Compositions and methods for treatment of fungal infections

Abstract

To develop peptide analogs of a θ-defensin that provide a biphasic effect in treating disseminated fungal disease and / or associated septic shock.SOLUTION: These analogs are active at concentrations below those needed to provide a fungicidal effect, and function by initially mobilizing effector cells of the immune system to address an infective pathogen and then regulating the immune system to down-regulate the inflammatory response. These θ-defensin analogs are protective at concentrations where naturally occurring θ-defensins have no apparent effect, and include a core set of structural and sequence features not found in native θ-defensins.SELECTED DRAWING: Figure 16

Description

[Technical field]

[0001] The technical field of the invention is biomedicine, in particular peptide pharmaceuticals. [Background technology]

[0002] The Background Discussion contains information that may be helpful in understanding the present invention. No admission is made that any information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Superficial fungal infections (e.g., of the oral and genital mucosa) are relatively common and rarely life-threatening. However, systemic or disseminated fungal infections can have mortality rates ranging from 30% to 50%. Fungal pathogens are a major cause of nosocomial infections, especially among surgical and catheterized patients. An increased risk of systemic fungal infections is also associated with decreased immune function, neutropenia, and diabetes. An increased risk of systemic or disseminated fungal infections is also associated with the use of biological therapies for the treatment of inflammatory or autoimmune diseases, which selectively suppress components of the immune response.

[0004] Systemic fungal infections are typically caused by Candida species (e.g., C. albicans), which are ubiquitous in nature and therefore not easily avoided. While antifungal drugs are available, resistant or multidrug-resistant strains are becoming increasingly prevalent. Unfortunately, systemic infections caused by multidrug-resistant fungi have become a global health concern. Approximately 1.5 million cases of disseminated mycoses occur annually and are associated with high mortality rates.

[0005] The rise of multidrug-resistant Candida infections contributes to the high mortality rate of systemic candidiasis. A major risk factor for systemic candidiasis is the presence of biofilms, which frequently occur on implanted medical devices such as intravenous catheters. Such biofilms are known to be resistant to antifungal treatment and are a common source of bloodborne spread of fungal pathogens.

[0006] The development of effective and relatively non-toxic antifungal drugs has proven challenging. There are currently only three classes of antifungal drugs used to treat invasive fungal infections: polyenes, azoles, and echinocandins. Of these, echinocandins are the most recently approved class of antifungal drugs, having first been introduced nearly 30 years ago. Limitations to the use of currently available antifungal drugs include a limited molecular target spectrum, serious adverse side effects, and lack of activity against biofilms. The emergence of multidrug-resistant fungal pathogens highlights the urgent need for the development of new approaches to the treatment of fungal infections.

[0007] Defensins are a diverse family of small antimicrobial proteins that are part of the body's non-specific defense against infection. There are three distinct and structurally distinct classes of defensin proteins: α, β, and θ defensins. α and β defensins are linear, tri-disulfide containing peptides with molecular weights of approximately 2.6 kDa or 4.5 kDa, respectively. In contrast, θ defensins are cyclic peptides (i.e., peptides in which the backbone is cyclic, formed by a series of peptide bonds that are not free at both the amino or carboxyl termini) consisting of 18 amino acids.

[0008] Theta defensins are expressed in tissues of rhesus macaques, baboons, and other Old World monkeys. They are absent in humans and other hominoids. Naturally occurring theta defensins consist of 18 backbone cyclized (i.e., via the alpha amine group rather than the side chain) peptides stabilized by three disulfide bonds. These three disulfide bonds are conserved in all known theta defensins. Theta defensins were originally discovered and classified as defensins based on the antimicrobial properties of the peptides. Recently, it has been found that theta defensins can have potent immunomodulatory effects.

[0009] International Patent Application Publication No. WO 2007 / 044998 (Lehrer et al.) describes the relationship between structure and biological activity for retrocyclin peptides and analogs of the peptides that include varying degrees of enantiomeric content in an attempt to derive structure / activity relationships. However, these analogs retain the length and structure of the native retrocyclin. In addition, the references only discuss antibacterial activity.

[0010] Peptide analogues of various defensins are being investigated. For example, European Patent Application EP2990415 (Colavita et al.) describes cyclic analogues of β-defensins that show improved antibiotic effects compared to the parent protein. However, such β-defensins have been shown to stimulate the release of proinflammatory cytokines, which poses safety concerns, limiting their practical utility.

[0011] US Patent Application Publication No. US2003 / 0022829 (Maury et al.) describes the synthesis and biological activity of chimeric theta defensins and speculates on possible conservative amino acid substitutions, but these appear to retain the length and structure of native theta defensins. US Patent No. 10,512,669 (Selsted et al.) describes various tetradecapeptide theta defensin analogs derived from RTD-1 and their biological properties.

[0012] Thus, there remains a need for safe and effective compounds for the management and / or treatment of fungal infections, particularly disseminated fungal infections. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] International Publication No. 2007 / 044998 [Patent Document 2] European Patent Application No. 2990415 [Patent Document 3] US Patent Application Publication No. 2003 / 0022829 [Patent Document 4] U.S. Pat. No. 10,512,669 Summary of the Invention

[0014] The present subject matter provides synthetic analogs of theta defensins that have improved activity in the treatment of fungal infections, particularly disseminated or systemic fungal infections, compared to naturally occurring theta defensins. These peptides act via a host-directed mechanism and are effective at concentrations below those of analogs that have direct bactericidal and / or fungistatic effects(s) against the same pathogens in vitro.

[0015] One embodiment of the inventive concept is a cyclic peptide consisting of 14 amino acids and having the structure shown in Figure 7A, which includes two disulfide bonds between two pairs of cysteines, AA3 and AA12 are cysteines linked by disulfide bonds, AA5 and AA10 are cysteines linked by disulfide bonds, AA4 is serine or a first hydrophobic amino acid, AA11 is serine or a second hydrophobic acid, AA6 is arginine, AA7 is arginine, and AA8 is arginine, and the cyclic peptide comprises five arginine residues that provide a positive charge content of at least about 36% at physiological pH. In some embodiments, the first hydrophobic amino acid and the second hydrophobic amino acid are leucine or isoleucine. In some embodiments, AA1 is glycine. In some embodiments, AA2 is a third hydrophobic amino acid (e.g., valine or leucine). In some embodiments, AA9 is a fourth hydrophobic amino acid (e.g., valine or phenylalanine). In some embodiments, AA13 and AA14 are arginines. In some embodiments, AA4 cannot be alanine or serine. In some embodiments, AA11 cannot be alanine.

[0016] In another embodiment, a cyclic peptide consisting of 14 amino acids and having the structure shown in FIG. 7A, which includes two disulfide bonds between two pairs of cysteines, where AA3 and AA12 are cysteines linked by a disulfide bond, AA5 and AA10 are cysteines linked by a disulfide bond, AA4 is an arginine, AA11 is an arginine, and two of AA6, AA7, and AA8 are arginines, said cyclic peptide comprising five or more arginine residues providing a positive charge content of at least about 36% at physiological pH.

[0017] Such cyclic peptides can be analogs of theta defensin that provide enhanced survival compared to theta defensin itself when applied systemically in a mouse model of disseminated fungal infection. In some embodiments, the cyclic peptides provide a biphasic response upon application to a mouse model of sepsis. Such a biphasic response includes a first phase of recruitment of host effector cells with antifungal activity and a second phase of attenuation of the host inflammatory response. In some embodiments, the cyclic peptides have TACE inhibitory activity and / or inhibit at least one of expression, processing, and release of TNF.

[0018] Such cyclic peptides retain activity after exposure to extreme environmental temperatures, low pH, freezing and / or thawing, and dissolution in biological matrices such as, for example, blood, plasma, or serum. In some embodiments, such cyclic peptides do not elicit an immune response in amounts effective to treat or prevent disseminated fungal disease and associated septic shock. Such cyclic peptides may activate the host immune system to enhance host pathogen clearance and may have activity in modulating inflammation to enhance disease resolution and survival in amounts effective to treat or prevent septic shock.

[0019] Another embodiment of the inventive concept is a method of treating or preventing septic shock and / or severe sepsis by administering the above-described cyclic peptides to an animal at risk for disseminated fungal disease.

[0020] Another embodiment of the inventive concept is the use of the above-mentioned cyclic peptides in treating or preventing disseminated fungal disease and / or associated septic shock and / or severe sepsis, or the use of such cyclic peptides in the preparation of a medicament effective in treating or preventing disseminated fungal disease and / or septic shock.

[0021] Various objects, features, aspects and advantages of the present subject matter will become apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings, in which like numerals refer to like elements and in which: [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows a schematic representation of the naturally occurring theta defensin RTD-1 (SEQ ID NO:1). [Diagram 2] FIG. 2 shows a schematic diagram of synthetic theta defensin analog cyclic peptide 1 (SEQ ID NO:2). [Diagram 3] FIG. 3 shows a schematic diagram of synthetic theta defensin analog cyclic peptide 2 (SEQ ID NO:3). [Figure 4] FIG. 4 shows a schematic diagram of synthetic theta defensin analog cyclic peptide 3 (SEQ ID NO:4). [Diagram 5] FIG. 5 shows a schematic diagram of synthetic theta defensin analog cyclic peptide 4 (SEQ ID NO:5). [Figure 6] FIG. 6 shows a schematic diagram of synthetic theta defensin analog cyclic peptide 5 (SEQ ID NO:6). [Figure 7A] FIG. 7A shows the numbering system used to designate particular amino acids within the cyclic tetradecapeptides described herein in the absence of the distinct amine and carboxy termini found in conventional linear peptides. [Figure 7B] FIG. 7B shows this numbering system applied to cyclic peptide 5 (SEQ ID NO:6). [Figure 8] FIG. 8 shows a schematic diagram of synthetic theta defensin analog cyclic peptide 6 (SEQ ID NO:7). [Figure 9]Figure 9 shows representative results of a study of the effect of RTD-1, the synthetic cyclic tetradecapeptide cyclic peptide 5, and two antifungal drugs in an in vivo model of disseminated candidiasis. Mice were infected intravenously at T=0 with 3×105 spores of the Candida albicans genetic reference strain SC5314. At T=24h, mice were treated intraperitoneally once daily for 7 days with saline, 5 mg / kg caspofungin (Caspo), 5 mg / kg fluconazole (Fluco), 5 mg / kg RTD-1, or 0.25 mg / kg cyclic peptide 5. Mice were observed for 26 days (pi) and survival rates of treated mice were compared against saline controls by log-rank test: RTD-1, Caspo, and Fluco, P=3.4×10-6; 0.25 mg / kg cyclic peptide 5, P=2.3×10-7. [Figure 10] Figure 10 shows representative results of a study of the effect of synthetic cyclic tetradecapeptide cyclic peptide 5 at 0.25 mg / kg and 0.1 mg / kg and fluconazole (Fluco) at 5 mg / kg in an in vivo model of disseminated candidiasis. Mice were infected with 3 x 105 spores of Candida albicans SC5314 by intravenous injection at T = 0. At T = 24 h, mice were treated by intraperitoneal administration once daily for 7 days. Mice were observed for 30 days (pi) and survival enhancement was analyzed by log-rank test. [Figure 11] FIG. 11 shows representative results from a study of the effects of the synthetic cyclic tetradecapeptide cyclic peptide 5 at 0.25 mg / kg and cyclic peptide 3 at 0.1 mg / kg and fluconazole (Fluco) at 5 mg / kg in an in vivo model of disseminated candidiasis, similar to the study shown in FIG. 10. [Figure 12] FIG. 12 shows representative results from a study of the effects of the synthetic cyclic tetradecapeptides Cyclic Peptide 5 at 0.25 mg / kg and Cyclic Peptide 4 at 0.1 mg / kg and fluconazole (Fluco) at 5 mg / kg in an in vivo model of disseminated candidiasis, similar to the study shown in FIG. [Figure 13]FIG. 13 shows representative results from a study of the effects of the synthetic cyclic tetradecapeptides Cyclic Peptide 5 at 0.25 mg / kg and Cyclic Peptide 1 at 0.1 mg / kg and fluconazole (Fluco) at 5 mg / kg in an in vivo model of disseminated candidiasis, similar to the study shown in FIG. [Figure 14] FIG. 14 shows representative results from a study of the effects of the synthetic cyclic tetradecapeptide cyclic peptide 5 at 0.25 mg / kg and cyclic peptide 2 at 0.1 mg / kg and fluconazole (Fluco) at 5 mg / kg in an in vivo model of disseminated candidiasis, similar to the study shown in FIG. [Figure 15] FIG. 15 shows representative results from a study of the effects of the synthetic cyclic tetradecapeptides cyclic peptide 5 at 0.25 mg / kg and cyclic peptide 6 at 0.1 mg / kg and fluconazole (Fluco) at 5 mg / kg in an in vivo model of disseminated candidiasis, similar to the study shown in FIG. [Figure 16] FIG. 16 shows the results of a fungal clearance study upon treatment with fluconazole (Fluco) and a synthetic cyclic tetradecapeptide of the present concept in a mouse model of disseminated candidiasis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The subject matter of the present invention provides new peptides that induce a two-phase effect in the treatment of fungal infections (e.g., disseminated fungal infections) using a host-mediated process. Such peptides may act by first recruiting effector cells of the immune system to combat the infecting fungal organisms and then modulating the immune system to regulate the inflammatory response. The new peptides are analogs of naturally occurring theta defensins with modified sequences to provide an indirect antifungal effect via recruitment of effector cells of the host immune system and prevent and / or treat sepsis / septic shock. These new theta defensin analogs are effective at sub-antifungal plasma concentrations that do not provide a direct antifungal effect (i.e., do not produce a bactericidal or fungistatic effect when applied at such concentrations in vitro) in the absence of host-specific immune effectors. Such theta defensin analogs may be protective at concentrations where natural theta defensins are ineffective and may contain a core set of structural and sequence features not found in natural theta defensins.

[0024] In the context of this application, a "sub-antifungal concentration" with respect to a fungal pathogen should be understood to be a concentration at which the compound so described does not have an antifungal effect when applied to the fungal pathogen in vitro (e.g., in liquid culture medium) in the absence of, for example, host immune effectors. For example, a sub-antifungal concentration of a compound with respect to Candida albicans is a concentration below the concentration that exhibits an antifungal effect against the fungus in an in vitro environment (e.g., in the absence of host immune effectors).

[0025] Basso et al. (Basso et al., “Rhesus monkey theta defensin 1 promotes long-term survival in systemic candidiasis by a host-directed mechanism” Nature Scientific Reports (2019) 9:16905) provide an example of the determination of sub-antifungal concentrations of the natural theta defensin RTD-1 for heterogeneous strains of Candida albicans. Cultures of heterogeneous strains of Candida albicans were established in RPMI medium or RPMI medium containing 50% serum. Different amounts of fluconazole (Fluco), caspofungin (Caspo), or RTD-1 were applied and fungal growth was monitored. The MFC was determined as the lowest concentration that exerted 99% killing activity against the added inoculum. The MIC was determined as the lowest concentration that inhibited growth. The results are shown in Table 1. [Table 1-1] [Table 1-2] Based on such data, the sub-antimycotic concentration of RTD-1 in the presence of serum for Candida albicans is less than 100 μg / mL. Such sub-antimycotic concentrations can be determined experimentally (e.g., by culture from patient samples) or, preferably, from historical data.

[0026] The following description contains information that may be helpful in understanding the present invention. No admission is made that any information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0027] It should be understood that in some embodiments, numbers expressing properties such as amounts of ingredients, concentrations, reaction conditions, and the like, used to describe and claim certain embodiments of the present invention may be modified by the term "about". Thus, in some embodiments, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practicable. The numerical values ​​set forth in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0028] As used throughout this description and the claims that follow, the meanings of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Also, as used throughout this description, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise.

[0029] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified to satisfy all Markush group descriptions used in the appended claims.

[0030] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually indicating each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided with respect to specific embodiments herein is intended merely to better clarify the invention and does not pose a limitation on the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0031] The following discussion provides a number of exemplary embodiments of the inventive subject matter. Although each embodiment represents a single combination of the invention, it is contemplated that the inventive subject matter includes all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, it is contemplated that the inventive subject matter also includes other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0032] It will be appreciated that the disclosed peptides provide many advantageous technical effects, including providing a two-phase response that is effective in reducing mortality from disseminated or systemic fungal infections and associated sepsis or shock when administered in low, sub-antifungal amounts.

[0033] Recently, Basso et al. (Basso et al., “Rhesus monkey theta defensin 1 promotes long-term survival in systemic candidiasis by a host-directed mechanism,” Nature Scientific Reports (2019) 9:16905) have shown that the naturally occurring theta defensin RTD-1 (SEQ ID NO:1) is effective in animal models of systemic candidiasis of both sensitive and multidrug-resistant strains of Candida albicans. This article is incorporated herein by reference. Although RTD-1 was effective in in vitro studies, the antifungal activity was abolished by the presence of serum and required concentrations more than 50-fold higher than those found to be effective in vivo in mouse animal model studies. These in vivo studies demonstrated both antifungal activity and a long-term reduction in the production of proinflammatory cytokines upon treatment with RTD-1, both of which contribute to recovery from disseminated fungal infections and a reduction in the potential deleterious sequelae of such infections. As shown below, new synthetic analogs of theta defensins may provide similar or improved activity.

[0034] The inventors have developed synthetic cyclic tetradecapeptide analogs of theta defensin RTD-1 that exhibit at least some of the antifungal activity of the parent peptide despite their smaller size and reduced number of disulfide bonds. The structure of RTD-1 is shown in Figure 1. As shown, RTD-1 (naturally expressed in rhesus monkeys) is a cyclic octadecapeptide that contains three pairs of cysteines linked by disulfide bonds that run through the cyclic primary structure of the peptide.

[0035] Numerous examples of synthetic (i.e., non-naturally occurring) analogs of RTD-1 are shown in Figures 2-6 and 8. Figure 2 shows the cyclic structure of theta defensin analog cyclic peptide 1 (SEQ ID NO:2). Figure 3 shows the cyclic structure of theta defensin analog cyclic peptide 2 (SEQ ID NO:3). Figure 4 shows the cyclic structure of theta defensin analog cyclic peptide 3 (SEQ ID NO:4). Figure 5 shows the cyclic structure of theta defensin analog cyclic peptide 4 (SEQ ID NO:5). Figure 6 shows the cyclic structure of theta defensin analog cyclic peptide 5 (SEQ ID NO:6), used as a model compound in these studies. Figure 8 shows the cyclic structure of theta defensin analog cyclic peptide 6 (SEQ ID NO:7). Each of the exemplary synthetic analogs is a tetradecapeptide that contains two pairs of cysteines linked by disulfide bonds. These disulfide bonds pass through the cyclic primary structure of the synthetic peptides to form a "box" structure that incorporates additional amino acids. It should be understood that these exemplary analogs exhibit varying degrees of sequence identity with RTD-1, and in some cases, conservative amino acid substitutions near and between the cysteine-defined "boxes" of the synthetic peptide analogs.

[0036] The inventors have prepared and screened a series of θ-defensin analogs that have substantial in vivo antifungal activity and result in long-term survival of mice in a model of disseminated candidiasis. These effects are at surprisingly low concentrations, far below those at which direct antifungal activity has been found in in vitro model pathogens. Without being bound by theory, the inventors believe that the observed antifungal effects are due to modulation of host immune effectors. It should be understood that long-term survival of disseminated fungal infections requires both control of the infecting organism and control of the shock induced by the host response to the infection, either of which may result in death.

[0037] Although examples of activity against disseminated fungal infections are provided, the inventors believe that the theta defensin analogs described herein may be effective in treating other fungal infections, such as localized fungal infections (e.g., thrush). In addition, the inventors believe that the theta defensin analogs described herein may be used to treat a variety of conditions, including chronic conditions, that result from dysregulation of immune or inflammatory responses. Examples of such chronic conditions include rheumatoid arthritis and inflammatory bowel disease.

[0038] The inventors note that theta defensins have been found to have antiviral activity, and believe that theta defensin analogs of the inventive concept may similarly provide antiviral activity and may prove useful in treating viral diseases and the inflammatory sequelae of viral infections. Such treatment includes prevention and / or ongoing disease. In some embodiments, the ongoing disease so treated is symptomatic. In other embodiments, the ongoing disease so treated is asymptomatic.

[0039] Surprisingly, it has been found that theta defensin analogs provide a two-phase response in modulating the immune system in response to systemic fungal infection. The initial effect is neutrophil recruitment, resulting in clearance of the fungal pathogen. This is effective against infection, and surprisingly, was found to occur at concentrations of theta defensin analogs that do not show antifungal effects against in vitro model pathogens. Following this initial recruitment effect, these synthetic theta defensin analogs show long-term immunomodulatory effects (e.g., reducing TNF, IL-6, and other inflammatory cytokines) that contribute to long-term survival and prevention of septic shock due to disseminated fungal infection.

[0040] As mentioned above, examples of naturally occurring theta defensins and exemplary theta defensin analogs are shown in Figures 1-6 and 8. It should be understood that these cyclic peptides are cyclized via a peptide backbone and thus lack traditional amino and carboxyl termini. Thus, the amino acid sequence information provided in the attached amino acid sequence listing should not be construed as a description of the individual N- or C-termini of these theta defensin analogs. Within the context of this application, amino acid positions are identified using numerical designations based on the common structural features of theta defensin analogs, as shown in Figure 7A. As shown, each position along the cyclic tetradecapeptide chain has a numerical designation. Application of this numbering scheme to the model synthetic cyclic tetradecapeptide cyclic peptide 5 (shown in Figure 6) is shown in Figure 7B. For such 14 amino acid analogs, it should be understood that their three-dimensional structures are designated using a numbering system adapted for use with cyclic theta defensins and their analogs, as shown in Figures 7A and 7B, and include a first β-turn formed by amino acids 6 to 9 and a second β-turn formed by amino acids 13, 14, 1, and 2.

[0041] Suitable cyclic tetradecapeptides can be identified by screening against a mouse model of disseminated candidiasis. Candida albicans SC5314 obtained from the American Type Culture Collection can be used as a suitable reference strain. In a preferred embodiment, resistant Candida albicans and / or one or more strains of Candida albicans that are resistant to two or more antifungal drugs can be used. Representative antifungal drugs include caspofungin and fluconazole. The cyclic tetradecapeptides and antifungal drugs to be tested can be suspended or dissolved in water or isotonic saline and administered by subcutaneous, intramuscular, intravenous and / or intraperitoneal injection.

[0042] In vitro activity of synthetic cyclic tetradecapeptides and antifungal compounds may be measured using conventional culture techniques known in the art, as described above for RTD-1, and may be used to measure subfungal concentrations. Systemic or disseminated candidiasis may be treated, for example, by infusing inbred BALB / c or outbred CD-1 female mice with 0.15 mL to 2 mL of Candida albicans (reference or resistant strains) at approximately 2 × 10 5 From about 2 × 10 7 Candidiasis can be modeled in vivo by administering up to 100 CFU / mL of bacteria. Animals can be treated with the candidate synthetic cyclic tetradecapeptide prior to, during, or after pathogen challenge. Antifungal agents and / or candidate synthetic cyclic tetradecapeptides can be administered subcutaneously, intramuscularly, intravenously, and / or intraperitoneally in such in vivo models of systemic or disseminated candidiasis.

[0043] The inventors have identified a number of new theta defensin analogs that exhibit significant antifungal activity in vivo. The amino acid sequences of exemplary cyclic peptides are shown in Table 2. It should be understood that the identity of the amino acids is indicated using the numerical designation of the corresponding positions within the cyclic structure established in Figure 7A. [Table 2] The peptides cyclic peptide 1, cyclic peptide 2, cyclic peptide 3, and cyclic peptide 4 exhibit common structural features with cyclic peptide 5, which the inventors believe are found in common with other synthetic cyclic tetradecapeptides that exhibit antifungal and anti-inflammatory activity in in vivo models of disseminated fungal disease.

[0044] Cyclic peptide 6 differs significantly from model peptide cyclic peptide 5 in that it sandwiches an arginine between the cysteines involved in the peptide's disulfide bonds (i.e., within a "CXC box") and does not contain a triplet of consecutive (i.e., adjacent) arginines within the first β-turn defined by amino acids 6, 7, 8, and 9 in Table 2. The inventors believe that cyclic peptide 6 represents a distinct family of synthetic cyclic tetradecapeptide theta defensin analogs from those represented by cyclic peptide 1, cyclic peptide 2, cyclic peptide 3, and cyclic peptide 4. The inventors further believe that synthetic cyclic tetradecapeptides that contain multiple arginines within a CXC box structure separated by two pairs of disulfide-bonded cysteines and / or lack the characteristic triplet of consecutive / adjacent arginines at positions 6, 7, and 8 may have significant antifungal activity in an in vivo model of disseminated fungal infection.

[0045] In activity studies, a synthetic cyclic tetradecapeptide, cyclic peptide 5 (SEQ ID NO: 6), which was initially identified as having significant antifungal activity, was used as a model peptide. Briefly, 7- to 8-week-old immunocompetent female BALB / c mice were inoculated with 3 × 10 5 CFU of Candida albicans SC5314 were administered intravenously at T=0. 24 hours after infection, mice were administered saline, fluconazole (Fluco), caspofungin (Caspo), or a synthetic cyclic tetradecapeptide intraperitoneally once daily for 7 days. The inventors had previously determined that the model peptide cyclic peptide 5 was substantially more potent than the native theta defensin RTD-1 in this in vivo model, such that 0.25 mg / kg of cyclic peptide 5 was more effective than 5 mg / kg of RTD-1. Both peptides were more effective than 5 mg / kg of fluconazole (see Figure 9). However, reducing the dose of cyclic peptide 5 peptide to 0.1 mg / kg did not result in improved survival (see Figure 10).

[0046] Candidate synthetic cyclic tetradecapeptides were pre-screened for tolerance by measuring lack of toxicity when administered at concentrations above 5 mg / kg. Candidate synthetic cyclic tetradecapeptides were screened for efficacy in the candidiasis model described above, comparing each candidate to the cyclic peptide 5 reference peptide and fluconazole, at daily doses of each peptide (0.1 and 0.5 mg / kg), for 7 days, beginning 24 hours after infection.

[0047] Under these study protocols, saline-treated Candida albicans-infected mice exhibited disheveled fur and significant weight loss and became moribund within 5 to 10 days, by which time they had lost more than 30% of their body weight. In contrast, long-term surviving Candida-septic mice treated with cyclic peptide 5 temporarily lost an average of 15% of their body weight, which plateaued by day 10, with 90% of this cohort regaining their initial body weight by day 3).

[0048] Using a Candida sepsis model and survival as an indication of efficacy, we identified a number of synthetic cyclic tetradecapeptides that were comparable or better than cyclic peptide 5. Among these were cyclic peptide 3, cyclic peptide 4, cyclic peptide 1, cyclic peptide 2, and cyclic peptide 6. Results for these in the in vivo disseminated candidiasis model are shown in Figures 10 through 15.

[0049] FIG. 10 shows representative results from a study using cyclic peptide 5 at 0.25 mg / kg or 0.1 mg / kg. Cyclic peptide 5 was found to be relatively ineffective, with results similar to saline administration. FIG. 11 shows representative results from a comparison between cyclic peptide 5 at 0.25 mg / kg and cyclic peptide 3 at 0.1 mg / kg. Cyclic peptide 3 was found to be effective at this relatively low dose. FIG. 12 shows representative results from a comparative study of cyclic peptide 5 and cyclic peptide 4, both peptides were used at 0.25 mg / kg. Cyclic peptide 4 is effective at this dose. FIG. 13 shows representative results from a comparison between cyclic peptide 5 and cyclic peptide 1, both peptides were applied at 0.25 mg / kg. Cyclic peptide 1 is effective at this dose. FIG. 14 shows representative results from a comparative study of cyclic peptide 5 at 0.25 mg / kg and cyclic peptide 1 at 0.1 mg / kg. Cyclic peptide 1 is effective at this relatively low dose.

[0050] As mentioned above, cyclic peptide 6 differs from other peptides cited herein in the lack of a triplet of consecutive (i.e., adjacent) arginine residues in the characteristic β-turn portion of the peptide (defined by AA6, AA7, AA8, and AA9) and in the presence of a highly basic arginine residue rather than a hydrophobic amino acid in the characteristic CXC box of this family of cyclic peptides. Figure 15 shows representative data from a comparative study between cyclic peptide 5 and cyclic peptide 6, where the peptides were used at 0.25 mg / kg. Cyclic peptide 6 was found to result in a survival rate that exceeded the results of both prior art techniques (antifungal drug (fluconazole) and cyclic peptide 5).

[0051] In each case, the specific synthetic cyclic tetradecapeptide enhanced survival, and the effect was highly significant (P < 1 × 10 -5 , log-rank test). Cyclic peptide 3, cyclic peptide 4, cyclic peptide 1, and cyclic peptide 6 were analyzed by end-point analysis (χ 2All of the identified synthetic tetradecapeptides significantly inhibited weight loss in this in vivo model.

[0052] Renal fungal burden was measured in kidney homogenates from moribund saline-treated controls (5 to 10 days (pi)) and long-term survivors treated with synthetic cyclic tetradecapeptide or fluconazole (30 days pi). As shown in Figure 16, synthetic cyclic tetradecapeptide (0.1 or 0.25 mg / kg) and fluconazole at 5 mg / kg reduced fungal burden. Cyclic peptide 5, cyclic peptide 3, cyclic peptide 4, and cyclic peptide 1 reduced fungal burden to a greater extent than fluconazole (asterisks in Figure 16; analyzed by Fisher's LSD test: cyclic peptide 5 (P=3×10 -3 ), cyclic peptide 3 (P = 7.4 × 10 -3 ), cyclic peptide 4 (P = 0.02), and cyclic peptide 1 (P = 3.5 × 10 -5 ).

[0053] A number of sequence features were identified that confer superior activity to analogs derived from RTD-1 and cyclic peptide 5 compared to these reference peptides. As discussed above, cyclic peptide 3, cyclic peptide 4, cyclic peptide 1, and cyclic peptide 2 represent a group of synthetic cyclic tetradecapeptide theta defensin analogs that exhibit distinct structural similarities. The inventors believe that theta defensin analogs within this family that have significant antifungal and / or anti-inflammatory activity may have at least the following: Two disulfide bonds (between cysteine ​​3 and cysteine ​​12, and between cysteine ​​5 and cysteine ​​10, respectively). a hydrophobic amino acid located between cysteine ​​3 and cysteine ​​5 and between cysteine ​​10 and cysteine ​​12 (i.e., positions 4 and 11) in the primary structure of a serine or theta defensin analog, the hydrophobic amino acid being preferably leucine or isoleucine. In combination with the disulfide bonds described above, this defines a feature within the cyclic primary structure of the peptide as a "CXC box", where "C" is cysteine ​​and "X" is serine, leucine, or isoleucine. A total of five arginine residues resulting in the peptide having a +5 charge at physiological pH. · A triplet of adjacent arginines present at positions 6, 7, and 8 (i.e., within the first β-turn). In some embodiments, active theta defensin analogs may also include one or more of the following features. Glycine present at position 1. A hydrophobic amino acid at position 2 and position 9, preferably valine or leucine. · An arginine pair within the second β-turn (eg, positions 13 and 14).

[0054] The toxicity of the candidate peptides suggests that an active theta defensin analog should not contain one or more of the following: · Alanine at position 4. · Alanine at position 11.

[0055] Thus, the inventors believe that synthetic cyclic tetradecapeptide theta defensin analogs containing the above-described "CXC box" structure, a triplet of adjacent arginine residues at positions 6, 7, and 8, a hydrophobic amino acid (e.g., valine or phenylalanine) at position 9, and having a net positive charge of +5 due to the arginine content (approximately 36% of the total amino acid content) may be effective in reducing mortality and / or improving long-term survival in disseminated fungal infections, and in treating other conditions characterized by dysregulation of inflammatory or immune responses.

[0056] The inventors believe that cyclic peptide 6 represents a distinct family of synthetic cyclic tetradecapeptide analogs of theta defensins that have all or some of the above characteristics with the following exceptions. · Presence of multiple amino acids with two or more positive charges (e.g., arginine, lysine) in the CXC box. Absence of consecutive (i.e. adjacent) arginine triplets within the second β-turn defined by AA6, AA7, AA8, and AA9.

[0057] As described herein, the synthetic cyclic tetradecapeptide analogs of theta defensins can be administered using any suitable method. For example, such analogs can be provided by injection or infusion. The high degree of efficacy observed for some theta defensin analogs indicates that they can be provided to individuals in need of administration in effective amounts by simple subcutaneous, intradermal, subcutaneous, and / or intramuscular injection.

[0058] Alternatively, the low molecular weight and high stability afforded by the presence of cyclic structures and disulfide bonds may allow oral administration of the theta defensin analogue of the inventive concept. Such oral administration may include administration of a solution or suspension of the theta defensin analogue in a liquid pharmaceutical carrier suitable for oral administration. In some embodiments, the theta defensin analogue may be provided in a dried or lyophilized form that is reconstituted in a liquid medium prior to oral administration. Such dried or lyophilized formulations may include stabilizers. Such stabilizers include sugars (e.g., mannitol, sucrose, trehalose) and / or proteins (e.g., albumin).

[0059] Alternatively, the analog of theta defensin may be provided in a tablet, capsule, pill, or other suitable solid and compact form for oral administration. Such formulations may include coatings, shells, or similar components that result in delayed release of theta defensin analog (e.g., delayed release until reaching the small intestine). Such formulations may include theta defensin in liquid form within an enclosure or coating. Alternatively, such formulations may include theta defensin analog in a dried or lyophilized form. Suitable dried or lyophilized forms include powders, granules, and compressed solids. Such dried or lyophilized formulations may include stabilizers. Suitable stabilizers include sugars (e.g., mannitol, sucrose, trehalose) and / or proteins (e.g., albumin).

[0060] As described above, the theta defensin analogs of the inventive concept can effectively treat disseminated fungal infections and associated sepsis and / or septic shock. In some embodiments, such treatment is responsive to an ongoing acute condition. In other embodiments, such treatment is prophylactic, for example, used to inhibit the development of disseminated fungal infections when an individual is suspected of having a disseminated fungal infection or is likely to develop this condition. Treatment can be provided by administration of the theta defensin analogs of the inventive concept in any suitable schedule. For example, the theta defensin analogs can be provided as a single dose, periodic doses, or continuous infusion. Periodic doses can be administered at any suitable interval. Periodic intervals can be hourly, every 2 hours, every 4 hours, 4 times a day, 3 times a day, twice a day, once a day, every 2 days, every 3 days, twice a week, every week, every 2 weeks, every 4 weeks, every 2 months, every 3 months, every 4 months, 3 times a year, twice a year, or annually.

[0061] In some embodiments, the method of administration of the theta defensin analog may be altered during treatment. For example, the theta defensin analog of the inventive concept may be administered initially by intravenous injection or infusion (e.g., to rapidly provide effective concentrations in acute disseminated fungal infections), and later by intradermal injection, intramuscular injection, and / or oral administration to maintain effective concentrations for the remainder of treatment.

[0062] For prophylactic use, theta defensin analogs can be administered before observable symptoms occur. For treatment of ongoing diseases or conditions, theta defensin analogs can be administered for a period of time appropriate to effectively treat the disease or condition. Such a period can be for a controlled period of time or can be long term (e.g., for treatment of chronic conditions).

[0063] In some embodiments of the inventive concept, the theta defensin analog may be used in combination with other pharmacologic active compounds. Suitable compounds include theta defensins, different theta defensin analogs, antifungal antibiotics, antibacterial antibiotics, antivirals, anti-inflammatory drugs (e.g., steroids, nonsteroidal anti-inflammatory drugs), hypertensives, and / or biologics (e.g., antibodies or antibody fragments). Such additional pharmaceutical compounds may be provided on the same schedule as the theta defensin analog or on an independent schedule. In some embodiments, a theta defensin analog-containing formulation may be provided incorporating one or more of such additional pharmacologic active compounds. The inventors believe that such a combination therapy may provide a synergistic effect in which the cumulative effect of administering a theta defensin analog in combination with an additional pharmacologic active compound exceeds the sum of the individual effects observed in treatment with the theta defensin analog and the additional pharmacologic active compound in amounts corresponding to the amounts used for the combination therapy.

[0064] It should be apparent to those skilled in the art that many other modifications beyond those already described are possible without departing from the inventive concept herein. Thus, the subject matter of the present invention should not be limited except in the spirit of the appended claims. In addition, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprising" should be interpreted as indicating elements, components, or steps in a non-exclusive manner, indicating that a referenced element, component, or step may be present, used, or combined with other elements, components, or steps not expressly referenced. When the specification and claims refer to at least one selected from the group consisting of A, B, C... and N, the language should be interpreted as requiring only one element from the group, not A+N, or B+N, etc.

Claims

1. A cyclic peptide consisting of 14 amino acids and having the following structure: 【Chemistry 1】 A cyclic peptide, wherein AA3 and AA12 are cysteines linked by a disulfide bond, AA5 and AA10 are cysteines linked by a disulfide bond, AA4 is arginine, AA11 is arginine, two of AA6, AA7, and AA8 are arginines, the β-turn portion of the cyclic peptide defined by AA6, AA7, AA8, and AA9 does not contain more than two adjacent arginines, and the cyclic peptide comprises five or more arginine residues that provide a positive charge content of at least about 36% at physiological pH.

2. The cyclic peptide of claim 1, wherein AA1 is glycine.

3. 2. The cyclic peptide of claim 1, wherein AA2 is the first hydrophobic amino acid.

4. The cyclic peptide of claim 3 , wherein the first hydrophobic amino acid is valine.

5. 2. The cyclic peptide of claim 1, wherein AA9 is the second hydrophobic amino acid.

6. The cyclic peptide of claim 5 , wherein the second hydrophobic amino acid is valine.

7. 2. The cyclic peptide of claim 1, wherein AA13 is arginine.

8. 2. The cyclic peptide of claim 1, wherein AA14 is arginine.

9. A method of use of a cyclic peptide in treating or preventing disseminated fungal disease and associated sepsis or septic shock, said cyclic peptide having the following common structural diagram: 【Chemistry 2】 AA3 and AA12 are cysteines linked by a disulfide bond, AA5 and AA10 are cysteines linked by a disulfide bond, AA4 is arginine, AA11 is arginine, two of AA6, AA7, and AA8 are arginines, the β-turn portion of the cyclic peptide defined by AA6, AA7, AA8, and AA9 does not contain more than two adjacent arginines, and the cyclic peptide comprises five or more arginine residues resulting in a positive charge content of at least about 36% at physiological pH.

10. 10. The method of claim 9, wherein AA1 is glycine, or AA2 is a first hydrophobic amino acid, or AA9 is a second hydrophobic amino acid, or AA13 is arginine, or AA14 is arginine.

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