Treatment initiated after the onset of symptoms may reduce the severity of symptoms of a condition or completely eliminate the symptoms.

[0072] Advantages of preventive or prophylactic treatment with the peptides listed in Table 1 or peptides of Formula I against currently available antifungals include a lower likelihood of pre-existing or developing antifungal resistance, which can lead to treatment failure or drug-related toxicity.

[0073] The ACP of the present invention can be introduced into a mammal or bird at any stage of fungal infection.

[0074] The peptides of the present invention may also be administered as therapeutic agents to treat subjects suffering from bacterial infections. Therapeutic treatment is initiated after diagnosis of bacterial infection or onset of symptoms consistent therewith. Exemplary bacterial infections include, but are not limited to, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella senftenberg, Shigella sonnei, and Mycobacterium species including Mycobacterium tuberculosis, as well as nontuberculous mycobacteria such as M. abscesses, M. chelonae, M. avium, and M. kansasii.

[0075] The ACPs of the present invention are useful in the treatment, control, or prevention of fungal or bacterial infections and diseases in animals as well as humans. The compounds may be administered to companion animals, domestic animals such as livestock, animals used in research, wild animals, or birds. Companion animals include, but are not limited to, dogs, cats, hamsters, rabbits, gerbils, birds (including chickens and turkeys), and guinea pigs. Domestic animals include, but are not limited to, cows, horses, pigs, goats, sheep, and llamas. Research animals include, but are not limited to, mice, rats, rabbits, dogs, pigs, apes, and monkeys.

[0076] Combination therapy The present invention therefore provides in a further aspect a combination comprising one or more peptides listed in Table 1, peptides of formula I, or combinations thereof, together with one or more therapeutically active agents, which in one non-limiting embodiment may be antibiotics, antifungals, antivirals, or other anti-infectives. As such, it will be understood that the pharmaceutical composition may further comprise at least one other pharmacologic active agent that is not necessarily an antibacterial or anti-infective. Suitably, the pharmacologic active agent may be selected from antibiotics, antibacterials, antifungals, and antivirals, or other anti-infectives. Non-limiting examples of therapeutic antifungal agents include polyenes, azoles, allylamines, echinocandins, and the like. Preferred examples of antifungal agents include amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, miconazole, posaconazole, voriconazole, caspofungin, ibrexafungalp, micafungin, and anidulafungin.

[0077] When a combination therapy is employed with one or more compositions of the present invention, the additional therapy may be administered prior to, concurrently with, and / or subsequent to the composition of the present invention.

[0078] kit Any of the compositions described herein may be provided in a kit. In a non-limiting example, the antifungal compositions of the present invention, such as one or more peptides listed in Table 1 or peptides of formula I, or combinations thereof, may be combined in a kit. The kit may include a suitably aliquoted composition of the present invention, packaged either in aqueous medium or in lyophilized form or as a solid dosage form in a blister pack, and optionally one or more additional agents. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which the components may be placed, or preferably suitably aliquoted. The kit may also include instructions for use.

[0079] Working Example Example 1 Peptides are synthesized using standard solid-phase peptide chemistry with FMOC-protected amino acids on the resin. Activation and coupling of the amino acids is performed, for example, with HBTU / HOBt and DIEA. The FMOC group is removed using 20% ​​piperidine in DMF. After completion of each peptide synthesis, the resin-bound sequence is then cleaved from the resin and deprotected with 80-90% trifluoroacetic acid (TFA) containing various scavengers that may include water, thioanisole, ethyl methyl sulfide, and ethanedithiol, and / or triisopropylsilane. The peptides are precipitated in ether and then isolated by centrifugation. The dried peptide pellet is reconstituted in a mixture of water and acetonitrile, lyophilized, and then purified by reverse-phase HPLC on a C18 column eluted with acetonitrile-water buffer containing 0.1% TFA. The peptides are analyzed and pure fractions are pooled and lyophilized. Analytical HPLC data is obtained with a 5 micron C18 analytical column and elution with acetonitrile-water buffer containing 0.1% TFA. Molecular weights are confirmed by MALDI-TOF analysis. For salt conversion, anion exchange resins were used in either acetate or chloride form. The purified peptides were dissolved in 20-50% acetonitrile in water, loaded onto the strong anion exchange resin (desired salt form) and eluted with 10% acetic acid in 30-50% acetonitrile in water for acetate form or simply 30-50% acetonitrile for chloride form. ACP results are shown in Table 14.

[0080] [Table 15]

[0081] Example 2 Arginine-containing peptides (ACPs) were tested for antifungal activity in a panel of fungal strains using an in vitro broth microdilution assay under assay conditions described by the Clinical Laboratory Standards Institute (CLSI). Yeasts and fungi were tested in medium RPMI-1640 buffered to pH 7.0 with 0.165 M 3-N-morpholinepropane sulfonic acid (MOPS). The minimum inhibitory concentration (MIC) is defined as the lowest concentration of drug that inhibits visible microbial growth. Test articles were dissolved in phosphate-buffered saline (PBS) and diluted in 2-fold serial dilutions in PBS for a total of 11 test concentrations. Using deep-well polypropylene 96-well plates, 10× serially diluted test article concentration solutions were first made, followed by 1.5-fold dilution in 125% medium (RPMI-1640 with MOPS) to make 2× test concentration solutions. 100 μL of each 2× test concentration solution was then added to each well of a separate 96-well plate, followed by 100 μL of the appropriate innocula prepared in medium, yielding approximately 0.4–5×10 3 Colony forming units (CFU) / mL (Candida spp., Cryptococcus spp., Coccidioides spp., and Rhizopus spp.), 0.4–5 × 10 4 CFU / mL (Fusarium spp., Scedosporium spp., and Paecilomyces variotii) and 1.5 × 10 3Final concentrations of CFU / mL (dermatophytes) were obtained. Plates were incubated aerobically at 35°C without agitation for 24 hours (Candida spp. and Rhizopus spp.), 48 hours (Fusarium spp. and P. variotii), 72 hours (Cryptococcus spp. and Scedosporium spp.), 48-72 hours (Coccidioides spp.) and 4-6 days for dermatophytes and MIC values ​​were reported as >50% inhibition for fungi, Coccidioides spp. and dermatophytes and complete (100%) inhibition for yeasts. For reference compounds, MICs were read as per CLSI guidelines as >50% inhibition for azoles and echinocandins and 100% inhibition for amphotericin B. Growth control wells contained 100 μL of fungal suspension and 100 μL of growth medium without the test article or positive control drugs (amphoterin B, fluconazole, voriconazole, posaconazole and / or caspofungin).ACP was assessed against 2–7 strains of C. albicans (including strains resistant to fluconazole and / or caspofungin), 2–8 strains of Candida glabrata (including strains resistant to fluconazole and / or caspofungin), 2–3 strains of Candida tropicalis (including strains resistant to fluconazole), 3–6 strains of Candida parapsilosis (including strains resistant to fluconazole), 2–3 isolates of Candida krusei (including strains resistant to fluconazole), 4–8 strains of Candida auris (including strains resistant to fluconazole), 1 strain of Candida dubliniensis, 2–5 strains of Cryptococcus neoformans (including strains resistant to fluconazole and / or caspofungin), 1 strain of Cryptococcus gattii (including resistant to caspofungin), 3-6 strains of Fusarium spp. (including F. falciforme, F. oxysporum, and F. solani and strains resistant to voriconazole, fluconazole, and / or caspofungin), 2-4 strains of Scedosporium spp. (including strains of S. boydii and S. apiospermum and strains resistant to fluconazole), 1 strain of Lomentospora prolificans (including a strain resistant to voriconazole), 3-10 strains of Coccidioides spp. (including C. immitis and C. posadaci and strains resistant to fluconazole), 1 strain of Paecilomyces variotii, 3 strains of Rhizopus arrhizus, and Trichophyton rubrum, Epidermophyton purpurea, and Microsporum gypseum. Each batch was tested at different times using one strain of each of the three strains, Lactobacillus casei (Lactobacillus gypseum).

[0082] The MIC data in Tables 3-6 demonstrate that ACPs have potent antifungal activity compared to positive reference compounds across a broad range of important fungal species, including strains that are resistant to current therapeutics.

[0083] Example 3 Time-kill kinetic studies were performed to evaluate the fungicidal activity of ACPs as described by Canton et al. (Canton E et al. Antimicrob Agents Chemother 2007,53(7):p.3108-11). SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:32 were tested at 2- and 8-fold MIC values ​​determined by broth microdilution.

[0084] For each test, wells of a deep well 96-well assay plate (Costar 3960) were filled with 900 μL of RPMI-1640, 100 μL of fungal inoculum (1–5 × 10 6 CFU / mL) and 2 μL of test agent. Drug-free control wells containing RPMI-1640, inoculum, and 2 μL of PBS served as growth controls for each isolate. After inoculation, deep-well plates were incubated at 35°C with shaking at 200 rpm. Viable yeast were quantified at 1, 2, 4, 6, and 24 hours post-inoculation for Candida albicans ATCC strain 90028 and at 1, 2, 6, 24, and 72 hours post-inoculation for Cryptococcus neoformans ATCC strain MYA-4564. At time = 0 hours, 0.1 mL aliquots were removed from each inoculum suspension and serially diluted 10-fold in chilled sterile PBS, and track dilutions were plated to determine CFU / mL at 0 hours. During track dilution plating, 10 μL aliquots of each dilution were spotted across the top of a square Sabouraud dextrose agar plate. The plates were then tilted at a 45-90° angle to allow a 10 μL aliquot to spread over the entire agar surface. The plates were laid flat and allowed to dry at room temperature, then inverted and incubated at 35° C. for approximately 24 hours for C. albicans or 48 hours for C. neoformans. CFU / mL was then determined from the average colony count of the replicates, with a detection limit of 50 CFU / mL. A reduction of at least 3 logs of CFU from the starting inoculum is considered fungicidal.

[0085] The results are shown in Figure 1A for C. albicans and Figure 1B for C. neoformans. In both cases, the three ACPs demonstrated rapid and significant fungicidal activity with a 3 log or greater reduction in CFU / mL compared to time 0, which is comparable to or exceeds the time-kill activity of approved antifungal agents for these species.

[0086] Example 4 Arginine-containing peptides (ACPs) were tested for antibacterial activity in a panel of bacterial species using an in vitro broth microdilution assay under assay conditions described by CLSI. Cation Adjusted Mueller Hinton broth (CAMHB) was used for MIC testing. The minimum inhibitory concentration (MIC) is defined as the lowest concentration of drug that inhibits visible microbial growth. Test articles were dissolved in phosphate-buffered saline (PBS) and diluted by two-fold serial dilutions in the same vehicle for a total of 11 test concentrations. Aliquots of 4 μL of each dilution were placed in wells of a 96-well plate (final bacterial counts: 2–8 × 10 per well). 5 The organism suspension was added at 196 μL of inoculated broth medium at 100 μl / mL (colony forming units). Plates were incubated at 35 or 36 °C for approximately 16-24 or 48 hours (Mycobacteria species). After incubation, the test plates were visually inspected and wells were scored for growth inhibition or complete growth inhibition to define the minimum inhibitory concentration. Vehicle controls and appropriate active reference drugs were used as blank and positive controls, respectively.

[0087] The MIC values ​​in Table 7 indicate that the ACPs have antibacterial activity.

[0088] Example 5 ACPs do not cause hemolysis of human red blood cells when tested at concentrations up to 300 μg / mL, which is substantially higher than their antifungal or antibacterial MICs. Hemolysis is prone to occur with many other cationic peptides, which has hindered their utility for treating systemic infections. The hemolytic potential of the peptides was tested using red blood cells taken from fresh human blood, washed three times with phosphate-buffered saline (pH 7.4) after centrifugation at room temperature, and then incubated in phosphate-buffered saline (PBS) at 37°C for 1 h with the peptides at concentrations between 3 and 300 μg / mL. Triton-X100 was used as a positive control and the vehicle (PBS) was used as a negative control. Amphotericin B and melittin, which are known to be hemolytic, were used as reference compounds. After incubation, the mixtures were centrifuged at room temperature and the supernatants were separated and analyzed for absorbance at a single wavelength of 410 nm. Background absorbance readings from the negative control were subtracted from all samples. A Triton-X100 sample was used to represent 100% lysis. All test compound and positive control samples were normalized to this value to determine the percentage of lysis caused by the test compound and positive control at each concentration. EC 50 Values ​​(concentration of test article resulting in 50% dissolution) were determined for each test compound where possible.

[0089] [Table 16]

[0090] The results showed that ACP (Table 15) had no detectable hemolytic activity. In comparison, the two positive controls produced a clear concentration-related increase in hemolysis, with ECs of 2.63 μg / mL and 6.95 μg / mL for melittin and amphotericin B, respectively. 50 had.

[0091] Example 6 ACPs have no or low cytotoxicity in human hepatoma (HepG2) cells when tested at concentrations up to 300 μg / mL, which are substantially higher than their antifungal or antibacterial MICs. Peptides were tested for cytotoxicity using changes in ATP levels in HepG2 cells as an indicator of cell viability. Changes in intracellular levels of ATP indicate cytotoxicity. ATP is the primary energy source for mammalian cells and tissues. Compounds that reduce cellular ATP have been shown to be cytotoxic. A human hepatoma cell line (HepG2) from the American Type Culture Collection (ATCC, Catalog No. HB 8065) ​​was used to assess cytotoxicity. This cell line is well characterized and has been used as an indicator of chemical toxicity for many years. Healthy cells have high levels of ATP. When cells are stressed by drug exposure, ATP levels decrease rapidly, which can indicate cytotoxic effects. ATP was monitored using the CellTiter Glo® Luminescent Cell Viability Assay (Promega, Cat. No. G7572) to detect intracellular ATP. HepG2 cells were seeded into 96-well culture plates at a density of 20,000 cells per 100 μL. Cells were cultured in Eagles Minimum Essential Medium (EMEM) with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. After an equilibration period of 18–22 h, the medium (with FBS) was removed and the cells were washed twice with medium without FBS. Then, 200 μL of medium without FBS and containing peptides (at 1–300 μg / mL) or without FBS and containing the positive controls melittin (0.01–10 μg / mL) and amphotericin B (1–100 μg / mL) was added. An internal control was performed using rotenone (at 0.1–100 μM) to verify that the ATP assay was performing within the range of historical values.Negative controls were vehicle, PBS, and EMEM medium without FBS for peptides and melittin, or DMSO (0.1%) and EMEM medium without FBS for amphotericin B and rotenone. Growth controls of cells exposed to vehicle in complete EMEM (with FBS) were also performed. Exposure to test and reference compounds was for 18-22 hours at 37°C with 5% CO2. After the exposure period, the medium was removed and 50 μL of fresh medium and 50 μL of lysis reagent (containing luciferase) were added to the cells and the plate was shaken for 10 minutes. The luminescence of the assay was read.

[0092] Raw data in relative luminescence units was obtained and cell viability was calculated using the following formula: Mean data was converted to cell viability relative to vehicle control without FBS. Exposure concentrations that resulted in 50% viability (EC50) were estimated using sigmoidal curve extrapolation in GraphPad Prism9 with Hill slope determination. Samples that reduced cell viability below 50% are considered cytotoxic in the concentration range tested.

[0093] Viability (%) = luminescence of sample / mean luminescence of vehicle control x 1

[0094] [Table 17]

[0095] The results showed that all but four of the ACPs tested had no detectable cytotoxicity (Table 16), and the EC 50 In comparison, the positive controls produced a clear concentration-related increase in cytotoxicity, with EC 50 The EC values ​​were 2.01–3.07 μg / mL and 5.79–14.13 μg / mL. 50 The values ​​were in the range of 0.129 to 0.594 μM.

[0096] Example 7 Acute and subacute toxicity of ACP was tested in CD-1 mice. In acute toxicity studies, groups of CD-1 male or female mice were administered single intravenous or intraperitoneal ascending doses (n=2-3 per dose) of peptide dissolved in normal saline or in phosphate buffered saline for injection. Intravenous doses were administered via the tail vein with a slow push over 15-20 seconds. Doses were titrated based on tolerance. Animals were observed for 15 minutes after injection for acute signs of intolerance (e.g., death, convulsions, tremors, ataxia, sedation, etc.) and autonomic effects (e.g., diarrhea, salivation, lacrimation, vasodilation, pilorection, etc.). Mice were then observed at least twice daily for 24 hours or in some cases up to 48-96 hours after injection for clinical signs and general health, including weight, wrinkled / matte fur, hunched posture, edema, decreased alertness, hypothermia, salivation, irritation / wounds at the injection site, inability to eat or drink, lethargy. The peptides (SEQ ID NO:32, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:14, SEQ ID NO:53, SEQ ID NO:33) were tolerated by mice following a single intravenous dose of up to 5-7.5 mg / kg or a single intraperitoneal dose of up to 10-15 mg / kg.

[0097] In the subacute toxicity study, groups of CD-1 male or female mice were administered daily intraperitoneal doses (n=3 per dose) of peptides (SEQ ID NO:7, SEQ ID NO:29, SEQ ID NO:53) dissolved in normal saline for injection for seven consecutive days. Each peptide was evaluated at two to three dose levels ranging from 2.5 to 7.5 mg / kg / day. Mice were weighed at least once daily and observed at least twice daily for abnormal findings and general health assessment. Twenty-four hours after the last dose, mice were humanely euthanized and blood was collected via cardiac puncture into K2EDTA microtainers to evaluate hematological parameters. All three peptides were tolerated at the highest dose evaluated (7.5 mg / kg / day) for seven days. No significant clinical or hematological adverse events were observed, including no evidence of hemolysis.

[0098] Example 8 Enzymatic digestion studies showed that the ACP of SEQ ID NO: 7 was resistant to trypsin digestion and that approximately 50% remained intact after 6 hours of incubation with Arg-targeted endoproteinase. The peptide (267 μg / mL) was incubated with trypsin (9 μg / mL) from porcine pancreas (Sigma Aldrich Cat. No. T6567) at 37° C. or the peptide (427 μg / mL) was incubated with 3 μg / mL endoproteinase Arg-C (Sigma Aldrich Cat. No. 11370529001) at 37° C. Samples were taken after 0.5, 1 and 6 hours of incubation to determine peptide concentrations using an LC-MS / MS method. The percentage of peptide remaining at each time point up to 6 hours after incubation with trypsin was approximately 100% of the peptide without incubation (time 0), indicating no degradation. The percentage of peptide remaining after 6 hours incubation with endoproteinase Arg-C was 50.5%, representing a degradation half-life of approximately 6 hours. When peptides SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:29, SEQ ID NO:53 (5 μM) were incubated with full strength human serum for 2 hours at 37° C., nearly 100% of the peptides remained intact for all peptides except SEQ ID NO:13, where only approximately 40% remained, indicating the importance of D-arginine substitution for improved serum stability.

[0099] The in vitro enzymatic and serum stability of the peptide is shown by the in vivo plasma pharmacokinetic (PK) profile. A single intravenous injection (via tail vein) of 5 mg / kg and a single intraperitoneal dose of 7.5 mg / kg of SEQ ID NO:7 were administered to two groups of CD-1 mice (n=3 per group). The peptide doses were well tolerated. Serial blood samples were collected from the saphenous vein in EDTA-K2 as anticoagulant up to 4 or 8 hours after intravenous or intraperitoneal administration. Plasma was separated and analyzed by LC-MS / MS method as follows. Due to the small amount of blood collected from each mouse, plasma was pooled from three mice before analysis. 50 μL of plasma was protein precipitated with 100 μL of a solution of 300 ng / mL TAT peptide (GRKKRRQRRRPQ; SEQ ID NO:99) as an internal standard in 5% trichloroacetic acid. After centrifugation, an aliquot of the supernatant was loaded onto an HPLC column (Waters ACQUITY UPLC HSS T3, 2.1 * Peptides were injected into a 50 mm, 1.8 μm column and eluted with a gradient of a mobile phase containing 0.1% perfluoropentanoic acid (PFPA) in water and 0.1% PFPA in acetonitrile. Peptides and internal standards were detected using a Triple Quad 6500+ mass spectrometer operated in positive ion SRM mode with electrospray ionization. The calibration curve range was 10-4000 ng / mL. The mean plasma concentrations of SEQ ID NO:7 after single intravenous and intraperitoneal doses are shown in Figure 2.

[0100] These results showed that the peptide reached a fairly high plasma concentration after 5 mg / kg intravenous administration in mice and had a fairly long half-life of 1.43 hours. The plasma profile after intraperitoneal injection showed that the peptide was substantially absorbed into the systemic circulation and escaped first-pass metabolism and presystemic degradation, with a bioavailability of about 75% (Table 17).

[0101] [Table 18]

[0102] In another PK study, peptides of SEQ ID NO:29 and SEQ ID NO:53 were administered intraperitoneally to CD-1 mice at 7.5 mg / kg once daily for 7 days. Plasma samples were collected from 3 mice at each time point up to 6 hours post-dose on day 1, and then from 3 mice at 6 hours post-dose on day 7. Samples were analyzed for peptide concentration using HPLC-MS / MS as described above. Good in vivo plasma exposure similar to that shown above was also observed with these two peptides, along with substantial peptide penetration into the kidney after 7 days of daily intraperitoneal (ip) administration of 7.5 mg / kg in mice. The AUC ratios of kidney to plasma on day 1 were 158 and 58.3 for SEQ ID NO:29 and SEQ ID NO:53, respectively.

[0103] [Table 19]

[0104] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a generic series of equivalent or similar features.

[0105] From the above description, those skilled in the art can easily grasp the essential features of the described embodiments, and can make various changes and modifications to the embodiments to suit various applications and conditions without departing from the spirit and scope thereof. Therefore, other embodiments are within the scope of the claims.

Claims

1. Formula I: S1-[Block-1] ｍ -x-[Block-2] ｎ -y-[Block-3] Ｏ -z-[Block-4] ｐ -S2 Formula I SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, wherein m, n, o, and p are independently 0 or 1, where 0 represents absence, 1 represents presence, and at least two of m, n, o, and p are 1; block-1, block-2, block-3, and block-4 independently comprise 2 to 7 amino acids each independently selected from L-arginine (R), D-arginine (r), and homoarginine (Har); S1 and S2 are each independently an amino acid or amino acid other than L-arginine (R), D-arginine (r), or homoarginine (Har), and are independently present or absent; x, y, and z are each a linker, each linker being independently present or absent, and selected from the group consisting of proline (P), glycine (G), 3-aminopropionic acid (β-alanine, Apr), 4-aminobutyric acid (Aba), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), 7-aminoheptanoic acid (Ahp), 8-aminooctanoic acid (Aoa), 9-aminononanoic acid (Ana), 10-amino Decanoic acid (Ada), 11-aminoundecanoic acid (Aun), 12-aminododecanoic acid (Ado), 13-aminotridecanoic acid (Atr), 14-aminotetradecanoic acid (Ata), 15-aminopentadecanoic acid (Apn), 16-aminohexadecanoic acid (Ahd), N-(3-aminopropyl)glycine (Apg), (S)-indoline-2-carboxylic acid (Ica), L-α-methylleucine (Leu( Me), and L-2-indanylglycine (Igl), 5-amino-3-oxapentanoic acid (Aea), N-(2-aminoethyl)glycine (Aeg or Aeg2), isonipecotic acid (Inp), 2-cyclohexylglycine, N-butylglycine (ButylGly), N-(4-piperidinyl)glycine (PipGly), 2-amino-3-guanidinopropionic acid (Agp), (4'-pyridyl ) composed of a single amino acid or amino acid selected from alanine (4-PyrAla), (S)—N-(1-phenylethyl)glycine (Feg), N-benzylglycine (Bng), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (Tiq), and 4-guanidino-phenylalanine (Phe(4-Ngu)); with the proviso that when m is 1 and n is 0 or m is 0 and n is 1, x is absent; when n is 1 and o is 0 or n is 0 and o is 1, y is absent; when o is 1 and p is 0 or o is 0 and p is 1, z is absent; The peptide has an N-terminal -NH ２ is -N(X １ ) (X ２ ) wherein (X １ ) and (X ２ ) are independently H, R １ , R ２ C(O), R ３ SO ２ , and R ４ R ５ NC(O), R １ , R ２ , and R ３ are independently an alkyl group or an alkaryl group, and R ４ and R ５ are independently H, an alkyl group, or an alkaryl group, wherein said alkyl group and said alkaryl group may be further substituted, independently, with halogen, alkyl, amino, and / or oxygen moieties; The peptide has a C-terminal —COOH of —CONH ２ may have a modified C-terminal amino acid (carboxamide) replaced with A peptide or a pharmaceutically acceptable salt thereof.

2. S1 and S2 are absent; or Either S1 or S2 is absent; The peptide of claim 1.

3. 2. The peptide of claim 1, having a sequence selected from SEQ ID NOs: 2 to 98.

4. 3. The peptide of claim 2, wherein at least two or three of m, n, o, and p are 1, and each of block-1, block-2, block-3, and block-4 has 2 to 7 amino acids independently selected from R, r, and Har, m, n, o and p may all be 1, and block-1, block-2, block-3 and block-4 may each have 3 or 4 amino acids independently selected from R, r and Har; peptide.

5. m, n, o, and p are all 1, block-1 and block-2 each have 3 amino acids, block-3 and block-4 each have 4 amino acids, x, y, and z are each independently selected from Aoa, P, Apr, Ada, and ButylGly, and each amino acid in block-1, block-2, block-3, and block-4 is independently selected from R, r, and Har; and the peptide is ２ 3. The peptide of claim 2, optionally having a modified C-terminal amino acid replaced with:

6. 6. The peptide of claim 5 having a sequence selected from SEQ ID NOs: 53-59, 29-35, 7-16, 88, and 90-92, wherein the sequence may be SEQ ID NO: 53, 29, 32, 33, 7, or 88.

7. m, n, o, and p are all 1, block-1 and block-2 each have 3 amino acids, block-3 and block-4 each have 4 amino acids, x, y, and z are each P or Aoa, and each amino acid in block-1, block-2, block-3, and block-4 is independently selected from R, r, and Har; and the peptide is ２ 6. The peptide of claim 5, wherein the peptide has a sequence of SEQ ID NO: 53, 29 or 88.

8. 5. The peptide of claim 4, having a sequence selected from SEQ ID NOs: 60-73, wherein the sequence may be SEQ ID NO: 60, 64, 63, 61 or 62.

9. 10. A peptide conjugate comprising the peptide of claim 1 and a group linked to the C-terminus or N-terminus, wherein the group is selected from a polyethylene glycol (PEG) group, a glycosyl group, a lipid group, a cholesterol group or a sterol group, a peptide or protein group, and an oligonucleotide group.

10. A pharmaceutical composition comprising the peptide of claim 1 and a pharmaceutically acceptable carrier, binder, diluent, or excipient.

11. 10. A pharmaceutical composition comprising the peptide conjugate of claim 9 and a pharmaceutically acceptable carrier, binder, diluent, or excipient.

12. 12. A pharmaceutical composition according to claim 10 or 11 for use in the treatment or prevention of a microbial infection in a subject, wherein the microbial infection may be a fungal infection.

13. The fungal infection is selected from the group consisting of Absidia spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Arthroglasphis spp., Aspergillus spp., Basidiobolus spp., Beauveria spp., Blastomyces spp., Blastoschizomyces spp., Candida spp., Chrysosporium spp., Cladophialophora spp., Coccidioides spp., Conidiobolus spp., Cryptococcus spp., Cusudacamacea spp., Emmonsia spp., Epidermophyton spp., Exophiala spp., Fonsecaea spp., Fusarium spp., Geotrichum spp., Graphium spp., Histoplasma spp., Lacazia spp., Leptosphaeria spp., Lomentospora spp., Malassezia spp., Microsporum spp.

13. The pharmaceutical composition of claim 12, wherein the fungal infection is caused by a fungus selected from the group consisting of Mucor species, Neotestudia species, Nocardia species, Nocardiopsis species, Paecilomyces species, Paracoccidioides species, Phialophora species, Fama species, Piedraia species, Pneumocystis species, Pseudoalescheria species, Pyrenochaeta species, Rhizomucor species, Rhizopus species, Rhodotorula species, Saccharomyces species, Scedosporium species, Scopulariopsis species, Sporobolomyces species, Sporothrix species, Syncephalastorum species, Chinea species, Trichoderma species, Trichophyton species, Trichosporon species, Urocladium species, Ustilago species, Verticillium species, and Wangiella species.

14. 14. The pharmaceutical composition of claim 13, wherein the fungal infection is an infection caused by one or more of Candida species, Coccidioides species, Cryptococcus species, Epidermophyton species, Fusarium species, Lomentospora species, Microsporum species, Paecilomyces species, Rhizopus species, Scedosporium species, and Trichophyton species.

15. The pharmaceutical composition of claim 11 , wherein the microbial infection is a bacterial infection.

16. 16. The pharmaceutical composition of claim 15, wherein the bacterial infection is an infection caused by a gram-positive bacterium, a gram-negative bacterium, or a mycobacterium, and the bacterium may be Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella senftenberg, Shigella sonnei, or a Mycobacterium species.

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