CN117897394A - Peptides with antimicrobial activity - Google Patents

Abstract

The present disclosure includes peptides of formula I: s1- [ block-1] m-x- [ block-2] n-y- [ block-3]o-z- [ block-4]p-S2. Also included are pharmaceutical compositions comprising the peptides and methods of using the peptides to treat microbial infections.

Description

Peptides with antimicrobial activity

Technical Field

The present invention relates to novel arginine-containing peptides (ACPs) as treatments for fungal infections. The novel peptides comprise 2 to 4 "blocks", each of which comprises 2 to 7 L-arginine and/or D-arginine and/or homoarginine amino acids connected by a "linker", wherein the linker comprises a single amino acid or amine acid between any two arginine and/or homoarginine "blocks". These peptides may also include modifications at the N-terminus and/or C-terminus.

Background Art

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

Approximately 7% of candida bacteremias are resistant to at least one class of antifungal agents (CDC, "Antibiotic Resistance Threats in the United States" CDC: Atlanta, GA, 2019, pp. 1-138). Candida glabrata is of particular concern due to the severity of the disease and the rising rate of echinocandin resistance in the context of high azole resistance (Vallabhaneni S et al., Open Forum Infect Dis 2015, 2(4): p. v163, PMC4677623). Despite the low overall prevalence, C. krusei also accounts for a large proportion of these resistant candida bacteremias, and the species is intrinsically resistant to fluconazole (Lockhart SR et al., J Clin Microbiol 2012, 50(11): p. 3435-42). Candida auris (C. auris) is another rare but concerning pathogen that has spread rapidly worldwide since its first appearance in 2009 and has high rates of drug resistance (90% resistant to 1 class, 30% resistant to 2 classes, and some resistant to all available antifungal agents) (Forsberg KK et al., Med Mycol. 2019, 57(1): pp. 1-12).

Antimicrobial peptides (AMPs) rich in cationic amino acids have been noted to have advantages such as rapid bactericidal activity (usually by interacting with negatively charged microbial membranes and causing destruction), low tendency to develop resistance, and low possibility of off-target or drug interactions (Hancock RE et al., Nat Biotechnol. 2006, 24(12): 1551-7. doi:10.1038/nbt1267; Gordon YJ et al., Curr Eye Res. 2005, 30(7): 505-15; Lau JL et al., Bioorg Med Chem 2018, 26(10): 2700-2707; Lewies A et al., Probiotics Antimicrob Proteins 2019, 11(2): 370-381). Due to structural differences and unique mechanisms, they are less likely to develop cross-resistance with traditional antimicrobial agents. Historically, the success of AMPs in clinical development has been largely limited by toxicity 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).

New antimicrobial therapeutics (especially new antifungal therapeutics) that are safe and effective and can overcome fungal resistance remain a major unmet medical need. The compounds of the present invention are intended to meet this unmet medical need, especially the need for new therapeutic agents for treating fungal infections. The compounds of the present invention have effective antifungal activity, tolerance, selectivity and stability.

Summary of the invention

The present invention relates to arginine-containing peptides (ACP) for use in treating microbial infections, particularly fungal infections.

In one aspect, the invention provides a peptide having the structure of Formula I:

S1-[segment-1]m-x-[segment-2]n-y-[segment-3]o-z-[segment-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, 0 represents absence, 1 represents presence, wherein at least two of m, n, o and p are 1;

Segment-1, segment-2, segment-3 and segment-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;

Each of x, y and z is a linker, and each linker is independently present or absent and is composed of a single amino acid or amino acids selected from the following:

Proline (P), glycine (G), 3-aminopropionic acid (β-alanine, Apr), 4-aminobutyric acid (Aba), 5-aminopentanoic 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- 2-dihydroindenylglycine (Igl), 5-amino-3-oxavalanic acid (Aea), N-(2-aminoethyl)glycine (Aeg or Aeg2), isohexadenic acid (Inp), 2-cyclohexylglycine, N-butylglycine (ButylGly), N-(4-piperidinyl)glycine (PipGly), 2-amino-3-guanidinopropionic acid (Agp), (4'-pyridinyl)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));

Provided that when m is 1 and n is 0, or when 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; and when o is 1 and p is 0, or o is 0 and p is 1, z is absent;

Optionally, the peptide has a modified N-terminal amino acid, wherein the _-NH2 at the N-terminus is replaced with -N( _X1 )( _X2 ), wherein ( _X1 ) and ( _X2 ) are independently selected from H, _R1 , _R2C (O), _R3SO2 and _R4R5NC (O), wherein _R1 , _R2 and _R3 are independently alkyl groups or alkaryl _groups , and _R4 and _R5 are independently H, alkyl groups or _alkaryl groups, and wherein the alkyl groups and alkaryl groups are independently further optionally substituted with halogen, alkyl, amine and/or oxygen moieties; and

Optionally, the peptide has a modified C-terminal amino acid, wherein the -COOH at the C-terminus is replaced by _-CONH2 (carboxamide).

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

Table 1: Peptides of Formula I

*Peptides in which the -COOH at the C-terminus is replaced by a carboxamide are represented by -NH _2. Peptides in which the -NH ₂ group at the N-terminus has one H atom replaced by another group, -NH is represented together with, for example, a CH ₃ CONH or CH ₃ SO ₂ NH group.

4-FPhNHC(O)＝4-fluorophenylaminocarbonyl

cHexC(O)＝cyclohexylcarbonyl

Morpholine CH ₂ C(O)＝2-morpholinoacetyl

In another aspect, the present invention provides a peptide conjugate comprising a peptide of Formula I or Table 1 and a group connected to the C-terminus or N-terminus or the peptide, wherein the group is selected from a polyethylene glycol (PEG) group, a glycosidic group, a lipid group, a cholesterol or sterol group, a peptide or protein group, and an oligonucleotide group.

In another aspect, the present 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 pharmaceutically acceptable carriers, binders, diluents and/or excipients.

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

In some embodiments, the microbial infection is a fungal infection. In some embodiments, the infection is an infection of a fungus selected from the group consisting of Absidia spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Arthrographis spp., Aspergillus spp., Basidiobolus spp., Beauveria spp., Blastomyces spp., Blastochizomyces spp., Candida spp., Chrysosporium spp., Cladophialophora spp., Coccidioides spp. 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., Lomentospora 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.

In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the infection is an infection with gram-positive bacteria, gram-negative bacteria or mycobacteria. For example, the bacteria can be Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella senftenberg, Shigella sonnei or Mycobacterium spp.

The details of one or more embodiments are described in the accompanying drawings and the following description. Other features, objects and advantages of the embodiments will be apparent from the description and drawings and from the scope of the claims. All publications cited herein are hereby incorporated by reference into this case.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a set of time-killing 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.

FIG. 2 is a graph showing mean plasma concentrations of SEQ ID NO:7 following single intravenous (IV) and intraperitoneal (IP) dose injections.

DETAILED DESCRIPTION

Abbreviations and definitions

As used herein, the term "amino acid" is understood to mean an organic compound containing a basic amine group and an acidic carboxyl group. This term includes traditional α-amino acids (e.g., L-amino acids), isomers of α-amino acids (e.g., D-amino acids), and known amino acids. α-amino acids include, but are not limited to, alanine [Ala (3-letter abbreviation); A (1-letter abbreviation)], arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamic acid (Glu; E), glutamic acid (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Methionine; (Met; M), phenylalanine (Phe; 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)), as well as L-2-dihydroindanylglycine (Igl) and L-2-cyclohexylglycine.

As used herein, the term "amino acid" includes 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- 2-dihydroindenylglycine (Igl), 5-amino-3-oxavalanic acid (Aea), N-(2-aminoethyl)glycine (Aeg or Aeg2), isohexadenic acid (Inp), 2-cyclohexylglycine, N-butylglycine (ButylGly), N-(4-piperidinyl)glycine (PipGly), 2-amino-3-guanidinopropionic acid (Agp), (4'-pyridinyl)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)).

The term "linker" or "linkage" means a single amino acid or amino acids as defined above (under Formula I) between any two stretches of arginine and/or homoarginine.

The term "peptide" as used herein refers to a plurality of amino acid residues and/or amino acids generally linked together by peptide bonds. It is used interchangeably and has the same meaning as polypeptide and protein. This term includes peptides containing a modified C-terminus or N-terminus.

The term "arginine-containing peptide (ACP)" means a peptide comprising 6 to 30 amino acid residues that are mainly arginine and/or homoarginine in a "segment" and further comprising a "linker" connecting the segments. The ACP may also be conjugated to polyethylene glycol (PEG), a glycosidic group, a lipid group, a cholesterol or sterol group, a peptide or protein group, and/or an oligonucleotide group at the C-terminus or N-terminus. In general, the ACP of the present invention is considered to be a linear peptide.

The ACPs of the present invention are particularly useful as antimicrobial peptides, for example, against bacteria, fungi, yeasts, parasites, protozoa and viruses. The term "antimicrobial peptide" may be used herein to define any peptide having microbicidal and/or microbial activity, and non-exclusively encompasses any peptide described as having antibacterial, antifungal, antifungal, antiparasitic, antiprotozoal, antiviral, anti-infective, anti-infective and/or bactericidal, algicidal, amoebicidal, microbicidal, bactericidal, fungicidal, parasiticidal and protozoal properties.

The term "mycosis" refers to infectious diseases of humans and animals caused by pathogenic fungi. Mycosis is very common and a variety of environmental and physiological conditions can lead to the development of mycosis.

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

The term "minimum inhibitory concentration (MIC)" means the lowest concentration of a therapeutic agent that prevents the visible growth of microorganisms, particularly fungi or bacteria in the case of an ACP of Formula I.

The term "treat" or "treatment" means administering an effective amount of a therapeutic agent to a subject in need thereof for the purpose of curing, alleviating, relieving, treating, ameliorating or preventing a disease, its symptoms or underlying properties. Such a subject may be identified by a healthcare professional based on the results of any suitable diagnostic method.

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

The term "subject" means an animal, preferably a mammal, and 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, orangutans, and humans. In addition, the subject may be a bird, including chickens and turkeys.

The phrase "pharmaceutically acceptable" as used herein means those agents, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with human tissues, or in animals, without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit/risk ratio.

The term "alkyl" means an alkane radical lacking one hydrogen. The general formula for linked non-cyclic alkyl radicals is _{CnH2n +1} . Alkyl radicals include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, and hexadecyl.

The term "alkaryl" refers to an alkyl group terminating in an aryl or heteroaryl group, which is optionally substituted, wherein optional substitution includes substitution with halogen, alkyl, amine and/or oxygen moieties.

The peptide of the present invention

In one aspect, the present invention provides compounds having the structure of Formula I:

S1-[segment-1]m-x-[segment-2]n-y-[segment-3]o-z-[segment-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, 0 represents absence, 1 represents presence, wherein at least two of m, n, o and p are 1;

Segment-1, segment-2, segment-3 and segment-4 independently comprise 2 to 7 (i.e., 1, 2, 3, 4, 5, 6 or 7) amino acids, each segment 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;

Each of x, y and z is a linker, and each linker is independently present or absent and is composed of a single amino acid or amino acids selected from the following:

Proline (P), glycine (G), 3-aminopropionic acid (β-alanine, Apr), 4-aminobutyric acid (Aba), 5-aminopentanoic 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- 2-dihydroindenylglycine (Igl), 5-amino-3-oxavalanic acid (Aea), N-(2-aminoethyl)glycine (Aeg or Aeg2), isohexadenic acid (Inp), 2-cyclohexylglycine, N-butylglycine (ButylGly), N-(4-piperidinyl)glycine (PipGly), 2-amino-3-guanidinopropionic acid (Agp), (4'-pyridinyl)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));

Provided that when m is 1 and n is 0, or when 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; and when o is 1 and p is 0, or o is 0 and p is 1, z is absent;

Optionally, the peptide has a modified N-terminal amino acid, wherein the _-NH2 at the N-terminus is replaced with -N( _X1 )( _X2 ), wherein ( _X1 ) and ( _X2 ) are independently selected from H, _R1 , _R2C (O), _R3SO2 and _R4R5NC (O), wherein _{R1 , R2} and _R3 are independently alkyl groups or _alkaryl groups, and _R4 and _R5 are independently H, alkyl groups or alkaryl groups, and wherein the alkyl groups and alkaryl groups are independently further optionally substituted with halogen, alkyl, amine and/or oxygen moieties; and

Optionally, the peptide has a modified C-terminal amino acid, wherein the -COOH at the C-terminus is replaced by _-CONH2 (carboxamide).

In some embodiments, the peptides disclosed herein may be provided in the form of pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable salt" means a salt of a compound that does not cause significant irritation or toxicity to an organism to which it is administered and does not eliminate the biological activity and properties of the peptide. In some embodiments, the salt is an acid additive to the peptide. Pharmaceutically acceptable salts may be obtained by reacting the peptide with a mineral acid or an organic acid (e.g., hydrochloric acid, hydrobromic acid, acetic acid, methanesulfonic acid, phosphoric acid, mesylate, oxalic acid, and the like).

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

Table 2: Chemical structures of representative amino acid linkers

In another aspect, the peptide of formula I is an ACP selected from Table 1.

biology

In a preferred aspect of the present invention, the microbial infection may be a fungal infection. Fungal infections can be 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 gattii, Fusarium spp., Scedosporium spp., including Lomentosporaprolificans, Coccidioides spp., Trichophyton spp.), Microsporum spp., Epidermophyton spp., Aspergillus spp., Mucoromycetes including Rhizopus arrhizus, and/or another fungal species. However, ACPs may provide treatment for other fungi, such as Exophiala spp., Tinea spp., Blastomyces spp., Blastochizomyces spp., Cryptococcus spp., Histoplasma spp., Paracoccidiomyces spp., Sporotrix spp., Absidia spp., Cladophialophora spp., Fonsecaea spp., Phialophora spp., Lacazia spp., Arthrographis spp., Acremonium spp. 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. (e.g., Malassezia furfur), Mucor spp., Neotestudina spp., Nocardia spp. 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., Verticillium spp., or Wangiella spp.

Invasive candidiasis is an infection caused by a type of yeast (a type of fungus) called Candida. Unlike Candida infections of the mouth and throat (oropharyngeal candidiasis, also called "thrush") or 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. Candidaemia is a bloodstream infection with Candida and is a common infection in hospitalized patients.

Cryptococcus is an invasive fungus that causes cryptococcosis, an infection that is usually associated with immunosuppressed individuals and is rare in healthy individuals. The two species of Cryptococcus commonly associated with human infection are Cryptococcus neoformans and Cryptococcus gattii. Cryptococcus can infect the meninges causing cryptococcal meningitis.

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 interaction with the net positive charge of the ACP, which results in cell wall and/or cell membrane lysis and death, similar to the action of antimicrobial peptides.

The ACP of the present invention is a positively charged cationic peptide, which is suitable for treating infections or diseases caused by a variety of pathogenic yeasts, molds, bacteria and other microorganisms. The peptides of the present invention may also be used to treat other conditions, including but not limited to conditions associated with mucosal infections, such as cystic fibrosis infections, gastrointestinal infections, urogenital infections, urinary tract infections (e.g., kidney infections or cystitis), vaginal infections, or respiratory tract infections.

Antifungal activity

Table 3 shows the minimum inhibitory concentrations (MICs) of the selected peptides listed in Table 1 against various Candida species and Cryptococcus species (see Example 2). Compared to the positive reference compounds fluconazole, caspofungin and amphotericin B, the peptides were found to have potent antifungal activity in Candida species and Cryptococcus species, including strains resistant to current antifungal therapy.

Table 3 Peptides of Table 1 and their antifungal activity against Candida species and Cryptococcus species

Activity of each Candida species against a representative strain of Cryptococcus species (ATCC 90028 or 90029 for Candida albicans, ATCC 90030 for Candida glabrata, ATCC 22019 for Candida parapsilosis, MMX 7135 for Candida krusei, CDC 386 for Candida auris, ATCC 90874 for Candida tropicalis, ATCC MYA-578 for Candida dubliniensis, ATCC 90112 for Cryptococcus neoformans, and ATCC MYA-4093 for Cryptococcus gattii). MIC: minimum inhibitory concentration; FLC: fluconazole; CAS: caspofungin; AMB: amphotericin B.

Table 4 shows the MIC values of selected peptides in Table 1 tested against Coccidioides species (see Example 2). Compared to the positive reference compound fluconazole, the peptides were found to have potent antifungal activity in Coccidioides.

Table 4 Peptides of Table 1 and their antifungal activity against Coccidioides species

Coccidioides species include C. immitis and C. posadasii. MIC: minimum inhibitory concentration; N: strain number; FLC: fluconazole

Table 5 shows the MIC values of the selected peptides in Table 1 against filamentous fungi (see Example 2). Compared with the positive reference compounds fluconazole, voriconazole, posaconazole, caspofungin and amphotericin B, the peptides were found to have effective antifungal activity in those species.

Table 5 Peptides of Table 1 and their antifungal activity against filamentous fungi

Scedosporium species include S. boydii and S. apiospermum. MIC: minimum inhibitory concentration; N: strain number; FLC: fluconazole; VOR: voriconazole; POS: posaconazole; CAS: caspofungin; AMB: amphotericin B

Table 6 shows the MIC values of SEQ ID NO: 5, SEQ ID NO: 2, SEQ ID NO: 34 and SEQ ID NO: 32 tested against three dermatophytes (see Example 2). Compared with the positive reference compounds fluconazole, caspofungin and amphotericin B, the peptides were found to have effective antifungal activity in these species.

Table 6 Peptides of Table 1 and their antifungal activity against dermatophytes

MIC: minimum inhibitory concentration; FLC: fluconazole; CAS: caspofungin; AMB: amphotericin B

As shown by SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:32 in Candida albicans (FIG. 1A) and Cryptococcus neoformans (FIG. 1B), ACP exhibited rapid fungicidal activity (defined as a 3 log reduction in CFU/mL from time zero in a time-kill kinetic assay).

Antibacterial activity

Table 7 shows the MIC values of selected peptides in Table 1 against Gram-positive and Gram-negative bacterial species and mycobacterial species (see Example 4).

Table 7 Peptides of Table 1 and their antibacterial activity

MIC: Minimum inhibitory concentration

Structure Activity Relationship

The role of C-terminal cysteine in the antifungal and antibacterial activity of arginine-containing peptides (ACPs) was studied and found to be unnecessary (Table 3 and Tables 7-8). SEQ ID NO:2 containing C-terminal cysteine has similar activity to SEQ ID NO:3 against Candida, which has the same sequence except for the lack of C-terminal cysteine. In Table 7, only SEQ ID NO:5 contains C-terminal cysteine, and its MIC value against bacteria is similar to that of the ACP structure without cysteine. Adding C-terminal cysteine can increase cytotoxicity (Table 9).

Table 8 Effect of C-terminal cysteine on MIC of Candida species (N=25)

Candida species included: 5 strains each of Candida albicans, Candida auris and Candida glabrata, 3 strains each of Candida krusei, Candida parapsilosis and Candida tropicalis, and 1 strain of Candida dubliniensis. MIC50: minimum inhibitory concentration at which 50% of fungal strains are inhibited; MIC90: minimum inhibitory concentration at which 90% of fungal strains are inhibited.

Table 9 Effect of C-terminal cysteine on cytotoxicity of HepG2 cells

_EC50 : The concentration at which 50% cell viability is observed. A lower _EC50 represents greater cytotoxicity.

The effect of arginine stereochemistry on the antifungal activity of ACPs was studied. The results showed that when all arginine amino acids in the ACP had L stereochemistry (i.e., L-arginine), the ACP lacked or significantly reduced antifungal activity (Table 10). The best antifungal activity was found in ACPs in which all arginines had D-stereochemistry (i.e., D-arginine), or in ACPs with a mixture of L- and D-arginine, or in ACPs with homoarginine (Har) (Table 10).

Table 10

Antifungal activity of ACPs containing all L, all D, or a mixture of L and D arginine and/or homoarginine (Har)

Activity screening of 15 strains of Candida species (4 strains of Candida auris, 2 strains each of Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis, and 1 strain of Candida dubliniensis). MIC50: minimum inhibitory concentration at which 50% of fungal strains are inhibited; MIC90: minimum inhibitory concentration at which 90% of fungal strains are inhibited.

The influence of the total number of arginine in ACP on antifungal or antibacterial activity was studied. The results show that the best activity is found in ACPs containing a total of 10 to 16 arginine amino acids in a mixture of L- and D-arginine. The ACP containing 14 arginine amino acids shows the lowest MIC50 value or MIC90 value (Table 11). In Candida (Table 11) and in Cryptococcus and filamentous fungi (Table 12), the ACP with less than 12 arginine amino acids is observed to have reduced activity. The ACP containing 14 arginines has antibacterial activity (Table 7), but the antibacterial activity of the ACP containing only 10 arginines disappears (SEQ ID NO:17, Table 7).

Table 11 Effect of the amount of arginine amino acid on the MIC of Candida species (N=15)

Candida species included 4 strains of C. auris, 2 strains each of C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis, and 1 strain of C. dubliniensis. MIC ₅₀ : Minimum inhibitory concentration at which 50% of fungal strains are inhibited; MIC ₉₀ : Minimum inhibitory concentration at which 90% of fungal strains are inhibited. Ava: 5-aminovaleric acid

Table 12

Effect of Arginine Amount on MIC of Cryptococcus and Filamentous Fungi

MIC: minimum inhibitory concentration; Cryptococcal species included 2 strains of Cryptococcus neoformans and 1 strain of Cryptococcus gattii; filamentous fungi included 3 strains of Clostridium and 2 strains of Scedosporium (Scedosporium bodenii and Scedosporium apiospermum).

When arginine was organized and arranged in 2 to 4 segments and the linker was placed between each of the two arginine and/or homoarginine segments, enhanced antifungal activity was observed (Table 13). Separating the arginine and/or homoarginine-containing segments by a linker between any two arginine or homoarginine segments is essential for enhancing antifungal activity, wherein ACPs containing 3 linkers and 4 arginine and/or homoarginine segments exhibited the best activity, i.e., the lowest MIC90 value (Table 13).

Table 13 Effect of the number of linkers on the MIC of Candida species (N=15)

Candida species included 4 strains of C. auris, 2 strains each of C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis, and 1 strain of C. dubliniensis. MIC ₅₀ : Minimum inhibitory concentration at which 50% of the strains are inhibited; MIC ₉₀ : Minimum inhibitory concentration at which 90% of the strains are inhibited; 14 polyarginines: rRrrRrrRrRrRrR-NH2

Pharmaceutical composition

The term "pharmaceutical composition" refers to a mixture of a therapeutic agent disclosed herein (ie, ACP) and other chemical components (eg, pharmaceutically acceptable diluents, carriers, binders and/or excipients). A pharmaceutical composition facilitates administration of the compound to a subject.

The present disclosure relates to a pharmaceutical composition comprising a physiologically acceptable carrier, binder, excipient and/or diluent for therapeutic use that is well known in the pharmaceutical art and described in, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, Pa. (1990), which is hereby incorporated by reference in its entirety.

The pharmaceutical compositions of the present invention can be manufactured in a manner that allows for a variety of routes of administration. For delivery by non-oral administration, such as by bolus injection or continuous infusion, an aqueous solution or suspension comprising a therapeutic agent may contain a substance that increases the viscosity of the suspension; optionally, the suspension may also contain a suitable stabilizer or agent that increases the solubility of the compound to allow for the preparation of a highly concentrated solution. The therapeutic arginine-containing peptide and/or homoarginine-containing peptide can be formulated as known in the art for direct topical application to the target area. For oral administration, the compound can be readily formulated by combining the active compound with a pharmaceutically acceptable carrier well known in the art to produce tablets, pills, dragees, capsules, sachets, liquids, gels, syrups, lozenges, slurries, suspensions and the like, which are orally ingested by the patient to be treated. In addition, the peptide can be formulated as an aerosolized liquid or dry powder, or drops (e.g., eye drops or nasal drops), or a mouthwash for administration to the respiratory tract.

Application method

Applicable routes of administration may include, but are not limited to, oral, sublingual, transmucosal, inhalation, transdermal, topical, vaginal or rectal administration; non-oral delivery, including intramuscular, subcutaneous, intravenous, intramedullary or intrathecal injection, and intranasal or intraocular injection. The compound may also be administered in a sustained-release or controlled-release dosage form, a storage preparation, continuous infusion via a pump, or pulse administration at a predetermined rate.

Pharmaceutical compositions suitable for administration include compositions in which the active ingredient is contained in a therapeutically effective amount to prevent, alleviate or improve disease symptoms or prolong the survival of the subject to be treated. The determination of the effective dosage level (i.e., the dosage level necessary to achieve the desired therapeutic result) can be accomplished by those skilled in the art using conventional pharmacological methods. The administration of the therapeutic agent of the present invention can be in a single dose, in multiple doses, in a continuous or intermittent manner; both systemic and local administration are contemplated.

Treatment

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

Preventive or prophylactic antifungal therapy is often used during treatment of cancer patients and high-risk liver transplant recipients (Rex JH et al., Healthcare Epidemiology CID 2001:32, pp. 1191-1200). The main fungi of concern for these patients or other transplant patients or immunocompromised patients are Candida species and various filamentous fungi, especially Aspergillus species. In addition, healthcare providers sometimes prescribe preventive or prophylactic antifungal therapy for patients at high risk for 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 infants (less than 2.2 pounds) in nurseries with high rates of invasive candidiasis.

The peptides of the invention can also be administered prophylactically, for example, before a subject exhibits symptoms of a fungal infection, to prevent or delay the development of a fungal infection. Treatment can be performed before, during or after the diagnosis or development of symptoms of infection. Treatment initiated after the onset of symptoms can reduce the severity of one of the symptoms or eliminate the symptoms completely.

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

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

The peptides of the present invention can also be administered as therapeutic agents to treat subjects with bacterial infections. Therapeutic agent treatment is initiated after diagnosis or the appearance of symptoms consistent with bacterial infection. Exemplary bacterial infections include, but are not limited to, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella senftonburg, Sonneiella and Mycobacterium, including Mycobacterium tuberculosis and non-tuberculous mycobacteria, such as Mycobacterium abscesses, Mycobacterium chelonae, Mycobacterium avium and Mycobacterium kansasii.

The ACPs of the present invention find use in treating, controlling or preventing fungal or bacterial infections, and the diseases occur not only in humans but also in animals. The compounds can be administered to companion animals, domesticated animals (e.g., farm animals), research animals, wild animals or birds. Companion animals include, but are not limited to, dogs, cats, hamsters, rabbits, gerbils, birds (including chickens, turkeys) and guinea pigs. Domesticated animals include, but are not limited to, cattle, horses, pigs, goats, sheep and llamas. Research animals include, but are not limited to, mice, rats, rabbits, dogs, pigs, apes and monkeys.

Combination therapy

Therefore, the present invention provides a combination in another aspect, which comprises one or more peptides listed in Table 1, peptides of Formula I or combinations thereof, and one or more therapeutically active agents, which in a non-limiting embodiment, may be antibiotics, antifungal agents, antiviral agents or other anti-infective agents. Therefore, it should be understood that the pharmaceutical composition may further comprise at least one other pharmaceutically active agent, not necessarily an antimicrobial or anti-infective agent. Suitably, the pharmaceutically active agent may be selected from antibiotics, antibacterial agents, antifungal agents and antiviral agents, or other anti-infective agents. Non-limiting examples of therapeutic antifungal agents include polyenes, azoles, allylamines, echinocandins, etc. Preferred examples of antifungal agents include amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, miconazole, posaconazole, voriconazole, caspofungin, ibrexafungerp, micafungin, and anidulafungin.

When a combination therapy is used with one or more compositions of the invention, the additional therapy may be administered prior to, concurrently with, and/or after the compositions of the invention.

Reagent test kit

Any composition described herein may be provided in the form of a kit. In a non-limiting embodiment, the antifungal composition of the present invention, such as one or more peptides listed in Table 1 or a peptide of Formula I or a combination thereof, may be incorporated into a kit. The kit may contain a composition of the present invention in appropriate equal parts, and in some cases, one or more additional reagents, packaged in an aqueous medium or lyophilized form, or as a solid dosage form in a blister pack. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container means, in which a component can be placed, and preferably, appropriately divided. The set may also contain instructions for use.

Example

Example 1

The peptides were synthesized using standard solid phase peptide chemistry, where the amino acids were protected on the resin with FMOC. For example, amino acid activation and coupling were performed with HBTU/HOBt and DIEA. The FMOC group was removed using DMF containing 20% piperidine. After the individual peptide synthesis was completed, the resin-bound sequence was then cleaved from the resin and deprotected with 80-90% trifluoroacetic acid (TFA) containing a variety of removal agents, including water, phenyl methyl sulfide, ethyl methyl sulfide and ethanedithiol, and/or triisopropylsilane. The peptides were precipitated in ether and then isolated by centrifugation. The dried peptide particles were reconstituted in a mixture of water and acetonitrile and lyophilized before purification by reverse phase HPLC on a C18 column eluted with acetonitrile-water buffer containing 0.1% TFA. The peptides were analyzed and the pure fractions were combined and lyophilized. HPLC analytical data were obtained on a 5 micron C18 analytical column eluted with water-acetonitrile buffer containing 0.1% TFA. The molecular weight was confirmed by MALDI-TOF analysis. For salt conversion, anion exchange resin was used, either in acetate or chloride form. The purified peptide was dissolved in 20-50% acetonitrile in water, loaded on a strong anion exchange resin (desired salt form), and eluted with 30-50% acetonitrile in water containing 10% acetic acid to obtain the acetate form, or eluted with 30-50% acetonitrile alone to obtain the chloride form. The results of ACP are shown in Table 14.

Table 14 Molecular weight and purity of ACP

Example 2

Arginine-containing peptides (ACPs) were tested for antifungal activity in multiple groups of fungal strains using an in vitro broth microdilution assay under experimental conditions described by the Clinical and Laboratory Standards Institute (CLSI). Yeast and fungi were tested in RPMI-1640 medium buffered to pH 7.0 with 0.165M 3-N-morpholinepropane sulfonic acid (MOPS). The minimum inhibitory concentration (MIC) is defined as the lowest concentration of an agent that inhibits visible microbial growth. The test article was dissolved in phosphate buffered saline (PBS) and diluted in PBS in a 2-fold serial dilution manner to obtain a total of 11 test concentrations. A 10X serially diluted test article concentration solution was first prepared using a deep-well polypropylene 96-well plate, followed by a 1:5 dilution into 125% medium (RPMI-1640 with MOPS) to obtain a 2X test concentration solution. Subsequently, 100 μL of each 2X test concentration solution was added to each well of another 96-well plate, followed by the addition of 100 μL of the appropriate inoculum prepared in culture medium to produce a final concentration of approximately 0.4 to 5×10 ³ colony forming units (CFU)/mL (Candida, Cryptococcus, Coccidioides, and Rhizopus), 0.4 to 5×10 ⁴ CFU/mL (Clostridium, Scedosporium, and Paecilomyces variotii), and 1.5×10 ³ CFU/mL (Dermatophytes). The plates were incubated aerobically at 35°C without agitation for 24 hours (Candida and Rhizopus), 48 hours (Clostridium and Paecilomyces), 72 hours (Cryptococcus and Scedosporium), 48-72 hours (Coccidioides), and 4-6 days (dermatophytes), with MIC values for filamentous fungi, Coccidioides, and dermatophytes reported as >50% inhibition and MIC values for yeasts reported as complete (100%) inhibition. For reference compounds, the MICs for azoles and echinocandin were interpreted as >50% inhibition and the MIC for amphotericin B was interpreted as 100% inhibition according to CLSI guidelines. Growth control wells contained 100 μL of fungal suspension and 100 μL of growth medium without test article or positive control agent (amphotericin B, fluconazole, voriconazole, posaconazole, and/or caspofungin). ACP was tested in batches at different times, each with 2-7 strains of Candida 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 strains 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), and 1 strain of Cryptococcus gattii (resistance to caspofungin) , 3-6 strains of Clostridium (including F. falciforme, F. oxysporum and F. solani, as well as strains resistant to voriconazole, fluconazole and/or caspofungin), 2-4 strains of Scedosporium (including strains of Scedosporium borrelioides and Scedosporium apiosporium, as well as strains resistant to fluconazole), 1 strain of Psoralea polytrichum (including strains resistant to voriconazole), 3-10 strains of Coccidioides (including C. immitis and C. posadasii, as well as strains resistant to fluconazole), 1 strain of Paecilomyces variabilis variottii), three strains of Rhizopus fissili, and one strain each of Trichophyton rubrum, Epidermophyton floccosum, and Microsporum gypseum.

The MIC data in Tables 3 to 6 show that ACP has potent antifungal activity compared to positive reference compounds in a wide range of important fungal species, including strains resistant to current therapies.

Example 3

As described by Canton et al. (Canton E et al., Antimicrob Agents Chemother 2007, 53(7): p. 3108-11), time-dependent toxicity studies were performed to evaluate the fungicidal activity of ACPs. SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 32 were tested at 2X and 8X MIC values (by microdilution of culture broth).

For each test, a deep well 96-well test plate (Costar 3960) contained 900 μL RPMI-1640, 100 μL fungal inoculum (1 to 5×10 ⁶ CFU/mL) and 2 μL test agent in each well. Drug-free control wells containing RPMI-1640, inoculum and 2 μL PBS were used as growth controls for each isolate. After inoculation, the deep well plates were incubated at 35°C with shaking at 200 rpm. Surviving yeast was quantified at 1, 2, 4, 6 and 24 hours post-inoculation for Candida albicans ATCC strain 90028 and 1, 2, 6, 24 and 72 hours post-inoculation for Cryptococcus neoformans ATCC strain MYA-4564. At time = 0 hours, a 0.1 mL aliquot was taken from each inoculum suspension, serially diluted 10-fold in cooled sterile PBS, and track dilutions were performed to determine the CFU/mL at 0 hours. During the track dilution plate, a 10 μl aliquot of each dilution was spotted on the top of a square Sabouraud Dextrose Agar plate. The plate was then tilted at a 45-90° angle to allow the 10 μL aliquot to track on the agar surface. The plate was laid flat, dried at room temperature, then inverted, and Candida albicans was cultured at 35°C for about 24 hours or Cryptococcus neoformans was cultured at 35°C for 48 hours. Subsequently, CFU/mL was determined from the average colony count of duplicates with a detection limit of 50 CFU/mL. A reduction of at least 3-logs from the starting inoculum was considered fungicidal.

Figure 1A shows the results for Candida albicans and Figure 1B shows the results for Cryptococcus neoformans. In both cases, the three ACPs showed rapid and significant fungicidal activity with a ≥3-log reduction in CFU/mL compared to time 0 hours, and this activity was comparable or superior to the time-toxin activity of approved antifungal agents against those species.

Example 4

Arginine-containing peptides (ACPs) were tested for antibacterial activity in a panel of bacterial species using an in vitro culture broth microdilution test under test 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 an agent that completely inhibits visible microbial growth. The test article was dissolved in phosphate buffered saline (PBS) and diluted in a 2-fold serial dilution in the same vehicle to obtain a total of 11 test concentrations. A 4 μL aliquot of each dilution was added to 196 μL of culture broth medium, which was placed in a well of a 96-well plate seeded with an organism suspension (final bacterial count: 2-8×105 colony forming units/mL per well). Each plate was cultured at 35 or 36° C. for approximately 16-24 hours or 48 hours (Mycobacterium). After incubation, the test plate was visually inspected and the growth or complete growth inhibition of each well was scored to define the minimum inhibitory concentration. Vehicle controls and appropriate active reference agents were used as blank and positive controls, respectively.

The MIC values in Table 7 show that ACP has antibacterial activity.

Example 5

ACP did not cause any hemolysis of human erythrocytes when tested at concentrations up to 300 μg/mL, which is substantially higher than its antifungal MIC or antibacterial MIC. Hemolysis is a problem for many other cationic peptides, which hinders their utility in treating systemic infections. The hemolytic potential of the peptides was tested using erythrocytes collected from fresh human blood, which were centrifuged at room temperature and washed three times in phosphate buffered saline (pH 7.4), followed by incubation with peptides at concentrations of 3-300 μg/mL in phosphate buffered saline (PBS) at 37°C for 1 hour. Triton-X100 was used as a positive control, while vehicle (PBS) was used as a negative control. Amphotericin B and melittin were used as reference compounds, both of which are known to be hemolytic. After incubation, the mixture was centrifuged at room temperature, the supernatant was separated, and the absorbance was analyzed at a single wavelength of 410 nm. The background absorbance readings of the negative control were subtracted for all samples. The Triton-X-100 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. The EC ₅₀ value (the concentration of the test article that produces 50% lysis) was determined for each test compound whenever possible.

Table 15 Arginine-containing peptides have no hemolytic potential in human erythrocytes

The results showed that ACP (Table 15) had no detectable hemolytic activity. In contrast, the two positive controls produced significant concentration-related increases in hemolysis, with the EC _50s of melittin and amphotericin B being 2.63 μg/mL and 6.95 μg/mL, respectively.

Example 6

When tested at concentrations up to 300 μg/mL, ACP had no or low cytotoxic potential in human hepatoma (HepG2) cells, which was substantially higher than its antifungal MIC or antibacterial MIC. The inventors used changes in ATP levels in HepG2 cells as an indicator of cell viability to test the cytotoxicity of peptides. Changes in intracellular ATP levels are an indicator of cytotoxicity. ATP is the main energy source for mammalian cells and tissues. Compounds that cause a decrease in cellular ATP have been shown to be cytotoxic. A human hepatoma cell line (HepG2) from the American Type Culture Collection (ATCC, Cat# HB 8065) was used to assess cytotoxicity. This cell line is well established and has been used as a sentinel for chemical toxicity for many years. Healthy cells have high ATP levels. If cells are stressed due to drug exposure, ATP levels can decrease rapidly, indicating cytotoxic effects. Using CellTiter ATP was monitored by the luminescent cell viability assay (Promega, Cat#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% CO _2. After an equilibration period of 18-22 hours, the culture medium (containing FBS) was removed and the cells were washed twice with medium without FBS. Subsequently, 200 μL of medium containing peptides (1-300 μg/mL) without FBS or medium containing positive control bee venom peptides (0.01 to 10 μg/mL) and amphotericin B (1-100 μg/mL) without FBS was added. Rotenone (0.1 to 100 μM) was used for internal control to confirm that the ATP assay was performed within historical values. Negative controls were vehicle, PBS plus EMEM medium without FBS for peptides and bee venom peptides, or DMSO (0.1%) plus EMEM medium without FBS for amphotericin B and rotenone. Cell growth controls exposed to vehicle in complete EMEM (with FBS) were also performed. Exposure to test and reference compounds was performed for 18-22 hours at 37°C and 5% CO2. After the exposure period, the medium was removed and 50 μL of fresh medium plus 50 μL of lysis reagent (which contained luciferase) was added to the cells, and each plate was shaken for 10 minutes. The experimental luminescence value was read.

Raw data of relative luminescence units were obtained and cell viability was calculated using the following equation. Average data were converted to percentage cell viability relative to the vehicle control without FBS. The exposure concentration that resulted in 50% viability (EC ₅₀ ) was assessed using GraphPad Prism 9 S-curve extrapolation and Hill Slope determination. Samples that resulted in cell viability below 50% were considered cytotoxic within the tested concentration range.

Table 16

Arginine-containing peptides lack cytotoxicity in a human hepatocellular carcinoma (liver) cell line (HepG2) using an intracellular ATP assay

SEQ ID NO. EC ₅₀ 2 40 34 162 32 >300 3 >300 4 >300 7 >300 8 >300 16 >300 17 >300 13 >300 14 >300 15 >300 29 >300 18 >300 53 >300 33 >300 66 >300 75 49.8 60 >300 76 138.6 65 >300 63 >300 64 >300 62 >300 88 >300

The results showed that except for four ACPs, all tested ACPs had no detectable cytotoxicity (Table 16), with EC ₅₀ values far above 300 μg/mL. In contrast, the positive control produced a significant concentration-related increase in cytotoxicity, with EC ₅₀ values of bee venom peptide and amphotericin B of 2.01-3.07 μg/mL and 5.79-14.13 μg/mL, respectively. The EC ₅₀ value of rotenone was 0.129-0.594 μM.

Example 7

The acute and subacute toxicity of ACP was tested in CD-1 mice. In the acute toxicity studies, a single intravenous or intraperitoneal increasing dose of the peptide dissolved in saline or phosphate buffer for injection was administered to groups of CD-1 male or female mice (n=2-3 per dose). The intravenous dose was administered slowly via the tail vein over 15-20 seconds. The dose was escalated according to tolerance. Animals were observed for 15 minutes after injection for acute signs of intolerance (e.g., death, convulsions, tremors, acoagulopathy, sedation, etc.) and autonomic effects (e.g., diarrhea, salivation, tearing, vasodilation, piloerection, etc.). Subsequently, the mice were observed at least twice a day for 24 hours, or in some cases, for up to 48-96 hours after injection for clinical signs and overall health, including body weight, ruffled/ruffled hair, hunched posture, edema, decreased alertness, hypothermia, salivation, injection site irritation/wounds, inability to eat or drink, and lethargy. Mice were tolerant to peptides (including 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) after a single intravenous dose of up to 5 to 7.5 mg/kg, or after a single intraperitoneal dose of up to 10-15 mg/kg.

In the subacute toxicity study, intraperitoneal doses of the peptides (SEQ ID NO:7, SEQ ID NO:29, SEQ ID NO:53) dissolved in saline for injection were administered to groups of CD-1 male or female mice (n=3 per dose) for 7 consecutive days. Each peptide was evaluated at 2 to 3 dose levels ranging from 2.5 to 7.5 mg/kg per day. The mice were weighed at least once a day and observed at least twice a day for any abnormal findings and to determine overall health. Twenty-four hours after the last dose, the mice were humanely euthanized and blood was collected in K2EDTA microcontainers by cardiac puncture to assess hematological parameters. At the highest dose evaluated (7.5 mg/kg per day), all three peptides were tolerated for 7 days. No significant clinical or hematological adverse reactions were observed, including no evidence of hemolysis.

Example 8

Enzyme cleavage studies showed that ACP (SEQ ID NO: 7) was resistant to trypsin cleavage and approximately 50% remained intact after 6 hours of incubation with an endoproteinase targeting Arg. The peptide (267 μg/mL) was incubated with trypsin (9 μg/mL) from porcine pancreas (Sigma Aldrich Cat T6567) at 37°C, or the peptide (427 μg/mL) was incubated with 3 μg/mL of endoproteinase Arg-C (SigmaAldrich Cat 11370529001) at 37°C. Samples were taken at 0.5, 1, and 6 hours after incubation, and the peptide concentration was determined 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 that without incubation (time 0), indicating no degradation. The percentage of peptide remaining 6 hours after incubation with endoproteinase Arg-C was 50.5%, indicating a degradation half-life of approximately 6 hours. When the 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 at 37°C for 2 hours, almost 100% of the peptides remained intact, except for SEQ ID NO:13 where only about 40% of the peptide remained, indicating the importance of D-arginine substitution in enhancing serum stability.

The in vitro enzyme and serum stability of the peptides was demonstrated by in vivo plasma pharmacokinetic (PK) curves. A single 5 mg/kg intravenous injection (via the tail vein) and a single 7.5 mg/kg intraperitoneal dose of SEQ ID NO:7 were administered to 2 groups of CD-1 mice (n=3 per group). The peptide doses were well tolerated. A series of blood samples collected from the saphenous vein were placed in containers with EDTA-K2 as an anticoagulant at time points up to 4 or 8 hours after the intravenous or intraperitoneal dose. The plasma was separated and analyzed by the following LC-MS/MS method. Due to the small amount of blood collected from each mouse, plasma from 3 mice was pooled before analysis. Protein precipitation of 50 μL of plasma was performed with 100 μL of 5% trichloroacetic acid solution containing 300 ng/mL TAT peptide (GRKKRRQRRRPQ; SEQ ID NO:99) (as an internal standard). After centrifugation, an aliquot of the supernatant was injected into an HPLC column (Waters ACQUITYUPLC HSS T3, 2.1*50 mm, 1.8 μm) and eluted with a mobile phase gradient of 0.1% perfluoropentanoic acid (PFPA) in water and 0.1% PFPA in acetonitrile. The peptides and internal standards were detected using a Triple Quad 6500+ mass spectrometer operated with electrospray ionization in positive ion SRM mode. The calibration curve range was 10 to 4000 ng/mL. FIG2 depicts the mean plasma concentrations of SEQ ID NO:7 after a single intravenous and intraperitoneal dose.

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

Table 17

Mean pharmacokinetic parameters of SEQ ID NO:7 after a single intravenous (IV) or intraperitoneal (IP) dose in mice

_C0 or _Cmax is the concentration at the time of IV dose injection or the maximum concentration after IP dose; _tmax is the time when _C0 or _Cmax is observed; t1 _/2 is the plasma half-life; AUC is the area under the plasma concentration vs. time curve; F is the absolute bioavailability.

In another PK study, peptides SEQ ID NO:29 and SEQ ID NO:53 were administered intraperitoneally once daily to CD-1 mice at 7.5 mg/kg for 7 days. Plasma samples were collected from 3 mice at each time point for up to 6 hours after the dose on day 1, followed by plasma samples from 3 mice 6 hours after the dose on day 7. The samples were analyzed for peptide concentration using HPLC-MS/MS as described above. Similar good in vivo plasma exposure as described above was also observed for both peptides, with substantial penetration of the peptides into the kidneys after daily 7.5 mg/kg intraperitoneal (ip) doses to mice for 7 days (Table 18). The day 1 AUC ratios (kidney to plasma) for SEQ ID NO:29 and SEQ ID NO:53 were 158 and 58.3, respectively.

Table 18 Kidney and plasma concentration ratios in mice

Compound Day 1 (n=3) Day 7 (n=3) SEQ ID NO:29 236±233 361±322 SEQ ID NO:53 179±106 916±36

Mean ± SD 6 hours after an intraperitoneal dose of 7.5 mg/kg.

Other embodiments

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an embodiment of a general series of equivalent or similar features.

From the above description, those skilled in the art can easily determine the basic features of the embodiments, and can make various changes and modifications to the embodiments without departing from the spirit and scope thereof to adapt them to various uses and conditions. Therefore, other embodiments also fall within the scope of the patent application.

Claims (20)

1. A peptide having a structure of formula I: S1-[segment-1]m-x-[segment-2]n-y-[segment-3]o-z-[segment-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, 0 represents absence, 1 represents presence, wherein at least two of m, n, o and p are 1; Segment-1, segment-2, segment-3 and segment-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 amine acid other than L-arginine (R), D-arginine (r) or homoarginine (Har), and are independently present or absent; Each of x, y and z is a linker, and each linker is independently present or absent and is composed of a single amino acid or amino acids selected from the following: Proline (P), glycine (G), 3-aminopropionic acid (β-alanine, Apr), 4-aminobutyric acid (Aba), 5-aminopentanoic 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- 2-dihydroindenylglycine (Igl), 5-amino-3-oxavalanic acid (Aea), N-(2-aminoethyl)glycine (Aeg or Aeg2), isohexadenic acid (Inp), 2-cyclohexylglycine, N-butylglycine (ButylGly), N-(4-piperidinyl)glycine (PipGly), 2-amino-3-guanidinopropionic acid (Agp), (4'-pyridinyl)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)); Provided that when m is 1 and n is 0, or when 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; and when o is 1 and p is 0, or o is 0 and p is 1, z is absent; Optionally, the peptide has a modified N-terminal amino acid, wherein the _-NH2 at the N-terminus is replaced by -N( _X1 )( _X2 ), wherein ( _X1 ) and ( _X2 ) are independently selected from H, _R1 , _R2C (O ₎ , _R3SO2 and _R4R5NC (O), wherein _{R1 , R2 and R3} are independently alkyl groups or alkaryl groups, and _R4 and _R5 are independently H, alkyl groups or alkaryl groups, and wherein the alkyl groups and the alkaryl groups are independently further optionally substituted by halogen, alkyl, amine and/or oxygen moieties; and Optionally, the peptide has a modified C-terminal amino acid, wherein the C-terminal -COOH is replaced by _-CONH2 (carboxamide). 2. The peptide of claim 1, wherein S1 and S2 are absent. 3. The peptide of claim 1, wherein S1 or S2 is absent. 4. The peptide according to any one of claims 1 to 3, wherein the peptide has a sequence selected from SEQ ID NO: 2-98. 5. The peptide of claim 2 or 3, wherein at least two or three of m, n, o and p are 1, and each of segment-1, segment-2, segment-3 and segment-4 has 2-4 amino acids independently selected from R, r and Har. 6. The peptide according to claim 5, wherein all of m, n, o and p are 1. 7. The peptide of claim 2 or 3, wherein: m, n, o and p are all 1; each of segment-1 and segment-2 has three amino acids; each of segment-3 and segment-4 has four amino acids; x, y and z are each Aoa; and each amino acid in segment-1, segment-2, segment-3 and segment-4 is independently selected from R, r and Har; optionally, the peptide has a modified C-terminal amino acid, wherein the -COOH at the C-terminus is replaced by _-CONH2 . 8. The peptide according to claim 7, wherein the peptide has the sequence of SEQ ID NO: 29. 9. The peptide of claim 2 or 3, wherein: m, n, o and p are all 1; each of segment-1 and segment-2 has three amino acids; each of segment-3 and segment-4 has four amino acids; x, y and z are each P; and each amino acid in segment-1, segment-2, segment-3 and segment-4 is independently selected from R, r and Har; optionally, the peptide has a modified C-terminal amino acid, wherein the -COOH at the C-terminus is replaced by _-CONH2 . 10. The peptide according to claim 9, wherein the peptide has the sequence of SEQ ID NO: 53. 11. A peptide conjugate comprising a peptide as claimed in any one of claims 1 to 10 and a group connected to the C-terminus or N-terminus, wherein the group is selected from a polyethylene glycol (PEG) group, a glycoside group, a lipid group, a cholesterol or sterol group, a peptide or protein group, and an oligonucleotide group. 12. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier, binder, diluent or excipient. 13. A pharmaceutical composition comprising the peptide conjugate according to claim 11 and a pharmaceutically acceptable carrier, binder, diluent or excipient. 14. A method of treating a microbial infection in a subject, comprising administering the pharmaceutical composition of claim 12 or 13 to a subject in need thereof. 15. The method of claim 14, wherein the microbial infection is a fungal infection. 16. The method of claim 15, wherein the fungal infection is selected from the group consisting of Absidia spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Arthrographis spp., Aspergillus spp., Basidiobolus spp., Beauveria spp., Blastomyces spp., Blastochizomyces spp., Candida spp., Chrysosporium spp., Cladophialophora spp., Coccidioides spp., Conidiobolus spp. 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., Lomentospora 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. spp.), Sporobolomyces spp., Sporotrix spp., Syncephalastrum spp., Tinea spp., Trichoderma spp., Trichophyton spp., Trichosporon spp., Ulocladium spp., Ustilago spp., Verticillium spp., and Wangiella spp. 17. The method of claim 16, wherein the fungal infection is an infection of one or more of the genera Candida, Coccidioides, Cryptococcus, Epidermophyton, Clostridium, Arthrosporum, Microsporum, Paecilomyces, Rhizopus, Scedosporium, and Trichophyton. 18. The method of claim 14, wherein the microbial infection is a bacterial infection. 19. The method of claim 18, wherein the bacterial infection is an infection with a gram-positive bacterium, a gram-negative bacterium, or a mycobacterium. 20. The method of claim 19, wherein the bacterium is Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella senftenberg, Shigella sonnei, or Mycobacterium spp.